Polymorphic and autoreactive H-2-specific monoclonal antibody isolated after injections of syngeneic sendai virus-coated lymphocytes

An H-2-specific monoclonal antibody (mAb Q-1) was obtained from B10.Q (H-2 ^q) mice injected with syngeneic Sendai virus-coated cells. The IgM monoclonal antibody recognizes the public determinant H-2.25 shared by H-2 ^k (K ^k) and H-2 ^r haplotypes and cross-reacts with H-2^d, H-2^s, H-2^p, and H-2^q cells, the latter being the haplotype of the challenged B-cell donor. The binding of mAb Q-1 to H-2^d, H-2^s, H-2^q, and H-2^p cells was lower than to H-2^k and H-2^r and of decreasing affinity but could be clearly distinguished from the negative reactions with H-2^b and H-2^f cells. MAb Q-1 distinguishes between Sendai virus-coated and uncoated lymphocytes only cells with low-affinity binding. On virus-coated or infected (H-2^p, H-2^q, H-2^d, H-2^s) cells lysis was stronger than on normal lymphocytes. We interpret the enhanced lysis of Sendai virus-positive cells by mAb Q-1 to be due to recognition of a modified exposure of public H-2 determinants induced by Sendai virus.On leave from The Institute of Immunology and Experimental Therapy, Wroclaw, Poland     

Recognition of Nonstructural Protein Peptides by Cytotoxic T Lymphocytes Raised against Japanese Encephalitis Virus

We have previously reported that Lyt2⁺ cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K^d-restricted CTL and to examine their cross-recognition of West Nile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K^d binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.     

Dissociation ofH-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2 ^b /H-2 ^b ). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (allH-2 ^b /H-2 ^b ), FY7 failed to induce the primary in vitro generation of anti-H-2^b CTL by (B10.A x A)F₁ (H-2 ^a /H-2 ^a or (B10.D2 x BALB/c)F₁ (H-2 ^d /H-2 ^d ) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2^b CTL. Quantitative absorption tests with H-2K^b and H-2D^b antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2K^b product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2K^b CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2K^b-gene product expressed by FY7.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PAGE polyacrylamide gel electrophoresis     

Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity

In preparation for functional analyses, a study of the binding of H-2K^b-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2^b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2K ^bm mutant cells and on FOR-treated H-2K^b cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2KP^b-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2^b cells as indicated by cold target inhibition studies. The H-2K^b-specific CTL were probably reactive with conformational determinants of H-2K^b, which are dependent on the integrity of both the 1 and the 2 domains of the H-2K^b molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the 1 domain without affecting conformational-type CTL determinants.Abbreviations used in this paper CTL cytotoxic T lymphocyte - FOR formaldehyde - PBS phosphatebuffered saline - FCS fetal calf serum - mAb monoclonal antibody - TNBS trinitrobenzene sulfonate     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

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 .     

Anti-MHC immunity detected prior to intentional alloimmunization

Cell fusion was performed between spleen cells from young BALB/cBy (H-2 ^d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2-specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2K^d, D^d, and H-2^s antigens, gave weak cross-reactions with H-2K^k, D^q, H-2^r, and H-2^v antigens and was negative with H-2^b, H-2^f, H-2^p, and H-2L^d antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B 10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2^d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2^d cells and by studies on H-2^d-transfected mouse L cells, the target molecules for McAb By-1 were H-2K^d and H-2D^d molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.Abbreviations McAb monoclonal antibody - MHC major histocompatibility complex - H-2 major histocompatibility complex of mice - CTLs cytotoxic T cells - FMF flow microfluorometry - FITC fluorescein isothiocyanate - LPS lipopolysaccharide W.E. coli 0111:134 - PBS phosphate-buffered saline - Iodogen 1,3,4,6,-tetrachloro-3,6-diphenylglycoluril - GAMIg goat-antimouse immunoglobulin - Staph-A Staphylococcus aureus Cowan I     

A new determinant,Qa-m208, detected on T lymphocytes and transfected L cells by a Kb-specific monoclonal antibody

A K^b-specific monoclonal antibody, 6.3.4, defining a new class I specificity, m208, reacts with some K and D region products and with a Qa determinant present on T lymphocytes but not detectable on thymocytes and B lymphocytes. The strain distribution of reactions indicates that this determinant is controlled by the Qa-Tla region, and shows no concordance with the strain distribution pattern of any of the known Qa antigens. The antibody reacts also with L cells expressing a cloned H-2 ^b class I gene mapping in the Qa-Tla region.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.     

