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.     

Cross-reactive lysis of trinitrophenyl (TNP)-derivatized H-2 incompatible target cells by cytolytic T lymphocytes generated against syngeneic TNP spleen cells

Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectiviely a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be medicated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-D surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.     

Virus and trinitrophenol hapten-specific T-cell-mediated cytotoxicity against H-2 incompatible target cells

Immune spleen cells from LCM virus-infected (CBA X C57BL/6)F1 radiation chimeras entirely repopulated with CBA-T6 lymphocytes were cytotoxic for allogeneic, LCM virus infected C57BL/6 mouse-derived target cells. Normal C57BL/6 targets were not lysed. CBA-T6 lymphocytes derived from (CBA X C57BL/6) radiation chimeras sensitized in vitro against TNP- conjugated C57BL/6 spleen cells lysed TNP-conjugated C57BL/6 targets. However normal C57BL/6 mouse-derived targets were not destroyed. The magnitude of virus-specific (or TNP-specific) cytotoxic responses against H-2 incompatible targets was lower compared to that against H-2 compatible targets. These data are considered to support and to extend the altered self concept, but are not consistent with the dual recognition concept.     

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.     

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.     

Regulation of T-cell-mediated lympholysis by the murine major histocompatibility complex. I. Preferential in vitro responses to trinitrophenyl-modified self K- and D-coded gene products in parental and F1 hybrid mouse strains

Spleen cells from H-2b,k,d C57Bl/10 congenic mice were sensitized in vitro to trinitrobenzenesulfonate (TNBS)-modified autologous spleen cells. Cold target competition studies at the lytic phase demonstrated three distinct patterns of cytotoxic responsiveness: (a) H-2b spleen cells generated approximately equivalent CTL responses against Kb and Db modified self products, (b) H-2d spleen cells generated preferential responses against Dd modified self products, and (c) H-2k spleen cells generated cytotoxic responses which could only be detected against Kk self products in association with TNP. F1 spleen cells were sensitized against autologous TNBS-treated cells. The results showed that, although H-2b parental cells generated approximately equivalent Kb-TNP- and Db-TNP-specific CTL, the presence of the H-2b haplotype did not result in the generation of (a) Dk-TNP CTL response by (H-2b x H-2k) spleen cells, nor (b) a Db CTL response by (H-2b x H-2a) F1 spleen cells. Additionally, (H-2d x H-2k) F1 cells failed to generate detectable Dd-TNP-specific CTL, although H-2d parental cells generated D-regional-specific CTL. The findings demonstrated that these F1 response patterns paralleled those of the H-2k and H-2a parents, i.e. weak or no D-region TNP-specific CTL were induced. Because (H-2d x H-2a) F1 responders stimulated with H-2d TNBS-treated cells did generate good Dd TNP responses, the results illustrated that the presence of responder genes was not sufficient to result in a D-region TNP CML. It is suggested that the absence of Kk alleles on the stimulating population is necessary for the generation of D-region TNP CTL in these F1's. Mechanisms which could account for these response patterns in parental F1 mice are discussed including immunodominance, suppression, T-cell response , and Ir-gene defects.     

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.     

T lymphocyte-mediated suppression of myeloma function in vitro. II. Evidence for regulation of hapten-binding myelomas by syngeneic hapten- specific cytolytic T lymphocytes

BALB/c splenocytes stimulated in vitro with trinitrophenyl (TNP)-modified syngeneic cells inhibit the secretion of antibody by the TNP-binding BALB/c myeloma MOPC 315 in the presence of soluble TNP-Keyhole limpet hemocyanin (KLH). The effector cells are hapten-specific, H-2-restricted, Thy-1.2-bearing, Ly-2-positive T lymphocytes whose precursors are resistant to pretreatment with cyclophosphamide. These phenotypic properties are typical of hapten-specific cytolytic T lymphocytes (CTL). The TNP-reactive CTL that inhibit MOPC 315 cells fail to suppress H-2d myelomas that do not bear TNP-specific surface receptors, and this is not attributable to differences in total binding of TNP-KLH to the different myeloma cells. Moreover, azobenzene arsonate (ABA)-specific CTL inhibit MOPC 315 cells in the presence of the double conjugate TNP-ABA-KLH, but not in the presence of soluble TNP-KLH or ABA-KLH. These results show that H-2-restricted, hapten-specific lymphocytes regulate the function of myeloma cells that bind the hapten only to specific surface receptors, and provide a model for associative recognition of surface H-2 determinants and receptor-bound antigen. The results are discussed with reference to the mechanisms of T lymphocyte-target cell interactions, and the possible physiologic role of hapten-reactive CTL in specifically regulating anti-hapten antibody responses.     

