Anti H-2Dd alloreactivity mediated by herpes-simplex-virus specific cytotoxic H-2k T lymphocytes is associated with H-2Dk

Herpes-simplex-virus (HSV) specific, H-2k-restricted, immune cytotoxic T lymphocytes also lyse noninfected H-2d target cells. Genetic mapping studies revealed that HSV-specific Dk-restricted CTL cross-react with allogeneic targets expressing Dd alloantigens. Cold target inhibition experiments indicate that only a minority of HSV-specific CTL mediate cross-reactive cytolysis. The data give an example of where the phenomenon of H-2-restricted versus nonrestricted responsiveness is not due to distinct subsets of T cells but solely depends on the antigenic determinants recognized.     

Anti H-2Dd alloreactivity mediated by herpes-simplex-virus specific cytotoxic H-2k T lymphocytes is associated with H-2Dk

Herpes-simplex-virus (HSV) specific, H-2^k-restricted, immune cytotoxic T lymphocytes also lyse noninfected H-2^d target cells. Genetic mapping studies revealed that HSV-specific D^k-restricted CTL cross-react with allogeneic targets expressing D^d alloantigens. Cold target inhibition experiments indicate that only a minority of HSV-specific CTL mediate cross-reactive cytolysis. The data give an example of where the phenomenon of H-2-restricted versus nonrestricted responsiveness is not due to distinct subsets of T cells but solely depends on the antigenic determinants recognized.This work was supported by the SFB 107 and the Stiftung Volkswagenwerk.     

Recognition by cytotoxic T lymphocytes of Qa-2 antigens. Sensitivity of Qa-2 molecules to phosphatidylinositol-specific phospholipase C

Con A splenic lymphoblasts were incubated with phosphatidyl-inositol specific phospholipase C (PIPLC) derived from Bacillus thuringiensis and subsequently analyzed for Qa-2 Ag with the Qa-2 reactive mAb Qa-m2. This treatment completely removed Qa-2 detectable Ag on lymphoblasts from H-2d animals, indicating that these molecules are likely anchored to the cell membrane through phosphatidyl inositol (PI). Although exposure of lymphoblasts from H-2b mice to PIPLC greatly reduced Qa-2 expression, a subpopulation of cells retained a limited quantity of the Ag. Bulk cultured anti-Qa-2 CTL generated against the Qa-2 region from H-2b haplotype mice lysed Qa-2+ targets from B6.K2 (H-2b) and BALB/cJ (H-2d) animals. Pretreatment of these lymphoblast targets with PIPLC completely abolished lysis of the BALB/cJ target cells, whereas lysis of B6 targets was reduced only slightly. Anti-Qa-2 CTL clones tested against PIPLC-treated B6 target cells revealed two patterns of reactivity. One group of clones was unaffected in its ability to lyse PIPLC-pretreated targets and cross-reacted on Q6d/Ld molecules expressed on transfected L cells. A second group was unable to lyse PIPLC-pretreated lymphoblasts and cross-reacted on Q7d/Ld targets. These data suggest that H-2b-derived lymphoblasts express two different types of Qa-2 molecules with respect to PIPLC sensitivity; one type is sensitive to PIPLC and cross-reactive with Q7d, the other type is resistant to PIPLC and cross-reactive with Q6d. In contrast, H-2d lymphoblasts express only the PIPLC-sensitive type of molecules. It was also noted that bulk cultured anti-Qa-2 CTL more readily lysed H-2b target cells expressing a smaller quantity of PIPLC-resistant Ag than H-2d targets expressing a larger amount of PIPLC-sensitive Ag. Further, anti-Qa-2 CTL clones readily lysed PIPLC-treated target cells expressing very low levels of serologically detectable Qa-2. This suggests that recognition of class I molecules anchored to the membrane via a PIPLC-resistant linkage may more readily activate CTL for expression of lytic activity than molecules anchored through PI.     

Biochemical and functional analyses of a secreted H-2Ld molecule.

