Lentivirus-Specific Cytotoxic T-Lymphocyte Respons
http://www.100md.com
病菌学杂志 2005年第13期
Department of Veterinary Biosciences
Center for Retrovirus Research
Comprehensive Cancer Center-Arthur G. James Cancer Hospital and Solove Research Institute, Ohio State University, Columbus, Ohio 43210
ABSTRACT
To what extent the thymus is needed to preserve the virus-specific cytotoxic T-lymphocyte (CTL) response of lentivirus-infected adults is unclear. Presented here is the first definitive study using thymectomized (ThX) animals to directly evaluate the contribution of thymic function to lentivirus-specific CTL response and the control of lentivirus infections. ThX and mock-ThX cats were inoculated with feline immunodeficiency virus (FIV) and monitored for their FIV-specific CTL responses. Early in infection, both FIV-ThX and FIV-mock-ThX cats produced similar CTL responses, but surprisingly, after 20 weeks, the FIV-ThX cats showed a statistically significant loss of FIV-specific CTL activity, while FIV-infected cats with intact thymuses continued to maintain FIV-specific CTL. The loss of CTL did not affect plasma virus load, which remained elevated for both groups. These results emphasize the importance of thymic integrity in maintaining immunity to lentiviruses, but also bring into question the notion that virus load is regulated predominantly by the virus-specific CTL response.
INTRODUCTION
The thymus is a primary lymphoid organ responsible for seeding secondary lymphoid tissues with T lymphocytes beginning during fetal development and extending into adolescence (22). Under normal circumstances, T-lymphocyte homeostasis in adults is independent of thymic output largely due to the expansion of the peripheral T-lymphocyte population in response to antigen stimulation. However, the demand for thymic-origin T lymphocytes may increase to resolve immunologic insufficiency as a consequence of toxic insult (e.g., radiation) or certain infections. For example, immunodeficiency-causing lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and feline immunodeficiency virus (FIV), deplete CD4+ T lymphocytes, and cause significant immune impairment leading to opportunistic infections and, occasionally, neoplastic disease (5, 25). A major complication of lentivirus infection is the accelerated thymic involution observed in HIV-1-infected humans and FIV-infected cats (24) with the associated loss of thymic function and subsequent CD4+ T-lymphocyte depletion (5, 24). Thymic changes are most pronounced in pediatric subjects where HIV-1 (18, 21) and FIV (9, 24) infections cause structural damage to the thymus, manifested by depletion of CD4+ CD8+ double-positive thymocytes, disruption of distinct corticomedullary junctions, and architectural distortion due to substantial B-cell follicle development. Furthermore, HIV-1 infection of adults has been associated with premature thymic atrophy (10, 11).
The contribution of the thymus to the maintenance of T-lymphocyte homeostasis in adults with HIV-1 infection is controversial. In highly active antiretroviral therapy-treated HIV-infected adults, the correlation of increased circulating naive T lymphocytes with the recovery of specific T-lymphocyte responses suggests active thymopoiesis (7). However, to what extent the thymus is needed to preserve the virus-specific CTL responses of lentivirus-infected adult hosts is unclear. Therefore, the purpose of this study was to evaluate the contribution of the thymus to the establishment and maintenance of the antiviral immune response in adult cats infected with FIV.
MATERIALS AND METHODS
Animals. Animal studies were performed in accordance with the University Laboratory Animal Care and Use Committee and Department of Health, Education, and Welfare publication no. NIH 74-23, Guide for the Care and Use of Laboratory Animals. Female specific-pathogen-free (SPF) cats, 12 weeks of age, were group housed under SPF conditions. Food and water were provided ad libitum, and animals were observed daily for any clinical problems. A total of 23 cats were divided into four groups: uninfected mock thymectomized (mock-ThX; n = 5), uninfected ThX (n = 6), FIV-infected mock-ThX (n = 6), and FIV-infected ThX (n = 6).
Thymectomy. ThX cats underwent surgical thoracotomy where one of the two internal thoracic arteries was ligated and the thymus surgically removed. Mock-ThX cats underwent thoracotomy and vessel ligation but the thymus was left intact. All cats recovered completely and without complication.
FIV inoculation. FIV challenge consisted of 1,000 50% tissue culture infective dose units of the Maryland isolate of FIV (FIV-MD) propagated in primary peripheral blood mononuclear cell (PBMC) culture (9).
FIV-specific CTL assay. Simian virus 40-immortalized autologous fibroblast cell lines (14) derived from each cat were used as target cells to detect virus-specific CTL responses in a 51Cr release assay (2). Target cells were infected with recombinant vaccinia virus (VV) expressing FIV-Maryland Env protein or FIV Gag protein or wild-type VV control for two hours after which autologous PBMCs from individual FIV-infected cats were added to target fibroblasts at 75:1 effector:target ratios and were incubated for four hours. Supernatants from quadruplicate wells were collected and 51Cr content determined to calculate the mean percent specific lysis for each sample using the formula [(mean 51Cr release from samples) – (mean 51Cr spontaneous release)]/[(mean 51Cr maximum release) – (mean 51Cr spontaneous release)] x 100.
