Role of self and foreign antigenic determinants in allogeneic and self- restricted cytotoxic T cell recognition

Murine spleen cells were sensitized in vitro to H-2 disparate allogeneic spleen cells and assayed on syngeneic target cells conjugated with the trinitrophenyl (TNP)-self or the fluorescein isothiocyanate (FITC)-self haptens, or on syngeneic target cells expressing the male H-Y antigen (H-Y self). The results indicated that allo-induced cytotoxic T lymphocytes (CTL) contained effectors that lysed both hapten-self but not H-Y self targets. Furthermore, it was demonstrated that separate populations of those allogeneic CTL were responsible for the lysis of TNP-self and FITC-self targets. This study also showed that cytotoxic effectors generated against the H-Y antigen with lytic activity equal to or greater than that of an allogeneically induced CTL response were unable to lyse hapten-self targets. These findings provide the first evidence that H-2 alloantigens may be unique in their ability to induce effectors that lyse hapten-conjugated autologous targets. These observations are discussed with respect to the self and foreign antigenic determinants involved in allogeneic and self-restricted CTL models.     

Specificity of cytotoxic effector cells directed against trinitrobenzene sulfonate-modified syngeneic cells. Failure to recognize cell surface- bound trinitrophenyl dextran

Mouse splenic lymphocytes and lymphoid tumor cells were modified with the trinitrophenyl (TNP) group either by treatment with trinitrobenzene sulfonate (TNBS) (which covalently modifies cell surface proteins) or with TNP stearoyl dextran (TSD) (which binds to the cell by noncovalent forces). These cell preparations were compared for their ability to: (a) sensitive syngeneic splenic lymphocytes leading to the generation of cytotoxic effector cells; (b) serve as lysable targets in a 4-h(51)Cr- release assay for effector cells generated in (a); and (c) act as blocking cells in the lysis of TNBS-medified targets lysed by TNP self effector cells generated in (a). In none of these three experimental systems did TSD-medified syngeneic spleen or H-2-matched tumor cells act either as a sensitizing immunogen or as a target antigen, despite the demonstration that quantitatively equivalent mounts of TNP were exposed on the cell surface in the TNBS- and TSD-modified cells. In contrast, TNBS-modified spleen cells sensitized syngeneic lymphocytes to generate effectors against TNBS-modified syageneic targets. Furthermore, TNBS- modified, H-2-matched cells served as specific lysable targets and as inhibiting cells for such effectors. These results indicate that the manner in which TNP is associated with the cell surface is important in the immunogenicity and antigenicity of hapten-modified syngeneic stimulating cells in generating H-2-associated cell-mediated lympholysis (CML) reactions. These findings raise the possibility that a covalent or at least a stable linkage with cell surface proteins (possibly H-2- controlled products) is important for immunological function. Furthermore, these observations do not favor the dual receptor model for H-2-restricted syngeneic CML if it is assumed in such a model that one receptor is specific for the TNP moiety and the second for unmodified self major histocompatibility products.     

Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Confirmation of genetic control of response to trinitrophenyl-modified H-2 antigens by the use of anti-H-2 and anti-Ia antibodies

Splenic lymphocytes from B10.A and B10.D2 mice were sensitized in vitro to trinitrophenyl (TNP)-modified autologous spleen cells. The effector cells generated were assayed in a 51Cr-release assay on TNP-modified syngeneic or congenic spleen target cells. Effector cells from B10.A donors lysed TNP-modified H-2Kk- but not H-2Dd-region products, whereas B10.D2 effectors reacted with modified products of both the H-2Kd and H-2Dd regions. As an independent confirmation that this selective K-end lysis by B10.A effector cells is due to an H-2-linked responder cell defect (4), anti-H-2Kk but not anti-H-2Dd sera were shown to inhibit the lysis of B10.A-TNP targets by B10.A effectors. In contrast, anti-H-2Dd sera inhibited the lysis of B10.A-TNP targets by B10.D2 effectors. Anti-Ia antibodies had no detectable effect on lysis. Anti-TNP-keyhole limpet hemocyanin sera blocked the lysis of TNP-modified targets, irrespective of whether the effector cells were directed against TNP-modified autologous H-2 products or H-2 alloantigens. These results independently verify that B10. A responding lymphocytes do not generate effector cells to TNP-modified H-2Dd products, whereas B10.D2 lymphocytes do (4), and suggest that some TNP groups are sterically close to (or part of) the serologically defined H-2K- and H-2D-region antigens.     

