Rapid accumulation of adoptively transferred CD8+ T cells at the tumor site is associated with long-term control of SV40 T antigen-induced tumors

We previously established a model to study CD8⁺ T cell (T_CD8)-based adoptive immunotherapy of cancer using line SV11 mice that develop choroid plexus tumors in the brain due to transgenic expression of Simian Virus 40 large T antigen (Tag). These mice are tolerant to the three dominant T_CD8-recognized Tag epitopes I, II/III and IV. However, adoptive transfer of spleen cells from naïve C57BL/6 (B6) mice prolongs SV11 survival following T_CD8 priming against the endogenous Tag epitope IV. In addition, survival of SV11 mice is dramatically increased following transfer of lymphocytes from Tag-immune B6 mice. In the current study, we compared the kinetics and magnitude of Tag-specific T_CD8 accumulation at the tumor site following adoptive transfer with a high dose of either Tag-immune or naïve donor cells or decreasing doses of Tag-immune lymphocytes. Following adoptive transfer of Tag-immune cells, epitope I- and IV-specific T_CD8 accumulated to high levels in the brain of SV11 mice, peaking at 5–7 days, while epitope IV-specific T_CD8 derived from naïve donors required three weeks to achieve peak levels. A similar delay in the peak of epitope IV-specific T_CD8 accumulation was observed when tenfold fewer Tag-immune donor cells were administered, reducing control of tumor progression. These results suggest that efficient and prolonged control of established autochthonous tumors is associated with high-level early accumulation of adoptively transferred T cells. We also provide evidence that although multiple specificities are represented in the Tag immune donor lymphocytes, epitope IV-specific donor T_CD8 play a predominant role in control of tumor growth.     

In vivo expansion of the residual tumor antigen-specific CD8+ T lymphocytes that survive negative selection in simian virus 40 T-antigen-transgenic mice

Mice that express the viral oncoprotein simian virus 40 (SV40) large T antigen (T-Ag) as a transgene provide useful models for the assessment of the state of the host immune response in the face of spontaneous tumor progression. Line SV11 (H2(b)) mice develop rapidly progressing choroid plexus tumors due to expression of full-length T-Ag from the SV40 promoter. In addition, T-Ag expression in the thymus of SV11 mice results in the deletion of CD8(+) T cells specific for the three H2(b)-restricted immunodominant epitopes of T-Ag. Whether CD8(+) T cells specific for the immunorecessive H2-D(b)-restricted epitope V of T-Ag survive negative selection in SV11 mice has not been determined. Immunization of SV11 mice with rVV-ES-V, a recombinant vaccinia virus expressing epitope V as a minigene, resulted in the induction of weak, but reproducible, epitope V-specific cytotoxic T-lymphocyte (CTL) responses. This weak lytic response corresponded with a decreased frequency of epitope V-specific CTL that could be recruited in SV11 mice. In addition, CTL lines derived from rVV-ES-V-immunized SV11 mice had reduced avidities compared to that seen with CTL derived from healthy mice. Despite this initial weak response, significant numbers of epitope V-specific CD8(+) T cells were detected in SV11 mice ex vivo following a priming-boosting approach and these cells demonstrated high avidity for epitope V. The results suggest that low numbers of tumor-reactive CD8(+) T cells with high avidity for epitope V survive negative selection in SV11 mice but can be expanded by specific boosting approaches in the tumor bearing host.     

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Accumulation of CD8+ T cells in advanced-stage tumors and delay of disease progression following secondary immunization against an immunorecessive epitope