Immunophenotyping. Standard flow cytometric procedures were used to determine phenotypes of PBMC and lymphoid tissues. Staining combinations were as follows: CD5(phycoerythrin [PE]-Cy5)/CD4(PE), CD5(PE-Cy5)/CD8(PE), CD21(PE), and CD45(PE). Anti-feline CD5, CD4, and CD8 monoclonal antibodies were obtained from Southern Biotechnologies (Birmingham, AL), anti-feline CD21 was obtained from Peter Moore (University of Southern California), and anti-human CD45 was obtained from Serotec (London, United Kingdom).
Real-time quantitative RT-PCR for FIV. A two-step reverse transcriptase PCR (RT-PCR) assay was developed for determination of viral load in plasma and tissue. Viral RNA was extracted from plasma samples using centrifugation and organic extraction as previously described (23). To control for extraction efficiency, at the virus lysis step, 108 copies of enhanced green fluorescent protein (EGFP) RNA were spiked into the sample (13). Extracted RNA samples were resuspended in 20 μl diethyl pyrocarbonate-treated water containing 1 ng/μl 7.5 kb synthetic RNA (Gibco BRL). RNA was reverse transcribed into cDNA as follows. Five microliters of template RNA (or standard curve RNA) was combined with 1x Thermoscript RT buffer, 0.8 mM deoxynucleoside triphosphates, 1.25 μM KH4, and 2 U Thermoscript RT in a total volume of 20 μl. Samples were incubated at 55°C for 1 h and then 85°C for 5 min. PCR was carried out using 4 μl cDNA in the following 20-μl reaction mixtures: 1x FastStart hybridization probe mixture (Roche), 4.2 mM MgCl2 (final), 0.625 μM KH3/KH4 primers (9), and 0.4 μM FIV probe. Cycling conditions were as follows in a Roche LightCycler: initial denaturation at 95°C for 10 min, followed by 5 cycles of 94°C for 15 s and 60°C for 30 s (no data acquisition) and then 45 cycles of 85°C for 15 s and 60°C for 30 s with data acquisition at the annealing/extension step.
FIV primers were KH3 M (5'-GAC CCA AAA ATG GTG TCC-3'), KH4 (5'-CCT ATT CCC ATA ATC TCT GC-3'), and FIV-2 Probe (6-carboxyfluorescein-TTG GAC TTC CTC ACC TCC TAG GG-6-carboxy tetramethylrhodamine).
Real-time RT-PCR assays for GAPDH and EGFP. Assays for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EGFP were adapted from the literature (16) for use in the LightCycler system as follows. The RT-PCR mixture consisted of 1x SybrGreen RT buffer (Quantitect RT-PCR kit; QIAGEN, Valencia, CA), 1.25 μM GAPDH.1/GAPDH.2 primers (or GFP-F/GFP-R primers), and 0.2 μl RT enzyme mixture. Two microliters of template RNA was added to the 18-μl reaction mixture, and RT-PCR was carried out in the LightCycler as follows: RT step of 50°C for 20 min followed by initial denaturation for 15 min at 95°C and then 25 cycles of 94°C for 0 s, 60°C for 5 s, and 72°C for 10 s with data acquisition at the extension step.
Necropsy and histopathology. Euthanasia and necropsy were performed at 10 months postinfection. Cats were placed under deep surgical plane anesthesia, exsanguinated, and perfused with saline. Tissue sections (spleen, mesenteric lymph node, peripheral lymph node, and thymus) were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, Calif.) and frozen in liquid nitrogen. Other tissue sections were fixed in 10% neutral buffered formalin for histologic analysis (liver, kidney, spleen, mesenteric and peripheral lymph nodes, adrenal gland, urinary bladder, bone marrow, skeletal muscle, heart, lung, brain, stomach, large and small intestines, and thymus [where available]).
Statistics. Statistical analysis was performed with the GraphPad statistical program (Instat). For virus load and CTL responses, the Mann-Whitney U test was performed. For CD4+/CD8+ T-lymphocyte ratios in tissues, the Kruskal-Wallis test with Dunn's comparison (four-group comparison) or the Mann-Whitney U test (two-group comparison) was used. Significance was set at a P value of 0.05.
RESULTS
Antigen-specific cytolytic responses are lost in thymectomized FIV-infected cats. Antigen-specific CTL responses to FIV-Maryland Gag and FIV-Maryland Env were measured over a period of 36 weeks after FIV infection in cats ThX or mock-ThX eight months prior to FIV infection. In this longitudinal study, both ThX and mock-ThX FIV-infected groups mounted CTL responses to FIV Gag and/or Env by three weeks postinfection (Fig. 1). Furthermore, there was no discernible difference between responses to Env and responses to Gag (Fig. 1). However, 20 to 24 weeks into the infection, the CTL responses to both Gag and Env for the FIV-ThX group declined sharply to undetectable levels for most animals (Fig. 1). These differences were significant at week 24 (P = 0.0374), week 32 (P = 0.0075), and week 36 (P = 0.0091). The FIV-mock-ThX group had consistently detectable CTL responses throughout the observation period of the study (Fig. 1).