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     

Definition of Amino Acid Residues on the Epitope Responsible for Recognition by Influenza A Virus H1-Specific, H2-Specific, and H1- and H2-Cross-Reactive Murine Cytotoxic T-Lymphocyte Clones

We defined the epitopes recognized by three influenza A virus-specific, H-2K^d-restricted CD8⁺ cytotoxic T-lymphocyte (CTL) clones: H1-specific clone A-12, H2-specific clone F-4, and H1- and H2-cross-reactive clone B7-B7. The A-12 and B7-B7 clones recognized the same peptide, which comprises amino acids 533 to 541 (IYSTVASSL) of A/PR/8 hemagglutinin (HA). The F-4 and B7-B7 clones both recognized the peptide which comprise amino acids 529 to 537 (IYATVAGSL) of A/Jap HA. Amino acids 533 to 541 of A/PR/8 HA are compatible with amino acids 529 to 537 of A/Jap HA. Amino acid S at positions 3 and 7 was responsible for recognition by H1-specific clone A-12, while amino acid G at position 7 was responsible for recognition by H2-specific clone F-4. Two conserved amino acids, T at position 4 and A at position 6, were responsible for recognition by H1-, and H2-cross-reactive clone B7-B7. These results indicate that a single nine-amino-acid region is recognized by HA-specific CTL clones of three different subtype specificities and that the amino acids responsible for the recognition by the CTL clones are different.     

Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

Variation of expression of histocompatibility antigens on tumor cells: absence of H-2Kk-gene products from a gross-virus-induced leukemia in BALB.K

The antigenic profile of the K-GV tumor of BALB.K origin, induced by Gross virus and maintained in vitro and in vivo, was investigated by serological and immunochemical methods and techniques of cell-mediated immunity. The H-2K^k-gene products were absent by several criteria: (1) monoclonal antibody and conventional alloantisera directed against the H-2K^k antigenic specificities were nonreactive by direct testing and by absorptions. (2) H-2K^k products could not be precipitated from glycoprotein or protein extracts of the radiolabeled K-GV tumor. (3) Cytotoxic effectors against H-2K^k produced by sensitization in vitro and in vivo failed to kill K-GV target cells. (4) The tumor could neither stimulate BALB.B congenic mice to produce cytotoxic effectors nor specific cytotoxic antibody against H-2K^k-gene products. In contrast, the H-2D^k antigen was readily detectable by all these criteria. These findings therefore describe a tumor which has selectively lost the H-2K-gene products. The K-GV tumor was able to generate Gross-virus-specific CTL, but had greatly reduced susceptibility to lysis by Gross-virus-specific CTL generated by H-2K expressing AKR (H-2 ^k) tumors. These findings have important implications for the associative recognition of tumor antigens and the immune surveillance of virally induced tumors.Abbreviations used in this paper MHS major histocompatibility system - LcH Lens culinaris hemagglutinin - SDS sodium dodecyl sulfate - CTL cytotoxic T lymphocytes - GCSA Gross-virus-induced cell-surface antigen - MuLV murine leukemia virus     

Interactions between MHC-encoded products and cloned T-cells. I. Fine specificity of induction of proliferation and lysis

To study the interactions between T cells and class I MHC products, we developed in vitro a T-cell line reactive to H-2K^b stimulating cells and derived T-cell clones from it. Although the T-cell line could proliferate in the absence of exogeneous T-cell growth factors when stimulated with H-2K^b spleen cells, each of the derived T-cell clones required both H-2K^b stimulating cells and an external source of T-cell growth factor for its propagation. Each of the T-cell clones was also cytolysic for H-2K^b target cells. Such T-cell clones allowed the comparison of the antigenic requirements for proliferation and cytolysis. By using H-2K ^b mutant mice, we found that while the original anti-H-2K^b T-cell line reacted with each of the six mutants tested, the individual T-cell clones could be distinguished in terms of their reactivity pattern. Similar fine specificity patterns were found when H-2K ^b mutant cells were used as stimulating or target cells for any given T-cell clone. Each of the three monoclonal H-2K^b-specific antibodies reacting with different epitopes of the H-2K^b molecule totally inhibited H-2K^b-induced proliferation and lysis by the T-cell clones. Further blocking studies involved use of Fab antibody fragments and definition of their reactivity on cells from the H-2K ^b mutants. We concluded that: (1) blocking with a monoclonal antibody does not prove identity of alloantigens recognized by the T-cells and the antibody; (2) a monoclonal antibody could either block or not block H-2K^b-CTL interactions depending on structural variations of the H-2K^b molecule not affecting the CTL-H-2K^b functional interaction; (3) blocking one type of H-2K^b-T-cell interaction (induction of proliferation) always affects the other type (cytolysis).Abbreviations used in this paper MHC major histocompatibility complex - CTL cytotoxic - T lymphocytes - Th T helper cells - PMA 4-phorbol 12-myristate 13-acetate - Con A Concanavalin A - LPS E. coli lipopolysaccharide - SCA Con A stimulated rat spleen-cells supernatant - SBD B6 anti-DBA/2 mixed lymphocyte culture supernatant - TCGF T-cell growth factors - IL-2 interleukin 2 - mAb monoclonal antibody - FCS fetal calf serum - PBS phosphate buffered saline - C complement     