Antigen presentation by hapten-specific B lymphocytes. I. Role of surface immunoglobulin receptors

The present study examines the ability of hapten-specific murine splenic B lymphocytes to present hapten-proteins to carrier-specific T cell hybridomas. BALB/cB cells specific for 2,4,6-trinitrophenyl (TNP) were isolated from spleens of immune mice by elution from TNP-gelatin-coated dishes. Such cells presented the TNP-modified terpolymer, GL phi, at concentrations as low as 0.1 microgram/ml, to a GL phi-specific, I-Ed-restricted, interleukin 2-producing T cell hybridoma. In contrast, the same B lymphocytes required 1,000-fold higher concentrations of unmodified GL phi to stimulate the same T cell hybridoma. The presentation of low concentrations of TNP-GL phi by TNP-specific B lymphocytes was significantly or completely blocked by anti-Ig antibody or TNP-proteins, indicating that surface Ig receptors were critically involved in this phenomenon. Finally, binding of TNP-proteins did not alter the ability of the B cells to present unrelated, unhaptenated proteins or to stimulate alloreactive T cells. These results suggest that surface Ig receptors serve to focus antigens onto specific B lymphocytes and that such cells are highly efficient at presenting linked antigenic determinants to T cells. The implications of these findings for the mechanisms of physiologic, histocompatibility-restricted T-B collaboration are discussed.     

H-2 restriction as a consequence of intentional priming. Frequency analysis of alloantigen-restricted, trinitrophenyl-specific cytotoxic T lymphocyte precursors within thymocytes of normal mice

An in vitro acute-depletion protocol was used to detect trinitrophenyl (TNP)-specific, allo-major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) within thymocytes of inbred mice. After removal of alloreactivity, the negatively selected cells could be sensitized to become TNP-specific, allo-MHC-restricted cytotoxic T cells. A precursors frequency analysis revealed a three- to ninefold lower frequency of allo-MHC-restricted CTL precursors (CTL-P) as compared to self-MHC-restricted CTL-P. The specificity analysis of clonally distributed allo-MHC-restricted CTL-P excluded cross- reactivity as an explanation of allo-MHC restriction. These results provide direct evidence that thymic T cells are composed of a mixture of self-MHC- and allo-MHC-restricted immunocompetent T cells and that antigen-driven selection of precommitted T cells dictates the H-2- restriction phenotype, i.e., H-2 restriction is a consequence of priming.     

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.     

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.     

Cellular and genetic control of antibody responses in vitro. III. Immune response gene regulation of accessory cell function

The possibility was investigated that Ir genes regulate the function of cells other than T or B cells in the primary IgM responses to the synthetic antigens trinitrophenylated poly-L-(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys [TNP-(T,G)-A--L]and trinitrophenylated poly-,-(His,Glu)-poly-D, L-Ala--poly-L-Lys [TNP-(H,G)-A--L]. The primary responses of (B10 × B10.A)F(1) spleen cells to both antigens were abrogated by Sephadex G-10 passage, and restored by the addition of spleen adherent cells. The cell type in the spleen adherent cell population active in reconstituting the responses to TNP-(T,G)-A--L and TNP-(H,G)-A--L was a non-T, non-B, radiation-resistant, glass-adherent spleen cell. The responses of Sephadex G-10-passed (responder x nonresponder)F(1) spleen cells to TNP-(T,G)-A--L or TNP-(H,G)-A--L were reconstituted by spleen adherent cells from only responder strains. Spleen adherent cells from F(1) mice reconstituted the responses to both antigens. Spleen adherent cells from each of the strains tested reconstituted the non- Ir gene-controlled response to a third antigen, TNP-keyhole limpet hemocyanin. The inability of spleen adherent cells from nonresponder strains to reconstitute the responses to either TNP-(T,G)-A--L or TNP-(H,G)-A--L was not a result of active suppression induced by the presence of nonresponder adherent cells, since a mixture of responder and nonresponder spleen adherent cells reconstituted the responses to both antigens. The use of spleen adherent cells from recombinant strains demonstrated that the autosomal dominant genes controlling the ability of spleen adherent cells to function as accessory cells in the responses to TNP-(T,G)-A--L and TNP-(H,G)-A--L are located in the K or I-A regions of the responder H-2 complex, the same region(s) of H-2 as the Ir genes controlling overall in vitro and in vivo responsiveness to these antigens.     

