Regulatory T Cells Suppress CD8+ T Cell Responses Induced by Direct Priming and Cross-Priming and Moderate Immunodominance Disparities
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免疫学杂志 2005年第6期
Abstract
Little is known regarding the participation of CD4+CD25+ regulatory T cells (Treg) in TCD8+ responses. In this study, we show that Treg depletion via treatment with anti-CD25 mAb (PC61) significantly enhances TCD8+ responses to influenza A virus, vaccinia virus, and SV40-transformed cells induced by either direct priming or cross-priming. PC61 did not enhance TCD8+ responses in CD4-deficient mice, providing the initial demonstration that PC61 acts on a subset of TCD4+, and not on other cells that express either CD25 or a fortuitously cross-reactive Ag. We further show that Treg selectively suppress responses to the most immunodominant TCD8+ determinants in the three systems examined. Therefore, Treg influence TCD8 immunodominance hierarchies by moderating disparities in responses to different determinants.
Introduction
Despite the presence of thousands to millions of potentially immunogenic peptides in complex pathogens, TCD8+ responses are dominated by clones responding to a very limited number of determinants. This phenomenon, known as immunodominance, has been observed in mouse and human TCD8+ responses to numerous viruses, and appears to apply to antibacterial responses as well. Among TCD8+ responding to different determinants, there is often large variation (up to several hundred-fold) in responses to immunodominant determinants (IDDs) 3 vs subdominant determinants (SDDs) (1).
The underlying mechanisms for immunodominance have been the subject of intense investigation due to its obvious importance for understanding TCD8+ biology and vaccine design. A number of factors are known to contribute to immunodominance in viral systems. In approximate order of importance, these include 1) peptide affinity for MHC class I molecules, 2) liberation of peptides by cellular proteases, 3) abundance of the viral source gene product, 4) TCD8+ repertoire, 5) specificity of the TAP, and 6) kinetics of determinant generation by virus-infected cells. An additional important contribution is made by immunodomination, the suppression of SDD-specific T cells by IDD-specific T cells. Immunodomination occurs at least in part at the level of individual APCs displaying multiple determinants (2).
However, these factors do not fully explain immunodominance. In many systems, no particular factors can account for the ascendance of IDDs. Precious little is known about possible contribution(s) of suppressive elements of the immune system to shaping immunodominance hierarchies of Ag-specific TCD8+. One such element is a subset of TCD4+ that constitutively coexpress IL-2R -chain (CD25).
Naturally occurring CD4+CD25+ regulatory T cells, which will hereafter be referred to as Treg, constitute 5–15% of peripheral TCD4+ in both mice and humans. Treg appear to function to prevent a host of autoimmune dyscrasias (3, 4). Treg play an important role in modulating immune responses to bacterial pathogens (5, 6, 7), parasitic protozoa (8, 9), opportunistic fungi (10, 11), and tumors (12, 13, 14). Treg have also been implicated in limiting allograft rejection (15, 16) and graft-vs-host disease (17, 18).
The involvement of Treg in TCD8+ responses is only now being scrutinized. Treg were recently reported to regulate TCD8+ responses directed against an IDD of HSV-1 following viral infection in mice (19). However, it is uncertain whether Treg-mediated suppression is a universal feature of antiviral TCD8+ responses, and their participation in establishing immunodominance hierarchies is essentially undefined.
In the present study, we have examined the contribution of Treg to the magnitude and composition of TCD8+ responses to SV40 large tumor Ag (Tag), vaccinia virus (VV), and influenza A virus (IAV).
Materials and Methods
Mice
Adult (6- to 10-wk-old) female mice were used in all experiments. CD4–/– mice on B6 background (H-2b) together with their age- and sex-matched wild-type controls (C57BL/6J) were purchased from Taconic Farms. All mice were housed in our animal care facility at National Institute of Allergy and Infectious Diseases (NIAID) under specific, pathogen-free conditions, and maintained on standard rodent chow and water supplied ad libitum.
Cell lines
The SV40-transformed cell lines C57SV (H-2b) and KD2SV (H-2d) and the methylcholanthrene-induced fibrosarcoma cell line MC57G (H-2b) were cultured in DMEM containing GLUTAMAX, glucose, sodium pyruvate, and pyridoxine · HCl, and supplemented with 10% FBS. The murine thymoma cell line EL4 (H-2b) was grown in RPMI 1640 medium containing GLUTAMAX plus 10% FBS. The immature murine dendritic cell line DC2.4 (H-2b) (20) was generously provided by Dr. K. L. Rock (University of Massachusetts Medical School, Worcester, MA) and maintained in IMDM containing GLUTAMAX, HEPES buffer (25 mM), and sodium bicarbonate, and supplemented with 10% FBS. Media and FBS were all from Invitrogen Life Technologies.
Antibodies
The hybridoma producing anti-mouse CD25 mAb (clone PC61, rat IgG1) was obtained from American Type Culture Collection. Culture supernatant was collected and PC61 was purified using protein G columns. Low endotoxin, azide-free rat IgG1 isotype control (with specificity for keyhole limpet hemocyanin) was purchased from Southern Biotechnology Associates. Anti-CD16/CD32 (clone 2.4G2, rat IgG2b, Fc Block), CyChrome-conjugated anti-mouse CD4 (clone RM4-5, rat IgG2a), CyChrome-conjugated anti-mouse CD8 (clone 53-6.7, rat IgG2a), FITC-conjugated anti-mouse CD25 (clone 7D4, rat IgM), and FITC-conjugated anti-mouse IFN- (clone XMG1.2, rat IgG1) mAbs were all from BD Pharmingen.
Peptides
Peptides used in this study (listed in Table I) were procured or synthesized, purified by HPLC, and analyzed by mass spectrometry by or under the supervision of the Biologic Resource Branch, NIAID (Rockville, MD). In each case, substances with the predicted mass constituted >95% of the material analyzed. Stock solutions of all peptides were prepared at 1 mM in DMSO and stored at –30°C.
Table I. Peptides used in this study
Viruses and immunization
Mice received i.p. a single 500-μg dose of purified PC61 anti-CD25 mAb. Control animals received either PBS or rat IgG1 isotype control. Four days later, animals were primed i.p. with our established immunizing doses of inocula, namely, 2 x 107 syngeneic, Tag-positive C57SV fibroblastic cells or allogeneic, Tag-positive KD2SV kidney cells, 5 x 106 PFU of wild-type (Western Reserve (WR)) VV or rVVs, or 600 hemagglutinating units (HAU) of IAV/Puerto Rico/8/34 (PR8). rVVs encoding SV40 full-length Tag (rVV-941T) and Tag404–411 (rVV-IV) minigene were kindly provided by Dr. S. S. Tevethia (Pennsylvania State University College of Medicine, Hershey, PA). VVs were propagated in the thymidine kinase-deficient human osteosarcoma cell line 143B. IAV was grown in 10-day-old embryonated chicken eggs and used as infectious allantoic fluid.
In vitro infection of DC2.4 cells for viral Ag presentation
For IAV infection, DC2.4 cells were extensively washed in prewarmed AIM medium containing BSA fraction V as well as 5 mM HEPES buffer, and resuspended in the same medium in a total volume of 1–2 ml along with 100 HAU of IAV per 106 cells. Cells were incubated for 1 h at 37°C while being mixed on a shaker before addition of 5 ml of Iscove’s medium (plus 10% FBS) and further incubation for a 4- to 5-h period. For VV infection of DC2.4, the protocol was the same except the WR strain of VV was added to the cells at a multiplicity of infection of 10 in balanced salt solution containing 0.1% BSA (BSS/BSA) during the first 30 min of incubation.
