Reducing the Stimulation of CD8+ T Cells during Infection with Intracellular Bacteria Promotes Differentiation Primarily into a Central (CD6
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免疫学杂志 2005年第9期
Abstract
During infection with lymphocytic choriomeningitis virus, CD8+ T cells differentiate rapidly into effectors (CD62LlowCD44high) that differentiate further into the central memory phenotype (CD62LhighCD44high) gradually. To evaluate whether this CD8+ T cell differentiation program operates in all infection models, we evaluated CD8+ T cell differentiation during infection of mice with recombinant intracellular bacteria, Listeria monocytogenes (LM) and Mycobacterium bovis (BCG), expressing OVA. We report that CD8+ T cells primed during infection with the attenuated pathogen BCG-OVA differentiated primarily into the central subset that correlated to reduced attrition of the primed cells subsequently. CD8+ T cells induced by LM-OVA also differentiated into central phenotype cells first, but the cells rapidly converted into effectors in contrast to BCG-OVA. Memory CD8+ T cells induced by both LM-OVA as well as BCG-OVA were functional in that they produced cytokines and proliferated extensively in response to antigenic stimulation after adoptive transfer. During LM-OVA infection, if CD8+ T cells were guided to compete for access to APCs, then they received reduced stimulation that was associated with increased differentiation into the central subset and reduced attrition subsequently. Similar effect was observed when CD8+ T cells encountered APCs selectively during the waning phase of LM-OVA infection. Taken together, our results indicate that the potency of the pathogen can influence the differentiation and fate of CD8+ T cells enormously, and the extent of attrition of primed CD8+ T cells correlates inversely to the early differentiation of CD8+ T cells primarily into the central CD8+ T cell subset.
Introduction
The CD8+ T cell is stimulated when peptides from endogenously derived Ags (intracellular bacteria, viruses, or tumors) are presented on MHC class I molecules (1). After differentiation, effector CD8+ T cells possess the unique ability to mediate specific cytotoxicity (by perforin and Fas-dependent pathways) toward infected cells and tumors (2, 3, 4). However, the vast majority (>95%) of Ag-specific T cells activated at the onset of the immune response die by apoptosis, and only a small portion of those T cells survives (<5%) for extended periods (5, 6, 7). These long-lived memory T cells possess the unique ability to respond rapidly and specifically to Ags (8, 9).
Various cell surface markers have been used to identify memory T cells (9, 10, 11); however, CD44 appears to be the most reliable marker that is expressed at high levels in all memory T cells of mice irrespective of their activation status (8, 12). During lymphocytic choriomeningitis virus (13) and Sendai virus (14) infection of mice, it was reported that memory CD8+ T cells segregate into two distinct populations: a CD44highCD62Llow population that is predominately located in the spleen and exerts a rapid effector function (effector memory), and a CD44highCD62Lhigh population that is found in the spleen and the lymph nodes with no immediate effector function (central memory). In humans, a phenotypic and functional model of the subsets of memory CD4+ and CD8+ T cells was proposed based on expression of CD62L and CCR7 (15). Based on this model, tissue-homing effector memory T cells (CD62LlowCCR7–), capable of immediate effector function, were separated from the lymph node-homing central memory T cells (CD62LhighCCR7+), which were devoid of effector activity in vitro. Several recent reports have confirmed the presence of distinct effector vs central memory T cell subsets (16, 17), and shown that effector memory T cells selectively extravasate into nonlymphoid compartment (18) to provide a first line of defense against pathogens. Both of the subsets of memory cells are present in the blood and spleen (8).
In this study, we have evaluated CD8+ T cell differentiation and fate during infection of mice with the intracellular bacteria, Listeria monocytogenes (LM)3 and Mycobacterium bovis (BCG). We show that BCG induces a highly attenuated stimulation of naive CD8+ T cells that differentiate primarily into the central CD8+ T cell subset and undergo minimal attrition subsequently. Similar differentiation program also operates when naive CD8 T cells compete for access to APCs, or they encounter APCs selectively during the waning phase of LM-OVA infection. Our results indicate that the differentiation and fate of naive CD8+ T cells are profoundly influenced by the potency of the immunogen.
Materials and Methods
Bacterial strains
BCG-OVA is a previously described recombinant strain (19) engineered with a partial sequence of the OVA gene (codons 230–359), downstream of the Ag 85B secretion signal (20), under the control of heat shock protein 60 promoter (21). Codons 230–359 of OVA gene encode the SIINFEKL epitope (OVA257–264) and its flanking sequences (22). Single colonies were used to inoculate liquid cultures, which were incubated at 37°C under constant shaking in 7H9 medium containing glycerol (0.2%), Tween 80 (0.05%), and albumin-dextrose supplement (10%; Difco Laboratories). At mid-log phase (OD600 = 1.0), bacteria were harvested and frozen at –80°C (in 20% glycerol). CFU were determined by plating serial dilutions in PBS-T (0.025% Tween 80) on Middlebrook 7H10 solid medium containing glycerol (0.5%) and oleic acid-albumin-dextrose supplement (10%; Difco Laboratories).
OVA-expressing LM (LM-OVA), as described previously (19), was grown to OD600 nm = 0.4. The bacteria were grown in brain heart infusion medium (Difco Laboratories), supplemented with 50 μg/ml streptomycin (Sigma-Aldrich). At mid-log phase (OD600 = 1.0), bacteria were harvested and frozen in 20% glycerol and stored at –80°C. CFUs were determined by performing serial dilutions in 0.9% NaCl, which were spread on brain heart infusion-streptomycin agar plates.
Mice and immunizations
Female C57BL/6 and BALB/c mice, 6–8 wk of age, were obtained from The Jackson Laboratory. Mice were maintained in the animal facility at the Institute for Biological Sciences (National Research Council of Canada) in accordance with the guidelines of the Canadian Council on Animal Care. For immunizations with LM-OVA, frozen stocks were thawed and diluted in 0.9% NaCl, and mice were inoculated with 1 x 104 LM-OVA suspended in 200 μl of 0.9% NaCl, via the lateral tail vein (i.v.). For BCG-OVA, frozen stocks were thawed and diluted in PBS containing 0.025% Tween 80, and injections were conducted through the i.v. route. Age-matched control mice were inoculated with 200 μl of PBS. B6.PL (Thy-1.1+), B6.SJL (CD45.2–), and OT-1 transgenic mice (CD45.2+Thy-1.2+ and expressing the TCR of OVA257–264) were obtained from The Jackson Laboratory.
Assessment of bacterial burden in spleen
Single cell suspensions from infected mice were tweezed in RPMI 1640, and an aliquot of the suspension was lysed with water for 30 s, and then evaluated for the numbers of viable bacteria. CFUs were determined by plating 100-μl aliquots of serial 10-fold dilutions in 0.9% saline on appropriate plates, as above.
CD8+ T cell purification
Single cell suspensions were prepared by tweezing the pooled spleens (n = 2–3) between the frosted ends of two sterile glass slides in RPMI 1640. Cells were subsequently passed through Falcon 2360 cell strainers (BD Discovery Labware), centrifuged, and resuspended (20 x 106/ml) in 0.5–1 ml of RPMI 1640 containing 8% FBS (HyClone) and 50 μg/ml gentamicin (Invitrogen Life Technologies), hereafter refer to as R8-A. CELLection Biotin Binder Dynabeads, precoated as per manufacturer’s instructions (Dynal Biotech) with biotin-conjugated rat anti-mouse CD8.2 mAb (53.5.8; BD Biosciences), were added to the resuspended cell pellet at a ratio of five beads per cell, and incubated for 15–20 min at 4°C in a rotating platform. CD8+ T cells were separated by magnetic isolation. Dynabead detachment was done using the CELLection Biotin Binder kit Releasing Buffer (DNase; 188 U/108 Dynabeads) in 37°C shaker for 15 min, followed by two to three rounds of washing/magnetic separation. This protocol resulted in >95% pure CD8+ T cells, as determined by follow-up analysis with PE-conjugated rat anti-mouse CD8 (BD Biosciences). Analysis was performed using EPICS XL flow cytometer and EXPO software (Beckman Coulter).
Assessment of the fate and phenotype of Ag-specific CD8+ T cells
For evaluation of the fate and phenotype of OVA257–264-specific CD8+ T cells in vivo, aliquots (10 x 106) of spleen cells were incubated in 200 μl of PBS plus 1% BSA with anti-CD16/32 at 4°C. After 10 min, cells were stained with various Abs (anti-CD8, anti-CD62L, anti-IL-2R, anti-IL-2R, anti-IL-2R, anti-IL-7R, anti-CD69, anti-mouse CD44, anti-Thy-1.2, and anti-CD45.2) for 30 min, on ice. All Abs were obtained from BD Biosciences. Cells were then washed with PBS-BSA and incubated for 30 min with PE-H-2KbOVA257–264 tetramer (Beckman Coulter) at room temperature. Cells were washed with PBS, fixed in 0.5% formaldehyde, and acquired on EPICS XL flow cytometer.
Assessment of intracellular IFN- in Ag-specific CD8+ T cells
Aliquots of spleen cells (10 x 106/ml) were stained with anti-CD8 and H-2KbOVA257–264 tetramer for 30 min, as described above. Cells were then washed, reconstituted in R8, plated into 96-well plates (2 x 106/well), and stimulated with OVA257–264 peptide (1 μg/ml) in the presence of Golgi-stop (BD Biosciences). After 1 or 4 h, cells were harvested, washed, permeabilized, and stained for intracellular IFN- using the IFN- staining kit (obtained from BD Biosciences). Cells were acquired on EPICS XL flow cytometer and analyzed using the EXPO software.
Results
Priming by the highly attenuated pathogen BCG causes minimal attrition
We have previously reported that BCG-OVA induces highly attenuated levels of inflammation (23) and CD8+ T cell memory (19) in comparison with that induced by LM-OVA. CD8+ T cell response induced by BCG-OVA peaked at day 21 in comparison with day 7 during LM-OVA infection model. Based on a consensus of a number of experiments conducted by us, it appears that when fold drop in the overall numbers of OVA257–264-specific memory CD8+ T cells is evaluated over time, a 10-fold attrition in the response is noted during BCG-OVA infection in comparison with >75-fold attrition that is noted during infection with LM-OVA. Similarly, we have reported that BCG-OVA infection results in a delayed and muted Ag presentation (24). LM-OVA induced a massive reduction in CFSE intensity of donor OT-1 CD8+ T cells (OVA257–264 TCR transgenic cells) within the first 4 days, with >80% of donor CD8+ T cells reducing their CFSE expression. When OT-1 donor CD8+ T cells were transferred on day 7 after LM-OVA infection, a significant reduction in CFSE staining was still detectable (20%). Thus, Ag presentation during LM-OVA infection occurs rapidly, and interestingly, it persists at reduced levels for 7–10 days. In contrast, BCG-OVA infection induced a low level Ag presentation during the first 4 days, peaked during the second week of infection, and declined to very low levels subsequently (24).
Phenotype and cytokine secretion of CD8+ T cells induced by BCG-OVA
Considering the attenuated Ag presentation and T cell priming during BCG-OVA infection, we sought to determine the phenotype of the primed CD8+ T cells. Because CD62L is considered as a key marker that segregates the effector and central CD8+ T cell subsets (25), we evaluated the expression of CD62L during CD8+ T cell differentiation. During both LM-OVA and BCG-OVA infections, the transferred OT-1 cells down-regulated CD62L expression only after multiple rounds of division (Fig. 1A), and interestingly, the majority of CD8+ T cells that differentiated during BCG-OVA infection did not down-regulate CD62L.