A truncated H-2Ld gene was constructed by deleting the transmembrane and cytoplasmic exons. The truncated H-2Ld gene was introduced into mouse L cells using the thymidine kinase gene as a selectable marker. Transformants were isolated and screened for the presence of truncated H-2Ld antigen. The truncated H-2Ld gene product was present in both the cytoplasm and culture medium, but not on the cell surface. The truncated H-2Ld antigen was stable in culture medium for at least 9 h and was secreted into the medium at a rate similar to the kinetics with which complete H-2 antigens reach the cell surface. Transformants expressing the truncated H-2Ld molecule were not recognized by cytotoxic T lymphocytes specific for the H-2Ld antigen.     

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

H-2M3 presents a Listeria monocytogenes peptide to cytotoxic T lymphocytes.

We report evidence that a major histocompatibility complex-encoded nonclassic class I molecule presents a foreign peptide to cytotoxic T lymphocytes (CTL) during an infection. Mice immunized with virulent Listeria monocytogenes generate CD8+ CTL with alpha beta receptors specific for a bacterial peptide presented by a conserved class I molecule encoded in the M region of the major histocompatibility complex. The Listeria peptide is digested by carboxypeptidase Y but resists aminopeptidase M, and only peptides with N-formyl methionine competitively block its presentation to CTL. Transfection with the H-2M3d gene enables a negative (H-2w17) cell line to present the bacterial peptide. One function, therefore, of H-2M3 is to present bacterial peptides to CTL during infection.     

Generation of cytotoxic T lymphocytes by the H-2-encoded E molecules

Primary CML was generated in strain combinations 4R anti-2R, R107 anti-3R, 7R anti-9R, and GD anti-R101 — combinations differing only in the chromosomal interval between the I-A subregion and the Ss locus. No CML could be obtained in any of the reciprocal combinations of these strains. This unidirectionality of the CML reaction correlates with the expression or nonexpression of the E molecules encoded by this interval: the reaction occurred in combinations in which the responder strain lacked and the stimulator strain expressed the E molecules in the cell membrane. The CML reaction was positive when tested on LPS-stimulated blast cells but weak on Con A-stimulated blasts and negative on la-negative tumor cells. The reaction could partially be inhibited by monoclonal antibodies to the Ia.m7 determinant presumably carried by the E chain; it was not inhibited by monoclonal antibodies specific for Ia determinants carried by the A molecule. Cytotoxic lymphocytes specific for a particular combination of E and E chains reacted with all cells expressing the particular E chain, no matter what the origin of the E chain associated with the E chain was. Attempts to generate cytotoxic lymphocytes specifically reactive with allotypic determinants on E chains failed. In F₁ hybrids expressing one type of E chain and two types of E chain, the single E chain was found to associate with both chains, producing two types of E molecule. We conclude from these experiments that the CML determinants detected in the strain combinations used are encoded by the same loci as those coding for the serologically detectable la determinants. The CML determinants are carried by the E chains; the E chain does not contribute in any way to the specificity of determinant recognition by the cytotoxic lymphocytes. No evidence for allotypic variation of the E chain as detected by the CML assay could be found in this study.     

Mapping of HLA epitopes recognized by H-2-restricted cytotoxic T lymphocytes specific for HLA using recombinant genes and synthetic peptides

Immunization of DBA/2 (H-2d) mice with syngeneic P815 tumor cell transfectants that express HLA class I genes elicits CTL that recognize HLA in the context of H-2Kd molecules. Anti-HLA-CW3 CTL cross-react to a variable extent on the related alleles A3 and A24. Using a panel of target cells expressing native or recombinant HLA genes, we could map the epitope recognized by a CTL clone specific for CW3 to the second external (alpha 2) domain of CW3. Moreover, the epitope recognized by this clone could be mimicked by incubating P815 (HLA negative) target cells with a synthetic peptide corresponding to the C-terminal 12 amino acids of the CW3 alpha 2 domain (residues 171 to 182). Other independent anti-CW3 CTL clones with different fine specificities recognized the same CW3 peptide. In contrast, CTL clones specific for HLA-A24 or HLA-A3 that did not lyse P815-CW3 transfectants did not recognize this peptide. The CW3 peptide could be recognized on other tumor cell targets that were also of H-2d origin, but not on those of H-2b or H-2k origin. The requirement for the expression of H-2Kd by the target cells was directly demonstrated using L cell Kd transfectants. Our results suggest that the CTL response of DBA/2 mice immunized with P815-CW3 transfectants is predominantly Kd restricted and focused on epitopes contained within the 12 C-terminal amino acids of the alpha 2 domain.     