Plasma virus loads were similar between the ThX and mock-ThX FIV-infected cats and did not correlate with CTL function. Plasma virus loads from Thx and mock-ThX cat blood samples collected during the 44-week observation period were determined by real-time RT-PCR. FIV copy numbers were similar for the FIV-mock and the FIV-ThX groups at all time points during the study (Fig. 2) and were compatible with levels determined in naturally infected cats in the asymptomatic/AIDS-related complex stages of infection (8). Statistical analysis of plasma virus loads versus CTL responses over time revealed no significant inverse correlation suggesting that virus-specific CTL responses in FIV-infected cats were less effective in controlling viremia than previously believed.
Tissue virus loads in ThX and mock-ThX FIV-infected cats. FIV RNA copy number was determined for thymus (FIV-mock-ThX only), lymph nodes, and spleen from terminal tissue samples collected at the time of euthanasia. All cats in the study were saline perfused prior to tissue collection at necropsy to minimize bias due to blood contamination. It should be noted that the small number of animals in the study and the inherent biologic variation between cats in individual tissues presented only trends rather than statistically significant differences for most of the tissue virus loads. Thymus tissue from FIV-mock-ThX cats carried the highest viral burden of all lymphoid tissues measured, with an average of 5.2 x104 RNA copies/50 ng total RNA (Fig. 3). Virus load in the popliteal and mesenteric lymph nodes from the FIV-mock-ThX cats were not significantly different in the FIV-ThX group. However, there was a significant increase in virus load in the spleen of the FIV-ThX cats when compared to the FIV-mock-ThX cats (P = 0.0467). In general, the rank order for virus expression was thymus > spleen > popliteal lymph node > mesenteric lymph node. Reextraction and RT-PCR of tissue RNA from a second tissue sample gave similar results. In agreement with our studies, relative quantitative data from other laboratories demonstrated that the lymph node compartment had the lowest level of viral RNA when compared to thymus and peripheral blood compartments (17).
Clinical and hematologic signs of FIV-related disease. At the termination of the study (44 weeks postinfection), only minor clinical signs of disease (lymphadenopathy, stomatitis) or histologic changes in collected tissue were apparent in any of the animals. The exception was thymus tissue from the FIV-mock-ThX group, which showed the typical pathology reported previously (24), including increased CD8+ T lymphocytes, formation of B-lymphocyte follicles, and disruption of corticomedullary architecture (data not shown). Ligation of one of the two internal thoracic arteries was necessary to perform the thymectomy. Therefore, the same vessel was ligated in the mock-ThX cats as a control for surgical trauma. This had no deleterious effect on the thymus as evidenced by normal histologic appearance of thymic tissue in the control-mock-ThX cats at necropsy.
Effects of thymectomy and/or FIV infection on peripheral blood CD4+ and CD8+ T lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD4+/CD8+ ratios in the blood segregated according to infection status rather than thymectomy, such that a slight decline in CD4+ T lymphocytes with a concomitant increase in CD8+ T lymphocytes was observed in the FIV-mock-ThX and FIV-ThX groups compared to the control-mock and control-ThX (Fig. 4). The differences in CD4+/CD8+ T-cell ratios or lymphocyte numbers between the control-mock-ThX and control-ThX were not significant at any time point during the study, although there was a trend towards an increase in the CD4+/CD8+ T-cell ratio in the control-ThX group compared to the control-mock-ThX group. This effect was due to a slight decrease in CD8+ T cells with no increase in CD4+ T cells in the control-mock-ThX group. Furthermore, the differences between the control-ThX and the FIV Thx groups were significant at all time points, supporting the conclusion that differences were due to FIV infection.
Similarly, there were no significant differences in CD4+ or CD8+ T-cell numbers or ratios between the FIV-mock-ThX and FIV-ThX groups. Additional analysis of the CD4+ and CD8+ T-cell numbers revealed (i) that the FIV-ThX group had less of an increase in CD8+ T-cells in response to FIV infection than did the FIV-mock-ThX group and (ii) that the CD4+ T-cell numbers declined to a lesser degree in the FIV-ThX group than in the FIV-mock-ThX group. As a result, the CD4+/CD8+ ratios were almost identical. These data suggest that differences in the peripheral blood lymphocytes were significantly correlated with FIV infection rather than thymectomy.
DISCUSSION
The decline of lentivirus-specific CTL has direct relevance to AIDS treatment strategies where protection of the thymic compartment from lentivirus-induced damage is of paramount importance. The longevity of the antiviral immune compensatory mechanisms, mediated by the peripheral memory T-lymphocyte pool, is not known. The data presented here suggest that without a functional thymus, the antiviral CTL response declines precipitously within a few months of infection.