FIGURE 1. Differentiation of Ag-specific CD8+ T cells during LM-OVA and BCG-OVA infection. B6.PL (Thy-1.2–) mice were injected with CFSE-labeled OT-1 CD8+ T cells (3 x 106) and 104 LM-OVA or BCG-OVA. The proliferation of donor Thy-1.2+ tetramer+ CD8+ T cells was evaluated on day 4 for LM-OVA and day 7 for BCG-OVA (A). C57BL/6J mice were injected with OT-1 cells (105, i.v.) and challenged (104, i.v.) with LM-OVA (B and D) or BCG-OVA (C and E), and the phenotypic distribution of the effector (CD62LlowCD44high) vs central subsets of (CD62LhighCD44high) tetramer+ CD8+ T cells was evaluated. Data are representative of two to three mice per group.
At day 5 after LM-OVA infection and OT-1 transfer, 42% of the OVA257–264-specific CD8+ T cells displayed a central phenotype (CD62LhighCD44high) (Fig. 1B), and the percentages of these cells decreased to 18% on day 7 and then increased progressively at subsequent time intervals. In contrast, the effector cells (CD62LlowCD44high) peaked at day 7 and then progressively decreased at subsequent time intervals, indicative of conversion into the central phenotype. The relative induction of the central and effector CD8+ T cell subsets during LM-OVA infection is indicated in Fig. 1D. During infection of mice with BCG-OVA, the majority of cells differentiated primarily into the central CD8+ T cell subset (Fig. 1, C and E), where they remained parked for prolonged periods without down-regulating CD62L expression.
We measured intracellular cytokine production by OVA-specific CD8+ T cells at day 60 after infection. OVA257–264-specific CD62Lhigh population induced by LM-OVA as well as BCG-OVA expressed IL-2 in comparison with the CD62Llow population that expressed IFN-, but little IL-2 (Fig. 2, A and B), suggesting that the OVA257–264-specific CD62Lhigh cells exhibit potent proliferative potential that is typical of central CD8+ T cell subset (15, 25). Cells in both the effector and central subset did not exhibit any differences in their ability to produce IFN-, which has also been reported by various investigators (25, 26). To further substantiate the difference in the IL-2 expression by the central vs effector CD8+ T cell subsets, we sorted the central and effector CD8+ T cell subsets of LM-OVA-infected mice and measured the production of IL-2 in the supernatants after stimulation with OVA257–264-pulsed dendritic cells. Central phenotype CD8+ T cells produced higher levels of IL-2 in comparison with the effector phenotype cells (Fig. 2C). We also measured in vitro cytolytic activity of purified CD8+ T cells of LM-OVA- and BCG-OVA-infected mice on day 30 in the absence or presence of IL-2. Addition of exogenous IL-2 resulted in a moderate enhancement of cytolytic activity of CD8+ T cells of both LM-OVA- and BCG-OVA-infected mice, whereas neutralization of endogenously produced IL-2 abrogated the cytolytic activity in both the groups of mice (Fig. 2, D and E).
FIGURE 2. Effector memory CD8+ T cells express reduced levels of IL-2. C57BL/6J mice were injected with 105 OT-1 cells, and challenged (104, i.v.) with LM-OVA (A) or BCG-OVA (B). At day 60, spleens were removed and stained with OVA-tetramer and anti-CD62L Ab. Cells were then stimulated in vitro with OVA257–264 for 1 h, and the expression of intracellular IL-2 and IFN- was evaluated on gated OVA-tetramer+ CD8+ T cells to determine the expression of these two cytokines by the CD62Llow vs CD62Lhigh subsets of CD8+ T cells (A and B). Tetramer+ cells were also sorted into CD62Llow and CD62Lhigh subsets and stimulated in vitro (1 x 105/well) with syngeneic bone marrow-derived dendritic cells (1 x 105/well) pulsed with OVA257–264 (10 ng/ml). Supernatants were collected after 12 h, and the production of IL-2 was measured by ELISA (C). Cytolytic activity of CD8+ T cells from the spleen cells of LM-OVA (D)- and BCG-OVA (E)-infected mice was evaluated on day 60. CD8+ T cells were purified and cultured (106/flask) with syngeneic naive spleen cells (25 x 106/flask) in the presence of OVA257–264 (10 ng/ml) and rat IgG (10 μg/ml), or anti-mouse IL-2 (10 μg/ml), or mouse IL-2 (100 pg/ml). After 5 days, cells were harvested, washed, and evaluated for cytolytic activity against EL-4 cells or EL-4 cells pulsed with OVA257–264. Cytotoxicity on EL-4 cells in the absence of OVA257–264 was <15%. Data are representative of two to three mice per group.
When the expression of intracellular IFN- was measured at various time intervals, BCG-OVA-induced CD8+ T cells expressed lesser levels of IFN- when the cells were stimulated for 1 h with OVA257–264 in vitro (Fig. 3A). When the in vitro stimulation was continued for 4 h, no differences in the levels of IFN- were noted (Fig. 3B) between LM-OVA- and BCG-OVA-primed CD8+ T cells.
FIGURE 3. Intracellular IFN--secreting Ag-specific CD8+ T cells induced by LM-OVA vs BCG-OVA. C57BL/6J mice were injected with OT-1 cells (105, i.v.) and LM-OVA (104, i.v.) or BCG-OVA (104, i.v.). At various time intervals, the numbers of IFN--secreting tetramer+ CD8+ T cells were evaluated after stimulation of cells for 1 h (A) or 4 h with OVA257–264 (B). Numbers in the panels indicate the percentage of IFN--secreting CD8+ T cells among the tetramer+ cells (A). The total number of OVA-specific (tetramer-positive vs IFN--positive) CD8+ T cells per spleen was enumerated (B). Data are representative of two to three mice per group.
Memory CD8+ T cells induced by BCG-OVA proliferate after antigenic stimulation in vivo
We sought to determine whether the OVA-specific memory CD8+ T cells induced by LM-OVA and BCG-OVA exhibit differences in their expansion in response to antigenic stimulus in vivo. To this end, we purified CD8+ T cells on day 30 after challenge of OT-1-parked mice with LM-OVA and BCG-OVA. The percentages of OVA-tetramer+ cells among the purified CD8+ T cells of LM-OVA- and BCG-OVA-infected mice were 10 and 1%, respectively. These cells were transferred into naive recipients that were challenged with LM-OVA or BCG-OVA, and the expansion of the transferred memory cells was measured at various time intervals. Upon transfer to naive mice, LM-OVA-generated memory CD8+ T cells proliferated enormously in response to LM-OVA with 50% of all splenic CD8+ T cells being OVA specific at day 7 after challenge (Fig. 4A, left panel). In contrast, the same LM-OVA-generated OVA-specific memory CD8+ T cells exhibited a delayed and muted proliferation in response to BCG-OVA rechallenge. Memory CD8+ T cells that were induced by BCG-OVA (10-fold less numbers transferred) also proliferated enormously upon transfer into naive recipients and challenge with LM-OVA (Fig. 4B, left panel), suggesting that BCG-OVA-induced memory CD8+ T cells are not functionally impaired in responding to Ag in vivo. When BCG-OVA-induced memory CD8+ T cells were challenged with BCG-OVA, the numbers of OVA-specific CD8+ T cells were below the detection limit, which is expected, considering the reduced numbers of cells transferred and the nature of stimulation induced by BCG-OVA.
FIGURE 4. BCG-OVA-induced memory CD8+ T cells proliferate normally after antigenic stimulation in vivo. C57BL/6J mice were injected with OT-1 cells (105, i.v.) and challenged with LM-OVA (A) or BCG-OVA (B) (104, i.v.). At day 30, spleens were removed and CD8+ T cells were purified. Ten percent of CD8+ T cells from LM-OVA-infected spleens were tetramer+ in comparison with CD8+ T cells from BCG-OVA-infected spleens that were 1% tetramer+. Purified CD8+ T cells (5 x 106/mouse) were then injected into normal B6.SJL (CD45.2–) mice that were challenged (104, i.v.) with LM-OVA or BCG-OVA. At various time intervals, the percentages of donor (CD45.2+) tetramer+ CD8+ T cells were enumerated. Data are representative of three mice per group.
Two important conclusions can be drawn from these data. First, as 10% of the transferred cells home to the spleen, the relative expansion of OVA-specific memory CD8+ T cells induced by LM-OVA (Fig. 4A, left panel) and BCG-OVA (Fig. 4B, left panel) in response to a challenge with LM-OVA appears to be identical (100-fold), suggesting that the memory CD8+ T cells induced by BCG-OVA exhibit the typical attributes of conventional memory CD8+ T cells. Second, memory CD8+ T cells (those induced by LM-OVA) respond to BCG-OVA with protracted kinetics (Fig. 4A, right panel), a situation that is similar to the response of naive CD8+ T cells to BCG-OVA.
CD8+ T cells compete for access to APCs
Considering the weak stimulation evoked by BCG-OVA that promoted differentiation of CD8+ T cells mainly into the central phenotype, we were interested in determining the consequence of reducing the extent of stimulation of naive CD8+ T cells during LM-OVA infection. To this end, we first evaluated the consequences of competition for access to APC on CD8+ T cell priming and stimulation. B6.PL (Thy-1.1+Thy-1.2–) mice were infected with LM-OVA and 104 or 105 or no OT-1 (OVA257–264 TCR transgenic) CD8+ T cells simultaneously (Fig. 5, A and B). Using an anti-Thy-1.2 Ab that binds only to the donor OT-1 cells, and H-2KbOVA257–264 tetramers that bind both endogenous and donor OVA257–264-specific CD8+ T cells, we followed the competition between donor and host OVA257–264-specific CD8+ T cells. In these adoptive transfer experiments, typically 5–10% of the transferred OT-1 cells home to the spleen (our unpublished results). On day 7 after LM-OVA infection, 5% of CD8+ T cells were OVA257–264 specific in the absence of OT-1 transfer (Fig. 5A). When 104 and 105 OT-1 cells were transferred, only 0.5 and 0.1% of OVA257–264-specific CD8+ T cells were of host origin (Thy-1.2–), respectively. Majority of the OVA257–264-specific CD8+ T cells (97–99%) in OT-1-transferred recipients were of donor origin (Thy-1.2+). There was an overall reduction (30- to 300-fold) in the numbers of host (Thy-1.2–) OVA257–264-specific CD8+ T cells in the spleens of OT-1-transferred recipients (Fig. 5B). Because the LM-OVA burden peaks between 48 and 72 h after infection (24) and the bacteria are undetectable in the spleens by days 5–7, we determined whether competition for Ag presentation would still occur during the declining phase of LM-OVA infection. Mice were infected with LM-OVA and injected with PBS or OT-1 CD8+ T cells at 72 h postinfection. Approximately 7% of CD8+ T cells in control mice were OVA257–264 specific, and the percentage of host OVA257–264-specific CD8+ T cells dropped to 0.8% in mice that received OT-1 cells at 72 h postinfection (Fig. 5C). Majority (91%) of OVA257–264-specific CD8+ T cells in OT-1-transferred mice were of donor origin (Thy-1.2+). Overall, there was a 10-fold reduction in the numbers of host OVA257–264-specific CD8+ T cells in the spleens of mice that received OT-1 cells at 72 h (Fig. 5D). OVA257–264-specific CD8+ T cells in the OT-1-transferred group of mice exhibited reduced forward and side scatter in comparison with the mice that received no OT-1 cells (data not shown).
