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PubMed1985

Functional distinctions between the LFA-1, LFA-2, and LFA-3 membrane proteins on human CTL are revealed with trypsin-pretreated target cells.

We asked whether we could distinguish the roles of the human lymphocyte membrane proteins LFA-1, LFA-2, and LFA-3 in the function of CTL-mediated killing. Little is known about the functions of these molecularly distinct proteins beyond the facts that i) binding of a monoclonal antibody (MAb) to any one of them is sufficient to inhibit killing, ii) that in each case inhibition involves prevention of CTL-target cell conjugate formation, and iii) that MAb to LFA-1 and LFA-2 inhibit best when bound to the CTL, whereas anti-LFA-3 inhibits only when bound to the target cell. This latter is despite the fact that (in our test system) LFA-1 and LFA-3 are expressed both on the CTL and on the target. When the target cells were pretreated with trypsin, the sensitivity of CTL-mediated killing was affected in a different way for each site. Inhibition of anti-LFA-1 was increased by approximately 20-fold. Inhibition by anti-LFA-2 was unaffected. Inhibition by anti-LFA-3 was abolished. Trypsin did not remove the specific antigens recognized by the various CTL, HLA-A,B,C or HLA-DR. Nor did it remove LFA-1 from the target cell. It did, however, selectively remove LFA-3 from the target cell. These results indicate, for the first time, that LFA-1 and LFA-2 have functionally distinct roles. They suggest that an unidentified trypsin-sensitive target cell molecule, operationally designated the "trypsin-sensitive counter blocker" (TSCB), plays an important role in the function of LFA-1, possibly by providing a target cell binding site for LFA-1 on the CTL. The hypothesis that this TSCB is identical to LFA-3 (and the related possibility that LFA-1 and LFA-3 are mutual ligands) is not favored by our data, but is not excluded. Finally, the data indicate that the mechanisms by which MAb inhibit killing differ at the LFA-1 and LFA-3 sites. They are consistent with LFA-1 providing adhesion strengthening by binding to another site (the TSCB?) and with LFA-3 delivering an inhibitory signal when provoked with MAb.

Antibodies, Monoclonal↗

PubMed1985

Blocking of CTL-mediated killing by monoclonal antibodies to LFA-1 and Lyt-2, 3. II. Evidence that trypsin pretreatment of target cells removes a non-H-2 molecule important in killing.

We sought additional evidence for an inverse relationship between functional CTL-target cell affinity on the one hand, and susceptibility of the CTL-mediated killing to inhibition by alpha LFA-1 and alpha Lyt-2,3 monoclonal antibodies on the other hand. Previously, we experimentally reduced affinity by pretreating the target cells with papain. This removed most of the class I H-2 antigens, had little effect on the ability of allospecific CTL to recognize and kill these targets, but dramatically reduced the initial strength of CTL-target cell adhesion, and increased by more than 10-fold the susceptibility of the killing to inhibition by alpha Lyt-2,3 and alpha LFA-1 MAb. In the present report, we find that pretreating the target cells with trypsin, like papain, does not significantly change the susceptibility of the target cells to killing by allospecific CTL in a 2-hr assay, and increases by about 10-fold susceptibility of the killing to inhibition by alpha LFA-1. Unlike papain, however, trypsin does not consistently increase blocking by alpha Lyt-2,3, does not remove class I H-2 antigens from the target cell, and does not substantially reduce the strength of initial CTL-target adhesion formation (estimated by post dispersion lysis after a 5-min conjugate-forming incubation). These results show a functional difference between LFA-1 and Lyt-2,3. Both papain and trypsin produced similar 10-fold increases in susceptibility to blocking by alpha LFA-1. In contrast, susceptibility to inhibition by alpha Lyt-2,3 was increased nearly 100-fold by papain, but was not consistently affected by trypsin. Thus, the above-mentioned inverse relationship holds for alpha Lyt-2,3 but not for alpha LFA-1. Our results are consistent with the hypothesis that Lyt-2,3 but not LFA-1 participates in recognition of class I H-2 antigens. Possibly LFA-1 participates in an adhesion-strengthening process that follows T cell recognition, and which may also be used by other LFA-1 expressing leucocytes in intercellular interactions. Finally, our results suggest (for the first time in the mouse system) that an unidentified non-H-2 "trypsin-sensitive counter blocking" molecule on the target cell plays an important role in CTL-target cell interaction.