Immunoselection of structural H-2Kb variants: Use of cloned cytolytic T cells to select for loss of a CTL-defined allodeterminant

Functional studies concerning the unique interaction between class I H-2 allodeterminants and cytolytic T lymphocyte (CTL) antigen receptors have benefitted from the development of H-2K^b mutant mouse strains and somatic H-2 variants selected with monoclonal antibody. Here, we describe the development of a novel approach to immunoselection of somatic H-2K^b variants employing a K^b-specific CTL clone as the negative selective agent. The rationale for this method is that the use of an alloreactive CTL clone as the selective agent should enable us to detect the emergence of structural K^b variants based on their loss of the relevant CTL-defined allodeterminant. Thus, these structural variants are well suited to an in-depth analysis of the functional relationship between H-2 antigens and receptor recognition by CTL. Using this approach, we successfully isolated two types of structural K^b variants, as well as numerous Kb-loss variants. The functional studies described in this paper indicate that these structural variants exhibit alterations in expression of both CTL-defined and serologically defined H-2K^b allodeterminants. The structural characterization of such variants should enable us to identify the precise amino acid residues responsible for the creation of the relevant CTL-defined K^b allodeterminants.     

Interaction between MHC-encoded products and cloned T cells

The interaction between class I major histocompatibility complex (MHC) products and T cells was studied using H-2K^b-specific alloreactive T-cell lines and clones obtained by repeated in vitro stimulation with allogeneic cells. Induction of proliferation of these T cells appeared to involve two signals: the H-2K^b alloantigen and interleukins. Immunopurified liposome-inserted H-2K^b, which stimulates specific secondary in vitro cytotoxic T lymphocyte (CTL) responses, could not replace cell-associated H-2K^b in the stimulation of these T-cell lines, even in the presence of feeder cells and interleukins. When T-cell lines were initiated in vitro and repeatedly stimulated with H-2K^b liposomes and feeder cells, it was possible to obtain T cells that could proliferate in response to H-2K^b liposomes in the presence of feeder cells and interleukin-2-containing supernatants or on H-2K ^b -expressing cells. Only stimulation with cells permitted maintenance of these T cells in culture for more than 12 weeks. Analyses of cell surface markers and of patterns of inhibition of proliferation by monoclonal antibodies (mAb) of T-cell lines induced in vitro with cell- or liposome-associated H-2K^b indicated that T-cell stimulation by class I antigen can occur in at least two ways. In the first, the H-2K^b-induced proliferation of Lyt-1^- Lyt-2⁺ T4^- T cells is inhibited by H-2K^b- and by Lyt-2-specific mAb, but not by Ia or T4-specific mAb. In the second, both Lyt-2⁺ and T4⁺ T cells are involved and the H-2K^b-induced proliferation is inhibited by H-2K^b- and Lyt-2-specific mAb and by Ia- and T4-specific mAb.Abbreviations used in this paper Ab antibody - mAb monoclonal antibody - C complement - i.p. intraperitoneally - PBS phosphate-buffered saline - PBS-B-N PBS containing bovine serum albumin and NaN₃ - CTL cytotoxic T lymphocyte - Th T helper cell - MHC major histocompatibility complex - PMA 4-phorbol 12-myristate 13-acetate - SCA concanavalin A-stimulated rat spleen cell supernatant - SC16 EL4 clone 16 supernatant - IL-1 interleukin-1 - IL-2 interleukin-2 (T-cell growth factor) - FCS fetal calf serum - H-2K^b-lip. H-2K^b inserted in liposomes - C. E. cell equivalents     

