Role of dendritic cells in the regulation of class I restricted cytotoxic T lymphocyte responses

Two class I MHC mutant mouse strains, bm14 and bm13, differ from the strain of origin B6 in one and three amino acids in the alpha 1 and alpha 2 domains of the H-2Db molecule, respectively. These alterations result in specific failure to generate a CTL (Tc) response to the male-specific Ag H-Y. Immunization and/or restimulation in vitro with syngeneic male dendritic cells (DC), expressing very high levels of class I MHC molecules, restored the H-Y-specific Tc response of bm14 but not of bm13 mice. Serologically Db determinants were lost in normal spleen cells of both mutants, because FACS analysis showed a decreased binding of Db domain-specific mAb. Although bm13 DC show a higher fluorescence than bm13 normal spleen cells it is still strongly reduced (30 to 50%) in comparison with B6 DC. Surprisingly, bm14 DC show an equally very strong binding compared with B6 DC with these mAb. The quantitative expression of class I molecules on APC thus appears to be a major determinant in the regulation of Tc responses. In addition, immunization with DC markedly influenced the target cell specificity of the ensuing Tc response. The combined data clearly demonstrate that besides the highly efficient class II-restricted presentation of Ag to Th, shown previously, DC are also superior in the presentation of Ag in the context of class I molecules to Tc. bm14 DC are capable of directly activating H-Y-specific Lyt-2+ Tc memory cells without the need for L3T4+ Th. These biologic effects of DC can at least in part be explained by their very high class I MHC expression. Moreover, these results reiterate that class I MHC Db mutants and different APC can be used to study the contribution of specific class I domains to Tc recognition and restriction specificity.     

Tumor-specific cytotoxic T lymphocyte responses against chondrosarcoma with HLA haplotype loss restricted by the remaining HLA class I allele

Although loss of HLA expression by malignant cells has also been demonstrated, it has not been clarified how the loss of HLA expression observed in vitro actually results in immune escape. We demonstrated two major findings: (i) a part of chromosome 6 coding for HLA haplotypes was deleted from the genome of chondrosarcoma cell line, OUMS-27; furthermore, immunohistostaining for HLA-A11 showed that the original chondrosarcoma tissue lost the expression of HLA-A11, implicating that HLA haplotype loss was already present in the original tumor tissue and (2) HLA class I-restricted and autologous tumor-specific cytotoxic T cells (CTL) were generated from peripheral blood lymphocytes of the patient with chondrosarcoma, from whom OUMS-27 originated. This CTL line was maintained by weekly stimulation with OUMS-27, and lysed OUMS-27 in an HLA-A24 dependent manner but did not either K562 or autologous (EBV)-transformed B cells. These observations indicated that OUMS-27 and its original tumor are still immunogenic and can present antigen peptides with the remaining HLA-A24, even if HLA expression is partially lost. Tumor specific immunotherapy can be applied to the treatment of malignancies, even if HLA expression is partially lost.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.     

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.     

MHC class I-restricted cytotoxic lymphocyte responses induced by enterotoxin-based mucosal adjuvants

The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257-264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257-264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257-264. Both 51Cr release assays and IFN-gamma enzyme-linked immunospot assays indicated that IFN-gamma-/- and IL-12 p40-/- gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-gamma and probably by a mechanism unrelated to cross-priming.     

Suppression of Ia-unrestricted primary anti-Qa-1 cytotoxic T lymphocyte responses by class II major histocompatibility complex-restricted cellular interactions

A model has been established for investigating the cellular interactions for the generation and regulation of primary cytotoxic T lymphocyte (CTL) responses to Qa-1 alloantigens. Although NZB anti-BALB/c one-way mixed leukocyte cultures (MLC) generate anti-Qa-1b CTL, anti-Qa-1 CTL responses are not generated during BALB/c anti-NZB one-way MLC or during two-way MLC with NZB and BALB/c spleen cells. However, depletion of L3T4+ cells from the spleens of BALB/c mice before two-way MLC with NZB spleen cells resulted in anti-Qa-1b CTL responses. Likewise, the addition of anti-L3T4 monoclonal antibody (mAb) or anti-I-Ad mAb to two-way MLC with NZB and BALB/c spleen cells resulted in the generation of anti-Qa-1b CTL. Conversely, anti-Lyt-2 mAb inhibited the generation of anti-Qa-1 CTL. These data indicate that class II major histocompatibility complex-restricted cellular interactions are capable of suppressing the generation of Ia-unrestricted anti-Qa-1 CTL responses by Lyt-2+ responder cells. This model provides a novel opportunity to both characterize the cellular interactions responsible for regulating primary CTL responses to the Qa/Tla-encoded class I molecule Qa-1, and determine the contribution of this L3T4+ Ts-dependent defect in NZB mice to the pathogenesis of autoimmunity.     

