Differences in HLA antigen recognition by human influenza virus-immune cytotoxic T cells.

The specificity of in vitro induced human influenza-immune cytotoxic effector cells was analyzed with respect to recognition of HLA-A and -B-linked gene products. The influenza-immune cytotoxic activity observed on panels of virus-infected targets demonstrated that virus-immune effectors preferentially lyse targets with which they share HLA-A or -B specificities. Virus-immune effectors from certain donors recognized virus in conjunction with some, but not all, of their self HLA-A and -B antigens. Among donors who share a given HLA antigen (such as A2 or B7), there are differences in the ability of their virus-immune T cells to recognize the shared antigen. Virus-infected target cells from HLA-A2 or -B7 "nonresponder" donors could be lysed by virus-immune T cells obtained from other donors who shared only the HLA-A2 or -B7 antigen with these target cells. These observations suggest that the absence of cytotoxic T cell responses by some donors to influenza virus in conjunction with HLA-A2 or -B7 is not due to control by the structural genes that code for these HLA antigens, but rather may result from control by regulatory genes that act at the level of the responder and/or stimulator cell. The results are discussed in the context of Ir gene regulation of human T cell responses.     

OKT4+ cytotoxic T cells can lyse targets via class I molecules and can be blocked by monoclonal antibody against T4 molecules

Human cytotoxic T lymphocytes (CTL) have been shown to recognize either class I or class II major histocompatibility (MHC) products. This recognition has been correlated with the expression of OKT antigens on the surface of the CTL. Thus, OKT4+ CTL have been shown to be reactive with class II products, whereas OKT8+ effectors recognize class I molecules. In this study, responder cells were separated according to their OKT4 or OKT8 cell surface phenotype on a fluorescence-activated cell sorter (FACS). The OKT4+ subsets were stimulated with an LCL mutant that did not express DR and MB/MT but did express SB and class I antigens. After 7 days in culture, the activated subsets were tested on a panel of class I matched or mismatched targets. The cytotoxicity observed could be correlated with the presence of matched class I antigens. In addition, monoclonal antibody (MCA) W6/32, directed at a monomorphic determinant on HLA-A and -B molecules, blocked lysis. Furthermore, six OKT4+ CTL clones were derived from the OKT4+ bulk cultures; three clones were found to be directed at class I molecules whereas the other three recognized class II determinants. The ability of these clones to lyse their relevant targets was blocked by OKT4 MCA, raising questions as to the role of the T4 molecule in antigen class-specific CTL recognition.     

Two subgroups of HLA Bw44 defined by cell-mediated lympholysis that differ in Bw44 expression on platelets and in patterns of genetic linkage disequilibrium

Possible immunogenic heterogeneity of the HLA-Bw44 antigen was investigated using cytotoxic T lymphocytes (CTL) generated between donors identical for HLA-A2,3,-B7,w44. Highly discriminatory CTL combinations were identified that defined two subgroups of Bw44, designated 44.1 and 44.2. Out of 47 Bw44-positive donors tested in a population study, 30 were lysed by the CTL defining 44.1, and 19 were lysed by the CTL defining 44.2. All Bw44 cells could be typed as either 44.1 or 44.2, except two Bw44-positive cells that were phenotypically homozygous for the serologically defined Bw44 antigen and were lysed by both CTL. No Bw44-negative donors (zero out of 37) expressed either 44.1 or 44.2, although cold target blocking was required to eliminate a contaminating reactivity of one CTL population on Bw35 and some Bw45 cells. CTL were also raised between responder/stimulator combinations mismatched for Bw44. These CTL lysed all Bw44-positive target cells, indicating a CML antigen shared by all Bw44 cells. But clear discrimination of the 44.1 and 44.2 subgroups was obtained when appropriate cold target blocking cells were added. All donors with 44.2 expressed high levels of serologically detectable Bw44 on their platelets, and all with 44.1 expressed low levels (p less than 0.005). Furthermore, population studies indicate that 44.1 is in positive linkage disequilibrium with HLA-A2 and possibly DR4, whereas 44.2 is in positive linkage disequilibrium with HLA-DR7 and possibly HLA-A23, -A26, and -A29. These data suggest the existence of two genetically and functionally different subgroups of Bw44 antigens.     

