Peptide variants of viral CTL epitopes mediate positive selection and emigration of Ag-specific thymocytes in vivo

During development, thymocytes carrying TCRs mediating low-affinity interactions with MHC-bound self-peptides are positively selected for export into the mature peripheral T lymphocyte pool. Thus, exogenous administration of certain altered peptide ligands (APL) with reduced TCR affinity relative to cognate Ags may provide a tool to elicit maturation of desired TCR specificities. To test this "thymic vaccination" concept, we designed APL of the viral CTL epitopes gp33-41 and vesicular stomatitis virus nucleoprotein octapeptide N52-59 relevant for the lymphocytic choriomeningitis virus-specific P14- and vesicular stomatitis virus-specific N15-TCRs, respectively, and examined their effects on thymocytes in vivo using irradiation chimeras. Injection of APL into irradiated congenic (Ly-5.1) mice, reconstituted with T cell progenitors from the bone marrow of P14 RAG2(-/-) (Ly-5.2) or N15 RAG2(-/-) (Ly-5.2) transgenic mice, resulted in positive selection of T cells expressing the relevant specificity. Moreover, the variants led to export of virus-specific T cells to lymph nodes, but without inducing T cell proliferation. These findings show that the mature T cell repertoire can be altered by in vivo peptide administration through manipulation of thymic selection.     

CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses

MHC class I-restricted T cell epitopes lack immunogenicity unless aided by IFA or CFA. In an attempt to circumvent the known inflammatory side effects of IFA and CFA, we analyzed the ability of immunostimulatory CpG-DNA to act as an adjuvant for MHC class I-restricted peptide epitopes. Using the immunodominant CD8 T cell epitopes, SIINFEKL from OVA or KAVYNFATM (gp33) from lymphocytic choriomeningitis virus glycoprotein, we observed that CpG-DNA conveyed immunogenicity to these epitopes leading to primary induction of peptide-specific CTL. Furthermore, vaccination with the lymphocytic choriomeningitis virus gp33 peptide triggered not only CTL but also protective antiviral defense. We also showed that MHC class I-restricted peptides are constitutively presented by immature dendritic cells (DC) within the draining lymph nodes but failed to induce CTL responses. The use of CpG-DNA as an adjuvant, however, initiated peptide presenting immature DC progression to professional licensed APC. Activated DC induced cytolytic CD8 T cells in wild-type mice and also mice deficient of Th cells or CD40 ligand. CpG-DNA thus incites CTL responses toward MHC class I-restricted T cell epitopes in a Th cell-independent manner. Overall, these results provide new insights into CpG-DNA-mediated adjuvanticity and may influence future vaccination strategies for infectious and perhaps tumor diseases.     

Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells

Appropriate activation of naive CD8(+) T cells depends on the coordinated interaction of these cells with professional APC that present antigenic peptides in the context of MHC class I molecules. It is accepted that dendritic cells (DC) are efficient in activating naive T cells and are unique in their capacity to prime CD8(+) T cell responses against exogenous cell-associated Ags. Nevertheless, it is unclear whether epitopes, derived from endogenously synthesized proteins and presented by MHC class I molecules on the surface of other APC including B cells and macrophages, can activate naive CD8(+) T cells in vivo. By infecting transgenic CD11c-DTR/GFP mice that allow conditional depletion of DC with lymphocytic choriomeningitis virus (LCMV), which infects all types of APC and elicits a vigorous CTL response, we unambiguously show that priming of LCMV-specific CD8(+) T cells is crucially dependent on DC, despite ample presence of LCMV-infected macrophages and B cells in secondary lymphoid organs.     