Thymectomy preempts resupply of naive CD4+ and CD8+ T lymphocytes to the peripheral immune compartment. As virus-specific memory T lymphocytes are exhausted by lentivirus infection, functional cytolytic T-lymphocyte losses become apparent. The surprising observation of this study was the rapid decline of CTL function (5 to 6 months postinfection) suggesting that immune compensatory mechanisms quickly fail in the absence of thymic influence. CTL escape mutants that evolve during the course of HIV infection may alter the specificity of the responder CTL population causing the appearance of loss of CTL function (3, 20). However, there is no compelling reason for this mechanism to be exclusively found in FIV-ThX cats and not in FIV-mock cats; therefore it is unlikely that escape mutants account for the sudden loss of CTL observed in the FIV-ThX cats.
We observed that there was a significant increase in virus load in the spleen of the FIV-ThX cats compared to the FIV-mock-ThX cats. Although the reason for this increase is not known, one could speculate that in the absence of a thymus, T lymphocytes may home to the spleen in greater numbers or that extrathymic lymphopoiesis may be taking place, thereby providing more target cells for infection. Furthermore, virus loads tended to be higher in the lymph nodes of the FIV-ThX cats as well, although the differences were not significant and presented only trends.
The lack of a difference in the plasma virus load between the FIV-ThX and FIV-mock-ThX groups was surprising based on the widely accepted perception that anti-HIV-1 CTL responses (or at least HIV-1-specific CD8+ lymphocyte numbers) inversely correlate with the temporal reduction in plasma virus load both during the acute phase of infection and after infection has been established (12, 15). Not all laboratories have been able to document this relationship (1, 6). However, Ogg et al. were able to demonstrate, using a tetramer-based assay, an inverse correlation between the frequency of HIV-1-specific CTL and the level of plasma viremia (19). The functional assay used in our study was the standard chromium release cytolytic assay using immortalized autologous fibroblasts target cells transduced with a vaccinia virus vector containing full-length FIV-Maryland gag or FIV env (14). While useful for determining overall CTL function, this assay did not allow enumeration of the frequency of virus-specific CTL. Therefore, the discrepancies between studies with respect to defining the association between CTL frequency and/or function and virus load (temporal or peak) reflect the different assay systems employed (functional versus nonfunctional), variations in the types and breadth of epitopes used, and inherent genetic differences in the population studied (1, 6, 19). During the 44-week observation period, no differences in disease parameters were observed between the FIV-ThX and FIV-mock-ThX groups. Given that the incubation period for FIV prior to the onset of an AIDS-like disease is estimated to be approximately five years (4), the lack of disease indicators was expected. We currently are pursuing additional studies to evaluate longer-term effects of thymectomy on the pathogenesis of FIV infection and the antiviral immune response.
Thymectomy had no significant effect on the CD4+ or CD8+ peripheral blood T-lymphocyte numbers in the absence of FIV infection during the 10-month observation period, although there was a trend toward an increased CD4+/CD8+ T-cell ratio in the control-ThX group relative to the control-mock-ThX group. This was due to a slight decrease in CD8+ T cells in the control-ThX group. In addition, the effect of thymectomy was not apparent in the two FIV-infected groups where the changes in CD4+ and CD8+ lymphocyte numbers were due to the FIV infection alone.
From this study, we discovered a remarkable role for the thymus during lentivirus infection in adult hosts. The thymus was necessary to maintain virus-specific CTL responses. In addition, in spite of the explosive virus infection within the thymus of the FIV-infected cats, the thymus continued to contribute to the peripheral blood CTL response in the FIV-mock-ThX group. We speculate that naive-T-lymphocyte production is sustained by the thymus during this relatively early stage of disease. The effect of accelerated thymic involution and associated loss of thymic function in advanced disease may resemble the effect of thymectomy with respect to revealing the necessity of the thymus for support of a sustained immune response to lentivirus infection of adult hosts.
Of equal importance is the observation that plasma virus loads did not increase dramatically with the loss of virus-specific CTL function as might have been predicted. The lack of increase in virus load in the FIV-ThX cats may be due to a compensatory antiviral process mediated by other immune mechanisms (e.g., NK cells). It also is possible that the systemic virus infection is at a maximal threshold that cannot be controlled effectively by CTL alone.
The importance of thymic function in adults with chronic lymphocyte depletion is just beginning to be established. How the thymus participates in the establishment and maintenance of the antiviral immune response to lentivirus infection remains to be determined. It is becoming apparent from our work and others that protection of the thymic compartment early in the course of lentivirus infection is of paramount importance.
ACKNOWLEDGMENTS
We acknowledge institutional support from the OSU Center for Retrovirus Research and the OSU Comprehensive Cancer Center. We thank Richard K. Meister for flow cytometry analysis.
This study was supported by NIH, NIAID grant RO1 AI40855 (L.E.M.), and NCI grant P30 CA16058 (L.E.M.).