FIGURE 5. CD8+ T cells compete for access to APCs. B6.PL (Thy-1.2–) mice were injected with LM-OVA (104, i.v.) or with LM-OVA (104, i.v.) and OT-1 CD8+ T cells simultaneously (A and B). Spleen cells were removed at day 7 postinfection, and the percentage (A) and numbers (B) of host (Thy-1.2–) and donor (Thy-1.2+) OVA 257–264-specific CD8+ T cells were evaluated. Numbers in A indicate the percentage of tetramer+ cells among CD8+ T cells. In another set of experiments, B6.PL (Thy-1.2–) mice were infected with LM-OVA (104, i.v.) first and injected at 72 h postinfection with PBS or OT-1 cells (105, i.v.). The percentages (C) and numbers (D) of host and donor OVA257–264-specific CD8+ T cells were evaluated. Data are representative of two to three mice per group.
Competition during priming decreases stimulation and increases the survival of CD8+ T cells
We transferred graded doses of OT-1 cells into mice that were simultaneously challenged with LM-OVA. Transfer of increasing doses of OT-1 cells resulted in increased numbers of OVA257–264-specific CD8+ T cells between days 5 and 7 (Fig. 6A). Regardless of the number of OT-1 cells transferred, day 7 was always the peak of the response. There was a dose-dependent correlation between the numbers of OT-1 cells transferred and the overall drop in the numbers of OVA257–264-specific CD8+ T cells subsequently (Fig. 6B). In the absence of any OT-1 transfer, OVA257–264-specific CD8+ T cells underwent maximal contraction, whereas contraction of OVA257–264-specific CD8+ T cells was substantially lower when 105 OT-1 cells were transferred. There was also a dose-dependent correlation between the numbers of OT-1 cells transferred and the blastogenic profile of OVA257–264-specific CD8+ T cells (Fig. 6C). In the absence of any OT-1 transfer, 33% of OVA257–264-specific CD8+ T cells displayed elevated forward and side scatter, and this number dropped to 13% with the transfer of 105 OT-1 cells. The forward and side scatter analysis was performed on gated OVA-tetramer+ CD8+ T cells. Not only was there a difference in the blastogenic profile of OVA-tetramer-positive cells, but the phenotypic profile of the cells was also shifted (Fig. 6D). At day 7 after LM-OVA infection, the numbers of OVA257–264-specific CD8+ T cells of effector phenotype (CD62LlowCD44high) were reduced from 90 to 70% as the initial OT-1 transfer dose was increased from 103 to 105 (Fig. 6D). This initial difference in the degree of stimulation of naive CD8+ T cells influenced the timing of their subsequent conversion into the central phenotype subsequently. At day 30 after LM-OVA infection, majority (65%) of OVA257–264-specific CD8+ T cells of mice that received 103 OT-1 cells still displayed effector (CD62LlowCD44high) phenotype, whereas majority (73%) of OVA257–264-specific CD8+ T cells of mice that received 105 OT-1 cells displayed central memory (CD62LhighCD44high) phenotype (Fig. 6D). Taken together, these results indicate that competition for access to APCs results in reduced stimulation and reduced generation of effector CD8+ T cells, which correlates with reduced contraction of CD8+ T cell response. Furthermore, CD8+ T cells that receive a relatively enhanced stimulation take longer to convert to a central memory state.
FIGURE 6. Transfer of increased numbers of OT-1 cells results in decreased stimulation. C57BL/6 mice were injected with LM-OVA (104, i.v.) and varying numbers of OT-1 cells. The total numbers of tetramer+ CD8+ T cells in the spleens were enumerated at various time intervals (A). Fold drop in the numbers of tetramer+ CD8+ T cells was enumerated by calculating the drop in the total number of OVA-tetramer-positive CD8+ T cells per spleen from day 7 to 30 (B). The percentage of cells exhibiting high forward and side scatter was evaluated on gated tetramer+ cells at day 7 postinfection (C). The relative distribution of effector (CD62LlowCD44high) vs central (CD62LhighCD44high) CD8+ T cell subsets was evaluated on gated tetramer+ CD8+ T cells at days 7 and 30 postinfection (D). Numbers in the panels indicate the percentage of cells within the indicated gate. Data are representative of two to three mice per group.
To determine the mechanisms involved, we first measured the bacterial burden in mice that received PBS or OT-1 cells and noted increased clearance of LM-OVA, particularly at later time periods in mice that received OT-1 cells (Fig. 7A). This was also associated with reduced inflammation because reduced whole spleen cell numbers were noted in mice that received OT-1 cells (Fig. 7B). OVA257–264-specific CD8+ T cells of mice that received 105 naive OT-1 cells expressed higher levels of IL-7R (Fig. 7C), again indicative of decreased activation. As naive OT-1 cells (before transfer) are all IL-7Rhigh, and it is possible that the transferred OT-1 cells exhibit a delayed decline in IL-7R expression, we evaluated the expression of IL-7R in OVA257–264-specific CD8+ T cells kinetically after LM-OVA infection. Results presented (Fig. 7, C and D) clearly indicate that the transferred OT-1 cells do not undergo delayed decline in IL-7R expression. Again, as was noted with the conversion of effectors into central memory cells, the timing of conversion of the IL-7Rlow cells back into IL-7Rhigh cells correlated with the degree of stimulation during priming.
FIGURE 7. Decreased down-regulation of IL-7R expression induced by reduced stimulation. C57BL/6 mice were injected with LM-OVA (104, i.v.) and OT-1 cells. LM-OVA burden (A) was enumerated at various time intervals, and the influence on total spleen cell numbers (B) was enumerated at day 7 postinfection. The expression of IL-7R+ on tetramer+ CD8+ T cells (C) and the percentages of IL-7R+ cells among tetramer+ CD8+ T cells (D) were enumerated at various time intervals after infection. Numbers in the panels indicate the percentage of IL-7Rhigh or IL-7Rlow cells among tetramer+ cells. Data are representative of three mice per group.
It may be suggested that the transferred OT-1 cells after differentiation in LM-OVA-infected mice are nonfunctional. We therefore measured the expression of intracellular IFN- in response to OVA257–264 peptide for 1 h (Fig. 8A) or for 4 h (Fig. 8B). OVA257–264-specific CD8+ T cells in OT-1-transferred group of mice expressed lesser levels of IFN- when the cells were stimulated for 1 h with OVA257–264 in vitro (Fig. 8A). When the in vitro stimulation was continued for 4 h, no differences were noted in the levels of IFN- (Fig. 8B) in the two groups of mice.
FIGURE 8. Intracellular IFN--secreting Ag-specific CD8+ T cells induced by reduced stimulation. C57BL/6 mice were injected with LM-OVA (104, i.v.), or with LM-OVA (104, i.v.) and OT-1 CD8+ T cells (105). At various time intervals, the numbers of IFN--secreting tetramer+ CD8+ T cells were evaluated after stimulation of cells for 1 h (A) or 4 h (B) with OVA257–264. Numbers in the panels indicate the percentage of IFN--secreting CD8+ T cells among the tetramer+ cells (A). The total number of OVA-specific (tetramer-positive vs IFN--positive) CD8+ T cells per spleen was enumerated (B). Data are representative of two to three mice per group.
Priming during the later stages of infection reduces attrition
We further examined whether naive CD8+ T cells that interact with Ag-bearing APCs during the declining phase of LM-OVA infection experience lesser stimulation, and hence may be susceptible to lesser attrition subsequently. To discriminate between the host and donor OVA-specific CD8+ T cells, we used B6.SJL mice recipients. B6.SJL mice (CD45.2–) were challenged with LM-OVA, and groups of mice received no OT-1 cells, or OT-1 cells (CD45.2+) simultaneously, or OT-1 cells at 96 h postinfection. Mice that received no OT-1 cells had 11% OVA257–264-specific CD8+ T cells (Fig. 9A) at day 7, and these cells underwent rapid attrition subsequently. Mice that received OT-1 and LM-OVA simultaneously had 39% donor OVA257–264-specific CD8+ T cells at day 7, and these cells experienced reduced attrition subsequently, as described before for the C57BL/6J strain. Interestingly, when OT-1 were transferred into mice at 96 h post-LM-OVA infection, only 0.5% OVA257–264-specific CD8+ T cells were of donor origin at day 7, and these cells experienced little, if any, attrition subsequently. What is interesting is that in the latter group of mice, endogenous (CD45.2–) OVA257–264-specific CD8+ T cells underwent a massive attrition from 10 to 0.3%, whereas the donor cells (CD45.2+) did not experience any attrition. Thus, it is interesting to note that CD8+ T cells against the same peptide differentiating in the same host can experience different level of attrition that correlates to the extent of stimulation during priming. Calculation of the overall numbers of OVA257–264-specific CD8+ T cells differentiating in the various groups of mice indicates that OVA257–264-specific CD8+ T cells underwent differential expansion and contraction (Fig. 9B). Interestingly, OT-1 cells that were transferred at 96 h post-LM-OVA infection underwent a 10-fold expansion (Fig. 9B), up-regulated CD44 (data not shown), but did not undergo significant attrition, a situation that mirrors the results obtained in the BCG-OVA model.
FIGURE 9. The timing of Ag recognition by naive CD8+ T cells influences their fate. B6/SJL (CD45.2–) mice were infected with LM-OVA (104, i.v.), and groups of mice were injected with OT-1 cells (105, i.v.) either at the same time or at 96 h post-LM-OVA infection. At various time intervals, the percentages of host (CD45.2–) and donor (CD45.2+) tetramer+ CD8+ T cells were evaluated (A). Numbers in the panels indicate the percentage of tetramer+ cells gated on CD8+ T cells. The overall numbers of splenic OVA-tetramer-positive CD8+ T cells in the various groups of mice were evaluated at various time intervals (B). Data are representative of three mice per group.
The level of attrition that was noted in the three groups of mice (Fig. 9) correlated to the relative induction of central vs effector CD8+ T cells at day 7 (Fig. 10A). Majority of OVA257–264-specific CD8+ T cells of mice that received no OT-1 cells were in an effector phenotype (CD62Llow) at day 7 (Fig. 10A), whereas OVA257–264-specific CD8+ T cells of mice that received OT-1 simultaneously with LM-OVA infection had an approximately equal distribution of central vs effector cells at day 7. Interestingly, the OT-1 donor cells that were transferred at 96 h post-LM-OVA infection differentiated primarily into a central phenotype. Again, in the same hosts, the host cells (CD45.2–) differentiated rapidly into an effector phenotype, whereas the donor cells (CD45.2+) that were transferred at 96 h differentiated mainly into a central phenotype (Fig. 10A). It could be argued that the CD8+ T cells that differentiated in the 96-h OT-1 transfer group did not differentiate properly and are consequently not functional. However, when intracellular IFN- expression was measured after 1 h (Fig. 10B) or 4 h (Fig. 10C) of in vitro stimulation with OVA257–264 peptide, they produced IFN-.
FIGURE 10. Phenotype of CD8+ T cells primed during the later stages of infection. Groups of B6/SJL (CD45.2–) mice were injected with LM-OVA (104, i.v.) and OT-1, as described in Fig. 9. At various time intervals, the proportion of effector (CD62LlowCD44high) vs central (CD62LhighCD44high) CD8+ T cell subsets was enumerated for the host (CD45.2–) and donor (CD45.2+) tetramer+ cells (A). Numbers in the panels indicate the percentage of cells gated on CD8+ tetramer+ T cells. Intracellular IFN--secreting CD8+ T cells were evaluated at days 7 and 11 postinfection after stimulation of cells for 1 h (B) or 4 h (C) with OVA257–264. Numbers in the panels indicate the percentage of IFN--secreting CD8+ T cells among the tetramer+ cells (B). The total number of OVA-specific (tetramer-positive vs IFN--positive) CD8+ T cells per spleen was enumerated (C). Data are representative of three mice per group.