Animals↗

PubMed1982

H-2Kb mutations limit the CTL response to SV40 TASA.

The cytotoxic T lymphocyte (CTL) responses directed towards SV40 tumor-associated specific antigen (TASA) in nine strains of spontaneously arising Kb mutant mice were analyzed. All nine mutants generated normal levels of H-2Db-restricted response, but the K-end-restricted CTL response varied. B6.C-H-2bm1 (bm1) did not produce K-end-restricted SV40 TASA-specific CTL upon immunization, and SV40-transformed bm1 cells were not lysed by intra-H-2 recombinant Kb [B10.A(5R)] CTL. Nonreciprocal cross-reactive lysis was seen between B6-H-2bm8 (bm8) and B10.A(5R). Strain B6-H-2bm8 mice produce highly specific Kbm8-restricted CTL that lyse SV40-transformed bm8 cells (Kbm8SV) but not B10.A(5R) target cells (K5RSV), although Kbm8SV targets can be partially lysed by B10.A(5R) CTL. The other seven Kb mutants cross-react with B10.A(5R). These experiments definitively show that genes mapping to the K and/or D region directly control the H-2-restricted CTL response to SV40 TASA.

Animals↗

PubMed1983

Secondary in vitro generation of CTL specific for MM antigen by stimulation with somatic tumor hybrid, but not with parental tumor cells.

Somatic tumor hybrid cells (L-FM3A#2), obtained by hybridization of MM tumor cells (FM3A/R) with HGPRT-less L cells, could induce CTL directed against MM antigen, a tumor-associated transplantation antigen that is expressed on some ascitic mammary tumor cell lines of C3H/He mice; parental tumor cells (FM3A/R) could not produce such CTL in syngeneic mice. In this study, the mechanisms of the generation of CTL by stimulation with L-FM3A#2 hybrid cells were investigated. In the secondary in vitro stimulation system, L cell component(s) that were introduced into hybrid cells by cell fusion play a role in the induction of CTL, as shown by the fact that stimulation with a mixture of L and FM3A/R cells could induce MM antigen-specific CTL, and that the killer helper effect of L cells could be replaced by cell free culture supernatants obtained from co-cultures of unprimed C3H spleen and L cells. IFN activity, but no IL 2 activity, was detected in the culture supernatants. Both IFN and killer helper activity were lost with pH 2 treatment; furthermore, CTL were generated by stimulation of primed spleen cells with FM3A/R cells in the presence of mouse beta-IFN, but not in its absence. These results suggest that IFN liberated by the helper cells that recognize L cell component(s) on the surfaces of tumor hybrid cells plays an essential role in the generation of CTL specific for MM antigen.

Animals↗

PubMed1984

Qa-like genes defined by CTL analysis of B10.W lines.

Twelve responder-stimulator combinations of mouse B10.W strains identical at K, D, and class II loci were tested for the generation of cytolytic T lymphocytes (CTL). No primary CTL could be obtained in any of the combinations but in nine combinations CTL were generated after priming in vivo. Six of these CTL are described. They define five antigenic determinants expressed exclusively in a small group of B10.W lines. The determinants appear to be part of the same system that resembles the Qa system originally defined in classic inbred strains. This resemblance rests on the observation that in vivo priming is necessary for the generation of the CTL, and that the CTL are not restricted in their reactivity by known H-2 loci. At least some of the determinants, however, appear to be controlled by a locus (or loci) associated with the K-rather than the D-end of the H-2 complex. Furthermore, some of the CTL directed against these Qa-like determinants cross-react with a molecule controlled by the K locus.

Animals↗

PubMed1995

Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides.

Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

Amino Acid Sequence↗

PubMed1995

Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.

MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.

Amino Acid Sequence↗

PubMed1994

TCR V beta 3+ and V beta 6+ CTL recognize tumor-associated antigens related to HER2/neu expression in HLA-A2+ ovarian cancers.