Genetic control of cytolytic T-lymphocyte responses. I. Ir gene control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells

The ability of cytotoxic T lymphocytes (CTL) induced in vitro to trinitrophenyl (TNP)-modified syngeneic cells to cross-reactively lyse a TNP allogeneic spleen target varies among inbred mouse strains. The cross-reactive CTL phenotype was found to be histocompatibility 2 (H-2) linked and to be dominant in F1 hybrid mice. All strains investigated demonstrated cross-reactivity except for some strains bearing portions of the H-2k haplotype. The gene(s) controlling this response maps to the K and/or I-A region of the H-2 complex. We have termed the immune response (Ir) gene responsible for controlling the specificity of CTL induced to TNP-modified syngeneic cells Ir-X-TNP.     

H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.     

Genetic control of cytolytic t-lymphocyte responses. II. The role of the host genotype in parental leads to F1 radiation chimeras in the control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells

Bone marrow cells from C3H (H-2k) mice, a strain that does not exhibit cross-reactive lysis of trinitrophenyl (TNP)-modified allogeneic targets, were allowed to mature in heavily irradiated (B6 times C3H)F1 (H-2b/k) recipients, an F1 hybrid that does demonstrate cross-reactive lysis. Spleen cells from these chimeric mice were removed after 3-4 mo and by H-2 typing shown to be of C3H origin. These cells were found to be tolerant to B6 alloantigens by mixed lymphocyte reaction and cell-mediated cytotoxicity and, when stimulated in vitro with TNP-modified syngeneic cells, now cross-reactively lysed TNP-modified allogeneic targets. These studies demonstrate that the host environment where T cells differentiate influences the specificity of the primary cytolytic T-lymphocyte (CTL) response to TNP-modified syngeneic antigens.     

On the role of the H-2 histocompatibility complex in determining the specificity of cytotoxic effector cells sensitized against syngeneic trinitrophenyl-modified targets

Spleen cells cultured with syngeneic trinitrophenyl (TNP)-modified stimulator cells display a cytotoxic effect against syngeneic TNP- modified targets, but not against modified targets from unrelated H-2 haplotypes. Targets that share the K and I region of the H-2 complex with the stimulator (or effector) cell are lysed to the same extent as the specific targets, while targets that share the I region only are not. When only the D region is shared, a weak cytotoxic effect is observed. Therefore, the stimulator (or effector) and target cell must share the K or D but not the I region of the H-2 complex in order for optimal cytotoxicity to occur. Spleen cells sensitized to irradiated TNP-modified H-2-allogeneic cells are cytotoxic to these specific cells. Coculture of F1 hybrid cells with irradiated TNP-modified parental cells result in a cytotoxic effect against only those specific parental cells and not TNP-modified cells from the other parent. The cytotoxic effect of the F1 effector cells in the cell-mediated lympholysis test is blocked by the addition of unlabeled TNP-modified targets that are H-2 syngeneic with the sensitizing parental strain, but not H-2 syngeneic with the other parental strain. These data demonstrate that the specificity of the effector cell in this syngeneic cytotoxicity system is directed against altered self H-2-controlled- gene products, rather than a requirement for sharing of histocompatibility genes between effector and target cell in order for lysis to occur. The role of H-2 antigens in determining the sensitivity of a target cell to T-cell-mediated lysis is discussed.     