Intracellular cytokine staining
Erythrocyte-depleted splenocytes were prepared and peritoneal exudate cells (PECs) were collected via peritoneal lavage using sterile PBS 6–7 days after infection with PR8 or VVs, or 9 days after priming with SV40-transformed cells, the time points at which specific TCD8+ responses are reportedly optimal (21, 22). Cells were resuspended in RPMI 1640 medium plus 10% FBS and 10 mM HEPES buffer and seeded at 1–2 x 106 cells/well in U-bottom, 96-well plates. Peptides were then added at a final concentration of 1 μM. Several wells received virus-infected DC2.4 or Tag-expressing C57SV cells instead of peptides for in vitro restimulation of TCD8+. After 2-h incubation at 37°C, brefeldin A (Sigma-Aldrich) was added at a final dose of 10 μg/ml, and plates were incubated for an additional 3–4 h to allow for IFN- accumulation in the endoplasmic reticulum of activated T cells (23). Cells were then washed and incubated with 5 μg/ml Fc Block to avoid nonspecific, FcR-mediated binding of Abs. CyChrome-labeled anti-CD8 mAb was added to the cells that were kept on ice for 30 min followed by washing and fixing cells with 1% paraformaldehyde for 20 min in dark. Cells were then washed again and incubated with FITC-conjugated anti-IFN- mAb diluted in PBS containing 0.1% permeabilizing agent saponin (Calbiochem). Cytofluorimetric evaluation of the cells was then performed using a FACSCalibur (BD Biosciences). Between 1 and 3 x 105 events were collected on the Calibur, and data were analyzed using FlowJo software (Tree Star). The percentage of IFN--positive cells was then determined after live gating on CD8+ events and used to calculate the absolute numbers of Ag-specific TCD8+ present in spleens or peritoneal cavities.
Ex vivo cytotoxicity assay
CTL induction in the presence or absence of Treg cells was assessed by conventional 51Cr release assay. In brief, mice were treated with PBS or PC61 followed by priming with C57SV cells as described above. Erythrocyte-depleted splenocytes were prepared on day 9 postpriming and used as effector cells at indicated ratios against either 51Cr-labeled C57SV or EL4 target cells. The latter cells were sensitized with 100 nM Tag-derived peptides. Target cells were seeded at 104 cells/well together with effector splenocytes in 96-well U-bottom plates. The plates were spun at 400 x g for 5 min at the end of a 6-, 9-, or 12-h incubation period at 37°C. A 100-μl aliquot of supernatant was then harvested from each well, and the 51Cr content of the samples was determined by gamma counting. Specific lysis of the target cells was determined using following formula: % specific lysis = [(ER – SR)/(TR – SR)] x 100, where ER (experimental release) is obtained from wells containing both effector and target cells, whereas SR (spontaneous release) and TR (total release) are determined from wells receiving only target cells plus medium or target cells plus a 1/7 dilution of 3.5% (w/v) cetrimide, respectively.
In vivo killing assay
This assay was performed as described elsewhere (24) with minor modifications. To prepare target cells, syngeneic splenocytes from naive C57BL/6 mice were depleted of erythrocytes, washed, and split into three populations, each of which was pulsed with indicated peptides at 37°C for 1 h and stained with a given concentration of CFSE (Molecular Probes). The population corresponding to the negative control was incubated with a mixture of noncognate peptides lymphocytic choriomeningitis virus (LCMV)-GP33 (Db-restricted) and HSV-gB498 (Kb-restricted) at 500 nM each and was stained with 0.02 μM CFSE (CFSElow). The CFSEint (0.2 μM CFSE) and CFSEhigh (2 μM CFSE) populations were preincubated with site I (1 μM) and site IV (1 μM) of Tag, respectively. After extensive washing, equal numbers of cells from each population were mixed, and each mouse (naive or previously primed with C57SV cells) received a total of 3 x 107 target cells in 500 μl of PBS i.v. Two or 4 h later, mice were sacrificed for their spleens, which were immediately transferred onto ice. Spleen cell preparations were then analyzed by flow cytometry using differential CFSE fluorescence intensities to distinguish CFSE-labeled target cells. Up to 1 x 104 CFSEhigh events were collected for each mouse for analysis and the percent specific killing was calculated as follows: 100 – [(% Tag peptide-pulsed in primed mouse/% control peptide-pulsed in primed mouse)/(% Tag peptide-pulsed in naive mouse/% control peptide-pulsed in naive mouse)] x 100.
Results
Treg depletion enhances Tag determinant-specific TCD8+ responses
SV40 Tag mediates neoplastic transformation of a variety of mammalian cell types (25). In H-2b mice, four determinants recognized by TCD8+, termed sites I, II/III, IV, and V (Table I), have been rigorously defined by Tevethia and colleagues (22). TCD8+ responses to these determinants, enumerated either by ex vivo evaluation of intracellular IFN- accumulation, tetramer staining, or in vitro killing, fall into the following dominance hierarchy: site IV >> I II/III >> V (22, 26, 27).
To examine the contribution of Treg cells to this immunodominance hierarchy, we treated mice with PC61 before i.p. immunization with C57SV cells, an SV40-transformed line generated from B6 mice. Treatment with PC61, but not with PBS or rat IgG1, led to nearly complete depletion of Treg from spleens as judged by cytofluorimetric analysis of splenocytes after staining for CD4 and CD25 (Fig. 1). (Note that we used a mAb to stain CD25 (7D4) that binds to CD25 noncompetitively with PC61 (28). Thus, the lack of staining for CD4+CD25+ cells following PC61 administration is not simply due to PC61 blocking of 7D4 mAb binding.) A profound depletion of Treg was similarly achieved among PBMCs and PECs of PC61-treated mice. Importantly, on the days when T cell functional assays were performed (e.g., 9 days after immunization with Tag-expressing cells), Treg were still dramatically missing from splenic and peritoneal cell preparations (data not shown).
FIGURE 1. Anti-CD25 mAb PC61 depletes CD4+CD25+ Treg. C57BL/6 mice were treated i.p. with either PBS (left panel) or a single, 500-μg dose of anti-CD25 mAb (PC61) (right panel). Four days later, splenocytes were isolated and incubated with Fc Block (5 μg/ml) followed by staining with CyChrome-conjugated anti-CD4 mAb and FITC-conjugated anti-CD25 mAb (clone 7D4). The value shown for the PBS-treated animal is the percentage of CD4+CD25+ T cells among total CD4+ T cell population.
TCD8+ specific for determinants I, II/III, or IV were readily detectable both locally (among PECs) and systemically (among splenocytes) by intracellular cytokine staining for IFN- 9 days after i.p. immunization of mice with C57SV cells. TCD8+ exhibited the classical Tag-specific immunodominance hierarchy (Fig. 2A). Treg depletion augmented the overall responses to C57SV by 2-fold. The increase was skewed toward the more dominant determinants sites IV and I, particularly the local response, in which the effect was highly specific for site IV. Consistent with this, PC61 treatment failed to raise responses to determinant V above background levels.
FIGURE 2. Treg depletion leads to augmented IFN- responses of Tag-specific TCD8+ generated via cross-priming. A, Three mice/group were injected i.p. with PBS or PC61 4 days before challenge with syngeneic, SV40-transformed C57SV cells. Nine days later, splenocytes and PECs were examined ex vivo for IFN- accumulation following restimulation with indicated peptides or C57SV cells used at 4 x 105 cells/well. LCMV-GP33 is an H-2b-binding immunodominant peptide of LCMV that was used as an additional negative control. Values are subtracted from background obtained from wells receiving no peptides and are expressed as mean ± SE of three individual mice in each group for splenic responses. For peritoneal responses, PECs were pooled and values were divided by the number of mice per group to give absolute cell numbers/mouse. Data are representative of three independent experiments yielding nearly identical results. B, Groups of two mice were injected with PBS or PC61 followed by i.p. immunization with KD2SV cells. Total Tag peptide-specific TCD8+ were then enumerated after restimulation ex vivo with Tag peptides or C57SV cells. Results are presented as the average number of IFN--positive TCD8+/spleen obtained from two mice/group. For peritoneal responses, PECs from two mice/group were pooled, and values were divided by 2 to give absolute cell numbers/mouse. There were no responses to any Tag-derived determinants following injection of mice with syngeneic, Tag-negative MC57G cells.
C57SV cells do not express costimulatory molecules and presumably are unable to activate naive TCD8+ directly. Nevertheless, to unambiguously examine the effect of Treg on TCD8+ activated by cross-priming, we immunized mice with SV40-transformed H-2d cells (KD2SV). Consistent with our previous report (27), this gave rise to robust Tag-specific TCD8+ response (Fig. 2B). PC61 treatment again enhanced responses. Now the enhancement was more evenly distributed among TCD8+ specific for IDD and SDDs. These data demonstrate that PC61 treatment enhances TCD8+ induced via cross-priming.