Regulatory mechanisms in cytotoxic T lymphocyte development. III. Induction, specificity, and genetic restriction of an in vitro suppressor T cell

We addressed questions pertaining to the immunogenetics of an in vitro alloinduced suppressor T cell (Ts) previously shown to inhibit cytotoxic T-lymphocyte (CTL) development by suppressing CTL precursor proliferation. Using intra-MHC recombinant strains of B10 congenic mice, the requirements for H-2 differences to induce Ts activity, the antigen specificity of the Ts, and the genetic restriction of Ts function were studied. It was found that differences at the K, D, or I regions alone can induce strong suppressor activity. Suppression of CTL development does not appear to be genetically restricted since the Ts inhibit CTL from responder cells disparate at K, K and D, I, or K and I. The alloinduced Ts is specific for the antigen stimulating its induction, but also inhibits CTL responses against immunologically unrelated determinants, even between class I and class II antigens, provided those determinants are carried on cells expressing the original inducing antigen. Ts can be triggered by antigens present on the responder cells but absent on the stimulator cells, indicating that the suppressive signal may be exerted directly on the responder population without specific interaction with stimulator cells.     

Role of beta2-microglobulin in the intracellular processing of HLA antigens

The biosynthesis of HLA-A, -B, and -C antigens was examined in the two lymphoblastoid cell lines DAUDI and RAJI. In RAJI cells the HLA-A, -B, and -C antigen heavy chains become core-glycosylated in the endoplasmic reticulum as evidenced by their sensitivity to endo-H digestion and tunicamycin treatment. Beta2-Microglobulin is present in excess in the endoplasmic reticulum of the RAJI cells and associates with the heavy chain at the time of synthesis of the heavy chain. Pulse-chase experiments demonstrated that the RAJI HLA-A, -B, and -C antigen heavy chains become terminally glycosylated since their changed characteristics included resistance to endo-H digestion, sensitivity to neuraminidase treatment, and incorporation fucose. DAUDI HLA-A, -B, and -C antigen heavy chains are synthesized normally and become core-glycosylated but not terminally glycosylated. Other glycosylated cell surface proteins, like the HLA-DR antigens, display normal glycosylation in DAUDI cells. Therefore it is unlikely that the absence of terminally glycosylated HLA-A, -B, and -C antigen heavy chains is the result of a general defect in the biosynthetic machinery of DAUDI cells. However, DAUDI cells lack the ability to synthesize beta2-microglobulin, the common subunit of all HLA-A, -B, and -C antigens. Therefore, it seems reasonable to conclude that beta2-microglobulin is of importance for intracellular transport of newly synthesized HLA-A, -B, and -C antigens.     

The Magnitude and Specificity of Influenza A Virus-Specific Cytotoxic T-Lymphocyte Responses in Humans Is Related to HLA-A and -B Phenotype

The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M1(58-66) epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2(+) donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M1(58-66) epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP(380-388) was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP(44-52) was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes.     

Preferential HLA usage in the influenza virus-specific CTL response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.     

Induction of Tc2 cells with specificity for prostate-specific antigen from patients with hormone-refractory prostate cancer

Prostate-specific antigen (PSA) is a potentially useful antigen for targeted T-cell immunotherapy of prostate cancer (CaP). Our laboratory has identified a synthetic nonamer peptide (PSA 146-154) homologue of PSA, which binds to the prevalent human leukocyte antigen, HLA-A2, and elicits specific cytotoxic T-lymphocyte (CTL) responses from normal individuals of the HLA-A2 phenotype. In the present study, we report on the induction of CTL from peripheral blood mononuclear cells (PBMC) of patients with hormone-refractory CaP, which exhibit the same specificity. T-cell lines were established from two patients by stimulation of PBMC with PSA 146-154 peptide in vitro. The T-cell lines exhibited specific cytolytic activity against T2 cells pulsed with PSA 146-154 peptide, but not a control HLA-A2 binding peptide (HIV-RT 476-484) via chromium release assay (CRA). The T-cell lines also showed PSA 146-154 peptide-specific IL-4 responses, but no detectable interferon-gamma (IFN-gamma) responses via enzyme-linked immuno-spot assays. Magnetic immuno-selection studies of one of the T-cell lines demonstrated that both cytolytic and interleukin-4 (IL-4) responses were mediated by CD8(+), but not by CD4(+) T cells. This Tc2 line was further characterized for the ability to recognize endogenously processed PSA epitopes. The line specifically secreted IL-4 in response to HLA-A2(+) target cells transfected to express PSA and specifically lysed the PSA(+) target cells, but not control transfected cells. The results indicate that the PSA 146-154 peptide emulates a naturally processed and presented peptide epitope of PSA that is within the T-cell repertoire of HLA-A2(+)patients with CaP.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Induction of CD4+ cytotoxic T cells by sensitization with allogeneic melanomas bearing shared or cross-reactive HLA-A.