Cell-mediated lympholysis of N-(3-nitro-4-hydroxy-5-iodophenylacetyl)- beta-anaylglycylglycyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by the H-2K and H-2D serological regions of the murine major histocompatibility complex

Splenic lymphocytes from four C57BL/10 congenic mouse strains were sensitized in vitro to N(-3-nitro-4-hydroxy-5-iodophenylacetyl)-beta-alanylglycylglycyl-(N) modified autologous lymphocytes. The effector cells generated after 5 days of culture were assayed on a series of either N-modified phytohemagglutinin-stimulated spleen cells or N-modified tumor cells. The results indicated in all cases that both N modification of the targets and H-2 homology between the modified stimulating and target cells are required for lysis to occur. In each case the effector cells were found to lyse N-modified target cells only when there was homology at either or both ends of the major histocompatibility complex (MHC) between the stimulator and target cells. B10.BR lysed targets sharing alleles at K (or K plus I-A) and/or at D. B10.A effector cell specificity was mapped to K (or K plus I-A) and/or the D half of the MHC (D or D plus I-C and/or S). The two regions of specificity determined for B10.D2 effector cells were D (or D plus S plus I-C) and a region not including D of the MHC. C57BL/10 effector cells lysed N-modified targets only if there was target cell H-2 homology at K, I-A, and I-B or at the D serological region. As in the trinitrophenyl (TNP) system (6) B10.BR and B10.A effector cells lysed targets sharing K end H-2 serological regions greater than target cells sharing D-end serological regions. The C57BL/10 effector cells were shown to react to the K end greater than the D end, which differed from the equal reactivity seen in the TNP system for this strain. The data are consistent with the hypothesis that the antigen recognized by the effector cell includes an altered H-2 serological cell surface product. That the reaction is not "hapten specific" and the H-2 homology is required only for effector:target cell interaction was excluded by the use of two F1 combinations in which lysis of only N-modified target cells sharing the H-2 haplotype with the stimulating parental strain was obtained. Finally, it was demonstrated that N and TNP modification create distinct new antigenic determinants, since an effector cell sensitized to one modifying agent will lyse only H-2 matched target modified with that same modifying agent.     

H-2-linked genetic control of murine T-cell-mediated lympholysis to autologous cells modified with low concentrations of trinitrobenzene sulfonate

Spleen cells from B10.BR and C57BL/10 (B10) mice were compared for their ability to generate primary in vitro cytotoxic responses to syngeneic cells modified with different concentrations (from 10 to 0.031 mM) of trinitrobenzene sulfonate (TNBS) (TNP-self). Although both strains generated effector cells to TNP-self in the range of 10-0.25 mM TNBS modification, effector activity of B10 cells was weaker than that of B10.BR cells. B10 spleen cells did not respond to syngeneic stimulating cells modified at 0.1 mM or lower, whereas B10.BR cells generated effector activity even when stimulated by TNP-self modified with as low as 0.031 mM TNBS. Fluorescence analysis of the modified cells using the FACS II indicated that equivalent quantities of TNP were conjugated to the surfaces of B10.BR and B10 spleen cells for any given concentration of TNBS modification. Similar strain-dependent differences were observed when the TNP was diluted out in the cultures by reducing the number of stimulating cells modified with 10 mM TNBS. These response patterns were verified by stimulating cultures of B10.BR and B10 spleen cells either with TNP conjugated to bovine serum albumin or bovine gamma globulin (B10.BR but not B10 cells responded to TNP-conjugated proteins) or with TNBS-modified glass-adherent spleen cells. The strain-dependent differences could also be detected at the effector phase, because optimally stimulated B10.BR, but not B10 effector cells, could lyse 0.1 mM TNBS-modified syngeneic target cells. The genetic parameters associated with the response and nonresponse patterns of B10.BR and B10 mice were further investigated by comparing the cytotoxic responses to low doses of TNP-self of spleen cells from the following strains: (a) C3H/HeJ (H-2k) and C3H.SW (H-2b); (b) BALB.K (H-2k) and BALb.b (h-2b); and (c) B10.A (H-2a) and B10.D2 (H-2d). The H-2k and H-2a, but not the H-2b and H-2d, strains generated cytotoxic responses to TNP-self when the syngeneic stimulators were modified with 0.1 mM TNBS. Further studies using (B10 X B10.BR)F1 responding cells and parental or F1-modified stimulating cells, indicated that the F1 cells generated cytotoxic activity to low doses of TNP in association with H-2k but not in association with H-2b self products. The results of this study indicate that H-2-linked genetic factors, expressed in the target as well as in the responding and/or stimulating cell populations, control the ability of inbred mouse strains to generate cytotoxic effector cells to low doses of TNP-self. Such dose-dependent genetic effects may be important in the regulation of immune responses activated in vivo by chronic exposure to infectious agents.     