Of note, using either C57SV or KD2SV cells as immunogens, PC61-mediated enhancement of both the absolute numbers and frequencies of Tag determinant-specific TCD8+ was consistently accompanied by heightened overall numbers of splenocytes and PECs (30 and 90% increases on average, respectively, compared with PBS-treated mice), as well as in percentages of total TCD8+ (up to 23% increase in spleens and 96% increase in peritoneal cavities).
Tag determinant-specific cytotoxicity is augmented by depleting Treg
IFN- secretion is but one of many TCD8+ functions that can differ widely among distinct functional TCD8+ subsets. To extend these findings to another central TCD8+ function, we examined the effect of Treg depletion on TCD8+-mediated Tag-specific cytotoxicity. Nine days following immunization of B6 mice with C57SV cells, we determined the capacity of splenocytes ex vivo to lyse 51Cr-labeled C57SV cells or EL4 cells sensitized with synthetic peptides corresponding to Tag determinants. PC61 treatment enhanced overall tumoricidal activity as determined by lysis of C57SV cells (40% increase in killing at a 30:1 splenocyte:C57SV ratio, compared with PBS-treated animals; data not shown). This reflected increased activities of TCD8+ specific for sites I, II/III, and IV (Fig. 3). By contrast, no specific lysis of site V-pulsed cells was induced. Although the absolute order of the hierarchy was maintained in PC61-treated mice, site I-specific killing was selectively enhanced.
FIGURE 3. TCD8+-mediated, ex vivo lysis of Tag determinant-displaying targets in Treg-depleted mice. Three mice/group were injected i.p. with PBS (, , , ) or PC61 (?, , , ) and subsequently immunized with Tag-positive C57SV cells. Nine days later, splenocytes from each group were pooled and used at indicated E:T ratios against EL4 target cells sensitized with indicated peptides in a 12-h 51Cr release assay. Data are from one experiment representative of two independent experiments with similar results. The background killing of EL-4 in wells receiving no Tag peptide, or containing dimethylsulfoxide or LCMV-GP33 was in the range of 4–14%. Each data point represents the average of quadruplicate samples after subtraction of background killing (no peptide) for each group. Each set of data exhibited <10% SD.
Next, we examined in vivo cytolytic activity of site I- and site IV-specific TCD8+ generated in the presence or absence of Treg (Fig. 4). Mice primed with C57SV cells destroy target splenocytes pulsed with the immunodominant site IV peptide more efficiently than those displaying the site I peptide injected simultaneously into the same animals. Consistent with the ex vivo data, site I-specific lysis was dramatically enhanced in Treg-depleted mice, whereas the lysis of splenocytes sensitized with site IV determinant was probably too robust to be further augmented. We also observed augmented destruction of site I-sensitized cells at several other time points following introduction of peptide-pulsed targets into mice (data not shown).
FIGURE 4. In vivo lysis of Tag determinant-displaying targets by TCD8+ induced in the presence or absence of Treg. Target splenocytes were pulsed with control peptides, Tag peptide I, and Tag peptide IV, and stained with CFSE at 0.02, 0.2, and 2 μM, respectively. These cells were washed extensively, mixed and injected into tail veins of naive or C57SV-primed mice pretreated with PBS or PC61. Two hours later, splenocytes from each mouse were harvested and analyzed by flow cytometry using differential CFSE fluorescence to distinguish CFSE-labeled target cells. The percent specific killing of each target cell population was calculated as described in Materials and Methods, and representative numbers are shown. Similar results were obtained in two additional experiments.
Together, these data demonstrate that Treg suppress the lytic activity of Tag-specific TCD8+, although in this case the suppressive effect is exerted with greatest force against site I, which ranks second in the hierarchy (only after site IV).
TCD8+ responses to rVV-encoded Tag determinants are elevated in the absence of Treg
We extended these findings to viral Ags by examining the contribution of Treg to day 7 IFN--producing TCD8+ responding to full-length Tag encoded by a rVV. PC61-treated animals immunized with rVV-Tag demonstrated clearly enhanced responses to site IV, whereas others remained almost unchanged (Fig. 5A).
FIGURE 5. Treg depletion results in enhanced IFN- responses of TCD8+ to VV-encoded Tag determinants. Mice in groups of two were pretreated with either PBS or PC61 mAb followed by i.p. injection of rVVs expressing full-length Tag (A) or Tag determinant IV minigene (B). Splenocytes were prepared and pooled on day 7 postinfection and total numbers of Tag-specific TCD8+/mouse were calculated after ex vivo restimulation with corresponding peptides or C57SV cells used at 4 x 105 cells/well. Note scale differences in B.
It is uncertain whether TCD8+ priming by rVV-encoded, metabolically stable Ags is due to cross-priming or direct priming. We recently presented evidence strongly suggesting that VV-encoded minigene products are exclusively presented by direct priming (29). This enabled us to determine in a relatively unambiguous manner whether PC61 administration can enhance direct priming. Following treatment with PBS or PC61, we immunized mice with a rVV encoding site IV determinant as cytosolic minigene product and enumerated peptide-specific, IFN--producing TCD8+. This experiment confirmed the notion that site IV-specific TCD8+ are more readily generated in the absence of Treg even when site IV is introduced to the immune system in the form of a rVV-encoded minigene (Fig. 5B). More importantly, Treg are capable of suppressing responses to determinants presented by direct priming.
In the same experiment, we evaluated the effect of PC61 treatment on VV-specific TCD8+ responses. We recently identified five VV determinants that account for approximately half of the primary VV-specific responses (30). Responses to these determinants fall into a typical immunodominance hierarchy in the order of B8R20 >> A47L138 K3L6 > A42R88 > A19L47. As seen in Fig. 5B, PC61 selectively enhanced responses to the two IDDs in this rVV, the aforementioned site IV and B8R20. Responses to these determinants were increased 1.8- and 4.4-fold, respectively, whereas responses to the SDDs were increased to a much lesser extent. In five of six additional experiments using rVVs or WR, B8R20-specific responses were enhanced by prior PC61 treatment to a greater extent than those directed against SDDs (data summarized in Table II) (A19L47 is not included in this analysis because responses were too close to background levels to allow for meaningful comparisons).
Table II. Summary of PC61 effects on VV and IAV immunodominance hierarchiesa
Immunodominance hierarchy of anti-IAV TCD8+ in the absence of Treg
To further generalize our findings, we examined the effects of PC61 treatment on TCD8+ responses to IAV in B6 mice, which exhibit a well-characterized immunodominance hierarchy (31, 32, 33, 34, 35). The typical hierarchy was observed in IFN--synthesizing TCD8+ among splenocytes and PECs examined ex vivo (Fig. 6). PC61 administration increased overall IAV-specific responses as measured by using IAV-infected DC2.4 cells and increased numbers of TCD8+ responding to individual peptides. The increase in PA224-specific TCD8+ was apparent in terms of both absolute numbers of responding TCD8+ (Fig. 6) and relative frequency among total TCD8+ recovered (data not shown).
FIGURE 6. Immunodominance hierarchy of IAV-specific TCD8+ generated in the absence of Treg. Three mice/group were injected i.p. with PBS or PC61 4 days before flu infection. On day 7 postinfection, spleen and peritoneal cells were examined ex vivo for IFN- accumulation following restimulation with indicated peptides or IAV-infected DC2.4 cells (used at 4 x 105 cells/well). Values were subtracted from background obtained from wells receiving no peptides and are expressed as mean ± SE of three individual mice in each group. For peritoneal responses, PECs were pooled and values were divided by the number of mice per group to give absolute cell numbers/mouse. Data are representative of three independent experiments yielding similar results.
Although determinants maintained their absolute ranking in the immunodominance hierarchy, PC61 treatment selectively enhanced responses to the two most dominant determinants PA224 and NP366, with the greatest effects exerted on PA224. Over three experiments in which we measured responses to PA224, NP366, and PB1-F262, PA224-specific TCD8+ responses were consistently enhanced to a greater extent than those against IAV SDDs. Thus, as with Tag and VV determinants, the overall effect of Treg is to moderate immunodominance disparities in suppressing overall responses.
Treg-mediated suppression of TCD8+ responses is absent in CD4–/– mice
To date, studies on the in vivo functions of Treg are based almost exclusively on the enhancing effects of PC61 anti-CD25 mAb administration on T cell responsiveness. Although these effects can be correlated with the depletion of Treg, it is possible that the real target is another cell that expresses either CD25 itself or an Ag that fortuitously cross-reacts with PC61.