Discussion
Memory T cells provide effective long-term protection against pathogens due to their unique ability to persist for extended periods (8, 27); however, the mechanisms that govern their induction and maintenance (6, 8, 28) and their differentiation are unclear (15, 25, 29, 30, 31, 32, 33). Pathogens vary in their intracellular habitat, survival, inflammation, and the extent of CD8+ T cell priming and memory generation. Mycobacteria are one of the highly studied intracellular bacteria, and one such organism, BCG, is one of the very few approved attenuated live vaccines used as a frontline prophylactic therapy against tuberculosis. However, the nature of Ag presentation and T cell memory induced by BCG has not been clear. In this study, we have evaluated CD8+ T cell priming during BCG-OVA infection, and show that BCG-OVA induces a unique CD8+ T cell differentiation program that is atypical of most viral infection models. We also show that this differentiation program operates selectively under conditions when CD8+ T cells receive reduced stimulation in vivo.
One of the surprising observations in this study is that BCG, despite being a chronic pathogen, induced CD8+ T cells that differentiated primarily into central CD8+ T cell phenotype early on where the primed cells remained parked for prolonged periods. Should chronic Ag presentation not convert primed cells into effectors eventually? Although it was previously thought that the chronicity of mycobacteria may be due to reduced proliferation of the bacterium in vivo, in a recent study in which the transcriptional profile of various mycobacterial genes was evaluated in vivo during the various stages of infection, it was shown that the bacteria go into a state of nonreplicating persistence as a survival strategy to counteract the development of host immune responses (34). Similarly, monocytes chronically infected with BCG fail to present mycobacterial Ags to T cells (35). These reports support our previous observations that the overall magnitude of Ag presentation during BCG-OVA infection is muted, which does not last appreciably beyond the first few weeks of infection (24). The reduction in Ag presentation during BCG-OVA infection, despite the persistence of the bacterium, was not due to the loss of the OVA gene by the bacterium in vivo (19). The doubling time of BCG-OVA is very long (29.5 h), in comparison with 45 min for LM-OVA, which would result in poor generation of Ag in vivo (24). Furthermore, we have used a relatively low dose (104) of BCG-OVA in this study, which does not cause splenomegaly, and fails to induce potent inflammation and CD8+ T cell activation. Most importantly, BCG resides within the phagosome of infected cells, in comparison with LM or other noncytopathic viruses that reside within the cytosol of infected cells, which might potentially prevent the availability of Ags for MHC class I presentation during BCG infection. Thus, BCG is a unique pathogen that needs to be viewed quite differently from the typical acute or chronic viral infection models.
We have previously reported that removal of BCG-OVA at day 15 of infection with antibiotic therapy resulted in decreased CD8+ T cell frequency, whereas the commencement of antibiotic therapy at day 60 had no impact (24). Thus, during the phase of peak BCG-OVA burden (day 15), CD8+ T cell response is dependent on pathogen persistence, whereas during the chronic phase (day 60), the response is independent of pathogen persistence. Considering that Ag presentation peaks only during the second week of BCG-OVA infection (24), it is possible that commencement of antibiotic therapy at day 15 reduces the generation of sufficient amount of Ag necessary for optimal priming of CD8+ T cell response. This problem may be more specific to BCG-OVA infection model because the doubling time (24–48 h) and hence the amount of Ag generated by this organism in vivo would be very low, and any further reduction by antibiotic therapy during the peak growth phase may seriously compromise the generation of CD8+ T cell response. Such early removal of BCG-OVA by antibiotics may thus limit CD8+ T cell priming, rather than influence the maintenance of CD8+ T cell memory.
We also noted in our previous study that the numbers of annexin V+ OVA-specific CD8+ T cells increased at day 30 of BCG-OVA infection, indicative of turnover of OVA-specific CD8+ T cells during BCG-OVA infection (24). However, LM-OVA induced more numbers of annexin V+ OVA-specific CD8+ T cells than those induced by BCG-OVA. Although it is not clear whether all annexin V+ CD8+ T cells die, it is, however, quite possible that some level of apoptosis might be occurring during BCG-OVA infection, particularly during the peak effector phase (days 15–30). Indeed, as is evident in Fig. 1E, not all CD8+ T cells induced by BCG-OVA exhibit a central memory phenotype, and a proportion of cells (albeit small in number in comparison with LM-OVA) that receive higher level of stimulation even during this infection might be committed to apoptosis.
We have previously reported that the CD8+ T cell response induced by BCG-OVA, but not LM-OVA, is CD4+ T cell-dependent even at day 30 after infection (19). The consensus from several reports now appears to be that CD8+ T cell responses are more dependent on CD4+ T cells in cases of immunization with weaker immunogens (36, 37, 38), or when the Ag amounts are limiting (39). Indeed, more recently, it was shown that the requirement for CD4+ T cell help was not a property intrinsic to the CD8+ memory T cells, but was linked to the infectious agent, and even in cases in which the immunogen was weak and the primary CD4+ T cell response was minimal, the CD8+ memory T cells produced mounted a robust recall response (38). This view resonates with our analysis of BCG-OVA infection model as being a weak immunogen, inducing a CD4+ T cell-dependent CD8+ T cell memory, which is highly protective in vivo.
In our adoptive transfer experiments involving OT-1 cell transfer, we transferred 104-105 OT-1 cells into recipients, and only 10% of the transferred cells home to the spleen as majority of cells home to nonlymphoid organs such as liver and lungs (our unpublished observations). Although it may appear that large numbers of OT-1 cells were transferred, however, considering that the frequency of naive Ag-specific CD8+ T cells in a normal mouse is 102 (1 in 105) (40), we have transferred only 10- to 102-fold more cells than what would be present in a normal mouse.
CD8+ T cells against the same epitope have been previously shown to compete for access to APCs (41, 42, 43, 44), and our results support this notion. However, other experimental models using peptide-loaded DCs to stimulate CD8+ T cells have reported little competition for access to APCs (45). The differences could relate to the amount of Ag used to stimulate naive precursors because dendritic cells can be loaded with high peptide levels that may not be similar to the levels generated during infection with live immunogens in vivo. What is interesting in our study is that the transfer of transgenic OT-1 cells even on day 3 after LM-OVA infection still curbed, albeit to a lesser degree, the proliferation of endogenous OVA-specific CD8+ T cells that were responding to LM-OVA from the first day of infection. These results indicate that endogenous CD8+ T cells continue to interact with Ag-bearing targets even on day 3 of infection. When OT-1 cells were transferred into mice at 96 h post-LM-OVA infection, they up-regulated CD44 expression (data not shown), increased in number 10-fold, and produced IFN-, indicating that these cells had differentiated in response to stimulation by Ag-bearing APCs. In this context, 20% of the CFSE-labeled donor OT-1 cells when transferred on day 7 of LM-OVA infection down-regulated CFSE expression, suggesting that Ag-bearing APCs were present during this time interval (24). It has been previously reported that Ag presentation in LM-infected BALB/c does not occur after a few days (46). This discrepancy in the extent of the duration of Ag presentation could be due to the two different strains of mice used, or may relate to the differences in the affinity and/or immunodominance of the Ags.
A crucial question that arises is how naive CD8+ T cells would get different levels of stimulation in vivo. When we evaluate Ag presentation using CFSE, it is clear that some OVA257–264-specific CD8+ T cells have completed more than seven divisions, and yet others are just beginning to go into cell cycle. Furthermore, not all Ag-specific CD8+ T cells down-regulate CD62L expression at day 7. After infectious challenge, the burden of pathogen, and hence the level of Ag, would not be the same everyday. Similarly, the level of inflammation would also vary with the timing of infection. In a recent report, contraction of the CD8+ T cell response was shown to be controlled by early inflammation (47), which correlates to the reduced contraction that we have shown in the various approaches in this study. Transfer of naive OT-1 cells into hosts reduces the bacterial burden and inflammation. Similarly, naive CD8+ T cells that interact with APCs during the late stages of infection may receive reduced inflammatory signals. BCG induces muted and delayed inflammation in comparison with LM (our unpublished observations) (23).
When we measured the expression of intracellular IFN- under conditions of reduced CD8+ T cell stimulation in vivo, lesser levels of IFN- were detected when the cells were stimulated for 1 h with OVA257–264 in vitro. When the in vitro stimulation was continued for 4 h, no differences in the levels of IFN- were noted. These results are in line with a recent report that indicated that immediate cytotoxicity, but not degranulation distinguishes effector and memory subsets of CD8+ T cells (48).
Our results indicate that if CD8+ T cells get higher level of stimulation in vivo, then they take longer to convert into the central memory phenotype. Similar results were obtained during lymphocytic choriomeningitis virus infection model in which CD8+ T cells induced by low dose of infection became CD62Lhigh more rapidly than those induced by higher dose (25).
Although we have used OT-1 transgenic CD8+ T cells in this study to enumerate the influence of the degree of stimulation of naive CD8+ T cells on the development of CD8+ T cell memory, it is not clear whether the endogenous OVA-specific CD8+ T cells would behave in a similar manner. However, we have previously reported that when mice are infected with BCG-OVA in the absence of OT-1 cells, then the CD8+ T cell response that is induced is also delayed and muted, and undergoes lesser attrition in comparison with that induced by LM-OVA (19), a situation that is similar to the one that occurs with transferred OT-1 cells.
Our results provide an explanation to the reported observation that naive and memory CD8+ T cells against the same Ag undergo differential apoptosis (49). As the frequency of naive CD8+ T cells against any given Ag is very low (40), this would result in massive pathogen growth, inflammation, and stimulation of naive CD8+ T cells culminating in an equally massive apoptosis of the effectors subsequently. In contrast, during pathogen rechallenge, very high numbers of responding memory CD8+ T cells that are present before the rechallenge would compete with each other and with fresh naive CD8+ T cells for Ag presentation (50), resulting in reduced pathogen growth, reduced inflammation, and reduced stimulation. Thus, during successive rechallenges, memory CD8+ T cells would experience lesser stimulation and attrition, which would result in greater memory expansion.
The functional segregation of effector vs central memory T cell subsets has been controversial, as some reports indicate that effector memory T cells exhibit more potent function (15, 48, 51, 52, 53), whereas other reports indicate no differences in function between central memory and effector memory subsets of CD8+ T cells in mice (25, 26, 54). Our results indicate that the two CD8+ T cell subsets exhibit differences in IL-2, but not IFN- production, reiterating the notion that the central memory CD8+ T cells may differ from effector memory cells mainly in terms of their superior proliferative ability. In a recent study, it was reported that the central memory CD8+ T cells mediate better protective efficacy due to their increased propensity to survive, proliferate, and convert into effector memory cells upon restimulation (25). What we have shown in this study is that CD8+ T cells of the central phenotype can be induced early on during BCG infection as a major population of primed CD8+ T cells and that these cells are functional. We have also previously reported that CD8+ T cells induced by BCG-OVA mediate potent, if not better protection than LM-OVA, against poorly immunogenic B16OVA tumor cells growing at a distal site (19). Thus, although BCG-OVA induces a muted activation of CD8+ T cells, the resulting CD8+ T cell memory that is generated is remarkably potent and functional. Our results highlight the flexibility of the immune system that seems to have evolved a compensatory differentiation program to achieve long-lasting protective memory even to an attenuated immunogen such as BCG.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by funds from the National Research Council of Canada.
2 Address correspondence and reprint requests to Dr. Subash Sad, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Room 4105, Ottawa, Ontario, Canada K1A 0R6. E-mail address: subash.sad@nrc-cnrc.gc.ca
3 Abbreviations used in this paper: LM, Listeria monocytogenes; BCG, Mycobacterium bovis.
Received for publication December 3, 2004. Accepted for publication February 18, 2005.