Impact of CCR7 on priming and distribution of antiviral effector and memory CTL

The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and dendritic cells (DC) into and their localization within secondary lymphoid organs. In this study we investigated the impact of CCR7 on CD8(+) T cell responses by infecting CCR7(-/-) mice with lymphocytic choriomeningitis virus (LCMV). We found that the absence of CCR7 affects the magnitude of an antiviral CTL response during the acute phase, with reduced numbers of virus-specific CTL in all lymphoid and nonlymphoid organs tested. On the single cell level, CCR7-deficient CTL gained full effector function, such that antiviral protection in CCR7-deficient mice was complete, but delayed. Similarly, adoptive transfer experiments using DC from CCR7-deficient or competent mice for the priming of CCR7-positive or CCR7-negative CD8(+) T cells, respectively, revealed that ectopic positioning of DC and CTL outside organized T cell zones results in reduced priming efficacy. In the memory phase, CCR7-deficient mice maintained a stable LCMV-specific CTL population, predominantly in nonlymphoid organs, and rapidly mounted protective CTL responses against a challenge infection with a vaccinia virus recombinant for the gp33 epitope of LCMV. Taken together, the CCR7-dependent organization of the T cell zone does not appear to be a prerequisite for antiviral effector CTL differentiation and the sustenance of antiviral memory responses in lymphoid or peripheral tissues.     

CD8+ T cell activation is governed by TCR-peptide/MHC affinity, not dissociation rate

Binding of peptide/MHC (pMHC) complexes by TCR initiates T cell activation. Despite long interest, the exact relationship between the biochemistry of TCR/pMHC interaction (particularly TCR affinity or ligand off-rate) and T cell responses remains unresolved, because the number of complexes examined in each independent system has been too small to draw a definitive conclusion. To test the current models of T cell activation, we have analyzed the interactions between the mouse P14 TCR and a set of altered peptides based on the lymphocytic choriomeningitis virus epitope gp33-41 sequence bound to mouse class I MHC D(b). pMHC binding, TCR-binding characteristics, CD8+ T cell cytotoxicity, and IFN-gamma production were measured for the peptides. We found affinity correlated well with both cytotoxicity and IFN-gamma production. In contrast, no correlation was observed between any kinetic parameter of TCR-pMHC interaction and cytotoxicity or IFN-gamma production. This study strongly argues for an affinity threshold model of T cell activation.     

Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus.

4-1BB ligand (4-1BBL) is a member of the TNF family expressed on activated APC. 4-1BBL binds to 4-1BB (CD137) on activated CD4 and CD8 T cells and in conjunction with strong signals through the TCR provides a CD28-independent costimulatory signal leading to high level IL-2 production by primary resting T cells. Here we report the immunological characterization of mice lacking 4-1BBL and of mice lacking both 4-1BBL and CD28. 4-1BBL-/- mice mount neutralizing IgM and IgG responses to vesicular stomatitis virus that are indistinguishable from those of wild-type mice. 4-1BBL-/- mice show unimpaired CTL responses to lymphocytic choriomeningitis virus (LCMV) and exhibit normal skin allograft rejection but have a weaker CTL response to influenza virus than wild-type mice. 4-1BBL-/-CD28-/- mice retain the CTL response to LCMV, respond poorly to influenza virus, and exhibit a delay in skin allograft rejection. In agreement with these in vivo results, allogeneic CTL responses of CD28-/- but not CD28+/+ T cells to 4-1BBL-expressing APC are substantially inhibited by soluble 4-1BB receptor as is the in vitro secondary response of CD28+ T cells to influenza virus peptides. TCR-transgenic CD28-/- LCMV glycoprotein-specific T cells are insensitive to the presence of 4-1BBL when a wild-type peptide is used, but the response to a weak agonist peptide is greatly augmented by the presence of 4-1BBL. These results further substantiate the idea that different immune responses vary in their dependence on costimulation and suggest a role for 4-1BBL in augmenting suboptimal CTL responses in vivo.     