If PC61 truly acts by depleting Treg, then it should have no effect on TCD8+ responses of mice lacking TCD4+. We therefore examined the effect of PC61 administration on site IV-specific TCD8+ responses in both wild-type and CD4–/– mice infected with rVV-IV. As originally reported by Schell et al. (36), CD4–/– mice mount a respectable site IV-specific response. The fairly CD4-independent nature of this response is particularly important to avoid potential complications in data interpretation due to the possible impact of Treg on effector TCD4+ (37, 38). Unlike wild-type mice, treatment with PC61 had no significant effect on the site IV-specific response of CD4–/– mice (Fig. 7). The enhancing effect of PC61 on TCD8+ responses to VV determinants was similarly reversed in CD4–/– mice in the same experiment (data not shown), further confirming that PC61 acts on a subset of TCD4+.
FIGURE 7. PC61-mediated enhancement of Ag-specific TCD8+ responses is absent in CD4–/– mice. Wild-type and CD4 knockout mice in groups of two were pretreated with PBS or PC61 4 days before i.p. challenge with rVV-IV. Seven days later, splenocyte from each group were pooled and total numbers of site IV-specific TCD8+ per spleen were calculated (A) after in vitro restimulation with site IV peptide. Values were also expressed as percent site IV-specific TCD8+ of total splenic TCD8+ pools (in comparison with PBS-treated animals in each group) (B).
Discussion
Observations of T cell-mediated immune suppression date back to the early 1970s (39, 40). Suppressor T cell studies suffered a severe setback when I-J, the genetic locus associated with suppression, proved to be illusory as determined by one of the initial applications of genome sequencing. T cell-mediated suppression was resurrected with the identification of CD4+CD25+ Treg cells (3), which have been implicated in limiting TCD4+ responses to foreign Ags in many systems. This mechanism appears to function to limit autoimmunity. Although much remains to be learned about Treg, knowledge of their interaction with TCD8+ is at a particularly rudimentary level. In this study, we provide the initial description of the effects of Treg on TCD8+ responses to multiple, well-characterized determinants in diverse systems presented by defined pathways.
Our approach, as in nearly all studies of Treg function in mice, entails the use of the PC61 mAb to deplete Treg. This approach is compromised by the troubling possibility that PC61 acts by affecting non-Treg cells. We provide here the initial evidence that the PC61-mediated enhancement of immune responsiveness requires the presence of TCD4+, because we failed to observe enhanced responses in CD4–/– mice. This finding is of prime importance because it categorically dispels the possibility that enhanced TCD8+ responses are secondary to increased survival of effector TCD8+ due to PC61-mediated inhibition of IL-2-IL-2R interactions (28, 41) that would otherwise initiate a program of activation-induced cell death in Ag-specific TCD8+ (42, 43). We further note that the residual PC61-mediated elimination of effector TCD8+ that up-regulate CD25 following TCR stimulation (44) is highly unlikely in our experiments because we consistently observed increases in absolute numbers of TCD8+ recovered from PC61-treated mice. Finally, our unpublished observations indicate that the mRNA encoding the transcription factor scurfin (also known as forkhead box P3 or FoxP3 for short), which is predominantly, if not exclusively, expressed by Treg (45, 46), is greatly reduced in the lymph nodes of PC61-treated mice. This confirms the elimination of Treg by PC61 in secondary lymphoid organs and indirectly rules out the possibility that the lack of CD25 staining in flow cytometry is due to CD25 down-regulation after PC61 binding.
We found that Treg depletion before cellular immunization with either MHC-compatible or -incompatible SV40-transformed cells results in elevated splenic (systemic) and peritoneal (local) TCD8+ responses to SV40 Tag determinants I, II/III, and IV. These data clearly indicate that Treg are operational in cross-priming settings for the following reasons. First, C57SV and KD2SV cells used to immunize mice lack viral genes required for the generation of virions. Second, these cells are derived from fibroblasts and epithelium, respectively, and not professional APCs and should not be capable of providing costimulatory signals required to activate naive TCD8+. Third, because KD2SV cells (H-2d) are allogeneic to the host (H-2b), priming must be based on transfer of Ag to the host professional APCs.
Treg depletion demonstrated a similar enhancement of TCD8+ responses to Ags encoded by IAV and VV. For viral proteins produced during the course of a natural infection, it is ambiguous whether presentation occurs via direct priming or cross-priming. However, it is clear that presentation of minigene products occurs via direct priming (47). Therefore, the clear effects of Treg depletion on the immunogenicity of rVV-encoded site IV minigene product provides a clear demonstration that Treg also interfere with direct presentation.
One of the principal goals of this study was to determine the contribution of Treg to establishing the immunodominance hierarchy, particularly whether Treg exacerbated differences in responses to IDDs vs SDDs. Previous work from our and others’ laboratories have identified several factors that contribute to immunodominance. A key factor is the generation of an appropriate number of peptide-class I complexes on the surface of APCs. Once this threshold is passed, however, other factors, including those yet to be defined, modulate TCD8+ responses to individual determinants. Our findings indicate that Treg activity is not responsible for the immunorecessive nature of site V in the Tag system nor does it dictate subdominance of SDDs harbored by VV and IAV. Furthermore, Treg do not significantly influence the rank order of determinants in immunodominance hierarchies in Tag, VV, or IAV systems in B6 mice.
The mechanisms by which Treg modify TCD8+ responses remain to be established. The similar effects of Treg on TCD8+ activated by cross-priming or direct priming implies (although not conclusively) that Treg act on TCD8+ and not on APCs, because the latter would be expected to differ in the different priming scenarios. The selective effects of Treg on TCD8+ responding to the most dominant determinants is consistent with the idea of direct interaction between Treg and TCD8+. The simplest possibility is that Treg limit TCD8+ expansion, and that this effect is greatest for cells dividing the most rapidly. In support of this idea, there is evidence that determinant-related differences in division time contributes to immunodominance (48). Thus, we propose that Treg moderate immunodominance hierarchies by retarding division of the most actively dividing TCD8+. It should be possible to directly address this hypothesis in future studies.
Although the effects of Treg on numbers of TCD8+ responding to individual determinants are complex, the overall effect of Treg depletion in the Tag system is to exacerbate the difference between TCD8+ responding to IDDs vs SDDs. Thus, following immunization with C57SV cells, responses to site IV are enhanced 2-fold whereas responses to site II/III increase only slightly, and responses to site V remain undetectable (Fig. 2).
The selective effect of Treg depletion on enhancing TCD8+ responses to IDDs was clear in the viral systems used. Thus, PC61 treatment selectively enhanced splenic TCD8+ responses to the IDDs, VV-B8R20 and IAV-PA224, in consistent manner over a series of experiments. Based on these data, we conclude that Treg are not principally responsible for immunodominance hierarchies among responding TCD8+ in these systems. Rather, Treg have the opposite effect, and function to narrow disparities among TCD8+ responding to different determinants. It will be interesting to determine the mechanism underlying this phenomenon, and whether it entails physical contact between Treg and their client cells. From the practical standpoint, those contemplating enhancing immunity by interfering with Treg function who are not daunted by potential difficulties with autoimmunity may also have to contend with difficulties with enhancing immunodominance disparities, which under some circumstances (e.g., viral infections), may enhance immune escape.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Deborah Tokarchick for providing outstanding technical assistance and Dr. Kari Irvine for valuable suggestions.
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 Current address: EBV Unit, Queensland Institute for Medical Research, Herston, Queensland 4006, Australia.
2 Address correspondence and reprint requests to Dr. Jack R. Bennink or Dr. Jonathan W. Yewdell, Room 211, 4 Center Drive, Mail Stop 0440, National Institutes of Health, Bethesda, MD 20892-0440. E-mail address: jbennink{at}nih.gov or E-mail address: jyewdell{at}nih.gov
3 Abbreviations used in this paper: IDD, immunodominant determinant; SDD, subdominant determinant; Treg, regulatory T cell; Tag, SV40 large tumor Ag; VV, vaccinia virus; IAV, influenza A virus; LCMV, lymphocytic choriomeningitis virus; PEC, peritoneal exudate cell; int, intermediate.