We have recently shown that HLA-A2-restricted, tumor-specific CTL can be isolated from tumor-infiltrating lymphocytes (TIL) in ovarian cancer, and that the sensitivity of ovarian tumors to these CTL is correlated with HER2/neu expression. Furthermore, utilizing PCR, we have documented previously that V beta 2, V beta 3, V beta 6, and V beta 7 are represented in increased proportions in ovarian tumor-specific CTL lines. Therefore, to correlate the interaction of these specific TCR V beta segments with the HLA-A2 molecule and potential tumor-associated Ags (TAA) related to HER2/neu expression, we have utilized available mAbs to V beta 2, V beta 3, and V beta 6. We found that V beta 2+, V beta 3+, and V beta 6+ CTL mediate antitumor activity, and a combination of these mAbs resulted in 83 to 95% inhibition of the cytotoxicity against autologous tumor from three separate patients. These mAbs also were capable of blocking HLA-A2-matched allogeneic cytotoxicity, suggesting that all three V beta families recognize TAA in the context of HLA-A2. An HLA-A2+ melanoma was transfected with the HER2/neu gene and became sensitive to HLA-A2+ ovarian cancer-specific CTL lysis. This cytotoxicity was mediated by V beta 3+ and V beta 6+ CTL, as demonstrated by mAb-blocking studies. FACS-depletion studies confirmed that CTL populations depleted of V beta 3 or V beta 6 no longer could recognize the HER2/neu transfectant. We conclude that V beta 3 and V beta 6 recognize some TAA that are either derived from the HER2/neu protein or induced by the expression of the HER2/neu gene and presented in the context of HLA-A2. Furthermore, V beta 2 seems to recognize an HER2/neu-unrelated Ag system also presented by HLA-A2.

Antigens, Neoplasm↗

PubMed1993

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. II. Differential effects of IL-6 on the helper cell-independent generation of CTL from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Although both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells that can develop into alloreactive CTL, these subsets differ in their ability to produce and use IL-2, a cytokine that is essential for T helper cell-independent CTL generation. In these studies, we have characterized the ability of these CD8+ subsets to undergo CTL differentiation in response to IL-6, another cytokine reported to be important or even essential for CTL generation. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic B cell lines, either alone or in the presence of exogenous cytokines. Alloactivated CD8+CD45RA- cells failed to differentiate into cytotoxic cells in the presence of IL-6 alone. In contrast, IL-6 stimulated the differentiation of antigen-specific CTL from alloactivated CD8+CD45RA+ precursors. The mechanism underlying this helper cell-independent process appeared to require IL-2, because IL-6-mediated CTL generation was abrogated by anti-CD25 or anti-IL-2 antibodies. Although CD8+CD45RA- cells did not respond to IL-6 alone, these cells were able to respond to IL-6 in a synergistic fashion in the presence of suboptimal concentrations of exogenous IL-2. These studies demonstrate that the CD8+ precursor subsets defined by CD45RA expression differ in their ability to undergo IL-6-mediated helper cell-independent CTL generation. Our data further suggest that this functional dissimilarity results from putative maturation-linked differences in the ability of these CD8+ precursor cells to produce IL-2.

Adult↗

PubMed1996

Human herpes simplex virus (HSV)-specific CD8+ CTL clones recognize HSV-2-infected fibroblasts after treatment with IFN-gamma or when virion host shutoff functions are disabled.

Herpes simplex virus (HSV)-specific CD8+ CTL cloned from individuals infected with HSV-2 efficiently lyse HSV-infected EBV-transformed B lymphoblastoid cells; however, these same CTL fail to lyse infected dermal fibroblasts. By 3 h after infection (early), class I MHC expression is reduced to less than 20% of that in uninfected fibroblasts, and expression is further reduced to less than 1% of the level in uninfected cells between 6 and 18 h after infection (late). We used an HSV-2 mutant that lacked the virion host shutoff (vhs) function to demonstrate that vhs plays a role in the loss of class I expression. While fibroblasts infected with this mutant are lysed by CTL that recognize virion proteins presented early as a consequence of introduction into the cytoplasm by the infecting virus, they are resistant to lysis by CTL that recognize viral proteins that must be synthesized de novo to be presented as class I Ags. Fibroblasts infected with a mutant that lacks the transporter-associated protein inhibitor ICP47 and is partially vhs defective are sensitive to CTL lysis. Pretreatment of fibroblasts with IFN-gamma prior to HSV infection sustained the level of class I expression for longer periods after infection, and these fibroblasts, infected with wild-type HSV-2, were partially sensitive to lysis by HSV-specific CTL. Taken together, these results suggest that the combined effects of the HSV-2 vhs and ICP47 gene products are to block Ag presentation by class I MHC. However, this effect can be transiently counteracted by IFN-gamma providing an early role for CD8+ CTL in the cellular immune response to HSV-2.