Participation of the H-2 antigens of tumor cells in their lysis by syngeneic T cells

Cytotoxic T lymphocytes were generated in vitro against H-2 compatible or syngeneic tumor cells. In vitro cytotoxic activity was inhibited by specific anti-H2 sera, suggesting that H-2 antigens are involved in cell lysis. Two observations directly demonstrated the participation of the H-2 antigens on the tumor cells in their lysis by H-2-compatible T cells. First, coating of the H-2 antigens on the target tumor cell reduced the number of cells lysed on subsequent exposure to cytotoxic T cells. Second, when cytotoxic T cells were activated against an H-2 compatible tumor and assayed against an H-2-incompatible tumor, anti-H-2 serum that could bind to the target cell, but not to the cytotoxic lymphocyte, inhibited lysis. H-2 antigens were also shown to be present on the cytotoxic lymphocytes. Specific antisera reacting with these H-2 antigens, but not those of the target cell, failed to inhibit lysis when small numbers of effector cells were assayed against H-2-incompatible target cells or when effector cells of F1-hybrid origin and bearing two H-2 haplotypes were assayed against a tumor cell of one of the parental strains. These findings suggest that it is the H-2 antigens on the tumor cell and not those on the cytotoxic lymphocytes that are important in cell-mediated lysis of H-2-compatible tumor cells.     

Production of target-specific effector cells using hetero-cross-linked aggregates containing anti-target cell and anti-Fc gamma receptor antibodies

Rabbit anti-2,4-dintrophenyl (DNP) antibodies or their F(ab')2 fragments were chemically cross-linked to the anti-mouse Fc gamma R monoclonal antibody 2.4G2 or to its Fab fragment. P388D1 cells were incubated with heteroaggregates between 2.4G2 and anti-DNP (anti-Fc gamma R X anti-DNP) and washed. The resulting cells lysed 2,4,6- trinitrophenyl chicken erythrocytes (TNP CRBC) in a hapten-specific manner. The lysis was inhibited by free hapten but was resistant to inhibition by immune complexes. Other cells coated with antibody heteroaggregates also mediated lysis of TNP-modified target cells. For example, mouse resident peritoneal exudate cells (PEC) lysed TNP CRBC and bacillus Calmette-Guerin-activated PEC lysed both TNP CRBC and TNP tumor targets. Human neutrophils, when incubated with heteroaggregates containing the anti-human neutrophil Fc gamma R antibody 3G8 and anti- DNP also lysed TNP CRBC and TNP-modified tumor cells. To test whether linkage to Fc gamma R was required for lysis, F(ab')2 fragments from the anti-KdDd monoclonal antibody 34-1-2 were cross-linked to anti-DNP F(ab')2 fragments. P388D1 cells (which express Kd and Dd) were then incubated with these heteroaggregates and washed, and their abilities to form conjugates and lyse TNP CRBC were compared with those of P388D1 cells treated with anti-Fc gamma R X anti-DNP. In both cases, P388D1 cells formed conjugates. However, only the cells treated with anti-Fc gamma R X anti-DNP mediated lysis to a significant extent. We conclude that heteroaggregates containing anti-Fc gamma R and anti-target cell antibodies can be used to create potent effector cells against red cell and tumor targets and that bridging of effectors with target cells directly to Fc gamma R on effector cells is required for lysis.     

Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.     

H-2 restriction of virus-specific cytotoxicity across the H-2 barrier. Separate effector T-cell specificities are associated with self-H-2 and with the tolerated allogeneic H-2 in chimeras

During infection with lymphocytic choriomeningitis or vaccinia virus, F1 irradiation chimeras reconstituted with bone marrow cells from or both parents generate cytotoxic T cells which can lyse targets across the H-2 barrier. However, activity of chimera T cells is H-2 restricted as shown by cold target competition experiments and selective restimulation of a secondary response in vitro; T cells of H-2k specificity which lyse tolerated infected H-2d target cells do not lyse infected H-2k or unrelated target cells and vice versa. Therefore, H-2 restriction of virus-specific cytotoxic T cells probably does not reflect need for like-like self-interactions for lysis to occur. The specificity of virus immune T cells is thus determined by the H-2K and H-2D specificities present in the infected animal and which are probably recognized unidirectionally by T cells. The results are compatible with the idea the T cells are specific for "altered alloantigen", i.e., a complex of cell surface marker and viral antigen. Alternatively, explained with a dual recognition model, T cells may possess two independently, clonally expressed receptors, a self-recognizer which is expressed for one of the syngeneic or tolerated allogeneic K or D "self" markers, and an immunologically specific receptor for viral antigen.     

T-cell-mediated cytotoxic immune responses to F9 teratocarcinoma cells: cytolytic effector T cells lyse H-2-negative F9 cells and syngeneic spermatogonia

Murine thymus derived (T) lymphocytes primed in vivo to mouse 129 (H-2bc) derived H-2-negative F9 embryonal carcinoma cells and rechallenged in vitro with X-irradiated F9 stimulator cells differentiated into anti-F9 cell immune cytotoxic T lymphocytes (CTL). Using CBA mouse derived splenic responder T cells, F9 stimulator cells triggered a primary cytotoxic anti-F9 response. The CTL generated lysed the F9 antigen-positive target cells F9. PCC3 and PCC4, but not the F9 antigen-negative mouse 129 derived PYS tumor cells, nor LPS induced H-2bc blast cells. Mouse 129 anti-F9 cell antisera but not H-2k anti-H-2bc antisera blocked the lytic interaction with F9 target cells. Similarily unlabeled F9 cells but not H-2bc blast cells inhibited the anti-F9 cell cytotoxicity H-2k anti-F9 cell immune CTL were found to be cytotoxic for syngeneic spermatogonia, known to express the F9 antigen. The results suggest not only that CTL can recognize and lyse H-2-negative target cells, but also that CTL precursors can be sensitized against H-2-negative stimulator cells. From the data available it may be inferred that anti-F9 Cell immune CTL recognize the F9 antigen, known to be linked with the T/t locus. Since anti-F9 cell immune CTL lyse syngeneic spermatogonia, the system may be useful to analyze in vitro the induction and effector phase of a T-cell-mediated cytotoxic autoimmune orchitis.     

Induction of virus-specific modifications recognized by cytotoxic T cells is not altered by prior substitution of target cells with trinitrophenol

Cytotoxic thymus-derived lymphocytes generated after interaction with trinitrophenyl (TNP)-substituted or virus-infected cells only lyse H-2 compatible target cells modified with the component used to immunize (TNP or virus). Prior saturation of TNP-reactive sites inhibits neither the infectivity of influenza A viruses, nor the capacity of infected cells to develop antigenic changes recognized by influenza-immune T cells. The two antigens are distinct entities on the cell membrane and do not obviously compete to form interactions with H-2 molecules.     