Received for publication October 7, 2004. Accepted for publication December 20, 2004.
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Little is known regarding the participation of CD4+CD25+ regulatory T cells (Treg) in TCD8+ responses. In this study, we show that Treg depletion via treatment with anti-CD25 mAb (PC61) significantly enhances TCD8+ responses to influenza A virus, vaccinia virus, and SV40-transformed cells induced by either direct priming or cross-priming. PC61 did not enhance TCD8+ responses in CD4-deficient mice, providing the initial demonstration that PC61 acts on a subset of TCD4+, and not on other cells that express either CD25 or a fortuitously cross-reactive Ag. We further show that Treg selectively suppress responses to the most immunodominant TCD8+ determinants in the three systems examined. Therefore, Treg influence TCD8 immunodominance hierarchies by moderating disparities in responses to different determinants.
Introduction
Despite the presence of thousands to millions of potentially immunogenic peptides in complex pathogens, TCD8+ responses are dominated by clones responding to a very limited number of determinants. This phenomenon, known as immunodominance, has been observed in mouse and human TCD8+ responses to numerous viruses, and appears to apply to antibacterial responses as well. Among TCD8+ responding to different determinants, there is often large variation (up to several hundred-fold) in responses to immunodominant determinants (IDDs) 3 vs subdominant determinants (SDDs) (1).
The underlying mechanisms for immunodominance have been the subject of intense investigation due to its obvious importance for understanding TCD8+ biology and vaccine design. A number of factors are known to contribute to immunodominance in viral systems. In approximate order of importance, these include 1) peptide affinity for MHC class I molecules, 2) liberation of peptides by cellular proteases, 3) abundance of the viral source gene product, 4) TCD8+ repertoire, 5) specificity of the TAP, and 6) kinetics of determinant generation by virus-infected cells. An additional important contribution is made by immunodomination, the suppression of SDD-specific T cells by IDD-specific T cells. Immunodomination occurs at least in part at the level of individual APCs displaying multiple determinants (2).
However, these factors do not fully explain immunodominance. In many systems, no particular factors can account for the ascendance of IDDs. Precious little is known about possible contribution(s) of suppressive elements of the immune system to shaping immunodominance hierarchies of Ag-specific TCD8+. One such element is a subset of TCD4+ that constitutively coexpress IL-2R -chain (CD25).
Naturally occurring CD4+CD25+ regulatory T cells, which will hereafter be referred to as Treg, constitute 5–15% of peripheral TCD4+ in both mice and humans. Treg appear to function to prevent a host of autoimmune dyscrasias (3, 4). Treg play an important role in modulating immune responses to bacterial pathogens (5, 6, 7), parasitic protozoa (8, 9), opportunistic fungi (10, 11), and tumors (12, 13, 14). Treg have also been implicated in limiting allograft rejection (15, 16) and graft-vs-host disease (17, 18).
The involvement of Treg in TCD8+ responses is only now being scrutinized. Treg were recently reported to regulate TCD8+ responses directed against an IDD of HSV-1 following viral infection in mice (19). However, it is uncertain whether Treg-mediated suppression is a universal feature of antiviral TCD8+ responses, and their participation in establishing immunodominance hierarchies is essentially undefined.
In the present study, we have examined the contribution of Treg to the magnitude and composition of TCD8+ responses to SV40 large tumor Ag (Tag), vaccinia virus (VV), and influenza A virus (IAV).
Materials and Methods
Mice
Adult (6- to 10-wk-old) female mice were used in all experiments. CD4–/– mice on B6 background (H-2b) together with their age- and sex-matched wild-type controls (C57BL/6J) were purchased from Taconic Farms. All mice were housed in our animal care facility at National Institute of Allergy and Infectious Diseases (NIAID) under specific, pathogen-free conditions, and maintained on standard rodent chow and water supplied ad libitum.
Cell lines
The SV40-transformed cell lines C57SV (H-2b) and KD2SV (H-2d) and the methylcholanthrene-induced fibrosarcoma cell line MC57G (H-2b) were cultured in DMEM containing GLUTAMAX, glucose, sodium pyruvate, and pyridoxine · HCl, and supplemented with 10% FBS. The murine thymoma cell line EL4 (H-2b) was grown in RPMI 1640 medium containing GLUTAMAX plus 10% FBS. The immature murine dendritic cell line DC2.4 (H-2b) (20) was generously provided by Dr. K. L. Rock (University of Massachusetts Medical School, Worcester, MA) and maintained in IMDM containing GLUTAMAX, HEPES buffer (25 mM), and sodium bicarbonate, and supplemented with 10% FBS. Media and FBS were all from Invitrogen Life Technologies.
Antibodies
The hybridoma producing anti-mouse CD25 mAb (clone PC61, rat IgG1) was obtained from American Type Culture Collection. Culture supernatant was collected and PC61 was purified using protein G columns. Low endotoxin, azide-free rat IgG1 isotype control (with specificity for keyhole limpet hemocyanin) was purchased from Southern Biotechnology Associates. Anti-CD16/CD32 (clone 2.4G2, rat IgG2b, Fc Block), CyChrome-conjugated anti-mouse CD4 (clone RM4-5, rat IgG2a), CyChrome-conjugated anti-mouse CD8 (clone 53-6.7, rat IgG2a), FITC-conjugated anti-mouse CD25 (clone 7D4, rat IgM), and FITC-conjugated anti-mouse IFN- (clone XMG1.2, rat IgG1) mAbs were all from BD Pharmingen.
Peptides
Peptides used in this study (listed in Table I) were procured or synthesized, purified by HPLC, and analyzed by mass spectrometry by or under the supervision of the Biologic Resource Branch, NIAID (Rockville, MD). In each case, substances with the predicted mass constituted >95% of the material analyzed. Stock solutions of all peptides were prepared at 1 mM in DMSO and stored at –30°C.
Table I. Peptides used in this study
Viruses and immunization
Mice received i.p. a single 500-μg dose of purified PC61 anti-CD25 mAb. Control animals received either PBS or rat IgG1 isotype control. Four days later, animals were primed i.p. with our established immunizing doses of inocula, namely, 2 x 107 syngeneic, Tag-positive C57SV fibroblastic cells or allogeneic, Tag-positive KD2SV kidney cells, 5 x 106 PFU of wild-type (Western Reserve (WR)) VV or rVVs, or 600 hemagglutinating units (HAU) of IAV/Puerto Rico/8/34 (PR8). rVVs encoding SV40 full-length Tag (rVV-941T) and Tag404–411 (rVV-IV) minigene were kindly provided by Dr. S. S. Tevethia (Pennsylvania State University College of Medicine, Hershey, PA). VVs were propagated in the thymidine kinase-deficient human osteosarcoma cell line 143B. IAV was grown in 10-day-old embryonated chicken eggs and used as infectious allantoic fluid.
In vitro infection of DC2.4 cells for viral Ag presentation
For IAV infection, DC2.4 cells were extensively washed in prewarmed AIM medium containing BSA fraction V as well as 5 mM HEPES buffer, and resuspended in the same medium in a total volume of 1–2 ml along with 100 HAU of IAV per 106 cells. Cells were incubated for 1 h at 37°C while being mixed on a shaker before addition of 5 ml of Iscove’s medium (plus 10% FBS) and further incubation for a 4- to 5-h period. For VV infection of DC2.4, the protocol was the same except the WR strain of VV was added to the cells at a multiplicity of infection of 10 in balanced salt solution containing 0.1% BSA (BSS/BSA) during the first 30 min of incubation.