Antigen Presentation↗

PubMed1996

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying viral CD8+ T cell epitopes.

Exogenous Ags enter the endosomal pathway and are presented to CD4+ T cells in association with class II molecules whereas endogenously synthesized Ags, such as viral proteins, are presented to CD8+ T cells in association with MHC class I molecules. Therefore, most CTL activation strategies use live vectors although an alternative possibility could be to deliver the epitope into the cytosol by targeting it to an invasive nonreplicative vector. The adenylate cyclase toxin of Bordetella pertussis is able to invade a large number of eukaryotic cells and to deliver its catalytic domain to the cytosol of the cells. In the present study, we have tested the in vivo immunogenicity of recombinant adenylate cyclase toxins expressing CTL epitopes either from the nucleoprotein of lymphocytic choriomeningitis virus or from the V3 region of HIV-1 gp120. BALB/c mice immunized with these toxins developed high specific CTL responses that were shown to be mediated by class I-restricted CD8+ CTL. The induction of CTL responses by recombinant toxins did not require CD4+ T cells and the cytotoxic activity persisted 2 mo after immunization. The activation of CTL responses by the recombinant adenylate cyclase toxin required the full-length invasive activity of the toxin but did not depend upon its catalytic adenylate cyclase activity as demonstrated with a genetically inactivated recombinant toxin expressing the lymphocytic choriomeningitis virus epitope. This genetically detoxified invasive toxin represents, therefore, an attractive new vector for CTL activation.

Adenylyl Cyclases↗

PubMed1996

DNA vaccination primes MHC class I-restricted, simian virus 40 large tumor antigen-specific CTL in H-2d mice that reject syngeneic tumors.

We investigated the stimulation of a MHC class I-restricted response of CTL to the SV40 large tumor Ag (T-Ag) by different vaccination strategies in H-2d and H-2b mice. Immunization with plasmid DNA or exogenous T-Ag, or infection with SV40, primed CTL to T-Ag in H-2b mice; these three different types of Ag delivery primed T-Ag-specific CTL populations with similar epitope/restriction specificities. In H-2d mice, i.m. immunization with plasmid DNA, but neither immunization with exogenous protein Ag nor SV40 infection, primed CTL to T-Ag. T-Ag-specific H-2d CTL were primed by DNA-based immunization in vivo, expressed the CD4-CD8+ phenotype, and were L(d)-restricted. In H-2d (DBA/2) mice, T-Ag-specific immune responses primed by plasmid DNA injection, but not those primed by exogenous T-Ag or SV40 infection, mediated CD8+ CTL-dependent rejection of T-Ag-expressing P815/T tumor grafts. The data indicate that immunization by plasmid DNA injection is an efficient strategy to induce class I-restricted CTL responses against oncogene-encoded Ags of low immunogenicity that mediate tumor rejection.

Amino Acid Sequence↗

PubMed1997

IL-1 beta convertase (ICE) does not play a requisite role in apoptosis induced in T lymphoblasts by Fas-dependent or Fas-independent CTL effector mechanisms.