K.A.H. and S.K. contributed equally to the work.
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Center for Retrovirus Research
Comprehensive Cancer Center-Arthur G. James Cancer Hospital and Solove Research Institute, Ohio State University, Columbus, Ohio 43210
ABSTRACT
To what extent the thymus is needed to preserve the virus-specific cytotoxic T-lymphocyte (CTL) response of lentivirus-infected adults is unclear. Presented here is the first definitive study using thymectomized (ThX) animals to directly evaluate the contribution of thymic function to lentivirus-specific CTL response and the control of lentivirus infections. ThX and mock-ThX cats were inoculated with feline immunodeficiency virus (FIV) and monitored for their FIV-specific CTL responses. Early in infection, both FIV-ThX and FIV-mock-ThX cats produced similar CTL responses, but surprisingly, after 20 weeks, the FIV-ThX cats showed a statistically significant loss of FIV-specific CTL activity, while FIV-infected cats with intact thymuses continued to maintain FIV-specific CTL. The loss of CTL did not affect plasma virus load, which remained elevated for both groups. These results emphasize the importance of thymic integrity in maintaining immunity to lentiviruses, but also bring into question the notion that virus load is regulated predominantly by the virus-specific CTL response.
INTRODUCTION
The thymus is a primary lymphoid organ responsible for seeding secondary lymphoid tissues with T lymphocytes beginning during fetal development and extending into adolescence (22). Under normal circumstances, T-lymphocyte homeostasis in adults is independent of thymic output largely due to the expansion of the peripheral T-lymphocyte population in response to antigen stimulation. However, the demand for thymic-origin T lymphocytes may increase to resolve immunologic insufficiency as a consequence of toxic insult (e.g., radiation) or certain infections. For example, immunodeficiency-causing lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and feline immunodeficiency virus (FIV), deplete CD4+ T lymphocytes, and cause significant immune impairment leading to opportunistic infections and, occasionally, neoplastic disease (5, 25). A major complication of lentivirus infection is the accelerated thymic involution observed in HIV-1-infected humans and FIV-infected cats (24) with the associated loss of thymic function and subsequent CD4+ T-lymphocyte depletion (5, 24). Thymic changes are most pronounced in pediatric subjects where HIV-1 (18, 21) and FIV (9, 24) infections cause structural damage to the thymus, manifested by depletion of CD4+ CD8+ double-positive thymocytes, disruption of distinct corticomedullary junctions, and architectural distortion due to substantial B-cell follicle development. Furthermore, HIV-1 infection of adults has been associated with premature thymic atrophy (10, 11).
The contribution of the thymus to the maintenance of T-lymphocyte homeostasis in adults with HIV-1 infection is controversial. In highly active antiretroviral therapy-treated HIV-infected adults, the correlation of increased circulating naive T lymphocytes with the recovery of specific T-lymphocyte responses suggests active thymopoiesis (7). However, to what extent the thymus is needed to preserve the virus-specific CTL responses of lentivirus-infected adult hosts is unclear. Therefore, the purpose of this study was to evaluate the contribution of the thymus to the establishment and maintenance of the antiviral immune response in adult cats infected with FIV.
MATERIALS AND METHODS
Animals. Animal studies were performed in accordance with the University Laboratory Animal Care and Use Committee and Department of Health, Education, and Welfare publication no. NIH 74-23, Guide for the Care and Use of Laboratory Animals. Female specific-pathogen-free (SPF) cats, 12 weeks of age, were group housed under SPF conditions. Food and water were provided ad libitum, and animals were observed daily for any clinical problems. A total of 23 cats were divided into four groups: uninfected mock thymectomized (mock-ThX; n = 5), uninfected ThX (n = 6), FIV-infected mock-ThX (n = 6), and FIV-infected ThX (n = 6).
Thymectomy. ThX cats underwent surgical thoracotomy where one of the two internal thoracic arteries was ligated and the thymus surgically removed. Mock-ThX cats underwent thoracotomy and vessel ligation but the thymus was left intact. All cats recovered completely and without complication.
FIV inoculation. FIV challenge consisted of 1,000 50% tissue culture infective dose units of the Maryland isolate of FIV (FIV-MD) propagated in primary peripheral blood mononuclear cell (PBMC) culture (9).
FIV-specific CTL assay. Simian virus 40-immortalized autologous fibroblast cell lines (14) derived from each cat were used as target cells to detect virus-specific CTL responses in a 51Cr release assay (2). Target cells were infected with recombinant vaccinia virus (VV) expressing FIV-Maryland Env protein or FIV Gag protein or wild-type VV control for two hours after which autologous PBMCs from individual FIV-infected cats were added to target fibroblasts at 75:1 effector:target ratios and were incubated for four hours. Supernatants from quadruplicate wells were collected and 51Cr content determined to calculate the mean percent specific lysis for each sample using the formula [(mean 51Cr release from samples) – (mean 51Cr spontaneous release)]/[(mean 51Cr maximum release) – (mean 51Cr spontaneous release)] x 100.