Self-reactive T cells that survive the process of positive and negative selection during thymocyte development represent potential effector cells against tumors that express these same self-Ags. We have previously shown that CD8+ T lymphocytes (T(CD8)) specific for an immunorecessive epitope, designated epitope V, from the SV40 large T Ag (Tag) escape thymic deletion in line SV11 Tag-transgenic mice. In contrast, these mice are tolerant to the three most dominant Tag epitopes. The majority of the residual epitope V-specific T(CD8) have a low avidity for the target epitope, but a prime/boost regimen can expand higher avidity clones in vivo. Whether higher avidity T(CD8) targeting this epitope are affected by Tag-expressing tumors in the periphery or can be recruited for control of tumor progression remains unknown. In the current study, we determined the fate of naive TCR-transgenic T(CD8) specific for Tag epitope V (TCR-V cells) following transfer into SV11 mice bearing advanced-stage choroid plexus tumors. The results indicate that TCR-V cells are rapidly triggered by the endogenous Tag and acquire effector function, but fail to accumulate within the tumors. Primary immunization enhanced TCR-V cell frequency in the periphery and promoted entry into the brain, but a subsequent booster immunization caused a dramatic accumulation of TCR-V T cells within the tumors and inhibited tumor progression. These results indicate that epitope V provides a target for CD8+ T cells against spontaneous tumors in vivo, and suggests that epitopes with similar properties can be harnessed for tumor immunotherapy.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes.

Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycoprotein B epitope (amino acids 498 to 505) was also expressed in SV40 T antigen at positions 350 and 650. Primary C57BL/6 mouse kidney cells were immortalized by transfection with the recombinant and wild-type T-antigen DNA. Clonal isolates of cells expressing chimeric T antigens were shown to be specifically susceptible to lysis by CTL clones directed to SV40 T-antigen site I and herpes simplex virus glycoprotein B epitopes, indicating that CTL epitopes restricted by two different elements can be processed, presented, and recognized by the epitope-specific CTL clones. Our results suggest that SV40 T antigen can be used as a carrier protein to express a wide variety of CTL epitopes.     

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.     

Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.     

SV40 Tag-specific cytotoxic T lymphocytes generated from the peripheral blood of malignant pleural mesothelioma patients

Malignant pleural mesothelioma (MPM) is an aggressive cancer, with survival of less than one year following diagnosis and treatment with current protocols. Recent studies have demonstrated the presence of the simian virus 40 (SV40)-like, large tumor antigen (Tag) in nearly 60% of MPMs. SV40 Tag is a viral-encoded tumor-specific antigen, and thus a potential target for the induction of anti-tumor immunity and the development of therapeutic vaccines. We describe here evidence for the existence of SV40 Tag-specific immune responses in patients with MPM whose tumors express Tag. Humoral immunity was demonstrated by the detection of IgG titers against Tag in serum samples from 1/3 of patients examined. CTLs were generated from the peripheral blood of an HLA-A2(+) MPM patient with a synthetic peptide representing an HLA-A2 binding epitope in SV40 Tag. The CTLs demonstrated epitope fine specificity, in that other peptides from SV40 Tag and a peptide from influenza virus were not recognized in the context of HLA-A2. Moreover, the CTLs were capable of recognizing mesothelioma tumor cells that expressed SV40 Tag, in an MHC class I restricted manner.     

Theiler's virus and Mengo virus induce cross-reactive cytotoxic T lymphocytes restricted to the same immunodominant VP2 epitope in C57BL/6 mice.

C57BL/6 mice develop a virus-specific cytotoxic T-lymphocyte (CTL) response after intraperitoneal inoculation with either the DA strain of Theiler's virus or Mengo virus, two members of the Cardiovirus genus. These CTLs contribute to viral clearance in the case of Theiler's virus but do not protect the mice from the fatal encephalomyelitis caused by Mengo virus. In this study we show that DA and Mengo virus-induced CTLs are cross-reactive. The cross-reactivity is due to a conserved, H-2Db-restricted epitope located between amino acid residues 122 and 130 of the VP2 capsid protein (VP2(122-130)). This epitope is immunodominant in C57BL/6 mice infected with Theiler's virus. The VP2(122-130) epitope, initially identified for Mengo virus, is the first CTL epitope described for Theiler's virus.     

