Signals delivered via the Qa-2 molecule can synergize with limiting anti-CD3-induced signals to cause T lymphocyte activation.

Qa-2 is a glycolipid anchored, MHC encoded class I molecule expressed at high levels on all murine peripheral T lymphocytes. Anti-Qa-2 antibodies have previously been found to stimulate T cells to proliferate in the presence of crosslinking antibody and PMA. We have examined the effect of anti-Qa-2 antibodies on T cells stimulated with a suboptimal concentration of immobilized anti-CD3. When anti-Qa-2 antibodies were co-immobilized with limiting anti-CD3, in the absence of PMA, a clear augmentation of T cell proliferation was seen. Interestingly, the co-stimulatory anti-Qa-2 antibodies could be directed against epitopes mapped to either the alpha 3 or the alpha 1/alpha 2 Qa-2 domains. As was the case with activation induced by soluble/crosslinked anti-Qa-2 antibodies plus PMA, CD8+ T cells were less able to be costimulated with anti-Qa-2 antibodies than CD4+ cells. Surprisingly, Ca2+ mobilization was only seen when two anti-Qa-2 antibodies reactive to separate structural domains were co-crosslinked on the surface of Indo-1 loaded T cells with a suboptimal concentration of anti-CD3. Collectively these results raise questions regarding the mechanism of Qa-2 mediated signaling and its potential role in T cell activation.     

Spontaneous in vivo retrovirus-infected T and B cells, but not dendritic cells, mediate antigen-specific Fas ligand/Fas-dependent apoptosis of anti-retroviral CTL

C57BL/6 (H-2b), but not spontaneous virus-expressing AKR.H-2b congenic, mice generate retrovirus-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL. AKR.H-2b non-responsiveness is mediated by a peripheral tolerance mechanism. When co-cultured with primed B6 antiviral pCTL, AKR.H-2b splenocytes are recognized by the antiviral TcR as "veto" cells, which inhibit by an exquisitely virus-specific, MHC-restricted, veto cell FasL/responder T cell Fas, mediated apoptotic mechanism. Here, AKR.H-2b thymus, lymph node, and bone marrow cells are also shown to inhibit antiviral CTL generation. Purified AKR.H-2b CD4+ and CD8+ T cells, and B cells, served effectively as FasL-dependent veto cells. In contrast, AKR.H-2b dendritic cells (DC) did not efficiently veto antiviral CTL responses, despite expressing sufficient MHC class I/viral peptide complexes for TcR recognition. AKR.H-2b DC also expressed FasL mRNA and cell surface protein, albeit at a lower level than AKR.H-2b T and B cells. These findings suggest a fail-safe escape mechanism by virus-infected cells for escape from CTL-mediated immunity.     

Lymphokine requirements for the development of specific cytotoxic T cells from single precursors

A high cloning efficiency, filler cell-free culture system was developed for the growth of single murine cytotoxic T lymphocyte precursors (CTLp) and their differentiation into cytotoxic T lymphocytes (CTL). The system used nonspecific stimulation with phorbol ester and calcium ionophore in the presence of recombinant lymphokines. The optimal lymphokine combination was interleukin 2 throughout, together with interferon-gamma during the first 6 days and interleukin 6 during the last 2 days of culture. Under these conditions half of all CD4-CD8+ T cells became CTL clones. The CTL were CD4-CD8+CD3+ TcR alpha/beta+ and were derived from CD4-CD8+Pgp-1- precursors.     

The activation of L3T4+ helper T cells assisting the generation of anti-tumor Lyt-2+ cytotoxic T lymphocytes: requirement of Ia-positive antigen-presenting cells for processing and presentation of tumor antigens

The present study investigates the mechanisms of the recognition of tumor antigens by L3T4+ helper T cells responsible for the generation of Lyt-2+ cytotoxic T lymphocytes (CTL) against a major histocompatibility complex (MHC) Class II (Ia) antigen-negative syngeneic X5563 plasmacytoma. Treatment of X5563-immunized spleen cells with anti-L3T4 antibody plus complement (C) diminished the generation of Lyt-2+ anti-X5563 CTL. Since the contribution of L3T4+ cells was completely replaced by the addition of exogenous lymphokines, it was demonstrated that L3T4+ cells functioned as helper T cells assisting the generation of anti-X5563 CTL responses. Elimination of Ia-positive accessory cells (AC) from X5563-immunized spleen cells resulted in the abrogation of CTL generation, whereas the addition of exogenous lymphokines to AC-depleted X5563 immunized spleen cells restored the CTL response. The addition of anti-self Ia antibody to the culture also eliminated CTL responses. These observations demonstrated the requirement of Ia-positive AC for and the involvement of self Ia antigens in the activation of helper T cells. Moreover, use of tumor cells pretreated with paraformaldehyde to cultures of X5563-immunized spleen cells or adding back of AC pretreated with chloroquine to cultures of AC-depleted immune spleen cells failed to generate CTL responses. Finally, the addition of exogenous lymphokines to the above cultures resulted in appreciable restoration of CTL responses. Taken collectively, these results indicate that L3T4+ helper T cells are activated with tumor antigens processed and presented by Ia-positive AC.     

Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4- CD8+ cytotoxic T lymphocytes in mice.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.     

Molecular complexity of Qa-2 antigens demonstrated by two-dimensional gel electrophoresis

Biosynthetically labelled Qa-2 antigens were isolated from mouse spleen cells by immunoprecipitation with anti-Qa-2 antisera. When newly synthesized Qa-2 molecules from several different inbred strains were analysed by two-dimensional (2D) gel electrophoresis; four different phenotypes were observed that differed in the number of polypeptides present. The ability to distinguish Qa-2+ phenotypes was used to map the recombination points in two congenic strains, B6.Tlaa and A.Tlab. No alternative Qa-2-like polypeptides were detected in B6.K1 (Qa-2-) cells using a polyspecific rabbit antiserum against mouse class I antigens, but a new molecule was detected in BALB/cBy (Qa-2-) cells. Pulse-chase and surface-labelling experiments showed that some, but not all, of the newly synthesized Qa-2 precursor forms are processed to mature cell surface molecules.     

Qa-1/Tla region alloantigen-specific CTL with alpha beta receptor

Recent studies involving T cells that express gamma delta T-cell receptor (gamma delta TcR) have raised the possibility that Qa-1/Tla region class I major histocompatibility complex (MHC)-like molecules are antigen-presenting molecules for gamma delta TcR. In this report, cytotoxic T lymphocyte (CTL) clones specific for a Qa-1/Tla region gene product were isolated from a bulk B10. QBR (Kb, Ib, Dq Qa-1/Tlab) anti-B10.MBR (Kb, Ik, Dq, Qa/Tlaa) CTL line. These CTL lysed blasts from all Qa-1a strains regardless of the H-2 haplotype, indicating that the recognition of the Qa-1 antigen by these CTL is not restricted by other class I molecules. In bulk populations, CTL activity of this specificity was found only in the CD8+CD4- subpopulation. Accordingly, all established CTL clones were phenotyped as Thy-1+, CD8+CD4-. Furthermore, these clones were shown to express alpha beta TcR rather than gamma delta TcR. Thus, the results indicate that Qa-1 antigen can be recognized by alpha beta TcR T cells in a manner similar to recognition of classical class I molecules.     

Acquired immunity to an intracellular pathogen:immunologic recognition of L. monocytogenes-infected cells

Summary: Listeria monocytogenes (L. monocytogenes) is a pathogenic bacterium, and subclinical infection in mice is utilized as a prototypic model to investigate the development and expression of acquired resistance to facultative intracellular organisms. A key virulence factor of L. monocytogenes is the hemolysin listeriolysin O (LLO), and BALB/c mice immunized with hemolysin-secreting strains of L. monocytogenes develop specific acquired resistance, while mice immunized with hemolysin-negative strains or non-viable preparations of L. monocytogenes do not develop a protective immune response. Adoptive transfer studies show that L. monocytogenes-Immune CD8+ T cells mediate acquired resistance. The L. monocytogenes-immune CD8+ population is cytotoxic, and target cells infected with hemolysin-secreting strains of L. monocytogenes are lysed, while target cells infected with hemolysin-negative strains or non-viable preparations of L. monocytogenes are not lysed. MHC dass la and Ib molecules present I. monocytogenes-derived peptides, and we have identified Qa-1^b, a T-region-encoded MHC class Ib molecule, as a restriction element for L, monocytogenes-specific CD8+ CTL. MHC class Ib-restricted CTL are stimulated following infection with L. monocytogenes and are a significant component of the total MHC class I-restricted CTL population. These findings support the observation that cytoplasmic L. monocytogenes-derived antigens are endogenously processed and presented in association with MHC class Ia and Ib molecules to CD8+ effector cells, and that both populations of effector cells contribute to the immune response to this intracellular pathogen.     