Immunophenotyping. Standard flow cytometric procedures were used to determine phenotypes of PBMC and lymphoid tissues. Staining combinations were as follows: CD5(phycoerythrin [PE]-Cy5)/CD4(PE), CD5(PE-Cy5)/CD8(PE), CD21(PE), and CD45(PE). Anti-feline CD5, CD4, and CD8 monoclonal antibodies were obtained from Southern Biotechnologies (Birmingham, AL), anti-feline CD21 was obtained from Peter Moore (University of Southern California), and anti-human CD45 was obtained from Serotec (London, United Kingdom).
Real-time quantitative RT-PCR for FIV. A two-step reverse transcriptase PCR (RT-PCR) assay was developed for determination of viral load in plasma and tissue. Viral RNA was extracted from plasma samples using centrifugation and organic extraction as previously described (23). To control for extraction efficiency, at the virus lysis step, 108 copies of enhanced green fluorescent protein (EGFP) RNA were spiked into the sample (13). Extracted RNA samples were resuspended in 20 μl diethyl pyrocarbonate-treated water containing 1 ng/μl 7.5 kb synthetic RNA (Gibco BRL). RNA was reverse transcribed into cDNA as follows. Five microliters of template RNA (or standard curve RNA) was combined with 1x Thermoscript RT buffer, 0.8 mM deoxynucleoside triphosphates, 1.25 μM KH4, and 2 U Thermoscript RT in a total volume of 20 μl. Samples were incubated at 55°C for 1 h and then 85°C for 5 min. PCR was carried out using 4 μl cDNA in the following 20-μl reaction mixtures: 1x FastStart hybridization probe mixture (Roche), 4.2 mM MgCl2 (final), 0.625 μM KH3/KH4 primers (9), and 0.4 μM FIV probe. Cycling conditions were as follows in a Roche LightCycler: initial denaturation at 95°C for 10 min, followed by 5 cycles of 94°C for 15 s and 60°C for 30 s (no data acquisition) and then 45 cycles of 85°C for 15 s and 60°C for 30 s with data acquisition at the annealing/extension step.
FIV primers were KH3 M (5'-GAC CCA AAA ATG GTG TCC-3'), KH4 (5'-CCT ATT CCC ATA ATC TCT GC-3'), and FIV-2 Probe (6-carboxyfluorescein-TTG GAC TTC CTC ACC TCC TAG GG-6-carboxy tetramethylrhodamine).
Real-time RT-PCR assays for GAPDH and EGFP. Assays for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EGFP were adapted from the literature (16) for use in the LightCycler system as follows. The RT-PCR mixture consisted of 1x SybrGreen RT buffer (Quantitect RT-PCR kit; QIAGEN, Valencia, CA), 1.25 μM GAPDH.1/GAPDH.2 primers (or GFP-F/GFP-R primers), and 0.2 μl RT enzyme mixture. Two microliters of template RNA was added to the 18-μl reaction mixture, and RT-PCR was carried out in the LightCycler as follows: RT step of 50°C for 20 min followed by initial denaturation for 15 min at 95°C and then 25 cycles of 94°C for 0 s, 60°C for 5 s, and 72°C for 10 s with data acquisition at the extension step.
Necropsy and histopathology. Euthanasia and necropsy were performed at 10 months postinfection. Cats were placed under deep surgical plane anesthesia, exsanguinated, and perfused with saline. Tissue sections (spleen, mesenteric lymph node, peripheral lymph node, and thymus) were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, Calif.) and frozen in liquid nitrogen. Other tissue sections were fixed in 10% neutral buffered formalin for histologic analysis (liver, kidney, spleen, mesenteric and peripheral lymph nodes, adrenal gland, urinary bladder, bone marrow, skeletal muscle, heart, lung, brain, stomach, large and small intestines, and thymus [where available]).
Statistics. Statistical analysis was performed with the GraphPad statistical program (Instat). For virus load and CTL responses, the Mann-Whitney U test was performed. For CD4+/CD8+ T-lymphocyte ratios in tissues, the Kruskal-Wallis test with Dunn's comparison (four-group comparison) or the Mann-Whitney U test (two-group comparison) was used. Significance was set at a P value of 0.05.
RESULTS
Antigen-specific cytolytic responses are lost in thymectomized FIV-infected cats. Antigen-specific CTL responses to FIV-Maryland Gag and FIV-Maryland Env were measured over a period of 36 weeks after FIV infection in cats ThX or mock-ThX eight months prior to FIV infection. In this longitudinal study, both ThX and mock-ThX FIV-infected groups mounted CTL responses to FIV Gag and/or Env by three weeks postinfection (Fig. 1). Furthermore, there was no discernible difference between responses to Env and responses to Gag (Fig. 1). However, 20 to 24 weeks into the infection, the CTL responses to both Gag and Env for the FIV-ThX group declined sharply to undetectable levels for most animals (Fig. 1). These differences were significant at week 24 (P = 0.0374), week 32 (P = 0.0075), and week 36 (P = 0.0091). The FIV-mock-ThX group had consistently detectable CTL responses throughout the observation period of the study (Fig. 1).