Major histocompatibility complex class I-restricted cytotoxic T lymphocyte responses during primary simian immunodeficiency virus infection in Burmese rhesus macaques

Major histocompatibility complex class I (MHC-I)-restricted CD8(+) cytotoxic T lymphocyte (CTL) responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. In particular, Gag-specific CTL responses have been shown to exert strong suppressive pressure on HIV/SIV replication. Additionally, association of Vif-specific CTL frequencies with in vitro anti-SIV efficacy has been suggested recently. Host MHC-I genotypes could affect the immunodominance patterns of these potent CTL responses. Here, Gag- and Vif-specific CTL responses during primary SIVmac239 infection were examined in three groups of Burmese rhesus macaques, each group having a different MHC-I haplotype. The first group of four macaques, which possessed the MHC-I haplotype 90-010-Ie, did not show Gag- or Vif-specific CTL responses. However, Nef-specific CTL responses were elicited, suggesting that primary SIV infection does not induce predominant CTL responses specific for Gag/Vif epitopes restricted by 90-010-Ie-derived MHC-I molecules. In contrast, Gag- and Vif-specific CTL responses were induced in the second group of two 89-075-Iw-positive animals and the third group of two 91-010-Is-positive animals. Considering the potential of prophylactic vaccination to affect CTL immunodominance post-viral exposure, these groups of macaques would be useful for evaluation of vaccine antigen-specific CTL efficacy against SIV infection.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Role of lymphokine-secreting CD8+ T cells in cytotoxic T lymphocyte responses against vaccinia virus

The present study has examined the relative role of CD4+ and CD8+ Th cells in the generation and reactivation of antivaccinia virus memory CTL responses. We show that mice primed in vivo to vaccinia virus generate in vitro antivaccinia virus memory CTL responses through both CD4+ and CD8+ Th cell pathways, with the CD4+ Th pathway being the more prominent of the two. In addition, we show that vaccinia virus-specific CD8+ Th cell function is mediated through production of lymphokines, including IL-2, and that the CD8+ Th cell component in the CTL response is labile, decreasing progressively with increasing time after in vivo priming. Thus, this study demonstrates the existence of two phenotypically distinct Th cell pathways in the generation of antivirus CTL responses.     

L3T4+ cytotoxic T lymphocytes specific for class I H-2 antigens are activated in primary mixed lymphocyte reactions

Thy-1+, L3T4+, Ly-2- cytotoxic lymphocytes (CTL) are generated in a primary anti-H-2d mixed lymphocyte reaction, by using responders depleted of Ly-2+ cells. In addition to expressing the L3T4 marker, as detected by anti-L3T4 antibody and complement-mediated elimination, the L3T4+ CTL are inhibited by L3T4 antibody. The observation of these L3T4+ CTL in cells recovered from primary mixed lymphocyte reactions confirms the previous reports. However it is demonstrated for the first time that a subpopulation of these are class I-specific by their specific inhibition with an antiserum to class I antigens. The class I specificity of the CTL was further shown by their ability to kill class II antigen negative P815 tumor cells. The lysis of this target cell by L3T4+ CTL was also specifically blocked by the class I antiserum. The data is consistent with the presence also of a class II-specific population of L3T4+ cytotoxic cells. The fact that a level of L3T4+ cell-mediated cytotoxic activity comparable to Ly-2+ cytolytic activity is generated in a primary mixed lymphocyte response, even though the precursor frequency of L3T4+ killer cells is 10 times lower than for Ly-2+ killers, is suggestive of their physiologic significance. It was also shown that the activation of these cells is not dependent on the presence of xenogeneic serum components or exogenous helper or mitogenic factors in the culture medium. The findings provide further evidence against both the phenotype-function and phenotype-major histocompatibility complex antigen specificity models of T cell diversity.     

Equilibrium binding of cytotoxic T lymphocytes to class I antigen

Cloned cytotoxic T lymphocytes specifically bind to purified alloantigen that has been immobilized on a surface. When the time course was examined, it was found that binding reached a plateau level within about 1 h at 37 degrees C, at which time about 30% of the CTL were tightly adhered to the surface. Analysis of the properties of binding demonstrated that this does not simply result because only a fraction of the cells in the clonal population are capable of binding. Instead, the binding is shown to result from an equilibrium involving tightly bound and unbound (or weakly bound) cells. Thus, the cells cycle between a tightly bound and unbound state, despite continuous contact with the Ag-bearing surface. The results suggest that dissociation of the bound cells may be an actively signaled event. A model that could account for these results based on activated CD8 binding is discussed.     

Cytotoxic T lymphocyte recognition of secreted HLA class I molecules

The cytolytic responses of DBA/2 mice against syngeneic transfected P815 mastocytoma cells expressing either membrane-associated (HLA-Cw3) or -secreted hybrid (HLA-Cw3 x H-2 Q10b) molecules were compared. In spite of the absence of serologically detectable hybrid molecules on their plasma membrane, cells secreting these molecules elicited a CTL response similar to that of cells expressing the membrane associated HLA-Cw3 molecules, in terms of both MHC-restriction and peptide specificity. Together with the observation that syngeneic mice were capable of rejecting the injected secreting cells, these results imply that secreted HLA class I molecules can function as minor histocompatibility Ag and suggest that processing of both the membrane-bound and the -secreted forms of a protein may follow common or overlapping pathways.     

Interaction between T cells and non-T cells in suppression of cytotoxic lymphocyte responses.