Structural identity between HLA-A2 antigens differentially recognized by alloreactive cytotoxic T lymphocytes

Alloreactive CTL raised against HLA-A2 Ag often display heterogeneous recognition of HLA-A2+ target cells. This heterogeneity has been found to reflect structural polymorphism among the corresponding target Ag, thus defining HLA-A2 subtypes. A previous study (van der Poel et al. 1986. Human Immunol. 16:247) established the existence of a new HLA-A2.4 variant, A2-SCHU, that was distinguished from A*0206 (A2.4a) by HLA-A2-specific alloreactive CTL. The same CTL subdivided HLA-A2.1 Ag into two subgroups. In the present study, the molecular basis of this heterogeneity has been examined by double-label comparative peptide mapping analysis of differentially recognized A2.1 and A2.4 Ag. In addition, we have determined the complete sequence of polymerase chain reaction-amplified full length cDNA from A2-SCHU. The results show that: 1) A2-SCHU is indistinguishable from A*0206 by peptide mapping; 2) the cDNA sequence of A2-SCHU is identical to that of A*0206; and 3) two differentially recognized A2.1 Ag are both indistinguishable from A*0201 by comparative peptide mapping. These results indicate that differential recognition by alloreactive CTL can occur among structurally identical class I HLA Ag and suggest that allorecognition by such CTL may involve corecognition of endogenous peptides, presumably derived from polymorphic proteins.     

The reovirus nonstructural protein sigma1NS is recognized by murine cytotoxic T lymphocytes.

The cytotoxic T-lymphocyte (CTL) response in reovirus-infected C3H mice was investigated by using reovirus-vaccinia virus recombinants. Results of cytotoxicity assays indicated that the nonstructural protein sigma1NS elicited a significant CTL response. Experiments with sigma1NS-specific CTL lines showed that both strain-specific and cross-reactive epitopes exist in the sigma1NS protein.     

MIs locus recognition by a cloned line of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

This report demonstrates the expression of strong MIs locus MIsd) recognition by a cloned line of H-2-restricted influenza virus-specific CTL. This clone of F1 (H-2b/d; MIsb) origin was found to specifically proliferate in response to uninfected cells of CBA/J (H-2k, MIsd) origin but not to uninfected B10.BR or CBA/CaJ cells (H-2k, MIsb). In addition, proliferation by this cTL line was observed in response to histocompatible cells expressing cross-reactive MIsa determinants (DBA/2, NZB; H-2d, MIsa). This recognition was observed only at the level of CTL proliferation. The CTL line exhibited no cytotoxic activity for target cells of these MIs types. These observations are contrasted with the response of another cloned H-2-restricted influenza-specific CTL line that simultaneously exhibits alloreactivity for H-2k. The significance of these results for T lymphocyte recognition is discussed.     

H-2 antigens incorporated into phospholipid vesicles interact specifically with allogeneic cytotoxic T lymphocytes

Subcellular fractions containing different H-2 antigens were tested for their ability to inhibit specific T cell-target cell conjugate formation. H-2-containing membrane vesicles, lentil-lectin-purified H-2 antigens solubilized with detergent (referred to in the text as high-density fraction) or incorporated into lipid vesicles, inhibited T cell-target cell conjugate formation effectively and specifically. However, two- to threefold more protein was required to inhibit T cell-target cell conjugate formation when detergent-solubilized lentil-lectin-purified H-2 antigens were tested. This suggests that a lipid matrix is advantageous for interaction with anti-H-2 T-cell receptors. Experiments were also undertaken to demonstrate specific binding of liposomes containing ¹²⁵I-labeled H-2 antigen to anti-H-2-specific cytotoxic T lymphocytes (CTLs). The binding of the ¹²⁵I-labeled H-2-containing liposomes was saturable and was specifically inhibited by unlabeled H-2 antigens. Monospecific anti-H-2 sera specifically inhibited the binding of liposomes containing H-2 antigen to the CTLs. The results suggest that a specific interaction can occur between serologically defined H-2 antigens and the receptor of anti-H-2 CTLs.     