Effects of an epitope-specific CD8+ T-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants

Both CD4(+) and CD8(+) T cells are required for clearance of the murine coronavirus mouse hepatitis virus (MHV) during acute infection. We investigated the effects of an epitope-specific CD8(+) T-cell response on acute infection of MHV, strain A59, in the murine CNS. Mice with CD8(+) T cells specific for gp33-41 (an H-2D(b)-restricted CD8(+) T-cell epitope derived from lymphocytic choriomeningitis glycoprotein) were infected with a recombinant MHV-A59, also expressing gp33-41, as a fusion protein with enhanced green fluorescent protein (EGFP). By 5 days postinfection, these mice showed significantly (approximately 20-fold) lower titers of infectious virus in the brain compared to control mice. Furthermore mice with gp33-41-specific CD8(+) cells exhibited much reduced levels of viral antigen in the brain as measured by immunohistochemistry using an antibody directed against viral nucleocapsid. More than 90% of the viruses recovered from brain lysates of such protected mice, at 5 days postinfection, had lost the ability to express EGFP and had deletions in their genomes encompassing EGFP and gp33-41. In addition, genomes of viruses from about half the plaques that retained the EGFP gene had mutations within the gp33-41 epitope. On the other hand, gp33-41-specific cells failed to protect perforin-deficient mice from infection by the recombinant MHV expressing gp33, indicating that perforin-mediated mechanisms were needed. Virus recovered from perforin-deficient mice did not exhibit loss of EGFP expression and the gp33-41 epitope. These observations suggest that the cytotoxic T-cell response to gp33-41 exerts a strong immune pressure that quickly selects epitope escape mutants to gp33-41.     

Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33-41) and the nucleoprotein (NP396-404), while the gp276-286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-gamma enhanced the presentation of gp33-41, whereas presentation of the gp276-286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-gamma treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276-286 epitope. The ability to generate gp276-286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.     

Determination of structural principles underlying three different modes of lymphocytic choriomeningitis virus escape from CTL recognition

Lymphocytic choriomeningitis virus infection of H-2(b) mice generates a strong CD8(+) CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2D(b) and H-2K(b) MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2D(b) and H-2K(b). Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.     

Addition of a prominent epitope affects influenza A virus-specific CD8+ T cell immunodominance hierarchies when antigen is limiting

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA(257-264)) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HKx31 (H3N2) influenza A viruses. Initial characterization determined that K(b)OVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering D(b) nucleoprotein (NP)366 presentation. There were similar levels of K(b)OVA257- and D(b)NP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant K(b)OVA257-specific response resulted in diminished D(b) acidic polymerase224- and K(b) basic polymerase subunit 1(703)-, but not D(b)NP366-specific responses and didn't alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of K(b)OVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.     

Competitive inhibition in vivo and skewing of the T cell repertoire of antigen-specific CTL priming by an anti-peptide-MHC monoclonal antibody.

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2D(d) and a decamer peptide corresponding to residues 311-320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8(+) CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2D(d). This inhibition is specific for this particular peptide Ag; another H-2D(d)-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2L(d)-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2D(d)/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vbeta usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vbeta7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8(+) T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.     

Affinity thresholds for naive CD8+ CTL activation by peptides and engineered influenza A viruses

High-avidity interactions between TCRs and peptide + class I MHC (pMHCI) epitopes drive CTL activation and expansion. Intriguing questions remain concerning the constraints determining optimal TCR/pMHCI binding. The present analysis uses the TCR transgenic OT-I model to assess how varying profiles of TCR/pMHCI avidity influence naive CTL proliferation and the acquisition of effector function following exposure to the cognate H-2K(b)/OVA(257-264) (SIINFEKL) epitope and to mutants provided as peptide or in engineered influenza A viruses. Stimulating naive OT-I CD8(+) T cells in vitro with SIINFEKL induced full CTL proliferation and differentiation that was largely independent of any need for costimulation. By contrast, in vitro activation with the low-affinity EIINFEKL or SIIGFEKL ligands depended on the provision of IL-2 and other costimulatory signals. Importantly, although they did generate potent endogenous responses, infection of mice with influenza A viruses expressing these same OVA(257) variants failed to induce the activation of adoptively transferred naive OT-I CTLps, an effect that was only partially overcome by priming with a lipopeptide vaccine. Subsequent structural and biophysical analysis of H2-K(b)OVA(257), H2-K(b)E1, and H2-K(b)G4 established that these variations introduce small changes at the pMHCI interface and decrease epitope stability in ways that would likely impact cell surface presentation and recognition. Overall, it seems that there is an activation threshold for naive CTLps, that minimal alterations in peptide sequence can have profound effects, and that the antigenic requirements for the in vitro and in vivo induction of CTL proliferation and effector function differ substantially.     

Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.     

New approaches to eliciting protective immunity through T cell repertoire manipulation: the concept of thymic vaccination

Conventional vaccines afford protection against infectious diseases by expanding existing pathogen-specific peripheral lymphocytes, both CD8 cytotoxic effector (CTL) and CD4 helper T cells. The latter induce B cell maturation and antibody production. As a consequence, lymphocytes within the memory pool are poised to rapidly proliferate at the time of a subsequent infection. The "thymic vaccination" concept offers a novel way to alter the primary T cell repertoire through exposure of thymocytes to altered peptide ligands (APL) with reduced T cell receptor (TCR) affinity relative to cognate antigens recognized by those same TCRs. Thymocyte maturation (i.e. positive selection) is enhanced by low affinity interaction between a TCR and an MHC-bound peptide in the thymus and subsequent emigration of mature cells into the peripheral T lymphocyte pool follows. In principal, such variants of antigens derived from infectious agents could be utilized for peptide-driven maturation of thymocytes bearing pathogen-specific TCRs. To test this idea, APLs of gp33-41, a Db-restricted peptide derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein, and of VSV8, a Kb-restricted peptide from the vesicular stomatitis virus (VSV) nucleoprotein, have been designed and their influence on thymic maturation of specific TCR-bearing transgenic thymocytes examined in vivo using irradiation chimeras. Injection of APL resulted in positive selection of CD8 T cells expressing the relevant viral specificity and in the export of those virus-specific CTL to lymph nodes without inducing T cell proliferation. Thus, exogenous APL administration offers the potential of expanding repertoires in vivo in a manner useful to the organism. To efficiently peripheralize antigen-specific T cells, concomitant enhancement of mechanisms promoting thymocyte migration appears to be required. This commentary describes the rationale for thymic vaccination and addresses the potential prophylactic and therapeutic applications of this approach for treatment of infectious diseases and cancer. Thymic vaccination-induced peptide-specific T cells might generate effective immune protection against disease-causing agents, including those for which no effective natural protection exists.     

HLA-A2.1/K(b) transgenic murine dendritic cells transduced with an adenovirus encoding human gp100 process the same A2.1-restricted peptide epitopes as human antigen-presenting cells and elicit A2.1-restricted peptide-specific CTL

HLA-A2.1/K(b) transgenic mice (A2.1/K(b) mice) were used to investigate the processing of human gp100 melanoma antigen by murine antigen presenting cells (APC). Bone marrow-derived dendritic cells (DC) from A2.1/K(b) mice were transduced with adenovirus encoding human gp100 (Ad2/hugp100v2). The Ad2/hugp100v2-transduced DC express human gp100, as documented by immunoperoxidase staining. Flow cytometric analysis demonstrates that Ad vector transduction does not downregulate expression of several markers, including MHC class I. We show that Ad2/hugp100v2-transduced DC are recognized by peptide-specific, A2.1-restricted CTL, suggesting correct processing and presentation of the hugp100 antigen by murine DC. To assess dominance among the various A2.1-restricted epitopes encoded by hugp100, A2.1/K(b) transgenic mice were immunized with Ad2/hugp100v2-transduced DC. Resulting effector cytotoxic T lymphocytes (CTL) were assayed for peptide specificity using a panel of six synthetic peptides known to encode A2.1-restricted epitopes of human gp100 (denoted G154, G177, G209, G280, G457, G476). CTL obtained from Ad2/hugp100v2-transduced DC immunized A2.1/K(b) mouse lysed target cells presenting five of the six epitopes, supporting the observation that murine cells correctly process the hugp100 antigen. The immunogenicity of individual gp100 epitopes correlates with their binding affinity to A2.1. CTL generated from A2.1/K(b) mice immunized with Ad2/hugp100v2-transduced DC also specifically recognize A2.1(+)/gp100(+) human melanoma cells. These data suggest that murine APC process and present the same set of HLA-restricted peptides, similar to human APC. HLA transgenic mice serve as a useful model system to study class I-restricted epitopes of human tumor-associated antigens.     

FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory

FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride) prolongs survival of solid organ allografts in animal models. Mechanisms of FTY720 immunomodulation were studied in mice infected with lymphocytic choriomeningitis virus (LCMV) to assess T cell responses or with vesicular stomatitis virus to evaluate Ab responses. Oral FTY720 (0.3 mg/kg/day) did not affect LCMV replication and specific CTL and B cells were induced and expanded normally. Moreover, the anti-viral humoral immune responses were normal. However, FTY720 treatment showed first a shift of overall distribution of CTL from the spleen to peripheral lymph nodes and lymphocytopenia was observed. This effect was reversible within 7-21 days. Together with unimpaired T and B cell memory after FTY720 treatment, this finding rendered enhancement of lymphocyte apoptosis by FTY720 in vivo unlikely. Secondly, the delayed-type hypersensitivity reaction to a viral MHC class I-presented peptide was markedly reduced by FTY720. These results were supported by impaired circulation of LCMV specific TCR transgenic effector lymphocytes in the peripheral blood and reduced numbers of tissue infiltrating CTL in response to delayed-type hypersensitivity reaction. Thirdly, in a CD8+ T cell-mediated diabetes model in a transgenic mouse expressing the LCMV glycoprotein in the islets of the pancreas, FTY720 delayed or prevented disease by reducing islet-infiltrating CTL. Thus, FTY720 effectively reduced recirculation of CD8+ effector T cells and their recruitment to peripheral lesions without affecting the induction and expansion of immune responses in secondary lymphoid organs. These properties may offer the potential to treat ongoing organ-specific T cell-mediated immunopathologic disease.     

Predominant utilization of V beta 8+ T cell receptor genes in the H-2Ld-restricted cytotoxic T cell response against the immediate-early protein pp89 of the murine cytomegalovirus

Cytotoxic T cell responses to the murine Cytomegalovirus (MCMV) were elicited in BALB/c mice (H-2d) by infectious virus. Eight days after infection, MCMV-primed local lymph node T cells were either depleted for T cells expressing a V beta 8+ TCR or separated into V beta 8+ and V beta 8- subpopulations by a cell sorter using the mAb F23.1. T cells were then expanded in vitro under limiting dilution conditions in the presence of IL-2 and in the absence of viral Ag to avoid selection by Ag in vitro. Frequencies of CTL precursors specific for the Immediate-Early-Ag 1 of MCMV and restricted to H-2Ld were determined. L cells of the endogenous haplotype H-2k cotransfected with the genes for MCMV-IE 1 and H-2Ld were used as target cells. Detection of a CTL response required previous priming of the animals by infection in vivo (less than 1/10(6) for nonimmunized animals). In primed animals CTL precursors of this specificity and restriction were three to fivefold more frequent in the V beta 8+ population (1/9.900 to 1/22.300) than in the V beta 8- population (1/57.000 to 1/87.200). Control experiments showed that frequencies were not influenced by the treatment with the anti-V beta 8-antibody and the fluorescein-labeled anti-Ig itself. V beta 8+ and V beta 8- T cells did not reveal any frequency differences when several other responses were determined (TNP-specific self-restricted CTL precursor; Th cells specific for keyhole limpet hemocyanin or Listeria monocytogenes).     