Both IL-1beta convertase (ICE) and other members of the ICE-like family of proteases have been reported to play a role in Fas-mediated apoptosis. Con A-stimulated T lymphoblasts generated from splenocytes isolated from ICE-deficient H-2b mice were found to be more susceptible than wild-type lymphoblasts to DNA fragmentation induced by H-2b-specific CTL derived from normal or Fas ligand-deficient gld/gld mice. Trinitrophenyl (TNP)-modified, H-2b target cell-specific CTL were generated from perforin-deficient mice and were found to induce DNA fragmentation only in target cells expressing functional Fas receptors. Similar rates of DNA fragmentation were induced in TNP-modified ICE -/- and ICE +/+ T lymphoblast targets by perforin -/- TNP-modified, H-2b target cell-specific CTL. In addition, anti-Fas Abs induced apoptosis in thymocytes, Con A-stimulated spleen T cells, LPS-stimulated spleen B cells, and thymocytes from ICE -/- mice. However, DNA fragmentation induced by either allospecific FasL-defective CTL, or by perforin-deficient, TNP-modified, H-2b target cell-specific CTL was prevented in ICE -/- target cells loaded by electroporation with Ac-DEVD-CHO, an inhibitor of CPP32 and related ICE family proteases. These findings indicate that ICE does not play a requisite role in Fas-dependent or Fas-independent mechanisms of apoptosis induced in peripheral T lymphoblasts by CTL. However, both major pathways of CTL-induced apoptosis appear to be dependent on the enzymatic activity of other ICE family proteases.

Animals↗

PubMed1997

CTL generation in the presence of IL-4 is inhibited by free p40: evidence for early and late IL-12 function.

The cytokine requirements for the generation of a CTL response were examined using thymocyte responders stimulated with Con A. In agreement with previous reports, IL-4 alone can generate a strong CTL response. To determine whether IL-12 was required for this response, we added either a neutralizing Ab against IL-12 or an antagonist of IL-12 function, p40. Both anti-IL-12 and p40 were found to inhibit the generation of CTL in the presence of IL-4. Consistent with these data, exogenous IL-12 was found to synergize with IL-4, particularly, when IL-4 was added at levels unable to generate a CTL response alone. This synergistic response was observed whether IL-12 was added at the beginning of culture along with IL-4 or after the first day to cultures initiated with IL-4. Likewise, the combination of IL-2 plus IL-12 evoked a strong synergistic response when cultures were initiated with IL-2 and IL-12 was added initially or after the first day of culture. Addition of either IL-2 or IL-12 alone did not generate a response. In contrast to these data showing that IL-12 was able to signal late in culture, p40 inhibited CTL generation in response to IL-4 only when added at the beginning of culture. These results imply that CTL induction involves a crucial early IL-12 signal, which once delivered permits cells to respond to a later IL-12 signal. These data are consistent with the idea that free circulating p40 could inhibit the generation of CTL by antagonizing the early IL-12 "differentiation" signal.

Animals↗

PubMed1997

Induction of protective CTL responses against the Plasmodium yoelii circumsporozoite protein by immunization with peptides.

To determine the optimum combination, concentration, and formulation of synthetic peptides and adjuvants to induce protective CTL responses against the Plasmodium yoelii circumsporozoite protein (PyCSP), BALB/c mice were immunized with linear and multiple antigen peptides (MAP) including PyCSP CTL and Th epitopes in Montanide's ISA51, Lipofectin, and Lipofectamine. An H-2K(d)-restricted PyCSP CTL epitope, SYVPSAEQI (amino acids (aa) 280-288), recognized by protective CTL clones, was included in the following peptides: a 9-aa linear peptide (SYVPSAEQI; PyCSP9), a 20-aa linear peptide (aa 280-299; SYVPSAEQILEFVKQISSL; PyCSP20), a MAP containing four branches of PyCSP20 (MAP(280-299)), and a linear peptide and a MAP(MAP(280-299)p2p30) in which PyCSP20 was colinearly synthesized with two universal Th epitopes from tetanus toxin (p2p30). A MAP containing the PyCSP Th epitope (aa 57-70; KIYNRNIVNRLLGD) was included in some experiments. The highest specific lytic activity against peptide-pulsed target cells was obtained with splenocytes from mice immunized with three doses at 3-wk intervals of MAP(280-299)p2p30 in Lipofectin or Lipofectamine. Forty percent of the mice immunized with MAP(280-299)p2p30 and Lipofectin were protected against sporozoite challenge. Immunization with CTL and Th epitopes co-linearly synthesized in a MAP induced significantly better CTL than did immunization with the same sequence as a linear peptide, or immunization with a mixture of two individual MAPs, one with the CTL epitope and the second with the Th epitope.

Adjuvants, Immunologic↗

PubMed1998

Activation requirements, lytic mechanism, and development of a novel anti-CD8-resistant CTL population.