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

Identification of the immunodominant H-2K(k)-restricted cytotoxic T-cell epitope in the Borna disease virus nucleoprotein

Borna disease virus (BDV)-induced immunopathology in mice is most prominent in strains carrying the major histocompatibility complex H-2k allele and is mediated by CD8(+) T cells that are directed against the viral nucleoprotein p40. We now identified the highly conserved octamer peptide TELEISSI, located between amino acid residues 129 and 136 of BDV p40, as a potent H-2K(k)-restricted cytotoxic T-cell (CTL) epitope. When added to the culture medium of L929 target cells, TELEISSI conferred sensitivity to lysis by CTLs isolated from brains of BDV-infected MRL mice with acute neurological disease. Vaccinia virus-mediated expression of a p40 variant with mutations in the two K(k)-specific anchor residues of the TELEISSI peptide (p40(E130K,I136T)) did not sensitize L929 target cells for lysis by BDV-specific CTLs, whereas expression of wild-type p40 did. Furthermore, unlike vaccination with wild-type p40, vaccination of persistently infected symptomless B10.BR mice with p40(E130K,I136T) did not result in central nervous system inflammation and neurological disease. These results demonstrate that TELEISSI is the immunodominant CTL epitope of BDV p40 in H-2k mice.     

Hierarchal Utilization of Different T-Cell Receptor Vβ Gene Segments in the CD8+-T-Cell Response to an Immunodominant Moloney Leukemia Virus-Encoded Epitope In Vivo

The CD8(+)-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) presented in the context of H-2D(b) and is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the Valpha3.2 and Vbeta5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 Vbeta(a) mice which are unable to express the dominant Valpha3.2(+) Vbeta5.2(+) T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the Vbeta5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 Vbeta(a) mice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8(+) cells utilizing the same Valpha3.2 gene segment in association with two different Vbeta segments (Vbeta3 and Vbeta17). Surprisingly, all these CTL recognized the same immunodominant M-MuLV gag epitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 x C57BL/6 Vbeta(a))F(1) mice revealed a clear hierarchy in Vbeta utilization, with a preferential usage of the Vbeta17 gene segment, whereas Vbeta3 and especially Vbeta5.2 were used to much lesser extents. Sequencing of TCRalpha- and -beta-chain junctional regions of CTL clones specific for the M-MuLV gag epitope revealed a diverse repertoire of TCRbeta chains in Vbeta(a) mice and a highly restricted TCRbeta-chain repertoire in Vbeta(b) mice, whereas TCRalpha-chain sequences were highly conserved in both cases. Collectively, our data indicate that the H-2D(b)-restricted M-MuLV gag epitope can be recognized in a hierarchal fashion by different Vbeta domains and that the degree of beta-chain diversity varies according to Vbeta utilization.     

Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+ Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8(+) CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8(+) CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/beta2m complexes. All SHIV-infected Mamu-A*01(+) rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8(+) CTL response is dominant and the p41A- and p68A-specific CD8(+) CTL responses are nondominant. These results indicate that CD8(+) CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8(+) CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.     

CD8+ T cells targeting a single immunodominant epitope are sufficient for elimination of established SV40 T antigen-induced brain tumors

Immunotherapy of established solid tumors is rarely achieved, and the mechanisms leading to success remain to be elucidated. We previously showed that extended control of advanced-stage autochthonous brain tumors is achieved following adoptive transfer of naive C57BL/6 splenocytes into sublethally irradiated line SV11 mice expressing the SV40 T Ag (T Ag) oncoprotein, and was associated with in vivo priming of CD8(+) T cells (T(CD8)) specific for the dominant epitope IV (T Ag residues 404-411). Using donor lymphocytes derived from mice that are tolerant to epitope IV or a newly characterized transgenic mouse line expressing an epitope IV-specific TCR, we show that epitope IV-specific T(CD8) are a necessary component of the donor pool and that purified naive epitope IV-specific T(CD8) are sufficient to promote complete and rapid regression of established tumors. While transfer of naive TCR-IV cells alone induced some initial tumor regression, increased survival of tumor-bearing mice required prior conditioning of the host with a sublethal dose of gamma irradiation and was associated with complete tumor eradication. Regression of established tumors was associated with rapid accumulation of TCR-IV T cells within the brain following initial priming against the endogenous T Ag in the peripheral lymphoid organs. Additionally, persistence of functional TCR-IV cells in both the brain and peripheral lymphoid organs was associated with long-term tumor-free survival. Finally, we show that production of IFN-gamma, but not perforin or TNF-alpha, by the donor lymphocytes is critical for control of autochthonous brain tumors.     