Human melanoma is an immunogenic neoplasm whereby enhancement of specific cell-mediated immunity can alter tumor progression. HLA-A2-restricted CTL have been demonstrated to kill allogeneic HLA-A2-matched melanoma. We investigated the ability of allogeneic melanoma cells sharing HLA-A antigens to sensitize melanoma patients' lymphocytes to induce HLA-A-restricted CTL to autologous melanoma. PBL from melanoma patients were cocultured with autologous melanoma cells in defined "cocktail medium" to generate melanoma-specific HLA-A-restricted CTL lines. CTL generated by sensitization with allogeneic melanoma bearing shared HLA-A2, A11, A24, or "cross-reactive" HLA-A antigens could kill almost as many autologous melanoma cells as CTL sensitized with autologous melanoma. There are HLA-A antigens that are immunogenically cross-reactive because they share determinant epitopes. CTL were not activated NK or LAK cells. The HLA restriction and melanoma cell specificity of the CTL were demonstrated by cold target inhibition with autologous and allogeneic melanoma and B lymphoblasts. Anti-CD3 and anti-HLA AB inhibited CTL killing of melanoma. The CTL were predominantly CD3+CD4+ TCR alpha/beta+. These studies demonstrate that melanomas being shared or cross-reactive HLA-A can be used for in vitro generation of HLA-restricted CTL that recognize melanoma-associated antigens. The findings have very important implications in human tumor immunotherapy.     

Cross-reactive cytotoxic T lymphocytes against human immunodeficiency virus type 1 protease and gamma interferon-inducible protein 30

The gamma interferon (IFN-gamma)-inducible protein 30 (IP-30) signal peptide -11 to -3 (LLDVPTAAV) is a prominent self peptide expressed with the class I human histocompatibility leukocyte antigen A2 (HLA-A2). Stimulation of peripheral blood mononuclear cells (PBMC) from HLA-A2 human immunodeficiency virus type 1 (HIV-1)-infected individuals with an HLA-A2-restricted HIV protease (PR) peptide 76-84 (LVGPTPVNI) activated cytotoxic T lymphocytes (CTL) against the IP-30 signal peptide. Since HIV-1 PR 76-84 stimulated CD8+ T cells from these individuals to secrete IFN-gamma, we tested whether the activation of IP-30-specific CTL in vitro resulted from T-cell cross-reactivity or from up-regulation of IP-30 by IFN-gamma. Neither high levels of exogenous IFN-gamma nor incubation of PBMC with other HIV peptides triggering substantial IFN-gamma release activated IP-30-specific CTL. Although the IP-30 signal peptide did not stimulate IFN-gamma release from freshly isolated PBMC, it activated CTL in vitro against itself and HIV PR 76-84. Peptide-stimulated IFN-gamma release, cold target inhibition, and HLA-A2/immunoglobulin dimer-mediated binding and depletion of effector cells all indicated that in vitro stimulation with HIV PR 76-84 or the IP-30 signal peptide activated a comparable population of cross-reactive effector cells. Neither IP-30 nor HIV PR 76-84 activated CTL against themselves following in vitro stimulation of PBMC from non-HIV-infected HLA-A2 individuals. Peptide titrations indicated higher-avidity T-cell interactions with HIV PR 76-84 than with the IP-30 signal peptide. These data indicate that HIV PR 76-84 is a heteroclitic variant of the IP-30 signal peptide -11 to -3, which has implications for immune memory and autoimmunity.     