Plasma virus loads were similar between the ThX and mock-ThX FIV-infected cats and did not correlate with CTL function. Plasma virus loads from Thx and mock-ThX cat blood samples collected during the 44-week observation period were determined by real-time RT-PCR. FIV copy numbers were similar for the FIV-mock and the FIV-ThX groups at all time points during the study (Fig. 2) and were compatible with levels determined in naturally infected cats in the asymptomatic/AIDS-related complex stages of infection (8). Statistical analysis of plasma virus loads versus CTL responses over time revealed no significant inverse correlation suggesting that virus-specific CTL responses in FIV-infected cats were less effective in controlling viremia than previously believed.
Tissue virus loads in ThX and mock-ThX FIV-infected cats. FIV RNA copy number was determined for thymus (FIV-mock-ThX only), lymph nodes, and spleen from terminal tissue samples collected at the time of euthanasia. All cats in the study were saline perfused prior to tissue collection at necropsy to minimize bias due to blood contamination. It should be noted that the small number of animals in the study and the inherent biologic variation between cats in individual tissues presented only trends rather than statistically significant differences for most of the tissue virus loads. Thymus tissue from FIV-mock-ThX cats carried the highest viral burden of all lymphoid tissues measured, with an average of 5.2 x104 RNA copies/50 ng total RNA (Fig. 3). Virus load in the popliteal and mesenteric lymph nodes from the FIV-mock-ThX cats were not significantly different in the FIV-ThX group. However, there was a significant increase in virus load in the spleen of the FIV-ThX cats when compared to the FIV-mock-ThX cats (P = 0.0467). In general, the rank order for virus expression was thymus > spleen > popliteal lymph node > mesenteric lymph node. Reextraction and RT-PCR of tissue RNA from a second tissue sample gave similar results. In agreement with our studies, relative quantitative data from other laboratories demonstrated that the lymph node compartment had the lowest level of viral RNA when compared to thymus and peripheral blood compartments (17).
Clinical and hematologic signs of FIV-related disease. At the termination of the study (44 weeks postinfection), only minor clinical signs of disease (lymphadenopathy, stomatitis) or histologic changes in collected tissue were apparent in any of the animals. The exception was thymus tissue from the FIV-mock-ThX group, which showed the typical pathology reported previously (24), including increased CD8+ T lymphocytes, formation of B-lymphocyte follicles, and disruption of corticomedullary architecture (data not shown). Ligation of one of the two internal thoracic arteries was necessary to perform the thymectomy. Therefore, the same vessel was ligated in the mock-ThX cats as a control for surgical trauma. This had no deleterious effect on the thymus as evidenced by normal histologic appearance of thymic tissue in the control-mock-ThX cats at necropsy.
Effects of thymectomy and/or FIV infection on peripheral blood CD4+ and CD8+ T lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD4+/CD8+ ratios in the blood segregated according to infection status rather than thymectomy, such that a slight decline in CD4+ T lymphocytes with a concomitant increase in CD8+ T lymphocytes was observed in the FIV-mock-ThX and FIV-ThX groups compared to the control-mock and control-ThX (Fig. 4). The differences in CD4+/CD8+ T-cell ratios or lymphocyte numbers between the control-mock-ThX and control-ThX were not significant at any time point during the study, although there was a trend towards an increase in the CD4+/CD8+ T-cell ratio in the control-ThX group compared to the control-mock-ThX group. This effect was due to a slight decrease in CD8+ T cells with no increase in CD4+ T cells in the control-mock-ThX group. Furthermore, the differences between the control-ThX and the FIV Thx groups were significant at all time points, supporting the conclusion that differences were due to FIV infection.
Similarly, there were no significant differences in CD4+ or CD8+ T-cell numbers or ratios between the FIV-mock-ThX and FIV-ThX groups. Additional analysis of the CD4+ and CD8+ T-cell numbers revealed (i) that the FIV-ThX group had less of an increase in CD8+ T-cells in response to FIV infection than did the FIV-mock-ThX group and (ii) that the CD4+ T-cell numbers declined to a lesser degree in the FIV-ThX group than in the FIV-mock-ThX group. As a result, the CD4+/CD8+ ratios were almost identical. These data suggest that differences in the peripheral blood lymphocytes were significantly correlated with FIV infection rather than thymectomy.
DISCUSSION
The decline of lentivirus-specific CTL has direct relevance to AIDS treatment strategies where protection of the thymic compartment from lentivirus-induced damage is of paramount importance. The longevity of the antiviral immune compensatory mechanisms, mediated by the peripheral memory T-lymphocyte pool, is not known. The data presented here suggest that without a functional thymus, the antiviral CTL response declines precipitously within a few months of infection.