Parainfluenza type 1 virus-infected cells are killed by both CD8+ and CD4+ cytotoxic T cell precursors.

The memory cytotoxic T lymphocyte (CTL) response to human parainfluenza type 1 virus (hPIV-1), a prominent cause of respiratory infection in young children, has been analysed for a panel of healthy adults. The CTL response to the parainfluenza viruses has not been investigated previously. Precursor CTL (CTLp) with activity against hPIV-1-infected Epstein-Barr virus (EBV)-transformed B lymphoblastoid target cells were found at a relatively high precursor frequency (approximately 1/2500-1/4700 CD8+ and CD4+ subsets respectively) in peripheral blood. Both CD4+ and CD8+ CTLp were detected by the analysis of individual microcultures set up under limiting dilution conditions from freshly isolated blood, the phenotype of the responder cell from individual wells being determined by flow cytometry. Further characterization of the CTL response demonstrated MHC restriction by the HLA-A2 glycoprotein in 3/4 HLA-A2+ donors. The presence of effective, hPIV-1-directed T cell memory may explain, in part, the protection observed in the adult population.     

Regulation of activated CD4+ T cells by NK cells via the Qa-1-NKG2A inhibitory pathway

The ability of natural-killer cells to regulate adaptive immunity is not well understood. Here we define an interaction between the class Ib major histocompatibility complex (MHC) molecule Qa-1-Qdm on activated T cells responsible for adaptive immunity and CD94-NKG2A inhibitory receptors expressed by natural-killer cells by using Qa-1-deficient and Qa-1 knockin mice containing a point mutation that selectively abolishes Qa-1-Qdm binding to CD94-NKG2A receptors. The Qa-1-NKG2A interaction protected activated CD4+ T cells from lysis by a subset of NKG2A+ NK cells and was essential for T cell expansion and development of immunologic memory. Antibody-dependent blockade of this Qa-1-NKG2A interaction resulted in potent NK-dependent elimination of activated autoreactive T cells and amelioration of experimental autoimmune encephalomyelitis. These findings extend the functional reach of the NK system to include regulation of adaptive T cell responses and suggest a new clinical strategy for elimination of antigen-activated T cells in the context of autoimmune disease and transplantation.     

Evidence of a role for TNF-alpha in cytolysis by CD4+, class II MHC-restricted cytotoxic T cells

Variants of the tumour necrosis factor (TNF)-sensitive cell lines BALB/c 3T3 and WEHI-164/13 were generated by selection in high concentrations of recombinant TNF-alpha. The selected cells were highly resistant to cytolysis by both TNF-alpha and TNF-beta (lymphotoxin); however, there was no significant difference in sensitivity to killing by class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). Resistance to TNF-mediated lysis was, however, accompanied by increased resistance to cytolysis by CD4+ class II-restricted CTL. Furthermore, cytolysis of WEHI-164/13 cells by CD4+ was completely inhibited by anti-TNF antiserum. These data strongly suggest a role for TNF in killing by CD4+ class II MHC-restricted CTL but not in class I MHC-restricted killing.     

Existence of mature human CD4+ T cells with genuine class I restriction.

A human T cell receptor (TcR) alpha/beta CD4+CD8-T cell clone (R416) is reactive with the minor histocompatibility antigen H-Y in the context of major histocompatibility complex (MHC) class I and not class II molecules. Therewith clone R416 violates the so-called specificity association of mature TcR alpha/beta+ T cells. R416 displays H-Y-specific, HLA-A2-restricted proliferation as well as cytotoxicity in vitro. Its fine specificity is identical to that of a classical H-Y-reactive CD4-CD8+ MHC class I-restricted CTL clone, showing that CTL expressing either CD4 or CD8 can display identical antigenic specificities. Exploiting the MHC class I restriction of this CD4+ T cells clone, it was found that interaction of CD4 with non-TcR-bound MHC class II molecules does not contribute to antigen specific activation of these CD4+ T cells. This coreceptor-mismatched T cell clone was not generated in vitro but obtained by expansion of CD8-depleted peripheral blood mononuclear cells of a female who had been immunized against H-Y. The existence of such MHC class I-restricted mature TcR alpha/beta+ T cells expressing CD4 and not CD8 is relevant because it indicates that the generally accepted model for thymic selection, in which the TcR specificity alone determines CD4/CD8 expression of mature thymocytes, may not be absolute.     