Mapping and characterization of the primary and anamnestic H-2(d)-restricted cytotoxic T-lymphocyte response in mice against human metapneumovirus

Cytotoxic T lymphocytes (CTLs) are important for the control of virus replication during respiratory infections. For human metapneumovirus (hMPV), an H-2(d)-restricted CTL epitope in the M2-2 protein has been described. In this study, we screened the hMPV F, G, N, M, M2-1, and M2-2 proteins using three independent algorithms to predict H-2(d) CTL epitopes in BALB/c mice. A dominant epitope (GYIDDNQSI) in positions 81 to 89 of the antitermination factor M2-1 and a subdominant epitope (SPKAGLLSL) in N(307-315) were detected during the anti-hMPV CTL response. Passive transfer of CD8(+) T-cell lines against M2-1(81-89) and N(307-315) protected Rag1(-/-) mice against hMPV challenge. Interestingly, diversification of CTL targets to include multiple epitopes was observed after repetitive infections. A subdominant response against the previously described M2-2 epitope was detected after the third infection. An understanding of the CTL response against hMPV is important for developing preventive and therapeutic strategies against the virus.     

Tissue-specific regulation of CD8+ T-lymphocyte immunodominance in respiratory syncytial virus infection

Cytotoxic T lymphocytes (CTLs) are critical for control of respiratory syncytial virus (RSV) infection in humans and mice. To investigate cellular immune responses to infection, it is important to identify major histocompatibility complex (MHC) class I-restricted CTL epitopes. In this study, we identified a new RSV-specific, H-2K(d)-restricted subdominant epitope in the M2 protein, M2(127-135) (amino acids 127 to 135). This finding allowed us to study the frequency of T lymphocytes responding to two H-2K(d)-presented epitopes in the same protein following RSV infection by enzyme-linked immunospot (ELISPOT) and intracellular cytokine assays for both lymphoid and nonlymphoid tissues. For the subdominant epitope, we identified an optimal nine-amino-acid peptide, VYNTVISYI, which contained an H-2K(d) consensus sequence with Y at position 2 and I at position 9. In addition, an MHC class I stabilization assay using TAP-2-deficient RMA-S cells transfected with K(d) or L(d) indicated that the epitope was presented by K(d). The ratios of T lymphocytes during the peak CTL response to RSV infection that were specific for M2(82-90) (dominant) to T lymphocytes specific for M2(127-135) (subdominant) were approximately 3:1 in the spleen and 10:1 in the lung. These ratios were observed consistently in primary or secondary infection by the ELISPOT assay and in secondary infection by MHC/peptide tetramer staining. The number of antigen-specific T lymphocytes dropped in the 6 weeks after infection; however, the proportions of T lymphocytes specific for the immunodominant and subdominant epitopes were maintained to a remarkable degree in a tissue-specific manner. These studies will facilitate investigation of the regulation of immunodominance of RSV-specific CTL epitopes.     

Characterization of the Fas ligand/Fas-dependent apoptosis of antiretroviral, class I MHC tetramer-defined, CD8+ CTL by in vivo retrovirus-infected cells

C57BL/6 (B6; H-2(b)) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8(+) CTL responses to the immunodominant K(b)-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2(b) congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2(b) spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2(b) cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2(b) veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8(+) CTL were identified by a labeled K(b)/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2(b) veto cell-mediated depletion of these CTL. A 65-93% reduction in the number of B6 K(b)/KSPWFTTL tetramer(+) CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2(b) veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2(b) veto cells) to block inhibition demonstrated that AKR.H-2(b) veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer(+) CTL cocultured with AKR.H-2(b) veto cells was annexin V positive and Fas(high), indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2(b) cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vbeta4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.     