A single amino acid substitution in HLA-A2 can alter the selection of the cytotoxic T lymphocyte repertoire that responds to influenza virus matrix peptide 55-73

Previous studies have demonstrated that certain amino acid substitutions in the alpha two domain at positions 152 and 156 in the alpha two helix of the HLA-A2 molecule can affect presentation of the influenza virus matrix peptide M1 55-73 without abolishing binding of the M1 peptide. HLA-A2.1-restricted M1 55-73 peptide-specific CTL lines obtained from almost all HLA-A2.1+ individuals fail to recognize the M1 peptide presented by site-directed mutants of HLA-A2 that have either a Val----Ala or Val----Gln substitution at position 152 or a Leu----Trp substitution at position 156. Only one HLA-A2+ individual (donor Q66, HLA-A2,-B53,-B63) has been found who is able to generate a unique repertoire of HLA-A2-restricted M1 peptide-specific CTL that can recognize peptide presented by HLA-A2 mutants with either an Ala or Gln substitution at position 152 or a Trp substitution at position 156. These Q66 M1 peptide-specific CTL could be selected by stimulation with M1 peptide-pulsed transfectants that express the mutant HLA-A2 gene with the Trp substitution at 156. To determine if the presence of the unique CTL repertoire could be attributed to a variant HLA-A2 molecule in Q66, sequences were determined from polymerase chain reaction-amplified segments of the HLA-A2 RNA. Two different HLA-A2 genes were found expressed in Q66 cells: one is identical to HLA-A2.1 and the other is identical to HLA-A2.2F (Gln----Arg at position 43, Val----Leu at position 95, and Leu----Trp at position 156). These results demonstrate that a different CTL repertoire specific for HLA-A2 plus the M1 55-73 peptide is generated in an individual that expresses both HLA-A2.1 and HLA-A2.2F compared to individuals who express HLA-A2.1 alone, and that the unique repertoire can be selected by the presence of an HLA-A2 molecule with a single amino acid substitution at position 156.     

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.     

Cell-mediated cytotoxicity against HLA-D-Region products expressed in monocytes and B lymphocytes

The cytotoxic effector cells that recognize HLA-D-region determinants and their precursors were characterized using monoclonal antibodies against human T lymphocytes and T-cell subsets. These studies were performed using MLC combinations giving rise to cytotoxic cells specific for both class I (HLA-A, B, C) and class 11 (HLA-D-region) antigens, and then tested against target cells displaying relevant antigens of only one class. Both class I and class II specific CTL (cytotoxic T lymphocytes) were inhibited by treatment with the OKT3 monoclonal antibody and complement, indicating that the effector cells were T lymphocytes. A major portion.of class II specific CTL, and their precursors, were inhibited by OKT4 and complement, while class I specific CTL from the same cultures were not. The T4+T8 — cell subset has previously been associated with helper or inducer functions, but not with cytotoxicity. The present findings indicate that class I and class 11 specific CTL, and their precursors, are different on the basis of the class of target antigen recognized and on the basis of surface phenotype detected by monoclonal antibodies.     

Genetics of cell-mediated lympholysis in man.

Cell-mediated lympholysis (CML) was studied in a family containing two siblings in who genetic recombinaiton had occurred in the HLA comples. In one sibling, recombination occurred between the HLA-A locus and the HLA-B locus. In the second sibling recombination occurred between the HLA-B locus and the HLA-D locus. Strong CML activity was generated in mixed lymphocyte cultures (MLC) when stimulator and responder cells differed in HLA-A, B, and D antigens. MLC involving HLA-D differences alone did not generate CML. Weak, but definite CML activity was generated during MLC with cells differing at HLA-A and HLA-B but sharing HLA-D. HLA-B antigens were good targets for lysis in all combinations studied. HLA-A antigens were poor targets in some but not in all combinations. However, combinations where HLA-A antigens seemed to be good targets could have involved HLA-B differences due to polymorphism of HLA-B7 antigens each inherited from a different parent. HLA-D antigens did not serve as targets for lysis. In three cell experiments, excellent CML activity was generated when responder cells were stimulated by HLA-D antigens and by HLA-A and B antigens present on separate stimulator cells.     