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During infection with lymphocytic choriomeningitis virus, CD8+ T cells differentiate rapidly into effectors (CD62LlowCD44high) that differentiate further into the central memory phenotype (CD62LhighCD44high) gradually. To evaluate whether this CD8+ T cell differentiation program operates in all infection models, we evaluated CD8+ T cell differentiation during infection of mice with recombinant intracellular bacteria, Listeria monocytogenes (LM) and Mycobacterium bovis (BCG), expressing OVA. We report that CD8+ T cells primed during infection with the attenuated pathogen BCG-OVA differentiated primarily into the central subset that correlated to reduced attrition of the primed cells subsequently. CD8+ T cells induced by LM-OVA also differentiated into central phenotype cells first, but the cells rapidly converted into effectors in contrast to BCG-OVA. Memory CD8+ T cells induced by both LM-OVA as well as BCG-OVA were functional in that they produced cytokines and proliferated extensively in response to antigenic stimulation after adoptive transfer. During LM-OVA infection, if CD8+ T cells were guided to compete for access to APCs, then they received reduced stimulation that was associated with increased differentiation into the central subset and reduced attrition subsequently. Similar effect was observed when CD8+ T cells encountered APCs selectively during the waning phase of LM-OVA infection. Taken together, our results indicate that the potency of the pathogen can influence the differentiation and fate of CD8+ T cells enormously, and the extent of attrition of primed CD8+ T cells correlates inversely to the early differentiation of CD8+ T cells primarily into the central CD8+ T cell subset.
Introduction
The CD8+ T cell is stimulated when peptides from endogenously derived Ags (intracellular bacteria, viruses, or tumors) are presented on MHC class I molecules (1). After differentiation, effector CD8+ T cells possess the unique ability to mediate specific cytotoxicity (by perforin and Fas-dependent pathways) toward infected cells and tumors (2, 3, 4). However, the vast majority (>95%) of Ag-specific T cells activated at the onset of the immune response die by apoptosis, and only a small portion of those T cells survives (<5%) for extended periods (5, 6, 7). These long-lived memory T cells possess the unique ability to respond rapidly and specifically to Ags (8, 9).
Various cell surface markers have been used to identify memory T cells (9, 10, 11); however, CD44 appears to be the most reliable marker that is expressed at high levels in all memory T cells of mice irrespective of their activation status (8, 12). During lymphocytic choriomeningitis virus (13) and Sendai virus (14) infection of mice, it was reported that memory CD8+ T cells segregate into two distinct populations: a CD44highCD62Llow population that is predominately located in the spleen and exerts a rapid effector function (effector memory), and a CD44highCD62Lhigh population that is found in the spleen and the lymph nodes with no immediate effector function (central memory). In humans, a phenotypic and functional model of the subsets of memory CD4+ and CD8+ T cells was proposed based on expression of CD62L and CCR7 (15). Based on this model, tissue-homing effector memory T cells (CD62LlowCCR7–), capable of immediate effector function, were separated from the lymph node-homing central memory T cells (CD62LhighCCR7+), which were devoid of effector activity in vitro. Several recent reports have confirmed the presence of distinct effector vs central memory T cell subsets (16, 17), and shown that effector memory T cells selectively extravasate into nonlymphoid compartment (18) to provide a first line of defense against pathogens. Both of the subsets of memory cells are present in the blood and spleen (8).
In this study, we have evaluated CD8+ T cell differentiation and fate during infection of mice with the intracellular bacteria, Listeria monocytogenes (LM)3 and Mycobacterium bovis (BCG). We show that BCG induces a highly attenuated stimulation of naive CD8+ T cells that differentiate primarily into the central CD8+ T cell subset and undergo minimal attrition subsequently. Similar differentiation program also operates when naive CD8 T cells compete for access to APCs, or they encounter APCs selectively during the waning phase of LM-OVA infection. Our results indicate that the differentiation and fate of naive CD8+ T cells are profoundly influenced by the potency of the immunogen.
Materials and Methods
Bacterial strains
BCG-OVA is a previously described recombinant strain (19) engineered with a partial sequence of the OVA gene (codons 230–359), downstream of the Ag 85B secretion signal (20), under the control of heat shock protein 60 promoter (21). Codons 230–359 of OVA gene encode the SIINFEKL epitope (OVA257–264) and its flanking sequences (22). Single colonies were used to inoculate liquid cultures, which were incubated at 37°C under constant shaking in 7H9 medium containing glycerol (0.2%), Tween 80 (0.05%), and albumin-dextrose supplement (10%; Difco Laboratories). At mid-log phase (OD600 = 1.0), bacteria were harvested and frozen at –80°C (in 20% glycerol). CFU were determined by plating serial dilutions in PBS-T (0.025% Tween 80) on Middlebrook 7H10 solid medium containing glycerol (0.5%) and oleic acid-albumin-dextrose supplement (10%; Difco Laboratories).
OVA-expressing LM (LM-OVA), as described previously (19), was grown to OD600 nm = 0.4. The bacteria were grown in brain heart infusion medium (Difco Laboratories), supplemented with 50 μg/ml streptomycin (Sigma-Aldrich). At mid-log phase (OD600 = 1.0), bacteria were harvested and frozen in 20% glycerol and stored at –80°C. CFUs were determined by performing serial dilutions in 0.9% NaCl, which were spread on brain heart infusion-streptomycin agar plates.
Mice and immunizations
Female C57BL/6 and BALB/c mice, 6–8 wk of age, were obtained from The Jackson Laboratory. Mice were maintained in the animal facility at the Institute for Biological Sciences (National Research Council of Canada) in accordance with the guidelines of the Canadian Council on Animal Care. For immunizations with LM-OVA, frozen stocks were thawed and diluted in 0.9% NaCl, and mice were inoculated with 1 x 104 LM-OVA suspended in 200 μl of 0.9% NaCl, via the lateral tail vein (i.v.). For BCG-OVA, frozen stocks were thawed and diluted in PBS containing 0.025% Tween 80, and injections were conducted through the i.v. route. Age-matched control mice were inoculated with 200 μl of PBS. B6.PL (Thy-1.1+), B6.SJL (CD45.2–), and OT-1 transgenic mice (CD45.2+Thy-1.2+ and expressing the TCR of OVA257–264) were obtained from The Jackson Laboratory.
Assessment of bacterial burden in spleen
Single cell suspensions from infected mice were tweezed in RPMI 1640, and an aliquot of the suspension was lysed with water for 30 s, and then evaluated for the numbers of viable bacteria. CFUs were determined by plating 100-μl aliquots of serial 10-fold dilutions in 0.9% saline on appropriate plates, as above.
CD8+ T cell purification
Single cell suspensions were prepared by tweezing the pooled spleens (n = 2–3) between the frosted ends of two sterile glass slides in RPMI 1640. Cells were subsequently passed through Falcon 2360 cell strainers (BD Discovery Labware), centrifuged, and resuspended (20 x 106/ml) in 0.5–1 ml of RPMI 1640 containing 8% FBS (HyClone) and 50 μg/ml gentamicin (Invitrogen Life Technologies), hereafter refer to as R8-A. CELLection Biotin Binder Dynabeads, precoated as per manufacturer’s instructions (Dynal Biotech) with biotin-conjugated rat anti-mouse CD8.2 mAb (53.5.8; BD Biosciences), were added to the resuspended cell pellet at a ratio of five beads per cell, and incubated for 15–20 min at 4°C in a rotating platform. CD8+ T cells were separated by magnetic isolation. Dynabead detachment was done using the CELLection Biotin Binder kit Releasing Buffer (DNase; 188 U/108 Dynabeads) in 37°C shaker for 15 min, followed by two to three rounds of washing/magnetic separation. This protocol resulted in >95% pure CD8+ T cells, as determined by follow-up analysis with PE-conjugated rat anti-mouse CD8 (BD Biosciences). Analysis was performed using EPICS XL flow cytometer and EXPO software (Beckman Coulter).
Assessment of the fate and phenotype of Ag-specific CD8+ T cells
For evaluation of the fate and phenotype of OVA257–264-specific CD8+ T cells in vivo, aliquots (10 x 106) of spleen cells were incubated in 200 μl of PBS plus 1% BSA with anti-CD16/32 at 4°C. After 10 min, cells were stained with various Abs (anti-CD8, anti-CD62L, anti-IL-2R, anti-IL-2R, anti-IL-2R, anti-IL-7R, anti-CD69, anti-mouse CD44, anti-Thy-1.2, and anti-CD45.2) for 30 min, on ice. All Abs were obtained from BD Biosciences. Cells were then washed with PBS-BSA and incubated for 30 min with PE-H-2KbOVA257–264 tetramer (Beckman Coulter) at room temperature. Cells were washed with PBS, fixed in 0.5% formaldehyde, and acquired on EPICS XL flow cytometer.
Assessment of intracellular IFN- in Ag-specific CD8+ T cells
Aliquots of spleen cells (10 x 106/ml) were stained with anti-CD8 and H-2KbOVA257–264 tetramer for 30 min, as described above. Cells were then washed, reconstituted in R8, plated into 96-well plates (2 x 106/well), and stimulated with OVA257–264 peptide (1 μg/ml) in the presence of Golgi-stop (BD Biosciences). After 1 or 4 h, cells were harvested, washed, permeabilized, and stained for intracellular IFN- using the IFN- staining kit (obtained from BD Biosciences). Cells were acquired on EPICS XL flow cytometer and analyzed using the EXPO software.
Results
Priming by the highly attenuated pathogen BCG causes minimal attrition
We have previously reported that BCG-OVA induces highly attenuated levels of inflammation (23) and CD8+ T cell memory (19) in comparison with that induced by LM-OVA. CD8+ T cell response induced by BCG-OVA peaked at day 21 in comparison with day 7 during LM-OVA infection model. Based on a consensus of a number of experiments conducted by us, it appears that when fold drop in the overall numbers of OVA257–264-specific memory CD8+ T cells is evaluated over time, a 10-fold attrition in the response is noted during BCG-OVA infection in comparison with >75-fold attrition that is noted during infection with LM-OVA. Similarly, we have reported that BCG-OVA infection results in a delayed and muted Ag presentation (24). LM-OVA induced a massive reduction in CFSE intensity of donor OT-1 CD8+ T cells (OVA257–264 TCR transgenic cells) within the first 4 days, with >80% of donor CD8+ T cells reducing their CFSE expression. When OT-1 donor CD8+ T cells were transferred on day 7 after LM-OVA infection, a significant reduction in CFSE staining was still detectable (20%). Thus, Ag presentation during LM-OVA infection occurs rapidly, and interestingly, it persists at reduced levels for 7–10 days. In contrast, BCG-OVA infection induced a low level Ag presentation during the first 4 days, peaked during the second week of infection, and declined to very low levels subsequently (24).
Phenotype and cytokine secretion of CD8+ T cells induced by BCG-OVA
Considering the attenuated Ag presentation and T cell priming during BCG-OVA infection, we sought to determine the phenotype of the primed CD8+ T cells. Because CD62L is considered as a key marker that segregates the effector and central CD8+ T cell subsets (25), we evaluated the expression of CD62L during CD8+ T cell differentiation. During both LM-OVA and BCG-OVA infections, the transferred OT-1 cells down-regulated CD62L expression only after multiple rounds of division (Fig. 1A), and interestingly, the majority of CD8+ T cells that differentiated during BCG-OVA infection did not down-regulate CD62L.