Identification of a viral antigen recognized by H-2-restricted cytolytic T lymphocytes on a murine leukemia virus-induced tumor

Monoclonal antibodies were produced against protein p30, a structural protein of murine leukemia viruses (MuLV) coded by the gag gene of MuLV. Three monoclonal antibodies of different isotypes (i.e., IgG-1, IgG-2a, and IgG-2b) were chosen for extensive analysis. These three antibodies bound to mouse tumor cells induced by Friend, Moloney, Rauscher, and Gross MuLV, but not to noninfected normal mouse spleen cells. The ability of these monoclonal antibodies to inhibit cytolytic T lymphocyte (CTL) activity by masking the antigens recognized by CTL on the target cell surface was studied in various CTL systems. It was found that the only CTL that were consistently inhibited in their lytic activity came from BALB.B (H-2b) mice immunized against syngeneic Gross MuLV-induced B.GV cells. These results thus showed that a subpopulation of BALB.B anti-Gross MuLV CTL recognized a Gross MuLV gag gene product expressed on the surface of B.GV cells.     

Characterization of determinants encoded by four Qa-1 genotypes and their recognition by cloned cytotoxic T lymphocytes

The alloantigens encoded by the four defined Qa-1 genotypes were characterized by cloned cytotoxic T lymphocyte (CTL) recognition. CTL clones specific for Qa-1a- and for Qa-1b-encoded antigens were generated. Examination of the reactivity of these clones with target cells from H-2r and H-2f strains provided the strongest evidence to date for the designation of the Qa-1c and Qa-1d genotypes, respectively, for these strains. Qa-1c-encoded antigens were recognized by most, but not all CTL clones that specifically lysed Qa-1b target cells, thus demonstrating that these antigens lack a Qa-1b-associated determinant. Similarly, Qa-1d encoded antigens were recognized by only half of the CTL clones that lysed Qa-1a target cells. In addition, one CTL clone that was cytotoxic for Qa-1b and Qa-1c target cells demonstrated low affinity, cross-reactive recognition of a Qa-1d encoded antigen. The reactivity patterns of the monoclonal CTL defined five Qa-1 determinants. Qa-1a, Qa-1b, and Qa-1d each encode multiple determinants. Two Qa-1d encoded determinants probably reside on different molecular species. Finally, large numbers of CTL clones tested on panels of target cells indicated that the Qa-1a strains expressed indistinguishable Qa-1.1 antigens and the Qa-1b strains expressed indistinguishable Qa-1.2 antigens. Therefore, additional polymorphism among these strains is improbable.     

Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys.

Infection of macaque monkeys with the simian immunodeficiency virus of macaques (SIVmac) results in disease similar to human AIDS. Therefore, the macaque monkey is proving to be an important model for testing the effectiveness of various AIDS vaccine approaches. A detailed analysis of the cellular immune responses is necessary for the evaluation of candidate vaccines. However, this has not been possible in macaques, due, in part, to the unknown nature of the MHC molecules that restrict their T lymphocytes. In our report we demonstrate that a particular MHC class I molecule involved in the rhesus monkey's effector T lymphocyte response to SIVmac is expressed at a high frequency in a colony of rhesus monkeys. SIVmac-infected monkeys that express this MHC class I molecule all develop CTL that are restricted by that molecule and recognize an identical nine amino acid epitope of the SIVmac gag protein. This MHC class I molecule has been defined as an HLA-A homolog by cDNA cloning and sequencing. It has also been expressed in an MHC class I-deficient cell line to demonstrate directly the cloned molecule's capacity to bind and present peptide Ag to CTL. These studies illustrate that AIDS virus-specific CTL can be characterized in detail in the rhesus monkey and lay the foundation for exploring novel approaches to AIDS virus vaccination in this species.     