Thymectomy preempts resupply of naive CD4+ and CD8+ T lymphocytes to the peripheral immune compartment. As virus-specific memory T lymphocytes are exhausted by lentivirus infection, functional cytolytic T-lymphocyte losses become apparent. The surprising observation of this study was the rapid decline of CTL function (5 to 6 months postinfection) suggesting that immune compensatory mechanisms quickly fail in the absence of thymic influence. CTL escape mutants that evolve during the course of HIV infection may alter the specificity of the responder CTL population causing the appearance of loss of CTL function (3, 20). However, there is no compelling reason for this mechanism to be exclusively found in FIV-ThX cats and not in FIV-mock cats; therefore it is unlikely that escape mutants account for the sudden loss of CTL observed in the FIV-ThX cats.
We observed that there was a significant increase in virus load in the spleen of the FIV-ThX cats compared to the FIV-mock-ThX cats. Although the reason for this increase is not known, one could speculate that in the absence of a thymus, T lymphocytes may home to the spleen in greater numbers or that extrathymic lymphopoiesis may be taking place, thereby providing more target cells for infection. Furthermore, virus loads tended to be higher in the lymph nodes of the FIV-ThX cats as well, although the differences were not significant and presented only trends.
The lack of a difference in the plasma virus load between the FIV-ThX and FIV-mock-ThX groups was surprising based on the widely accepted perception that anti-HIV-1 CTL responses (or at least HIV-1-specific CD8+ lymphocyte numbers) inversely correlate with the temporal reduction in plasma virus load both during the acute phase of infection and after infection has been established (12, 15). Not all laboratories have been able to document this relationship (1, 6). However, Ogg et al. were able to demonstrate, using a tetramer-based assay, an inverse correlation between the frequency of HIV-1-specific CTL and the level of plasma viremia (19). The functional assay used in our study was the standard chromium release cytolytic assay using immortalized autologous fibroblasts target cells transduced with a vaccinia virus vector containing full-length FIV-Maryland gag or FIV env (14). While useful for determining overall CTL function, this assay did not allow enumeration of the frequency of virus-specific CTL. Therefore, the discrepancies between studies with respect to defining the association between CTL frequency and/or function and virus load (temporal or peak) reflect the different assay systems employed (functional versus nonfunctional), variations in the types and breadth of epitopes used, and inherent genetic differences in the population studied (1, 6, 19). During the 44-week observation period, no differences in disease parameters were observed between the FIV-ThX and FIV-mock-ThX groups. Given that the incubation period for FIV prior to the onset of an AIDS-like disease is estimated to be approximately five years (4), the lack of disease indicators was expected. We currently are pursuing additional studies to evaluate longer-term effects of thymectomy on the pathogenesis of FIV infection and the antiviral immune response.
Thymectomy had no significant effect on the CD4+ or CD8+ peripheral blood T-lymphocyte numbers in the absence of FIV infection during the 10-month observation period, although there was a trend toward an increased CD4+/CD8+ T-cell ratio in the control-ThX group relative to the control-mock-ThX group. This was due to a slight decrease in CD8+ T cells in the control-ThX group. In addition, the effect of thymectomy was not apparent in the two FIV-infected groups where the changes in CD4+ and CD8+ lymphocyte numbers were due to the FIV infection alone.
From this study, we discovered a remarkable role for the thymus during lentivirus infection in adult hosts. The thymus was necessary to maintain virus-specific CTL responses. In addition, in spite of the explosive virus infection within the thymus of the FIV-infected cats, the thymus continued to contribute to the peripheral blood CTL response in the FIV-mock-ThX group. We speculate that naive-T-lymphocyte production is sustained by the thymus during this relatively early stage of disease. The effect of accelerated thymic involution and associated loss of thymic function in advanced disease may resemble the effect of thymectomy with respect to revealing the necessity of the thymus for support of a sustained immune response to lentivirus infection of adult hosts.
Of equal importance is the observation that plasma virus loads did not increase dramatically with the loss of virus-specific CTL function as might have been predicted. The lack of increase in virus load in the FIV-ThX cats may be due to a compensatory antiviral process mediated by other immune mechanisms (e.g., NK cells). It also is possible that the systemic virus infection is at a maximal threshold that cannot be controlled effectively by CTL alone.
The importance of thymic function in adults with chronic lymphocyte depletion is just beginning to be established. How the thymus participates in the establishment and maintenance of the antiviral immune response to lentivirus infection remains to be determined. It is becoming apparent from our work and others that protection of the thymic compartment early in the course of lentivirus infection is of paramount importance.
ACKNOWLEDGMENTS
We acknowledge institutional support from the OSU Center for Retrovirus Research and the OSU Comprehensive Cancer Center. We thank Richard K. Meister for flow cytometry analysis.
This study was supported by NIH, NIAID grant RO1 AI40855 (L.E.M.), and NCI grant P30 CA16058 (L.E.M.).
K.A.H. and S.K. contributed equally to the work.
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