Intracellular cytokine staining
Erythrocyte-depleted splenocytes were prepared and peritoneal exudate cells (PECs) were collected via peritoneal lavage using sterile PBS 6–7 days after infection with PR8 or VVs, or 9 days after priming with SV40-transformed cells, the time points at which specific TCD8+ responses are reportedly optimal (21, 22). Cells were resuspended in RPMI 1640 medium plus 10% FBS and 10 mM HEPES buffer and seeded at 1–2 x 106 cells/well in U-bottom, 96-well plates. Peptides were then added at a final concentration of 1 μM. Several wells received virus-infected DC2.4 or Tag-expressing C57SV cells instead of peptides for in vitro restimulation of TCD8+. After 2-h incubation at 37°C, brefeldin A (Sigma-Aldrich) was added at a final dose of 10 μg/ml, and plates were incubated for an additional 3–4 h to allow for IFN- accumulation in the endoplasmic reticulum of activated T cells (23). Cells were then washed and incubated with 5 μg/ml Fc Block to avoid nonspecific, FcR-mediated binding of Abs. CyChrome-labeled anti-CD8 mAb was added to the cells that were kept on ice for 30 min followed by washing and fixing cells with 1% paraformaldehyde for 20 min in dark. Cells were then washed again and incubated with FITC-conjugated anti-IFN- mAb diluted in PBS containing 0.1% permeabilizing agent saponin (Calbiochem). Cytofluorimetric evaluation of the cells was then performed using a FACSCalibur (BD Biosciences). Between 1 and 3 x 105 events were collected on the Calibur, and data were analyzed using FlowJo software (Tree Star). The percentage of IFN--positive cells was then determined after live gating on CD8+ events and used to calculate the absolute numbers of Ag-specific TCD8+ present in spleens or peritoneal cavities.
Ex vivo cytotoxicity assay
CTL induction in the presence or absence of Treg cells was assessed by conventional 51Cr release assay. In brief, mice were treated with PBS or PC61 followed by priming with C57SV cells as described above. Erythrocyte-depleted splenocytes were prepared on day 9 postpriming and used as effector cells at indicated ratios against either 51Cr-labeled C57SV or EL4 target cells. The latter cells were sensitized with 100 nM Tag-derived peptides. Target cells were seeded at 104 cells/well together with effector splenocytes in 96-well U-bottom plates. The plates were spun at 400 x g for 5 min at the end of a 6-, 9-, or 12-h incubation period at 37°C. A 100-μl aliquot of supernatant was then harvested from each well, and the 51Cr content of the samples was determined by gamma counting. Specific lysis of the target cells was determined using following formula: % specific lysis = [(ER – SR)/(TR – SR)] x 100, where ER (experimental release) is obtained from wells containing both effector and target cells, whereas SR (spontaneous release) and TR (total release) are determined from wells receiving only target cells plus medium or target cells plus a 1/7 dilution of 3.5% (w/v) cetrimide, respectively.
In vivo killing assay
This assay was performed as described elsewhere (24) with minor modifications. To prepare target cells, syngeneic splenocytes from naive C57BL/6 mice were depleted of erythrocytes, washed, and split into three populations, each of which was pulsed with indicated peptides at 37°C for 1 h and stained with a given concentration of CFSE (Molecular Probes). The population corresponding to the negative control was incubated with a mixture of noncognate peptides lymphocytic choriomeningitis virus (LCMV)-GP33 (Db-restricted) and HSV-gB498 (Kb-restricted) at 500 nM each and was stained with 0.02 μM CFSE (CFSElow). The CFSEint (0.2 μM CFSE) and CFSEhigh (2 μM CFSE) populations were preincubated with site I (1 μM) and site IV (1 μM) of Tag, respectively. After extensive washing, equal numbers of cells from each population were mixed, and each mouse (naive or previously primed with C57SV cells) received a total of 3 x 107 target cells in 500 μl of PBS i.v. Two or 4 h later, mice were sacrificed for their spleens, which were immediately transferred onto ice. Spleen cell preparations were then analyzed by flow cytometry using differential CFSE fluorescence intensities to distinguish CFSE-labeled target cells. Up to 1 x 104 CFSEhigh events were collected for each mouse for analysis and the percent specific killing was calculated as follows: 100 – [(% Tag peptide-pulsed in primed mouse/% control peptide-pulsed in primed mouse)/(% Tag peptide-pulsed in naive mouse/% control peptide-pulsed in naive mouse)] x 100.
Results
Treg depletion enhances Tag determinant-specific TCD8+ responses
SV40 Tag mediates neoplastic transformation of a variety of mammalian cell types (25). In H-2b mice, four determinants recognized by TCD8+, termed sites I, II/III, IV, and V (Table I), have been rigorously defined by Tevethia and colleagues (22). TCD8+ responses to these determinants, enumerated either by ex vivo evaluation of intracellular IFN- accumulation, tetramer staining, or in vitro killing, fall into the following dominance hierarchy: site IV >> I II/III >> V (22, 26, 27).
To examine the contribution of Treg cells to this immunodominance hierarchy, we treated mice with PC61 before i.p. immunization with C57SV cells, an SV40-transformed line generated from B6 mice. Treatment with PC61, but not with PBS or rat IgG1, led to nearly complete depletion of Treg from spleens as judged by cytofluorimetric analysis of splenocytes after staining for CD4 and CD25 (Fig. 1). (Note that we used a mAb to stain CD25 (7D4) that binds to CD25 noncompetitively with PC61 (28). Thus, the lack of staining for CD4+CD25+ cells following PC61 administration is not simply due to PC61 blocking of 7D4 mAb binding.) A profound depletion of Treg was similarly achieved among PBMCs and PECs of PC61-treated mice. Importantly, on the days when T cell functional assays were performed (e.g., 9 days after immunization with Tag-expressing cells), Treg were still dramatically missing from splenic and peritoneal cell preparations (data not shown).
FIGURE 1. Anti-CD25 mAb PC61 depletes CD4+CD25+ Treg. C57BL/6 mice were treated i.p. with either PBS (left panel) or a single, 500-μg dose of anti-CD25 mAb (PC61) (right panel). Four days later, splenocytes were isolated and incubated with Fc Block (5 μg/ml) followed by staining with CyChrome-conjugated anti-CD4 mAb and FITC-conjugated anti-CD25 mAb (clone 7D4). The value shown for the PBS-treated animal is the percentage of CD4+CD25+ T cells among total CD4+ T cell population.
TCD8+ specific for determinants I, II/III, or IV were readily detectable both locally (among PECs) and systemically (among splenocytes) by intracellular cytokine staining for IFN- 9 days after i.p. immunization of mice with C57SV cells. TCD8+ exhibited the classical Tag-specific immunodominance hierarchy (Fig. 2A). Treg depletion augmented the overall responses to C57SV by 2-fold. The increase was skewed toward the more dominant determinants sites IV and I, particularly the local response, in which the effect was highly specific for site IV. Consistent with this, PC61 treatment failed to raise responses to determinant V above background levels.
FIGURE 2. Treg depletion leads to augmented IFN- responses of Tag-specific TCD8+ generated via cross-priming. A, Three mice/group were injected i.p. with PBS or PC61 4 days before challenge with syngeneic, SV40-transformed C57SV cells. Nine days later, splenocytes and PECs were examined ex vivo for IFN- accumulation following restimulation with indicated peptides or C57SV cells used at 4 x 105 cells/well. LCMV-GP33 is an H-2b-binding immunodominant peptide of LCMV that was used as an additional negative control. Values are subtracted from background obtained from wells receiving no peptides and are expressed as mean ± SE of three individual mice in each group for splenic responses. For peritoneal responses, PECs were pooled and values were divided by the number of mice per group to give absolute cell numbers/mouse. Data are representative of three independent experiments yielding nearly identical results. B, Groups of two mice were injected with PBS or PC61 followed by i.p. immunization with KD2SV cells. Total Tag peptide-specific TCD8+ were then enumerated after restimulation ex vivo with Tag peptides or C57SV cells. Results are presented as the average number of IFN--positive TCD8+/spleen obtained from two mice/group. For peritoneal responses, PECs from two mice/group were pooled, and values were divided by 2 to give absolute cell numbers/mouse. There were no responses to any Tag-derived determinants following injection of mice with syngeneic, Tag-negative MC57G cells.
C57SV cells do not express costimulatory molecules and presumably are unable to activate naive TCD8+ directly. Nevertheless, to unambiguously examine the effect of Treg on TCD8+ activated by cross-priming, we immunized mice with SV40-transformed H-2d cells (KD2SV). Consistent with our previous report (27), this gave rise to robust Tag-specific TCD8+ response (Fig. 2B). PC61 treatment again enhanced responses. Now the enhancement was more evenly distributed among TCD8+ specific for IDD and SDDs. These data demonstrate that PC61 treatment enhances TCD8+ induced via cross-priming.