FIGURE 1. Differentiation of Ag-specific CD8+ T cells during LM-OVA and BCG-OVA infection. B6.PL (Thy-1.2–) mice were injected with CFSE-labeled OT-1 CD8+ T cells (3 x 106) and 104 LM-OVA or BCG-OVA. The proliferation of donor Thy-1.2+ tetramer+ CD8+ T cells was evaluated on day 4 for LM-OVA and day 7 for BCG-OVA (A). C57BL/6J mice were injected with OT-1 cells (105, i.v.) and challenged (104, i.v.) with LM-OVA (B and D) or BCG-OVA (C and E), and the phenotypic distribution of the effector (CD62LlowCD44high) vs central subsets of (CD62LhighCD44high) tetramer+ CD8+ T cells was evaluated. Data are representative of two to three mice per group.
At day 5 after LM-OVA infection and OT-1 transfer, 42% of the OVA257–264-specific CD8+ T cells displayed a central phenotype (CD62LhighCD44high) (Fig. 1B), and the percentages of these cells decreased to 18% on day 7 and then increased progressively at subsequent time intervals. In contrast, the effector cells (CD62LlowCD44high) peaked at day 7 and then progressively decreased at subsequent time intervals, indicative of conversion into the central phenotype. The relative induction of the central and effector CD8+ T cell subsets during LM-OVA infection is indicated in Fig. 1D. During infection of mice with BCG-OVA, the majority of cells differentiated primarily into the central CD8+ T cell subset (Fig. 1, C and E), where they remained parked for prolonged periods without down-regulating CD62L expression.
We measured intracellular cytokine production by OVA-specific CD8+ T cells at day 60 after infection. OVA257–264-specific CD62Lhigh population induced by LM-OVA as well as BCG-OVA expressed IL-2 in comparison with the CD62Llow population that expressed IFN-, but little IL-2 (Fig. 2, A and B), suggesting that the OVA257–264-specific CD62Lhigh cells exhibit potent proliferative potential that is typical of central CD8+ T cell subset (15, 25). Cells in both the effector and central subset did not exhibit any differences in their ability to produce IFN-, which has also been reported by various investigators (25, 26). To further substantiate the difference in the IL-2 expression by the central vs effector CD8+ T cell subsets, we sorted the central and effector CD8+ T cell subsets of LM-OVA-infected mice and measured the production of IL-2 in the supernatants after stimulation with OVA257–264-pulsed dendritic cells. Central phenotype CD8+ T cells produced higher levels of IL-2 in comparison with the effector phenotype cells (Fig. 2C). We also measured in vitro cytolytic activity of purified CD8+ T cells of LM-OVA- and BCG-OVA-infected mice on day 30 in the absence or presence of IL-2. Addition of exogenous IL-2 resulted in a moderate enhancement of cytolytic activity of CD8+ T cells of both LM-OVA- and BCG-OVA-infected mice, whereas neutralization of endogenously produced IL-2 abrogated the cytolytic activity in both the groups of mice (Fig. 2, D and E).
FIGURE 2. Effector memory CD8+ T cells express reduced levels of IL-2. C57BL/6J mice were injected with 105 OT-1 cells, and challenged (104, i.v.) with LM-OVA (A) or BCG-OVA (B). At day 60, spleens were removed and stained with OVA-tetramer and anti-CD62L Ab. Cells were then stimulated in vitro with OVA257–264 for 1 h, and the expression of intracellular IL-2 and IFN- was evaluated on gated OVA-tetramer+ CD8+ T cells to determine the expression of these two cytokines by the CD62Llow vs CD62Lhigh subsets of CD8+ T cells (A and B). Tetramer+ cells were also sorted into CD62Llow and CD62Lhigh subsets and stimulated in vitro (1 x 105/well) with syngeneic bone marrow-derived dendritic cells (1 x 105/well) pulsed with OVA257–264 (10 ng/ml). Supernatants were collected after 12 h, and the production of IL-2 was measured by ELISA (C). Cytolytic activity of CD8+ T cells from the spleen cells of LM-OVA (D)- and BCG-OVA (E)-infected mice was evaluated on day 60. CD8+ T cells were purified and cultured (106/flask) with syngeneic naive spleen cells (25 x 106/flask) in the presence of OVA257–264 (10 ng/ml) and rat IgG (10 μg/ml), or anti-mouse IL-2 (10 μg/ml), or mouse IL-2 (100 pg/ml). After 5 days, cells were harvested, washed, and evaluated for cytolytic activity against EL-4 cells or EL-4 cells pulsed with OVA257–264. Cytotoxicity on EL-4 cells in the absence of OVA257–264 was <15%. Data are representative of two to three mice per group.
When the expression of intracellular IFN- was measured at various time intervals, BCG-OVA-induced CD8+ T cells expressed lesser levels of IFN- when the cells were stimulated for 1 h with OVA257–264 in vitro (Fig. 3A). When the in vitro stimulation was continued for 4 h, no differences in the levels of IFN- were noted (Fig. 3B) between LM-OVA- and BCG-OVA-primed CD8+ T cells.
FIGURE 3. Intracellular IFN--secreting Ag-specific CD8+ T cells induced by LM-OVA vs BCG-OVA. C57BL/6J mice were injected with OT-1 cells (105, i.v.) and LM-OVA (104, i.v.) or BCG-OVA (104, i.v.). At various time intervals, the numbers of IFN--secreting tetramer+ CD8+ T cells were evaluated after stimulation of cells for 1 h (A) or 4 h with OVA257–264 (B). Numbers in the panels indicate the percentage of IFN--secreting CD8+ T cells among the tetramer+ cells (A). The total number of OVA-specific (tetramer-positive vs IFN--positive) CD8+ T cells per spleen was enumerated (B). Data are representative of two to three mice per group.
Memory CD8+ T cells induced by BCG-OVA proliferate after antigenic stimulation in vivo
We sought to determine whether the OVA-specific memory CD8+ T cells induced by LM-OVA and BCG-OVA exhibit differences in their expansion in response to antigenic stimulus in vivo. To this end, we purified CD8+ T cells on day 30 after challenge of OT-1-parked mice with LM-OVA and BCG-OVA. The percentages of OVA-tetramer+ cells among the purified CD8+ T cells of LM-OVA- and BCG-OVA-infected mice were 10 and 1%, respectively. These cells were transferred into naive recipients that were challenged with LM-OVA or BCG-OVA, and the expansion of the transferred memory cells was measured at various time intervals. Upon transfer to naive mice, LM-OVA-generated memory CD8+ T cells proliferated enormously in response to LM-OVA with 50% of all splenic CD8+ T cells being OVA specific at day 7 after challenge (Fig. 4A, left panel). In contrast, the same LM-OVA-generated OVA-specific memory CD8+ T cells exhibited a delayed and muted proliferation in response to BCG-OVA rechallenge. Memory CD8+ T cells that were induced by BCG-OVA (10-fold less numbers transferred) also proliferated enormously upon transfer into naive recipients and challenge with LM-OVA (Fig. 4B, left panel), suggesting that BCG-OVA-induced memory CD8+ T cells are not functionally impaired in responding to Ag in vivo. When BCG-OVA-induced memory CD8+ T cells were challenged with BCG-OVA, the numbers of OVA-specific CD8+ T cells were below the detection limit, which is expected, considering the reduced numbers of cells transferred and the nature of stimulation induced by BCG-OVA.
FIGURE 4. BCG-OVA-induced memory CD8+ T cells proliferate normally after antigenic stimulation in vivo. C57BL/6J mice were injected with OT-1 cells (105, i.v.) and challenged with LM-OVA (A) or BCG-OVA (B) (104, i.v.). At day 30, spleens were removed and CD8+ T cells were purified. Ten percent of CD8+ T cells from LM-OVA-infected spleens were tetramer+ in comparison with CD8+ T cells from BCG-OVA-infected spleens that were 1% tetramer+. Purified CD8+ T cells (5 x 106/mouse) were then injected into normal B6.SJL (CD45.2–) mice that were challenged (104, i.v.) with LM-OVA or BCG-OVA. At various time intervals, the percentages of donor (CD45.2+) tetramer+ CD8+ T cells were enumerated. Data are representative of three mice per group.
Two important conclusions can be drawn from these data. First, as 10% of the transferred cells home to the spleen, the relative expansion of OVA-specific memory CD8+ T cells induced by LM-OVA (Fig. 4A, left panel) and BCG-OVA (Fig. 4B, left panel) in response to a challenge with LM-OVA appears to be identical (100-fold), suggesting that the memory CD8+ T cells induced by BCG-OVA exhibit the typical attributes of conventional memory CD8+ T cells. Second, memory CD8+ T cells (those induced by LM-OVA) respond to BCG-OVA with protracted kinetics (Fig. 4A, right panel), a situation that is similar to the response of naive CD8+ T cells to BCG-OVA.
CD8+ T cells compete for access to APCs
Considering the weak stimulation evoked by BCG-OVA that promoted differentiation of CD8+ T cells mainly into the central phenotype, we were interested in determining the consequence of reducing the extent of stimulation of naive CD8+ T cells during LM-OVA infection. To this end, we first evaluated the consequences of competition for access to APC on CD8+ T cell priming and stimulation. B6.PL (Thy-1.1+Thy-1.2–) mice were infected with LM-OVA and 104 or 105 or no OT-1 (OVA257–264 TCR transgenic) CD8+ T cells simultaneously (Fig. 5, A and B). Using an anti-Thy-1.2 Ab that binds only to the donor OT-1 cells, and H-2KbOVA257–264 tetramers that bind both endogenous and donor OVA257–264-specific CD8+ T cells, we followed the competition between donor and host OVA257–264-specific CD8+ T cells. In these adoptive transfer experiments, typically 5–10% of the transferred OT-1 cells home to the spleen (our unpublished results). On day 7 after LM-OVA infection, 5% of CD8+ T cells were OVA257–264 specific in the absence of OT-1 transfer (Fig. 5A). When 104 and 105 OT-1 cells were transferred, only 0.5 and 0.1% of OVA257–264-specific CD8+ T cells were of host origin (Thy-1.2–), respectively. Majority of the OVA257–264-specific CD8+ T cells (97–99%) in OT-1-transferred recipients were of donor origin (Thy-1.2+). There was an overall reduction (30- to 300-fold) in the numbers of host (Thy-1.2–) OVA257–264-specific CD8+ T cells in the spleens of OT-1-transferred recipients (Fig. 5B). Because the LM-OVA burden peaks between 48 and 72 h after infection (24) and the bacteria are undetectable in the spleens by days 5–7, we determined whether competition for Ag presentation would still occur during the declining phase of LM-OVA infection. Mice were infected with LM-OVA and injected with PBS or OT-1 CD8+ T cells at 72 h postinfection. Approximately 7% of CD8+ T cells in control mice were OVA257–264 specific, and the percentage of host OVA257–264-specific CD8+ T cells dropped to 0.8% in mice that received OT-1 cells at 72 h postinfection (Fig. 5C). Majority (91%) of OVA257–264-specific CD8+ T cells in OT-1-transferred mice were of donor origin (Thy-1.2+). Overall, there was a 10-fold reduction in the numbers of host OVA257–264-specific CD8+ T cells in the spleens of mice that received OT-1 cells at 72 h (Fig. 5D). OVA257–264-specific CD8+ T cells in the OT-1-transferred group of mice exhibited reduced forward and side scatter in comparison with the mice that received no OT-1 cells (data not shown).