Class I MHC molecules rather than other mouse genes dictate influenza epitope recognition by cytotoxic T cells

Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147–161 as the epitope seen in conjunction with K ^d , the only H-2^d class I responder allele for NP restriction. H-2 ^d /H-2 ^b F₁ mice (C57BL × DBA/2) primed by influenza infection lyse only H-2^d target cells treated with peptide 147–161 while H-2^b targets are recognized only after treatment with NP residues 365–379 (previously found to be recognized by D^b restricted Tc cells). Tc cell recognition of NP peptide 147–161 is entirely dictated by expression of K ^d and not by other B10 or OH background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.     

Cytotoxic T lymphocytes recognize determinants on the BALB/c-H-2Ld molecule controlled by α1 and α2 but not α3 external domains

We have shown that cytotoxic T lymphocytes (CTL) raised in H-2 ^dmice use H-2L^d but not H-2D^d or H-2K^d antigens as restricting elements in lymphocytic choriomeningitis virus (LCMV) and vesicular stomatis virus (VSV) infections. To localize the regions of H-2L^d protein recognized by CTL, we constructed a recombinant H-2L ^d/D ^dgene encoding a hybrid antigen with 1 and 2 external domains of H-2L^d and 3, transmembrane and cytoplasmic domains of H-2D^d. The recombinant gene was transfected into mouse cells and the hybrid molecules were characterized serologically, biochemically and functionally. In all assays, H-2L^d/D^d molecules were recognized by LCMV- and VSV-specific H-2L^d-restricted CTL in a manner similar to that of wild-type H-2L^d antigens. Analogous results were obtained with alloreactive CTL. Hybrid antigens containing the 3 domain of H-2L^d fused to 1 and 2 domains of a Qa-2,3 region-encoded antigen were not used as restricting elements by LCMV-specific CTL. These results suggest that H-2L^d-restricted CTL directed against LCMV and VSV recognize determinants controlled by the 1 and/or 2 domains of the H-2L^d molecule.Abbreviations used in this paper CTL cytotoxic T lymphocytes - VSV vesicular stomatitis virus - LCMV lymphocytic choriomeningitis virus - tk thymidine kinase - HAT hypoxanthine, aminopterine, thymidine - HSV herpes simplex virus - FCS fetal calf serum - SAC Staphylococcus aureus Cowan I strain - TM transmembrane - CYT cytoplasmic     

Fine specificity of cytotoxic T lymphocytes directed againstH-2L d

The fine specificity of cytotoxic T lymphocytes (CTL) directed againstH-2L ^d was analyzed by studying the lytic activity of BALB/cH- 2^dm2 (H-2L ^d loss mutant) anti-BALB/c-H-2 ^d CTL, generated in secondary mixed lymphocyte culture (MLC) against a panel of target cells of differentH-2 haplotypes. Target cells of allH-2 haplotypes tested, except that of the MLC responder, were lysed by anti-L^d CTL, although to a widely varying extent. The genes coding for antigens detected by anti-L ^d CTL were mapped to distinct regions in theH-2 ^d ,H- 2^dm1,H-2 ^q ,H-2 ^k , andH-2 ^b haplotypes. The sequence of lysis intensity against the variousH-2 haplotypes and theH-2 regions involved were as follows:L ^d ,D ^q L ^q ,D ^dm1 L^dm1,K ^k ,D ^b L ^b ,r, p, f, s, C3H.OH (K ^d D ^k L ^k ), strong lysis occurring againstL ^d and weak lysis againstH-2 ^s and C3H.OH.By monolayer adsorption and cold target inhibition experiments, it was shown that anti-L ^d CTL contained a CTL subset directed against a private L^d specificity, hitherto undetected by anti-L ^d antibodies. This subset of CTL was separate from the CTL subsets reacting againstH-2 ^q and against the mutant haplotypeH- 2^dm1. The reactions against the latter two haplotypes were also mediated by separate CTL subsets. It is concluded that the L^d molecule, to a varying extent, shares target antigens for CTL with K- and/or D-end H-2 molecules of all haplotypes tested. These antigens are detected by multiple subsets of anti-L ^d CTL. One CTL subset is directed against a target structure unique forL ^d (L^d private specificity).     

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.