Almost all conventional CD8+ CTL and their CD8+ precursors are inhibited by anti-CD8 mAb. This requirement for CD8 function reflects both an avidity-augmentation role and a signal-transduction role for CD8 on T cells. We have, however, previously identified and partially characterized a novel functional population of CD8+, but anti-CD8-resistant, MHC class I-allospecific CTL. These CTL have unusual activation requirements in that their efficient generation in vitro requires inhibition of the CD8 avidity contribution (but not the CD8 signaling contribution), by anti-CD8 mAb. In this study, we have investigated the relationship of anti-CD8-sensitive and anti-CD8-resistant CTL by several criteria. These CTL populations share the phenotypic markers we have tested to date, they have similar but not identical Ag-specific repertoires, and they both appear to be generated from naive unprimed T cells. However, anti-CD8-sensitive and anti-CD8-resistant CTL populations exhibit important functional differences. They differ in their kinetics of activation in vitro, their dependence on exogenous cytokines, their use of lytic effector mechanisms, and their tissue distribution during ontogeny. Based on these results, we favor the hypothesis that these CTL populations represent distinct T cell lineages or subsets, and not merely different TCR avidity ranges within a single T cell lineage or subset.

Animals↗

PubMed1998

TCR usage by homocysteine-specific human CTL.

We have recently demonstrated that homocysteine can modify HLA class I Ags and induce homocysteine-specific CTL (Hom-CTL) responses in humans. Here, we have investigated TCR usage by Hom-CTL from five patients with ankylosing spondylitis or reactive arthritis. TCR of HLA-A68-restricted Hom-CTL from two unrelated donors share the same TCR Valpha, Vbeta, and Jbeta gene segments (AV4, BV23, and BJ2S1, respectively) with similar third complementarity determining regions (CDR3) of the beta-chains. Interestingly, the Va and Vbeta gene segments employed by an HLA-B27-restricted Hom-CTL clone are also closely related to AV4 and BV23, indicating strong selection pressure for AV4, BV23, and related gene products in the homocysteine-specific TCR. An arginine or lysine residue frequently appeared at position alpha93 in the CDR3 of the TCR alpha-chains from Hom-CTL restricted by HLA-A68 or -B8. This may suggest a potential salt bridge between the carboxyl group of homocysteine and specific TCR. TCR usage by HLA-B27-restricted Hom-CTL from unrelated individuals appears to be less conserved, although two T cell clones from one individual rearranged the same V gene segments with identical lengths of CDR3. Implications of these data for the molecular mechanisms for homocysteine modification of HLA Ags are also discussed.

Amino Acid Sequence↗

PubMed1998

TAP-independent MHC class I peptide antigen presentation to alloreactive CTL is enhanced by target cell incubation at subphysiologic temperatures.

We investigated the peptide dependency of a group of CD8+ anti-HLA-B7 alloreactive CTL. The CTL killed target cells after acid denaturation of more than 98% of target cell surface peptide/MHC class I complexes. The CTL also killed TAP- HLA-B7-transfected T2 (T2B7) cells. The killing was enhanced by target cell incubation at 26 degrees C. Despite these findings, which suggested peptide-independent allorecognition, CTL-mediated cytolysis was reduced or abolished by several point mutations affecting the HLA-B7 peptide-binding groove. Acid denaturation of HLA complexes on T2B7 cells prohibited CTL recognition. CTL recognition was restored by T2B7 cell incubation with beta2-microglobulin and a single HPLC fraction containing peptides extracted from TAP+HLA-B7+ cells, but not by any of a panel of 17 synthetic HLA-B7-binding peptides. These findings indicated that CTL allorecognition was peptide specific. Sensitizing peptide was extracted from T2B7 cells only after incubation at 26 degrees C. The amount of peptide detected in TAP+ cells was at least 10-fold and 100-fold greater than that detected in TAP- cells incubated at 26 degrees C and at 37 degrees C, respectively. TAP-independent peptide epitope presentation was sensitive to treatment with brefeldin A, but not sensitive to treatment with chloroquine, consistent with an endogenous peptide source. We propose that subphysiologic temperature incubation can enhance peptide/MHC class I presentation in the total absence of TAP function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