The influence of allo-class II MHC-specific Th2 cells on the generation of CD4 and CD8 cytotoxic T cells to associated class I and class II MHC alloantigen

There is considerable interest in whether CD4 T cell function can affect the outcome of allogeneic transplants. In mice tolerant to an isolated class II MHC disparity, the normal Th1 activity in vitro associated with graft rejection is switched to Th2 in tolerant animals. Because clinical transplants involve multiple class I and II MHC disparities we tested how the switch to Th2 activity of tolerant mice would affect the generation of CD4 and CD8 cytotoxic T cells (CTL) against MHC alloantigens to which the mice were not tolerant. A.TH mice (K^kI^sD^d) were rendered neonatally tolerant of A.TL (K^kI^kD^d) and the generation of CD4 or CD8 CTL measured in a mixed lymphocyte reaction (MLR) against (A.TLxB6)F₁ stimulators. Normal mice generated CD4 CTL against both A.TL and B6 (K^bI^bD^b), but tolerant mice were unable to generate cytotoxicity against either A.TL or B6. However, tolerant cells were able to generate CD8 CTL against B6. IL-4 inhibited the generation of CD4, but not CD8, CTL by normal cells and anti-IL-4 antibody was shown to increase the generation of CD4 CTL against B6 in F₁ stimulated cultures. Overall the results showed that a Th2 response could inhibit the generation of CD4 CTL against concomitant alloantigen in a process at least partially involving IL-4, but that, conversely, tolerant Th2 cells could help in the generation of CD8 CTL. The results suggest that with whole MHC disparities a simple change of CD4 T cells to Th2 would not be enough to procure graft acceptance.     

Progressive loss of IL-2-expandable HIV-1-specific cytotoxic T lymphocytes during asymptomatic HIV infection

In HIV-1 infection, circulating HIV-1-specific cytotoxic T lymphocytes (CTL) exist in different states of activation, including activated cytotoxic cells and memory cells. We report that a subpopulation of HIV-1-specific CTL is capable of clonal expansion upon culture with IL-2 without exogenous antigen. The IL-2-expandable HIV-1-specific CTL precursor frequency was reduced in patients with advancing infection, although HIV-1-specific memory CTL could still be detected by stimulation in vitro with allele-specific HIV-1 peptide. Longitudinal analysis during advancing infection showed a progressive decline in the IL-2-expandable HIV-1-specific CTL precursor (CTLp) frequency without a decline in Epstein-Barr virus (EBV)-specific or allo-specific CTLp frequencies. To address mechanisms that may contribute to the decline in the IL-2-expandable HIV-specific CTL response, the requirements for in vitro generation of HIV-1-specific and EBV-specific effector CTL were examined. In the absence of exogenous IL-2 in limiting dilution, generation of EBV-specific CD8⁺ effector CTL was dependent upon help from CD4⁺ cells. CD4⁺ help for EBV-specific CD8⁺ CTL was observed in asymptomatic HIV infection but not in advanced infection. In the presence of exogenous IL-2, CD4⁺ cells could also provide help for the optimal generation of HIV-1 peptide-specific CD8⁺ CTL, because in vitro depletion of CD4⁺ cells prior to culture using stimulation with an MHC class I-restricted HIV-1 peptide reduced the peptide-specific CD8⁺ CTL response. We conclude that there is a decline in the IL-2-expandable HIV-1-specific CTL response during advancing infection. There are a number of possible mechanisms for this decline, including a reduction in CD4⁺ T cell help for in vivo antigen-activated CD8⁺ T cells.     

Reduced lysis by CD8+ cytotoxic T cells in mixed lymphocyte reactions induced via CD4+ T cells exposed to chemically modified antigen presenting cells.

The resistance by T lymphocytes to activation by antigen (anergy) is well documented for CD4+ T-helper (Th) cells, although less is known about CD8+ cytotoxic T lymphocytes (CTL). One widely used method of inducing anergy of CD4+Th is presentation of antigen by ECDI (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide)-fixed antigen-presenting cells (APCs). We report here that in murine mixed lymphocyte reactions (MLRs), a marked reduction in detected cytotoxicity (which is mediated predominantly by CD8+ CTL) occurs on day 7 if the bulk cultures are restimulated 2 days previously with ECDI-fixed allogeneic splenocytes. No differences were seen between untreated cultures on days 5 and 7, or on day 7 of cultures to which were added unfixed allogeneic splenocytes, fixed or unfixed syngeneic splenocytes, or 'third-party' allogeneic splenocytes, 2 days previously. The effect is not mediated directly on CD8+ cells, since MLRs depleted of CD4+ cells immediately prior to exposure to fixed allogeneic splenocytes fail to show reduced lysis. On the other hand, reduced lysis did occur if CD4+ cells, purified from the MLRs on day 4, were exposed to ECDI-fixed allogeneic splenocytes and then returned to MLRs previously depleted of CD4+ cells. Moreover the effect is overcome using exogenous interleukin-2 (IL-2). We propose that CD4+ cells, restimulated by a regimen shown previously to induce their anergy, can cause a reduction in CD(8+)-mediated cytotoxicity in MLRs.     