A third class I major histocompatibility complex antigen encoded by a gene in the D region of the H-2 d haplotype recognized by cytotoxic T lymphocytes

The D region of the H-2 ^d haplotype contains five class I genes: H-2D ^d , D2 ^d , D3 ^d , D4 ^d and H-2L ^d . Although previous studies have suggested the presence of D-end encoded class I molecules in addition to H-2D^d and H-2L^d, segregation of genes encoding such molecules has not been demonstrated. In this report we have used cãtotoxic T lymphocytes (CTL) to examine the D region of the H-2 ^d haplotype for the presence of additional class I molecules. CTL generated in (C3H × B6.K1)F₁ (K ^k D ^k , K ^b D ^b ) mice against the hybrid class I gene product Q10^d/L^d expressed on L cells cross-react with H-2L^d but not H-2D^d molecules, as determined by lysis of transfected cells expressing H-2L^d but not H-2D^d. Although H-2L^d-specific monoclonal antibodies (mAb) completely inhibit H-2L^d-specific CTL from killing B10.A(3R) (K ^b D ^d L ^d ) target cells, only partial inhibition of anti-Q10 CTL-mediated lysis was observed, suggesting the presence of an additional D-end molecule as a target for these latter CTL. To identify the region containing the gene encoding the Q10 cross-reactive molecule, we show that anti-Q10 CTL lyse target cells from a D-region recombinant strain B10.RQDB, which has H-2D ^d , D2 ^d , D3 ^d , D4 ^d , and H-2D ^b but not the H-2L ^d H-2 ^d , and H-2L ^d (including D2 ^d , D3 ^d , and D4 ^d , lacks this anti-Q10 CTL target molecule. Together, these data demonstrate that a class I gene mapping between H-2D ^d and H-2L ^d encodes an antigen recognozed by anti-Q10 CTL. A likely candidate for this gene is D2 ^d , D3 ^d or D4 ^d .     

Clonal analysis of the anti-Qa-1 cytotoxic T lymphocyte repertoire: definition of the Qa-1d and Qa-1c alloantigens and cross-reactivity with H-2

To characterize the four common Qa-1 allelic products, we examined in detail the CTL-defined determinants encoded by Qa-1. In previous studies with anti-Qa-1 CTL and alloantisera, investigators have described antigenic determinants present on Qa-1a and Qa-1b antigens, but they have defined Qa-1c and Qa-1d exclusively by their cross-reactivity with Qa-1a and/or Qa-1b determinants. To delineate further the CTL-defined determinants encoded by Qa-1d, we generated CTL clones with Qa-1d specificity and demonstrated that the Qa-1d molecule expressed determinants that were not detected on Qa-1a, Qa-1b, or Qa-1c target cells. Other CTL clones derived from anti-Qa-1d MLC recognized new antigenic determinants on Qa-1c that cross-reacted with Qa-1d. Each of the four common Qa-1 phenotypes was shown to exhibit unique antigenic determinants. In addition, Qa-1d anti-Qa-1a and Qa-1d anti-Qa-1b CTL confirmed extensive cross-reactivity among these Qa-1 alloantigens. Analysis of CTL from these four immunizations also resulted in the isolation of Qa-1a-specific and Qa-1d-specific CTL clones that cross-reacted with H-2Df and H-2Ks, respectively.     

A single retroviral gag precursor signal peptide recognized by FBL-3 tumor-specific cytotoxic T lymphocytes.

Several dominant T-cell receptors of cytotoxic T-lymphocyte (CTL) clones specific for FBL-3 tumor antigen were clonally amplified in mixed lymphocyte tumor cell cultures derived from an individual immune mouse. Every CTL clone analyzed had a common specificity for a single epitope in the precursor to cell membrane-associated nonstructural gag-encoded protein, Pr75gag, which can be minimally identified by nine amino acid residues, SIVLCCLCL. This epitope is located within the hydrophobic signal sequence motif that mediates translocation of the protein into the endoplasmic reticulum. These novel observations suggest that expression of Pr75gag in FBL-3 tumor cells led to the amplification of CTLs which recognize the signal sequence of the nonstructural gag-encoded glycoprotein precursor.