Of note, using either C57SV or KD2SV cells as immunogens, PC61-mediated enhancement of both the absolute numbers and frequencies of Tag determinant-specific TCD8+ was consistently accompanied by heightened overall numbers of splenocytes and PECs (30 and 90% increases on average, respectively, compared with PBS-treated mice), as well as in percentages of total TCD8+ (up to 23% increase in spleens and 96% increase in peritoneal cavities).
Tag determinant-specific cytotoxicity is augmented by depleting Treg
IFN- secretion is but one of many TCD8+ functions that can differ widely among distinct functional TCD8+ subsets. To extend these findings to another central TCD8+ function, we examined the effect of Treg depletion on TCD8+-mediated Tag-specific cytotoxicity. Nine days following immunization of B6 mice with C57SV cells, we determined the capacity of splenocytes ex vivo to lyse 51Cr-labeled C57SV cells or EL4 cells sensitized with synthetic peptides corresponding to Tag determinants. PC61 treatment enhanced overall tumoricidal activity as determined by lysis of C57SV cells (40% increase in killing at a 30:1 splenocyte:C57SV ratio, compared with PBS-treated animals; data not shown). This reflected increased activities of TCD8+ specific for sites I, II/III, and IV (Fig. 3). By contrast, no specific lysis of site V-pulsed cells was induced. Although the absolute order of the hierarchy was maintained in PC61-treated mice, site I-specific killing was selectively enhanced.
FIGURE 3. TCD8+-mediated, ex vivo lysis of Tag determinant-displaying targets in Treg-depleted mice. Three mice/group were injected i.p. with PBS (, , , ) or PC61 (?, , , ) and subsequently immunized with Tag-positive C57SV cells. Nine days later, splenocytes from each group were pooled and used at indicated E:T ratios against EL4 target cells sensitized with indicated peptides in a 12-h 51Cr release assay. Data are from one experiment representative of two independent experiments with similar results. The background killing of EL-4 in wells receiving no Tag peptide, or containing dimethylsulfoxide or LCMV-GP33 was in the range of 4–14%. Each data point represents the average of quadruplicate samples after subtraction of background killing (no peptide) for each group. Each set of data exhibited <10% SD.
Next, we examined in vivo cytolytic activity of site I- and site IV-specific TCD8+ generated in the presence or absence of Treg (Fig. 4). Mice primed with C57SV cells destroy target splenocytes pulsed with the immunodominant site IV peptide more efficiently than those displaying the site I peptide injected simultaneously into the same animals. Consistent with the ex vivo data, site I-specific lysis was dramatically enhanced in Treg-depleted mice, whereas the lysis of splenocytes sensitized with site IV determinant was probably too robust to be further augmented. We also observed augmented destruction of site I-sensitized cells at several other time points following introduction of peptide-pulsed targets into mice (data not shown).
FIGURE 4. In vivo lysis of Tag determinant-displaying targets by TCD8+ induced in the presence or absence of Treg. Target splenocytes were pulsed with control peptides, Tag peptide I, and Tag peptide IV, and stained with CFSE at 0.02, 0.2, and 2 μM, respectively. These cells were washed extensively, mixed and injected into tail veins of naive or C57SV-primed mice pretreated with PBS or PC61. Two hours later, splenocytes from each mouse were harvested and analyzed by flow cytometry using differential CFSE fluorescence to distinguish CFSE-labeled target cells. The percent specific killing of each target cell population was calculated as described in Materials and Methods, and representative numbers are shown. Similar results were obtained in two additional experiments.
Together, these data demonstrate that Treg suppress the lytic activity of Tag-specific TCD8+, although in this case the suppressive effect is exerted with greatest force against site I, which ranks second in the hierarchy (only after site IV).
TCD8+ responses to rVV-encoded Tag determinants are elevated in the absence of Treg
We extended these findings to viral Ags by examining the contribution of Treg to day 7 IFN--producing TCD8+ responding to full-length Tag encoded by a rVV. PC61-treated animals immunized with rVV-Tag demonstrated clearly enhanced responses to site IV, whereas others remained almost unchanged (Fig. 5A).
FIGURE 5. Treg depletion results in enhanced IFN- responses of TCD8+ to VV-encoded Tag determinants. Mice in groups of two were pretreated with either PBS or PC61 mAb followed by i.p. injection of rVVs expressing full-length Tag (A) or Tag determinant IV minigene (B). Splenocytes were prepared and pooled on day 7 postinfection and total numbers of Tag-specific TCD8+/mouse were calculated after ex vivo restimulation with corresponding peptides or C57SV cells used at 4 x 105 cells/well. Note scale differences in B.
It is uncertain whether TCD8+ priming by rVV-encoded, metabolically stable Ags is due to cross-priming or direct priming. We recently presented evidence strongly suggesting that VV-encoded minigene products are exclusively presented by direct priming (29). This enabled us to determine in a relatively unambiguous manner whether PC61 administration can enhance direct priming. Following treatment with PBS or PC61, we immunized mice with a rVV encoding site IV determinant as cytosolic minigene product and enumerated peptide-specific, IFN--producing TCD8+. This experiment confirmed the notion that site IV-specific TCD8+ are more readily generated in the absence of Treg even when site IV is introduced to the immune system in the form of a rVV-encoded minigene (Fig. 5B). More importantly, Treg are capable of suppressing responses to determinants presented by direct priming.
In the same experiment, we evaluated the effect of PC61 treatment on VV-specific TCD8+ responses. We recently identified five VV determinants that account for approximately half of the primary VV-specific responses (30). Responses to these determinants fall into a typical immunodominance hierarchy in the order of B8R20 >> A47L138 K3L6 > A42R88 > A19L47. As seen in Fig. 5B, PC61 selectively enhanced responses to the two IDDs in this rVV, the aforementioned site IV and B8R20. Responses to these determinants were increased 1.8- and 4.4-fold, respectively, whereas responses to the SDDs were increased to a much lesser extent. In five of six additional experiments using rVVs or WR, B8R20-specific responses were enhanced by prior PC61 treatment to a greater extent than those directed against SDDs (data summarized in Table II) (A19L47 is not included in this analysis because responses were too close to background levels to allow for meaningful comparisons).
Table II. Summary of PC61 effects on VV and IAV immunodominance hierarchiesa
Immunodominance hierarchy of anti-IAV TCD8+ in the absence of Treg
To further generalize our findings, we examined the effects of PC61 treatment on TCD8+ responses to IAV in B6 mice, which exhibit a well-characterized immunodominance hierarchy (31, 32, 33, 34, 35). The typical hierarchy was observed in IFN--synthesizing TCD8+ among splenocytes and PECs examined ex vivo (Fig. 6). PC61 administration increased overall IAV-specific responses as measured by using IAV-infected DC2.4 cells and increased numbers of TCD8+ responding to individual peptides. The increase in PA224-specific TCD8+ was apparent in terms of both absolute numbers of responding TCD8+ (Fig. 6) and relative frequency among total TCD8+ recovered (data not shown).
FIGURE 6. Immunodominance hierarchy of IAV-specific TCD8+ generated in the absence of Treg. Three mice/group were injected i.p. with PBS or PC61 4 days before flu infection. On day 7 postinfection, spleen and peritoneal cells were examined ex vivo for IFN- accumulation following restimulation with indicated peptides or IAV-infected DC2.4 cells (used at 4 x 105 cells/well). Values were subtracted from background obtained from wells receiving no peptides and are expressed as mean ± SE of three individual mice in each group. For peritoneal responses, PECs were pooled and values were divided by the number of mice per group to give absolute cell numbers/mouse. Data are representative of three independent experiments yielding similar results.
Although determinants maintained their absolute ranking in the immunodominance hierarchy, PC61 treatment selectively enhanced responses to the two most dominant determinants PA224 and NP366, with the greatest effects exerted on PA224. Over three experiments in which we measured responses to PA224, NP366, and PB1-F262, PA224-specific TCD8+ responses were consistently enhanced to a greater extent than those against IAV SDDs. Thus, as with Tag and VV determinants, the overall effect of Treg is to moderate immunodominance disparities in suppressing overall responses.
Treg-mediated suppression of TCD8+ responses is absent in CD4–/– mice
To date, studies on the in vivo functions of Treg are based almost exclusively on the enhancing effects of PC61 anti-CD25 mAb administration on T cell responsiveness. Although these effects can be correlated with the depletion of Treg, it is possible that the real target is another cell that expresses either CD25 itself or an Ag that fortuitously cross-reacts with PC61.