The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity

Two groups of human and murine cytotoxic T lymphocyte (CTL) clones specific for human leukocyte antigen (HLA)-A2 or -B7 can be distinguished based on their ability to kill murine transfectants expressing these molecules. The clones which do not recognize murine transfectants exhibited greatly reduced conjugate formation with these cells, indicating that the inability to lyse these cells occurs in recognition and binding. No systematic differences in inhibitory titer between the two types of CTL clones were seen with anti-CD8 (Lyt-2), anti-LFA-1, or monoclonal antibodies against HLA class I molecules. However, blocking with anti-HLA class I monoclonal antibodies suggested that different CTL clones recognized spatially separate epitopes on HLA-A2 and -B7. In addition, a correlation between the inability to recognize murine transfectants and fine specificity was seen. Eight of nine clones which did not lyse murine transfectants also failed to recognize human cells expressing HLA-A2.2 or -A2.3. In contrast only 5 of 12 clones which lysed transfectants failed to recognize the variant molecules. Analogous data were obtained with human CTL clones raised against HLA-A2.1. These findings suggest that CTL clones that do not lyse murine cells expressing appropriate antigens recognize epitopes that have been altered or lost as a consequence of expression on the murine cell surface. It is suggested that the loss of HLA-associated epitopes on the murine cell surface may be due to differences between mouse and human cells in the processing or presentation of class I-associated peptides.     

Role of accessory cell processing and presentation of shed H-2 alloantigens in allospecific cytotoxic T lymphocyte responses

The present study was undertaken to evaluate the role of accessory cell processing of MHC alloantigens in the initiation of primary allospecific CTL responses. To first determine whether antigen processing by accessory cells is involved in the initiation of allospecific CTL responses, accessory cells were retreated with the lysosomotropic drug chloroquine before their addition to culture. It was found that chloroquine pretreatment abrogated their ability to function as accessory cells only when they were of responder haplotype and had no effect when the accessory cells were of stimulator haplotype. Although accessory cells of either responder or stimulator haplotype can initiate allospecific CTL responses, we have previously demonstrated that they do so by activating distinct classes of T helper TH) cells. Indeed, the differential effects of chloroquine on accessory cells of responder or stimulator haplotypes were shown to reflect the fact that chloroquine pretreatment markedly impaired the ability of accessory cells to activate self-Ia-restricted TH cells, but had little effect on the ability of the same accessory cells to activate either allo-class I- or allo-class II-specific TH cells. We next examined the possibility that accessory cells of responder haplotype mediate alloresponses by acquiring and processing shed MHC alloantigens derived from the stimulator cell population. In these experiments, accessory cell-depleted stimulator cells were fixed with paraformaldehyde to inhibit shedding of their surface MHC alloantigens. It was observed that even though mixed stimulator cells were recognized normally by allospecific CTL precursors, they completely failed to stimulate CTL responses mediated by responder haplotype accessory cells, indicating that the function of such accessory cells is dependent upon their acquisition of shed MHC alloantigens. Taken together, the data presented in this report demonstrate that accessory cells of responder haplotype function in allospecific CTL responses by acquiring and processing shed class I MHC alloantigens, and by then presenting the processed alloantigens in association with self-Ia determinants to self-Ia-restricted TH cells. Thus, these data indicate that the self-Ia-restricted TH cells that are involved in allospecific CTL responses recognize processed class I alloantigens in association with self-Ia determinants.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.     

CTL adhesion and antigen recognition are discrete steps in the human CTL-target cell interaction

Th initial step in cytolytic T lymphocyte (CTL)-mediated cytolysis involves target cell adhesion and antigen recognition. To investigate these initial events in the CTL-target interaction, we used HLA-A2- and HLA-B7-specific human CTL clones and HLA-typed B lymphoblastoid target cells. By using two different adhesion assays, we demonstrated antigen nonspecific CTL-target cell adhesion. To more precisely define the contribution of the antigen-specific receptor to CTL-target cell adhesion, we used the HLA-A2, HLA-B7, and mock transfected RD target cells. Consistent with the results when using B lymphoblastoid target cells, the CTL clones demonstrated equivalent adhesions to the RD target cells whether or not they expressed HLA-A2 or HLA-B7. These results suggested that CTL-target cell adhesion occurred independent of the T cell receptor. By using the calcium-sensitive dye Indo-1 and flow cytometry, we assessed CTL-target cell adhesion and CTL activation. Simultaneous measurement of adhesion and intracellular free calcium demonstrated that CTL-target cell adhesion alone did not activate CTL clones. Both CTL-target cell adhesion and the presence of the appropriate HLA target molecule were necessary for the efficient activation of human CTL. MAb inhibition studies indicated that antigen nonspecific adhesion is largely regulated by the LFA-1, CD2 (LFA-2/T11), and LFA-3 cell surface molecules. These antigen nonspecific cell-cell interaction molecules appear to play an important role in facilitating antigen recognition and subsequent target cell lysis.