FIGURE 5. CD8+ T cells compete for access to APCs. B6.PL (Thy-1.2–) mice were injected with LM-OVA (104, i.v.) or with LM-OVA (104, i.v.) and OT-1 CD8+ T cells simultaneously (A and B). Spleen cells were removed at day 7 postinfection, and the percentage (A) and numbers (B) of host (Thy-1.2–) and donor (Thy-1.2+) OVA 257–264-specific CD8+ T cells were evaluated. Numbers in A indicate the percentage of tetramer+ cells among CD8+ T cells. In another set of experiments, B6.PL (Thy-1.2–) mice were infected with LM-OVA (104, i.v.) first and injected at 72 h postinfection with PBS or OT-1 cells (105, i.v.). The percentages (C) and numbers (D) of host and donor OVA257–264-specific CD8+ T cells were evaluated. Data are representative of two to three mice per group.
Competition during priming decreases stimulation and increases the survival of CD8+ T cells
We transferred graded doses of OT-1 cells into mice that were simultaneously challenged with LM-OVA. Transfer of increasing doses of OT-1 cells resulted in increased numbers of OVA257–264-specific CD8+ T cells between days 5 and 7 (Fig. 6A). Regardless of the number of OT-1 cells transferred, day 7 was always the peak of the response. There was a dose-dependent correlation between the numbers of OT-1 cells transferred and the overall drop in the numbers of OVA257–264-specific CD8+ T cells subsequently (Fig. 6B). In the absence of any OT-1 transfer, OVA257–264-specific CD8+ T cells underwent maximal contraction, whereas contraction of OVA257–264-specific CD8+ T cells was substantially lower when 105 OT-1 cells were transferred. There was also a dose-dependent correlation between the numbers of OT-1 cells transferred and the blastogenic profile of OVA257–264-specific CD8+ T cells (Fig. 6C). In the absence of any OT-1 transfer, 33% of OVA257–264-specific CD8+ T cells displayed elevated forward and side scatter, and this number dropped to 13% with the transfer of 105 OT-1 cells. The forward and side scatter analysis was performed on gated OVA-tetramer+ CD8+ T cells. Not only was there a difference in the blastogenic profile of OVA-tetramer-positive cells, but the phenotypic profile of the cells was also shifted (Fig. 6D). At day 7 after LM-OVA infection, the numbers of OVA257–264-specific CD8+ T cells of effector phenotype (CD62LlowCD44high) were reduced from 90 to 70% as the initial OT-1 transfer dose was increased from 103 to 105 (Fig. 6D). This initial difference in the degree of stimulation of naive CD8+ T cells influenced the timing of their subsequent conversion into the central phenotype subsequently. At day 30 after LM-OVA infection, majority (65%) of OVA257–264-specific CD8+ T cells of mice that received 103 OT-1 cells still displayed effector (CD62LlowCD44high) phenotype, whereas majority (73%) of OVA257–264-specific CD8+ T cells of mice that received 105 OT-1 cells displayed central memory (CD62LhighCD44high) phenotype (Fig. 6D). Taken together, these results indicate that competition for access to APCs results in reduced stimulation and reduced generation of effector CD8+ T cells, which correlates with reduced contraction of CD8+ T cell response. Furthermore, CD8+ T cells that receive a relatively enhanced stimulation take longer to convert to a central memory state.
FIGURE 6. Transfer of increased numbers of OT-1 cells results in decreased stimulation. C57BL/6 mice were injected with LM-OVA (104, i.v.) and varying numbers of OT-1 cells. The total numbers of tetramer+ CD8+ T cells in the spleens were enumerated at various time intervals (A). Fold drop in the numbers of tetramer+ CD8+ T cells was enumerated by calculating the drop in the total number of OVA-tetramer-positive CD8+ T cells per spleen from day 7 to 30 (B). The percentage of cells exhibiting high forward and side scatter was evaluated on gated tetramer+ cells at day 7 postinfection (C). The relative distribution of effector (CD62LlowCD44high) vs central (CD62LhighCD44high) CD8+ T cell subsets was evaluated on gated tetramer+ CD8+ T cells at days 7 and 30 postinfection (D). Numbers in the panels indicate the percentage of cells within the indicated gate. Data are representative of two to three mice per group.
To determine the mechanisms involved, we first measured the bacterial burden in mice that received PBS or OT-1 cells and noted increased clearance of LM-OVA, particularly at later time periods in mice that received OT-1 cells (Fig. 7A). This was also associated with reduced inflammation because reduced whole spleen cell numbers were noted in mice that received OT-1 cells (Fig. 7B). OVA257–264-specific CD8+ T cells of mice that received 105 naive OT-1 cells expressed higher levels of IL-7R (Fig. 7C), again indicative of decreased activation. As naive OT-1 cells (before transfer) are all IL-7Rhigh, and it is possible that the transferred OT-1 cells exhibit a delayed decline in IL-7R expression, we evaluated the expression of IL-7R in OVA257–264-specific CD8+ T cells kinetically after LM-OVA infection. Results presented (Fig. 7, C and D) clearly indicate that the transferred OT-1 cells do not undergo delayed decline in IL-7R expression. Again, as was noted with the conversion of effectors into central memory cells, the timing of conversion of the IL-7Rlow cells back into IL-7Rhigh cells correlated with the degree of stimulation during priming.
FIGURE 7. Decreased down-regulation of IL-7R expression induced by reduced stimulation. C57BL/6 mice were injected with LM-OVA (104, i.v.) and OT-1 cells. LM-OVA burden (A) was enumerated at various time intervals, and the influence on total spleen cell numbers (B) was enumerated at day 7 postinfection. The expression of IL-7R+ on tetramer+ CD8+ T cells (C) and the percentages of IL-7R+ cells among tetramer+ CD8+ T cells (D) were enumerated at various time intervals after infection. Numbers in the panels indicate the percentage of IL-7Rhigh or IL-7Rlow cells among tetramer+ cells. Data are representative of three mice per group.
It may be suggested that the transferred OT-1 cells after differentiation in LM-OVA-infected mice are nonfunctional. We therefore measured the expression of intracellular IFN- in response to OVA257–264 peptide for 1 h (Fig. 8A) or for 4 h (Fig. 8B). OVA257–264-specific CD8+ T cells in OT-1-transferred group of mice expressed lesser levels of IFN- when the cells were stimulated for 1 h with OVA257–264 in vitro (Fig. 8A). When the in vitro stimulation was continued for 4 h, no differences were noted in the levels of IFN- (Fig. 8B) in the two groups of mice.
FIGURE 8. Intracellular IFN--secreting Ag-specific CD8+ T cells induced by reduced stimulation. C57BL/6 mice were injected with LM-OVA (104, i.v.), or with LM-OVA (104, i.v.) and OT-1 CD8+ T cells (105). At various time intervals, the numbers of IFN--secreting tetramer+ CD8+ T cells were evaluated after stimulation of cells for 1 h (A) or 4 h (B) with OVA257–264. Numbers in the panels indicate the percentage of IFN--secreting CD8+ T cells among the tetramer+ cells (A). The total number of OVA-specific (tetramer-positive vs IFN--positive) CD8+ T cells per spleen was enumerated (B). Data are representative of two to three mice per group.
Priming during the later stages of infection reduces attrition
We further examined whether naive CD8+ T cells that interact with Ag-bearing APCs during the declining phase of LM-OVA infection experience lesser stimulation, and hence may be susceptible to lesser attrition subsequently. To discriminate between the host and donor OVA-specific CD8+ T cells, we used B6.SJL mice recipients. B6.SJL mice (CD45.2–) were challenged with LM-OVA, and groups of mice received no OT-1 cells, or OT-1 cells (CD45.2+) simultaneously, or OT-1 cells at 96 h postinfection. Mice that received no OT-1 cells had 11% OVA257–264-specific CD8+ T cells (Fig. 9A) at day 7, and these cells underwent rapid attrition subsequently. Mice that received OT-1 and LM-OVA simultaneously had 39% donor OVA257–264-specific CD8+ T cells at day 7, and these cells experienced reduced attrition subsequently, as described before for the C57BL/6J strain. Interestingly, when OT-1 were transferred into mice at 96 h post-LM-OVA infection, only 0.5% OVA257–264-specific CD8+ T cells were of donor origin at day 7, and these cells experienced little, if any, attrition subsequently. What is interesting is that in the latter group of mice, endogenous (CD45.2–) OVA257–264-specific CD8+ T cells underwent a massive attrition from 10 to 0.3%, whereas the donor cells (CD45.2+) did not experience any attrition. Thus, it is interesting to note that CD8+ T cells against the same peptide differentiating in the same host can experience different level of attrition that correlates to the extent of stimulation during priming. Calculation of the overall numbers of OVA257–264-specific CD8+ T cells differentiating in the various groups of mice indicates that OVA257–264-specific CD8+ T cells underwent differential expansion and contraction (Fig. 9B). Interestingly, OT-1 cells that were transferred at 96 h post-LM-OVA infection underwent a 10-fold expansion (Fig. 9B), up-regulated CD44 (data not shown), but did not undergo significant attrition, a situation that mirrors the results obtained in the BCG-OVA model.
FIGURE 9. The timing of Ag recognition by naive CD8+ T cells influences their fate. B6/SJL (CD45.2–) mice were infected with LM-OVA (104, i.v.), and groups of mice were injected with OT-1 cells (105, i.v.) either at the same time or at 96 h post-LM-OVA infection. At various time intervals, the percentages of host (CD45.2–) and donor (CD45.2+) tetramer+ CD8+ T cells were evaluated (A). Numbers in the panels indicate the percentage of tetramer+ cells gated on CD8+ T cells. The overall numbers of splenic OVA-tetramer-positive CD8+ T cells in the various groups of mice were evaluated at various time intervals (B). Data are representative of three mice per group.
The level of attrition that was noted in the three groups of mice (Fig. 9) correlated to the relative induction of central vs effector CD8+ T cells at day 7 (Fig. 10A). Majority of OVA257–264-specific CD8+ T cells of mice that received no OT-1 cells were in an effector phenotype (CD62Llow) at day 7 (Fig. 10A), whereas OVA257–264-specific CD8+ T cells of mice that received OT-1 simultaneously with LM-OVA infection had an approximately equal distribution of central vs effector cells at day 7. Interestingly, the OT-1 donor cells that were transferred at 96 h post-LM-OVA infection differentiated primarily into a central phenotype. Again, in the same hosts, the host cells (CD45.2–) differentiated rapidly into an effector phenotype, whereas the donor cells (CD45.2+) that were transferred at 96 h differentiated mainly into a central phenotype (Fig. 10A). It could be argued that the CD8+ T cells that differentiated in the 96-h OT-1 transfer group did not differentiate properly and are consequently not functional. However, when intracellular IFN- expression was measured after 1 h (Fig. 10B) or 4 h (Fig. 10C) of in vitro stimulation with OVA257–264 peptide, they produced IFN-.
FIGURE 10. Phenotype of CD8+ T cells primed during the later stages of infection. Groups of B6/SJL (CD45.2–) mice were injected with LM-OVA (104, i.v.) and OT-1, as described in Fig. 9. At various time intervals, the proportion of effector (CD62LlowCD44high) vs central (CD62LhighCD44high) CD8+ T cell subsets was enumerated for the host (CD45.2–) and donor (CD45.2+) tetramer+ cells (A). Numbers in the panels indicate the percentage of cells gated on CD8+ tetramer+ T cells. Intracellular IFN--secreting CD8+ T cells were evaluated at days 7 and 11 postinfection after stimulation of cells for 1 h (B) or 4 h (C) with OVA257–264. Numbers in the panels indicate the percentage of IFN--secreting CD8+ T cells among the tetramer+ cells (B). The total number of OVA-specific (tetramer-positive vs IFN--positive) CD8+ T cells per spleen was enumerated (C). Data are representative of three mice per group.