Generation of cytotoxic T lymphocytes (CTL) in mixed leukocyte cultures was suppressed by a factor elaborated by alloantigen-activated T cells. This suppressor factor, CTL-TsF, in contrast to a factor that suppresses proliferative responses in mixed leukocyte reactions (MLR-TsF), was effective only when added during the first 24 hr of a 6-day-culture period. Moreover, removal of CTL-TsF 24 hr after culture initiation failed to restore CTL responses. CTL activity could be rescued from suppressed cultures, however, by addition of 2-mercaptoethanol on days 3 or 4. Similarly, transfer of nonadherent cells at 3 or 4 days from cultures treated with CTL-TsF to cultures of adherent cells initiated in control factor restored CTL responses. Mixing experiments with cells pulsed with CTL-TsF for 4 hr at culture initiation identified a target of CTL-TsF as a Thy-1 negative cell that was adherent to plastic and to Sephadex G-10. Suppression was not due to interference with physiologic accessory cell function, but more likely was accomplished via a negative signal from CTL-TsF-pulsed cells. The results thus suggest that CTL-TsF acts early, but reversibly, in the CTL differentiative process via a second suppressor effector cell, possibly a macrophage.     

Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T lymphocyte responses.

The ability of a saponin adjuvant, QS-21, to induce OVA-specific, class I MHC Ag-restricted CTL was investigated using different forms of soluble OVA and OVA adsorbed onto alum as immunogens. C57BL/6 mice were immunized with soluble native or denatured OVA in formulations that contained increasing quantities of QS-21, and CTL responses were measured using EL4 and E.G7-OVA cells as targets and splenic mononuclear cells as effectors. Ag-specific CTL responses were produced but only if the QS-21 adjuvant was used. Similar responses were induced using alum-adsorbed OVA when mixed with the QS-21 adjuvant but not when used alone. The CTL were specific for an epitope present on the OVA258-276 synthetic peptide, which contains the dominant CTL epitope recognized by C57BL/6 mice. The CD8+ subpopulation of lymphocytes in immune mice was not increased in spleens but increased significantly in vitro after culture with soluble OVA. The CTL activity of splenic mononuclear cell preparations was totally destroyed by treatment with mAb specific to the CD8 Ag plus complement. The ability of the QS-21 adjuvant to induce class I MHC Ag-restricted CTL after immunization with soluble proteins is a characteristic unique to saponin adjuvants.     

Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline.

Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T: residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6: residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment. However, helix association is accompanied by loss of overall helical content. Thermal-dependence studies show that the unfolding of self-associated alpha-helices is significantly more pronounced than the unfolding of isolated alpha-helices. Circular dichroism data, together with tryptic limited proteolysis, suggest the presence of a charged amino acid within the hydrophobic core. This study should provide a basis for engineering more effective immunogenic peptides against the measles virus by increasing the stability of the TT-NP6 peptide.     

Functional clonal deletion of class I-specific cytotoxic T lymphocytes by veto cells that express antigen

Intravenous injection of class I incompatible spleen cells into mice results in a drastic reduction of the recipient's cytotoxic T lymphocyte (CTL) response against the injected, but not against third party, class I antigens when measured in bulk cultures initiated 5 to 6 days after the injection. This specific suppressive effect is partly due to T cells but can also be seen when high numbers of anti-Thy-1 and complement-treated spleen cells of nude mice are injected. Such cells suppressing CTL responses against self histocompatibility antigens are called "veto cells." The precursor frequency of CTL specific for the injected class I antigen is found to be reduced greater than 200-fold at days 5 to 6 after the injection, whereas the frequencies of CTL specific for third party class I antigens are not significantly changed. These results indicate that there is a functional clonal deletion of the CTL recognizing class I incompatible veto cells in vivo. The role of such a veto phenomenon in the induction and maintenance of self tolerance and allograft tolerance is discussed.     

Regulation of interleukin 2 receptor expression on murine cytotoxic T lymphocyte clones

We have previously reported that influenza virus-specific cytotoxic T lymphocyte (CTL) clones require antigen and exogenous growth factors for continued proliferation in culture. In this report we show that after stimulation with specific antigen, cloned CTL are capable of limited proliferation in response to interleukin 2 (IL 2) alone but with time these large blast-like cells revert to smaller, quiescent cells that are no longer responsive to IL 2. The IL 2-unresponsive CTL can not be driven to proliferate by supra-optimal concentrations of IL 2, and unresponsiveness correlates with decreased ability to absorb IL 2 from conditioned medium at 0 degrees C, suggesting that unresponsiveness is due to diminished IL 2 receptors. Stimulation of the unresponsive CTL with antigen leads to re-expression of the IL 2 receptor. Decreased absorbing capacity of the unresponsive cells could not be accounted for by their smaller surface area, and the IL 2-unresponsive cells seemed not to down-regulate all their immune functions, as they remained cytotoxic. These results provide a basis for the role of specific antigen in maintaining CTL clones in vitro. Furthermore, these results suggest that antigen-dependent CTL lines can be regulated and that antigen and IL 2 both play a role in their regulation.