Antiretroviral cytolytic T-lymphocyte nonresponsiveness: FasL/Fas-mediated inhibition of CD4(+) and CD8(+) antiviral T cells by viral antigen-positive veto cells

C57BL/6 (H-2(b)) mice generate type-specific cytolytic T-lymphocyte (CTL) responses to an immunodominant Kb-restricted epitope, KSPWFTTL located in the membrane-spanning domain of p15TM of AKR/Gross murine leukemia viruses (MuLV). AKR.H-2(b) congenic mice, although carrying the responder H-2(b) major histocompatibility complex (MHC) haplotype, are low responders or nonresponders for AKR/Gross MuLV-specific CTL, apparently due to the presence of inhibitory AKR. H-2(b) cells. Despite their expression of viral antigens and Kb, untreated viable AKR.H-2(b) spleen cells cause dramatic inhibition of the C57BL/6 (B6) antiviral CTL response to in vitro stimulation with AKR/Gross MuLV-induced tumor cells. This inhibition is specific (AKR.H-2(b) modulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility antigen-specific CTL production), dependent on direct contact of AKR.H-2(b) cells in a dose-dependent manner with the responder cell population, and not due to soluble factors. Here, the mechanism of inhibition of the antiviral CTL response is shown to depend on Fas/Fas-ligand interactions, implying an apoptotic effect on B6 responder cells. Although B6.gld (FasL-) responders were as sensitive to inhibition by AKR.H-2(b) modulator cells as were B6 responders, B6.lpr (Fas-) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL stimulation cultures, relieved the inhibition caused by the AKR.H-2(b) cells if the primed responders were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2(b) cells express FasL. Because of the antigen specificity of the inhibition, these results collectively implicate a FasL/Fas interaction mechanism: viral antigen-positive AKR.H-2(b) cells expressing FasL inhibit antiviral T cells ("veto" them) when the AKR.H-2(b) cells are recognized. Consistent with this model, inhibition by AKR.H-2(b) modulator cells was MHC restricted, and resulted in approximately a 10- to 70-fold decrease in the in vitro expansion of pCTL/CTL. Both CD8(+) CTL and CD4(+) Th responder cells were susceptible to inhibition by FasL+ AKR.H-2(b) inhibitory cells as the basis for inhibition. The CTL response in the presence of inhibitory cells could be restored by several cytokines or agents that have been shown by others to interfere with activation-induced cell death (e.g. , interleukin-2 [IL-2], IL-15, transforming growth factor beta, lipopolysaccharide, 9-cis-retinoic acid) but not others (e.g., tumor necrosis factor alpha). These results raise the possibility that this type of inhibitory mechanism is generalized as a common strategy for retrovirus infected cells to evade immune T-cell recognition.     

HLA-A11-restricted epitope polymorphism among Epstein-Barr virus strains in the highly HLA-A11-positive Chinese population: incidence and immunogenicity of variant epitope sequences

An individual's CD8(+)-cytotoxic-T-lymphocyte (CTL) response to Epstein-Barr virus (EBV) latent cycle antigens focuses on a small number of immunodominant epitopes often presented by just one of the available HLA class I alleles; for example, HLA-A11-positive Caucasians frequently respond to two immunodominant HLA A11 epitopes, IVTDFSVIK (IVT) and AVFDRKSDAK (AVF), within the nuclear antigen EBNA3B. Here, we reexamine the spectrum of EBV strains present in the highly HLA-A11-positive Chinese population for sequence changes in these epitopes relative to the Caucasian type 1 prototype strain B95.8. The IVT epitope was altered in 61 of 64 Chinese type 1 viruses, with four different sequence variants being observed, and the AVF epitope was altered in 46 cases with six different sequence variants; by contrast, all 10 Chinese type 2 viruses retained the prototype 2 epitope sequences. All but one of the type 1 epitope variants were poorly recognized by IVT- or AVF-specific CTLs in pulse-chase assays of peptide-mediated target cell lysis. More importantly, we screened HLA-A11-positive Chinese donors carrying viruses with known epitope mutations for evidence of epitope-specific CTL memory by enzyme-linked immunospot assays: none of the type 1 variants tested, nor the type 2 prototype, appeared to be immunogenic in vivo. The data remain consistent with the possibility that, during virus-host coevolution, pressure from the host CTL-mediated immune response has given A11 epitope-loss viruses a selective advantage.