Discussion
Memory T cells provide effective long-term protection against pathogens due to their unique ability to persist for extended periods (8, 27); however, the mechanisms that govern their induction and maintenance (6, 8, 28) and their differentiation are unclear (15, 25, 29, 30, 31, 32, 33). Pathogens vary in their intracellular habitat, survival, inflammation, and the extent of CD8+ T cell priming and memory generation. Mycobacteria are one of the highly studied intracellular bacteria, and one such organism, BCG, is one of the very few approved attenuated live vaccines used as a frontline prophylactic therapy against tuberculosis. However, the nature of Ag presentation and T cell memory induced by BCG has not been clear. In this study, we have evaluated CD8+ T cell priming during BCG-OVA infection, and show that BCG-OVA induces a unique CD8+ T cell differentiation program that is atypical of most viral infection models. We also show that this differentiation program operates selectively under conditions when CD8+ T cells receive reduced stimulation in vivo.
One of the surprising observations in this study is that BCG, despite being a chronic pathogen, induced CD8+ T cells that differentiated primarily into central CD8+ T cell phenotype early on where the primed cells remained parked for prolonged periods. Should chronic Ag presentation not convert primed cells into effectors eventually? Although it was previously thought that the chronicity of mycobacteria may be due to reduced proliferation of the bacterium in vivo, in a recent study in which the transcriptional profile of various mycobacterial genes was evaluated in vivo during the various stages of infection, it was shown that the bacteria go into a state of nonreplicating persistence as a survival strategy to counteract the development of host immune responses (34). Similarly, monocytes chronically infected with BCG fail to present mycobacterial Ags to T cells (35). These reports support our previous observations that the overall magnitude of Ag presentation during BCG-OVA infection is muted, which does not last appreciably beyond the first few weeks of infection (24). The reduction in Ag presentation during BCG-OVA infection, despite the persistence of the bacterium, was not due to the loss of the OVA gene by the bacterium in vivo (19). The doubling time of BCG-OVA is very long (29.5 h), in comparison with 45 min for LM-OVA, which would result in poor generation of Ag in vivo (24). Furthermore, we have used a relatively low dose (104) of BCG-OVA in this study, which does not cause splenomegaly, and fails to induce potent inflammation and CD8+ T cell activation. Most importantly, BCG resides within the phagosome of infected cells, in comparison with LM or other noncytopathic viruses that reside within the cytosol of infected cells, which might potentially prevent the availability of Ags for MHC class I presentation during BCG infection. Thus, BCG is a unique pathogen that needs to be viewed quite differently from the typical acute or chronic viral infection models.
We have previously reported that removal of BCG-OVA at day 15 of infection with antibiotic therapy resulted in decreased CD8+ T cell frequency, whereas the commencement of antibiotic therapy at day 60 had no impact (24). Thus, during the phase of peak BCG-OVA burden (day 15), CD8+ T cell response is dependent on pathogen persistence, whereas during the chronic phase (day 60), the response is independent of pathogen persistence. Considering that Ag presentation peaks only during the second week of BCG-OVA infection (24), it is possible that commencement of antibiotic therapy at day 15 reduces the generation of sufficient amount of Ag necessary for optimal priming of CD8+ T cell response. This problem may be more specific to BCG-OVA infection model because the doubling time (24–48 h) and hence the amount of Ag generated by this organism in vivo would be very low, and any further reduction by antibiotic therapy during the peak growth phase may seriously compromise the generation of CD8+ T cell response. Such early removal of BCG-OVA by antibiotics may thus limit CD8+ T cell priming, rather than influence the maintenance of CD8+ T cell memory.
We also noted in our previous study that the numbers of annexin V+ OVA-specific CD8+ T cells increased at day 30 of BCG-OVA infection, indicative of turnover of OVA-specific CD8+ T cells during BCG-OVA infection (24). However, LM-OVA induced more numbers of annexin V+ OVA-specific CD8+ T cells than those induced by BCG-OVA. Although it is not clear whether all annexin V+ CD8+ T cells die, it is, however, quite possible that some level of apoptosis might be occurring during BCG-OVA infection, particularly during the peak effector phase (days 15–30). Indeed, as is evident in Fig. 1E, not all CD8+ T cells induced by BCG-OVA exhibit a central memory phenotype, and a proportion of cells (albeit small in number in comparison with LM-OVA) that receive higher level of stimulation even during this infection might be committed to apoptosis.
We have previously reported that the CD8+ T cell response induced by BCG-OVA, but not LM-OVA, is CD4+ T cell-dependent even at day 30 after infection (19). The consensus from several reports now appears to be that CD8+ T cell responses are more dependent on CD4+ T cells in cases of immunization with weaker immunogens (36, 37, 38), or when the Ag amounts are limiting (39). Indeed, more recently, it was shown that the requirement for CD4+ T cell help was not a property intrinsic to the CD8+ memory T cells, but was linked to the infectious agent, and even in cases in which the immunogen was weak and the primary CD4+ T cell response was minimal, the CD8+ memory T cells produced mounted a robust recall response (38). This view resonates with our analysis of BCG-OVA infection model as being a weak immunogen, inducing a CD4+ T cell-dependent CD8+ T cell memory, which is highly protective in vivo.
In our adoptive transfer experiments involving OT-1 cell transfer, we transferred 104-105 OT-1 cells into recipients, and only 10% of the transferred cells home to the spleen as majority of cells home to nonlymphoid organs such as liver and lungs (our unpublished observations). Although it may appear that large numbers of OT-1 cells were transferred, however, considering that the frequency of naive Ag-specific CD8+ T cells in a normal mouse is 102 (1 in 105) (40), we have transferred only 10- to 102-fold more cells than what would be present in a normal mouse.
CD8+ T cells against the same epitope have been previously shown to compete for access to APCs (41, 42, 43, 44), and our results support this notion. However, other experimental models using peptide-loaded DCs to stimulate CD8+ T cells have reported little competition for access to APCs (45). The differences could relate to the amount of Ag used to stimulate naive precursors because dendritic cells can be loaded with high peptide levels that may not be similar to the levels generated during infection with live immunogens in vivo. What is interesting in our study is that the transfer of transgenic OT-1 cells even on day 3 after LM-OVA infection still curbed, albeit to a lesser degree, the proliferation of endogenous OVA-specific CD8+ T cells that were responding to LM-OVA from the first day of infection. These results indicate that endogenous CD8+ T cells continue to interact with Ag-bearing targets even on day 3 of infection. When OT-1 cells were transferred into mice at 96 h post-LM-OVA infection, they up-regulated CD44 expression (data not shown), increased in number 10-fold, and produced IFN-, indicating that these cells had differentiated in response to stimulation by Ag-bearing APCs. In this context, 20% of the CFSE-labeled donor OT-1 cells when transferred on day 7 of LM-OVA infection down-regulated CFSE expression, suggesting that Ag-bearing APCs were present during this time interval (24). It has been previously reported that Ag presentation in LM-infected BALB/c does not occur after a few days (46). This discrepancy in the extent of the duration of Ag presentation could be due to the two different strains of mice used, or may relate to the differences in the affinity and/or immunodominance of the Ags.
A crucial question that arises is how naive CD8+ T cells would get different levels of stimulation in vivo. When we evaluate Ag presentation using CFSE, it is clear that some OVA257–264-specific CD8+ T cells have completed more than seven divisions, and yet others are just beginning to go into cell cycle. Furthermore, not all Ag-specific CD8+ T cells down-regulate CD62L expression at day 7. After infectious challenge, the burden of pathogen, and hence the level of Ag, would not be the same everyday. Similarly, the level of inflammation would also vary with the timing of infection. In a recent report, contraction of the CD8+ T cell response was shown to be controlled by early inflammation (47), which correlates to the reduced contraction that we have shown in the various approaches in this study. Transfer of naive OT-1 cells into hosts reduces the bacterial burden and inflammation. Similarly, naive CD8+ T cells that interact with APCs during the late stages of infection may receive reduced inflammatory signals. BCG induces muted and delayed inflammation in comparison with LM (our unpublished observations) (23).
When we measured the expression of intracellular IFN- under conditions of reduced CD8+ T cell stimulation in vivo, lesser levels of IFN- were detected when the cells were stimulated for 1 h with OVA257–264 in vitro. When the in vitro stimulation was continued for 4 h, no differences in the levels of IFN- were noted. These results are in line with a recent report that indicated that immediate cytotoxicity, but not degranulation distinguishes effector and memory subsets of CD8+ T cells (48).
Our results indicate that if CD8+ T cells get higher level of stimulation in vivo, then they take longer to convert into the central memory phenotype. Similar results were obtained during lymphocytic choriomeningitis virus infection model in which CD8+ T cells induced by low dose of infection became CD62Lhigh more rapidly than those induced by higher dose (25).
Although we have used OT-1 transgenic CD8+ T cells in this study to enumerate the influence of the degree of stimulation of naive CD8+ T cells on the development of CD8+ T cell memory, it is not clear whether the endogenous OVA-specific CD8+ T cells would behave in a similar manner. However, we have previously reported that when mice are infected with BCG-OVA in the absence of OT-1 cells, then the CD8+ T cell response that is induced is also delayed and muted, and undergoes lesser attrition in comparison with that induced by LM-OVA (19), a situation that is similar to the one that occurs with transferred OT-1 cells.
Our results provide an explanation to the reported observation that naive and memory CD8+ T cells against the same Ag undergo differential apoptosis (49). As the frequency of naive CD8+ T cells against any given Ag is very low (40), this would result in massive pathogen growth, inflammation, and stimulation of naive CD8+ T cells culminating in an equally massive apoptosis of the effectors subsequently. In contrast, during pathogen rechallenge, very high numbers of responding memory CD8+ T cells that are present before the rechallenge would compete with each other and with fresh naive CD8+ T cells for Ag presentation (50), resulting in reduced pathogen growth, reduced inflammation, and reduced stimulation. Thus, during successive rechallenges, memory CD8+ T cells would experience lesser stimulation and attrition, which would result in greater memory expansion.
The functional segregation of effector vs central memory T cell subsets has been controversial, as some reports indicate that effector memory T cells exhibit more potent function (15, 48, 51, 52, 53), whereas other reports indicate no differences in function between central memory and effector memory subsets of CD8+ T cells in mice (25, 26, 54). Our results indicate that the two CD8+ T cell subsets exhibit differences in IL-2, but not IFN- production, reiterating the notion that the central memory CD8+ T cells may differ from effector memory cells mainly in terms of their superior proliferative ability. In a recent study, it was reported that the central memory CD8+ T cells mediate better protective efficacy due to their increased propensity to survive, proliferate, and convert into effector memory cells upon restimulation (25). What we have shown in this study is that CD8+ T cells of the central phenotype can be induced early on during BCG infection as a major population of primed CD8+ T cells and that these cells are functional. We have also previously reported that CD8+ T cells induced by BCG-OVA mediate potent, if not better protection than LM-OVA, against poorly immunogenic B16OVA tumor cells growing at a distal site (19). Thus, although BCG-OVA induces a muted activation of CD8+ T cells, the resulting CD8+ T cell memory that is generated is remarkably potent and functional. Our results highlight the flexibility of the immune system that seems to have evolved a compensatory differentiation program to achieve long-lasting protective memory even to an attenuated immunogen such as BCG.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by funds from the National Research Council of Canada.
2 Address correspondence and reprint requests to Dr. Subash Sad, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Room 4105, Ottawa, Ontario, Canada K1A 0R6. E-mail address: subash.sad@nrc-cnrc.gc.ca
3 Abbreviations used in this paper: LM, Listeria monocytogenes; BCG, Mycobacterium bovis.
Received for publication December 3, 2004. Accepted for publication February 18, 2005.
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