Suppression of virus-specific antibody production by CD8+ class I-restricted antiviral cytotoxic T cells in vivo.

The question of whether virus-induced immunosuppression includes the antibody response against the infecting virus itself was evaluated in a model situation. Transgenic mice expressing the T-cell receptor (TCR) specific for peptide 32-42 of lymphocytic choriomeningitis virus (LCMV) glycoprotein 1 presented by Db reacted with a strong transgenic cytotoxic T-lymphocyte (CTL) response starting on day 3 after infection with a high dose (10(6) PFU intravenously [i.v.]) of the WE strain of LCMV (LCMV-WE); LCMV-specific antibody production in the spleen was suppressed in these mice. Low-dose (10(2) PFU i.v.) infection resulted in an antiviral antibody response comparable to that of the transgene-negative littermates. The induction of suppression of LCMV-specific antibody responses was specifically mediated by CD8+ TCR transgenic CTLs, since the LCMV-8.7 variant virus (which is not recognized by transgenic TCR-expressing CTLs because of a point mutation) did not induce suppression. In addition, treatment with CD8 monoclonal antibody in vivo abrogated suppression. Once suppression had been established, it was found to be nonspecific. The abrogation of antibody responses depended on the relative kinetics of the antibody response involved and the kinetics of the anti-LCMV CTL response. Analysis of T- and B-cell subpopulations showed no significant changes, but immunohistochemical analysis of spleens revealed extensive destruction of follicular organization in lymphoid tissue by day 4 in transgenic mice infected with LCMV-WE but not in those infected with the CTL escape mutant LCMV-8.7. Impairment of antigen presentation rather than of T or B cells was also suggested by adoptive transfer experiments, showing that transferred infected macrophages may improve the anti-LCMV antibody response in LCMV-immunosuppressed transgenic recipients; also, T and B cells from suppressed transgenic mice did respond in irradiated and virus-infected nontransgenic mice with antibody formation to LCMV. Such virus-triggered, T-cell-mediated immunopathology causing the suppression of B cells and of protective antibody responses, including those against the infecting virus itself, may permit certain viruses to establish persistent infections.     

IFN-gamma promotes Fas ligand- and perforin-mediated liver cell destruction by cytotoxic CD8 T cells

To study liver cell damage by CTL, CD8 T cells from P14 TCR transgenic (tg) mice specific for the gp33 epitope of lymphocytic choriomeningitis virus with either deficiency in IFN-gamma (P14.IFN-gamma(null)), functional Fas ligand (P14.gld), or perforin (P14.PKO) were transferred into H8 tg mice ubiquitously expressing gp33 Ag. Treatment of H8 recipient mice with agonistic anti-CD40 Abs induced vigorous expansion of the transferred P14 T cells and led to liver cell destruction determined by increase of glutamate dehydrogenase serum levels and induction of caspase-3 in hepatocytes. Liver injury was mediated by the Fas/Fas ligand (FasL) pathway and by perforin, because P14.gld and P14.PKO T cells failed to induce increased glutamate dehydrogenase levels despite strong in vivo proliferation. In addition, H8 tg mice lacking Fas were resistant to the pathogenic effect of P14 T cells. Besides FasL and perforin, IFN-gamma was also required for liver cell damage, because P14.IFN-gamma(null) T cells adoptively transferred into H8 mice failed to induce disease. Moreover, Fas expression on hepatocytes from H8 recipient mice was increased after transfer of wild-type compared with P14.IFN-gamma(null) T cells, and wild-type P14 T cells expressed higher levels of FasL than P14 T cells lacking IFN-gamma. Thus, our data suggest that IFN-gamma released by activated CD8 T cells upon Ag contact facilitates liver cell destruction.