Suppression of inducible CD4 regulatory cells by MHC class I-restricted human tumor epitope specific TCR engineered multifunctional CD4 T cells

Regulatory T cells (Treg) can interfere with the generation and function of anti-tumor immune effectors. Accordingly, ways that could block Treg function would be useful in cancer immunotherapy. We have previously shown that incorporation of CD4+CD25-ve T cells in an in vitro cytolytic T lymphocyte (CTL) generation assay leads to generation of induced regulatory T cells (iTregs), and that these iTreg block the generation of productive CTL response (Chattopadhyay et al., 2006). We here show that human CD4 T cells engineered to express MHC class I-restricted human melanoma associated epitope, MART-1_27–35, specific T cell receptor (TCR), that can simultaneously exhibit helper as well as cytolytic effector functions (Chhabra et al., 2008, Ray et al., 2010), can interfere with the generation of inducible Treg, block iTreg-mediated suppression, and allow the activation and expansion of MART-1_27–35 specific CTL responses, in vitro. We also show that mitigation of Treg generation by TCR engineered CD4 T cells is not mediated by a soluble factor and may involve “licensing/conditioning” of the dendritic cells (DC). Our data offer novel insights on the biology of MHC class I restricted TCReng CD4 T cells and have translational implications.     

Foreign antigenic peptides delivered to the tumor as targets of cytotoxic T cells.

Cytotoxic T cells (CTL) are readily activated by immunogenic peptides and they exert potent anti-tumor activity if the same peptides are displayed on class I major histocompatibility complex (MHC) of the tumor cells. A handful of tumor-associated antigens have been identified and many of them are weak antigens. As an alternative strategy, strongly antigenic foreign peptides are delivered to the tumor, marking them for CTL recognition. To establish the principle of this new strategy, in vitro and in vivo tumor destruction was tested with BALB/c CTL to L(d)-associated beta-galactosidase (beta-gal) peptide p876. In vitro, anti-p876 CTL destroyed tumor cells in a single-cell suspension or in 3-D tumor boluses when exogenous p876 was added. Exogenous IL-2 was required to sustain CTL activity for complete destruction of tumor boluses. In vivo, BALB/c mice were immunized with p876 and a CD4 activating Pan DR reactive epitope (PADRE). PADRE, which binds to several different MHC class II antigen and activates CD4 T cells, induced delayed-type hypersensitivity and stimulated T cell proliferation. Immunized mice were injected with tumor cells loaded with p876 and mixed with PADRE. Starting from the day after tumor injection, mice received five rounds of peptide injection at the tumor sites and all tumors were rejected. Injection with saline had no effect. Injection with PADRE had minor anti-tumor activity. Immunization and treatment with p876 alone was not protective. Therefore, by delivering CD4 and CD8 reactive foreign peptides to the tumor, peptide-specific T cells rejected the tumors as demonstrated by the in vitro and in vivo tests.     

Specific epitope-induced conversion of CD8+ memory cells into effector cytotoxic T lymphocytes in vitro: presentation of peptide antigen by CD8+ T cells.