Antigen receptors on major histocompatibility complex-restricted T lymphocytes. I. Preparation and characterization of syngeneic antisera against trinitrophenyl-activated T cell blasts and demonstration of their specificity for idiotypes on cytotoxic T lymphocytes

This paper describes the specificity of AKR anti-(a) [AKR anti-trinitrophenylated AKR (AKR-TNP)] [AKRa (AKRaAKR TNP)] antisera raised in syngeneic AKR mice against AKRaAKR-TNP cell populations enriched for H-2-restricted aTNP cytotoxic lymphocytes (CTL) by blast-cell isolation. The activity of the antisera resided in the Ig fraction. All antisera were shown to reproducibly react with AKRaAKR-TNP-CTL-containing cell populations in indirect immunofluorescence and all removed the major fraction of CTL in complement-dependent lysis causing a considerable depression of cell-mediated lympholysis. The antisera were nonreactive with alloreactive AKRaC57BL/6 CTL and other H-2-restricted AKR CTL against fluorescein-isothiocyanate-conjugated AKR-target cells. It could be excluded that the antisera contained contaminating antibodies against TNP, TNP-neoantigenic determinants (NAD), or processed CTL-receptor-bound TNP-NAD, thus demonstrating specificity for determinants on T cell receptors of AKRaAKR-TNP CTL. These receptors were produced by the CTL themselves. These observations are interpreted to suggest that AKRa (AKRaAKR-TNP) antisera contain anti-idiotypic antibodies directed against specificity-associated determinants (idiotypes) on T cell receptors of H-2-restricted AKRaAKR-TNP CTL. The antisera provide a new tool to study the genetic control of idiotype expression on H-2-restricted CTL, the biochemistry of T cell receptors, and the regulation of the generation of H-2 restricted CTL on the idiotype level.     

Hybrid resistance to EL-4 lymphoma cells. I. Characterization of natural killer cells that lyse EL-4 cells and their distinction from marrow-dependent natural killer cells

Natural killer (NK) cells from nonimmunized mice capable of lysing EL-4 (C57BL/6 strain H-2b) tissue culture-adapted lymphoma cells have been analyzed and compared with NK cells which lyse YAC-1 (A-strain, H-2a) lymphoma cells. A correlation was seen in the ability of inbred and B6D2F1 mice to reject C57BL/6 (B6) bone-marrow grafts and the ability of their spleen cells to lyse EL-4 cells in vitro. This suggests that hybrid or hemopoietic histocompatibility antigens, (Hh-1b), relevant in the rejection of B6 stem cells may also be the relevant target structures for the anti-EL-4 NK cells. Certain features of these NK cells are similar to the NK cells reactive against YAC-1 cells. Both types of NK cells are present in athymic nude mice, are not affected by treatment with anti-immunoglobulin plus complement, and are not depleted by techniques that remove macrophages. NK activity against both targets is stimulated 3 d after injection of Corynebacterium parvum, and 24 h after challenge with polyinosinic:polycytidylic acid. Hydrocortisone acetate and cyclophosphamide lead to reduction of NK activity within 2-3 d after administration. However, the anti-YAC and anti-EL-4 NK reactivities differed in several important respects. Treatment of mice with 89Sr, the bone-seeking isotope, to deplete marrow-dependent cells, depleted the anti-YAC-1 but not anti-EL-4 cell functions. Anti-EL-4 NK cells were unaffected by silica particles in vivo or in vitro; the NK cells reactive to EL-4 cells matured functionally much earlier in life (5 d of age) and the function did not decline with age. Irradiated mice reconstituted with syngeneic marrow or spleen cells developed functional NK cells against EL-4 targets before they developed anti-YAC-1 NK cells in their spleen. Thus anti-EL-4 NK cells that express hybrid resistance in vitro appear to differ from anti-YAC-1 NK cells and do not require an intact marrow microenvironment for functional differentiation. Despite differences in the NK-cell types involved in the lysis of YAC-1 and EL-4 cells, these two tumor cells share certain common determinants. This was ascertained both by cold competition and by utilization of YAC-1 and EL-4 cell monolayers as immunoadsorbents. We conclude that Hh-1b is the common antigen present in EL-4 and YAC-1 cells, because B6D2F1 anti-B6 (anti-Hh-1b) cytotoxic T lymphocytes lysed both the tumor cells. Our data suggest that Hh-1b antigen is recognized by both types of NK cells, but that additional determinants must be present on YAC-1 cells. Two models of NK cell lysis compatible with the data are presented.     