If PC61 truly acts by depleting Treg, then it should have no effect on TCD8+ responses of mice lacking TCD4+. We therefore examined the effect of PC61 administration on site IV-specific TCD8+ responses in both wild-type and CD4–/– mice infected with rVV-IV. As originally reported by Schell et al. (36), CD4–/– mice mount a respectable site IV-specific response. The fairly CD4-independent nature of this response is particularly important to avoid potential complications in data interpretation due to the possible impact of Treg on effector TCD4+ (37, 38). Unlike wild-type mice, treatment with PC61 had no significant effect on the site IV-specific response of CD4–/– mice (Fig. 7). The enhancing effect of PC61 on TCD8+ responses to VV determinants was similarly reversed in CD4–/– mice in the same experiment (data not shown), further confirming that PC61 acts on a subset of TCD4+.
FIGURE 7. PC61-mediated enhancement of Ag-specific TCD8+ responses is absent in CD4–/– mice. Wild-type and CD4 knockout mice in groups of two were pretreated with PBS or PC61 4 days before i.p. challenge with rVV-IV. Seven days later, splenocyte from each group were pooled and total numbers of site IV-specific TCD8+ per spleen were calculated (A) after in vitro restimulation with site IV peptide. Values were also expressed as percent site IV-specific TCD8+ of total splenic TCD8+ pools (in comparison with PBS-treated animals in each group) (B).
Discussion
Observations of T cell-mediated immune suppression date back to the early 1970s (39, 40). Suppressor T cell studies suffered a severe setback when I-J, the genetic locus associated with suppression, proved to be illusory as determined by one of the initial applications of genome sequencing. T cell-mediated suppression was resurrected with the identification of CD4+CD25+ Treg cells (3), which have been implicated in limiting TCD4+ responses to foreign Ags in many systems. This mechanism appears to function to limit autoimmunity. Although much remains to be learned about Treg, knowledge of their interaction with TCD8+ is at a particularly rudimentary level. In this study, we provide the initial description of the effects of Treg on TCD8+ responses to multiple, well-characterized determinants in diverse systems presented by defined pathways.
Our approach, as in nearly all studies of Treg function in mice, entails the use of the PC61 mAb to deplete Treg. This approach is compromised by the troubling possibility that PC61 acts by affecting non-Treg cells. We provide here the initial evidence that the PC61-mediated enhancement of immune responsiveness requires the presence of TCD4+, because we failed to observe enhanced responses in CD4–/– mice. This finding is of prime importance because it categorically dispels the possibility that enhanced TCD8+ responses are secondary to increased survival of effector TCD8+ due to PC61-mediated inhibition of IL-2-IL-2R interactions (28, 41) that would otherwise initiate a program of activation-induced cell death in Ag-specific TCD8+ (42, 43). We further note that the residual PC61-mediated elimination of effector TCD8+ that up-regulate CD25 following TCR stimulation (44) is highly unlikely in our experiments because we consistently observed increases in absolute numbers of TCD8+ recovered from PC61-treated mice. Finally, our unpublished observations indicate that the mRNA encoding the transcription factor scurfin (also known as forkhead box P3 or FoxP3 for short), which is predominantly, if not exclusively, expressed by Treg (45, 46), is greatly reduced in the lymph nodes of PC61-treated mice. This confirms the elimination of Treg by PC61 in secondary lymphoid organs and indirectly rules out the possibility that the lack of CD25 staining in flow cytometry is due to CD25 down-regulation after PC61 binding.
We found that Treg depletion before cellular immunization with either MHC-compatible or -incompatible SV40-transformed cells results in elevated splenic (systemic) and peritoneal (local) TCD8+ responses to SV40 Tag determinants I, II/III, and IV. These data clearly indicate that Treg are operational in cross-priming settings for the following reasons. First, C57SV and KD2SV cells used to immunize mice lack viral genes required for the generation of virions. Second, these cells are derived from fibroblasts and epithelium, respectively, and not professional APCs and should not be capable of providing costimulatory signals required to activate naive TCD8+. Third, because KD2SV cells (H-2d) are allogeneic to the host (H-2b), priming must be based on transfer of Ag to the host professional APCs.
Treg depletion demonstrated a similar enhancement of TCD8+ responses to Ags encoded by IAV and VV. For viral proteins produced during the course of a natural infection, it is ambiguous whether presentation occurs via direct priming or cross-priming. However, it is clear that presentation of minigene products occurs via direct priming (47). Therefore, the clear effects of Treg depletion on the immunogenicity of rVV-encoded site IV minigene product provides a clear demonstration that Treg also interfere with direct presentation.
One of the principal goals of this study was to determine the contribution of Treg to establishing the immunodominance hierarchy, particularly whether Treg exacerbated differences in responses to IDDs vs SDDs. Previous work from our and others’ laboratories have identified several factors that contribute to immunodominance. A key factor is the generation of an appropriate number of peptide-class I complexes on the surface of APCs. Once this threshold is passed, however, other factors, including those yet to be defined, modulate TCD8+ responses to individual determinants. Our findings indicate that Treg activity is not responsible for the immunorecessive nature of site V in the Tag system nor does it dictate subdominance of SDDs harbored by VV and IAV. Furthermore, Treg do not significantly influence the rank order of determinants in immunodominance hierarchies in Tag, VV, or IAV systems in B6 mice.
The mechanisms by which Treg modify TCD8+ responses remain to be established. The similar effects of Treg on TCD8+ activated by cross-priming or direct priming implies (although not conclusively) that Treg act on TCD8+ and not on APCs, because the latter would be expected to differ in the different priming scenarios. The selective effects of Treg on TCD8+ responding to the most dominant determinants is consistent with the idea of direct interaction between Treg and TCD8+. The simplest possibility is that Treg limit TCD8+ expansion, and that this effect is greatest for cells dividing the most rapidly. In support of this idea, there is evidence that determinant-related differences in division time contributes to immunodominance (48). Thus, we propose that Treg moderate immunodominance hierarchies by retarding division of the most actively dividing TCD8+. It should be possible to directly address this hypothesis in future studies.
Although the effects of Treg on numbers of TCD8+ responding to individual determinants are complex, the overall effect of Treg depletion in the Tag system is to exacerbate the difference between TCD8+ responding to IDDs vs SDDs. Thus, following immunization with C57SV cells, responses to site IV are enhanced 2-fold whereas responses to site II/III increase only slightly, and responses to site V remain undetectable (Fig. 2).
The selective effect of Treg depletion on enhancing TCD8+ responses to IDDs was clear in the viral systems used. Thus, PC61 treatment selectively enhanced splenic TCD8+ responses to the IDDs, VV-B8R20 and IAV-PA224, in consistent manner over a series of experiments. Based on these data, we conclude that Treg are not principally responsible for immunodominance hierarchies among responding TCD8+ in these systems. Rather, Treg have the opposite effect, and function to narrow disparities among TCD8+ responding to different determinants. It will be interesting to determine the mechanism underlying this phenomenon, and whether it entails physical contact between Treg and their client cells. From the practical standpoint, those contemplating enhancing immunity by interfering with Treg function who are not daunted by potential difficulties with autoimmunity may also have to contend with difficulties with enhancing immunodominance disparities, which under some circumstances (e.g., viral infections), may enhance immune escape.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Deborah Tokarchick for providing outstanding technical assistance and Dr. Kari Irvine for valuable suggestions.
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 Current address: EBV Unit, Queensland Institute for Medical Research, Herston, Queensland 4006, Australia.
2 Address correspondence and reprint requests to Dr. Jack R. Bennink or Dr. Jonathan W. Yewdell, Room 211, 4 Center Drive, Mail Stop 0440, National Institutes of Health, Bethesda, MD 20892-0440. E-mail address: jbennink{at}nih.gov or E-mail address: jyewdell{at}nih.gov
3 Abbreviations used in this paper: IDD, immunodominant determinant; SDD, subdominant determinant; Treg, regulatory T cell; Tag, SV40 large tumor Ag; VV, vaccinia virus; IAV, influenza A virus; LCMV, lymphocytic choriomeningitis virus; PEC, peritoneal exudate cell; int, intermediate.
Received for publication October 7, 2004. Accepted for publication December 20, 2004.
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