The requirements for the conversion of CD8+ memory T cells into effector class I major histocompatibility complex (MHC) Kd-restricted cytotoxic T (Tc) cells in vitro have been studied. Purified CD8+ splenocytes from influenza A/WSN-primed BALB/c (H-2d) mice stimulated with a synthetic nucleoprotein peptide 147-158 R156- (NPP) alone generated Tc cells specific for influenza virus-infected target cells. No additional requirements for accessory cells or their lymphokine products were necessary indicating that peptide antigen (Ag) in association with Kd was presented on CD8+ T cells. The evidence for presentation of NPP by CD8+ T cells was supported by the use of CD8+ memory T cells from semiallogeneic bone marrow radiation chimeras of P1----F1 type (H-2b----[H-2d x H-2b]F1). Memory CD8+ splenocytes from A/WSN-immune chimeras did not develop into secondary effector Tc cells as a result of a 4-day culture with NPP alone, however, were able to do so if NPP was presented by Kd-bearing Ag-presenting cells. In addition, these results exclude the possibility of direct recognition of free NPP molecules by the specific T cell receptor of CD8+ memory T cells. CD8+ memory splenocytes (H-2b) from chimeras were also able to develop into functionally active Tc cells as a result of presentation of Db-restricted synthetic peptide (NP 366-374) with a sequence derived from influenza virus nucleoprotein with high affinity for Db MHC class I molecules. Blockade of endogenously produced interleukin 2 (IL-2) activity by anti-IL-2 or anti-IL-2 receptor monoclonal antibody in the culture of CD8+ memory T cells during a 4-day NPP stimulation completely abolished Tc cell generation, indicating that the utilization of this lymphokine is absolutely required for the secondary Tc cell development. These findings demonstrate that CD8+ memory T cells per se are able to recognize the restimulating epitope as a result of its presentation by CD8+ T cells and develop into cytolytically active and highly specific Tc cells with no requirements for other cellular helper components or their lymphokine products.     

Search for class II major histocompatibility complex molecular involvement in the response of Lyt-2+ cytotoxic T lymphocyte precursors to alloantigen

A possible requirement for class II major histocompatibility complex (Ia) molecules in the initial activation of cytotoxic T lymphocyte precursors (CTLp) for allocytotoxic responses was investigated. To avoid possible interaction with other alloreactive cell types, a highly purified population of Lyt-2+ splenocytes was used as a source of CTLp. In the light of preliminary results indicating that Lyt-2+ CTLp, even in the presence of interleukin 2 (IL2), could best be triggered into mature CTL in vitro by cells known to be Ia+, we examined whether an interaction of CTLp with Ia antigens (either on syngeneic accessory cells or on allogeneic stimulators) played a role in the development of allocytotoxicity. Results from experiments done with C57BL/6 Lyt-2+ splenocytes co-cultured with P815 stimulator cells and IL 2 showed that the early activation of CTLp was independent of Ia+ syngeneic accessory cells: (a) flow microfluorometry analysis of the responder population at the beginning or after 1 or 3 days of co-culture did not reveal the presence of Ia+ cells; (b) procedures for removal of residual Ia+ cells or of dendritic cells from the responder population before co-culture did not affect the development of cytotoxicity; (c) co-culture with monoclonal antibodies against syngeneic Iab antigens did not inhibit the CTLp activation. By comparing an Ia+ P815 tumor line with its Ia- clone as allogeneic stimulator cells, it was found that the CTLp activation was also independent of Ia alloantigen on the stimulator cells. The response against both the Ia+ and the Ia- stimulator cell types was not inhibited by monoclonal anti-L3T4 present in the co-culture, indicating that these responses were not affected by residual L3T4 helper cells.     

T cell- and perforin-dependent depletion of B cells in vivo by staphylococcal enterotoxin A.

Bacterial superantigens bind to major histocompatibility complex (MHC) class II and subsequently activate both CD4+ and CD8+ T lymphocytes expressing certain T-cell receptor (TCR)-Vbeta chains. In response to superantigen exposure these subsets proliferate, produce large amounts of proinflammatory cytokines and in addition CD8+ cytotoxic T lymphocytes (CTL) are induced. Previous studies in vitro have shown that these CTL effectively lyse MHC class II-expressing cells presenting the proper superantigen. However, it is unknown whether superantigens induce a similar response towards MHC class II+ antigen-presenting cells in vivo. In this study we demonstrate that administration of repeated injections of the superantigen staphylococcal enterotoxin A (SEA) to TCR-Vbeta3 transgenic mice results in a loss of MHC class II-expressing cells in the spleen. Analysis of different MHC class II+ subsets revealed a selective depletion of CD19+ B cells, while F4/80+ macrophages increased in number. Depletion of T cells with anti-CD4 or anti-CD8 monoclonal antibody indicated that CD8+ T cells were crucial for SEA-induced cytotoxicity in vivo. Repeated injections of SEA to perforin-deficient mice resulted in significantly less B-cell depletion compared with control mice. This suggests that superantigen-activated CD8+ T cells lyse MHC class II+ antigen-presenting cells in a perforin-dependent manner in vivo. It is suggested that this represents a novel bacterial immune escape mechanism, which may particularly impair local humoral immune responses.