Primary cell-mediated lympholysis response to a maternally transmitted antigen

Mta-specific cytotoxic T lymphocyte (CTL) can be generated in primary cultures of (NZB X B10.D2)F1 spleen cells with H-2-compatible BALB/c stimulator cells. The CTL lyse reciprocal Mta+ (B10.D2 X NZB)F1 as well as H-2-disparate targets, such as B10, B6, and B6-Tlaa; they do not lyse targets from NZB or any F1 hybrid of an NZB mother. The lysis of 51Cr-labeled B10 targets is completely inhibited by unlabeled targets from Mta+ (B10.D2 X NZB)F1, but not from the reciprocal Mta- F1, thus demonstrating H-2-unrestricted lysis of Mta.     

H-2 compatibility requirement for virus-specific T-cell-mediated cytolysis. The H-2K structure involved is coded by a single cistron defined by H-2Kb mutant mice

Lymphocytic choriomeningitis or vaccinia virus-immune spleen cells of H-2 mutant mice carrying a point mutation in the K region (B6 H-2ba, B6 H-2bf) cannot lyse infected wild-type H-2Kb targets and vice versa. Yet, cytotoxic T cells specific for infected H-2Kba or H-2Kbf targets are generated during virus infections as shown by cold target competition experiments. The critical structure for the apparent restriction by the K or D regions of the H-2 gene complex of cytolytic interactions between T cells and virus-infected target cells are therefore each coded, at least as shown for the K region, by a single cistron. This finding is most readily accommodated within the altered self concept (postulating that T cells are specific for virus-modified self structures) but cannot exclude the possibility of a physiological interaction mechanism being responsible for the apparent H-2 restriction of virus-specific cytotoxic T cells.     

Exclusive involvement of H-2Db or H-2Kd product in the interaction between T-killer lymphocytes and syngeneic H-2b or H-2d viral lymphomas.

It was demonstrated previously that the cytolysis of murine viral lymphoma cells by anti-murine sarcoma virus (MSV) syngeneic T-killer lymphocytes was restricted by some products of the H-2 complex. The respective role of the products of different regions of the H-2 complex were studied with six H-2(b) and three H-2(d) lymphomas induced by five different type C viruses. They were tested in a classical chromium release test against anti-MSV T-killer cells obtained from different inbred strains of mice, including several H-2 recombinants. Tumors o£ the H-2(b) haplotype were lysed only when effectors and target cells have in common the D(b) region. On the contrary an identity limited to the K end of the H-2 complex is necessary and sufficient in the H-2(d) haplotype. An in vitro restimulation of the spleen cells with concanavalin A strongly increased the activity of in vivo-primed T lymphocytes but did not provide any response for in vivo-primed but nonresponder cells. Preincubation of the tumor cells with anti-H-2 sera abolished the lysis by syngeneic anti-MSV effector lymphocytes. The same results were obtained by preincubating the H-2(b) targets with anti-H-2D(b), or the H-2(d) target with anti-H-2K(d). Preincubation with anti-H-2K(b) or anti- H-2D(d) were ineffective. These results show that the T-killer/target cells interaction in the MSV system involved some products of the H-2 complex which might be different with the various H-2 haplotypes and could possibly vary according to the antigenic specificity. A specific association of a viral product with a normal cellular structure, directed by the H-2 region during the viral budding could explain the observed results.