An antigen-specific signal is required for the activation of second- order suppressor T cells in the regulation of delayed-type hypersensitivity to 2,4,6-trinitrobenzene sulfonic acid

Suppressor T cells (Ts-1) induced with trinitrophenyl (TNP)-conjugated syngeneic spleen cells (TNP-SC) can be enriched on antigen-coated plates and are afferent suppressors. In addition, these suppressor cells produced soluble suppressor factors (TsF) that were active in vivo. Therefore, the Ts-1 cells in the TNP system are very similar to the Ts-1 cells in other systems we have studied earlier. Further characterization of these TsF-1 revealed that TsF-1 obtained from TNP-SC-induced Ts-1 is major histocompatibility complex restricted in its activity. Injection of TNP-specific TsF-1 into naive mice did not induce Ts-2 unless additional corresponding antigen was provided. Moreover, the Ts-2 cells induced by administration of both TsF-1 and trinitrobenzene sulfonic acid were antigen specific rather than antiidiotypic.     

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.     

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.     

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.     

Allorestricted cytotoxic T cells. Large numbers of allo-H-2Kb- restricted antihapten and antiviral cytotoxic T cell populations clonally develop in vitro from murine splenic precursor T cells

Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.     

Bifunctional major histocompatibility-linked genetic regulation of cell- mediated lympholysis to trinitrophenyl-modified autologous lymphocytes

Murine thymus-derived lymphocytes can be sensitized in vitro to trinitrophenyl (TNP)-modified autologous spleen cells (1, 2). Cytotoxic effector cells were generated which were specific for TNP-modified target cells expressing the same H-2K and H-2D serological regions as the modified stimulator cells (3, 7). Spleen cells from two C57BL/10 congenic strains of mice sharing common I-C, S, and D regions, but differing at K, I-A, and I-B regions, generated different levels of lytic responses to the shared modified H-2Dd products upon sensitization with auto logous TNP-modified cells. Lymphocytes from an F1 between responder and nonresponder strain generated a level of cytolysis toward the H-2Dd modified specificity which was of the same order of magnitude as that obtained with the high responder, irrespective of whether F 1 or either parental strain of modified stimulator cell was used. These results suggest that the modification of H-2Dd products resulted in formation of new antigenic determinants in both parental strains. However, the difference observed in responsiveness appeared to be due to a gene or genes mapping in the K, I-A, or I-B region which influenced the ability of the responding lymphocytes to react to these modified H-2Dd products. Responsiveness was expressed as a dominant trait in the F1.     

Cytotoxic T lymphocyte responses in allogeneic radiation bone marrow chimeras. The chimeric host strictly dictates the self-repertoire of Ia- restricted T cells but not H-2K/D-restricted T cells

The present report has used fully H-2 allogeneic radiation bone marrow chimeras to assess the role of host restriction elements in determining the self-specificity of Ia- and H-2K/D-restricted T cells that participate in the generation of trinitrophenyl (TNP)-specific cytotoxic T lymphocytes (CTL). It was demonstrated that there exists a stringent requirement for the recognition of host thymic-type Ia determinants, but there exists only a preference for host thymic-type H-2K/D determinants. Indeed, once the stringent requirement for recognition of host Ia determinants was fulfilled, anti-TNP CTL were generated in response to TNP-modified stimulators that expressed either donor-type or host-type H-2K/D determinants. The CTL that were generated in response to TNP-modified donor-type stimulators were shown to be specific for TNP and restricted to the non-thymic H-2K/D determinants of the chimeric donor. Thus, these results demonstrate in a single immune response that the thymic hypothesis accurately predicts the self-specificity expressed by Ia-restricted T cells, but does not fully account for the self-specificity expressed by H-2K/D-restricted T cells. These results are consistent with the concept that H-2K/D-restricted T cells, but not Ia-restricted T cells, can differentiate into functional competence either intrathymically or extra-thymically. The present results are also informative for understanding the cellular interactions that are required for the generation of antigen-specific CTL responses. The Ia-restricted T cells that are required for the generation of H-2K/D-restricted anti-TNP CTL were shown to be helper T (TH) cells since (a) like TH cells functioning in antibody responses, they were specific for Ia determinants expressed by accessory cells, and (b) their function could be replaced by either TNP-primed, irradiated TH cells or by nonspecific soluble helper factors. It was also shown that the T-T cell interaction between Ia-restricted TH cells and H-2K/D-restricted precursor CTL (pCTL) is not Ia restricted. Rather, the results demonstrate that the generation of anti-TNP CTL responses involve two parallel sets of major histocompatibility complex-restricted cell interactions, an Ia-restricted TH-accessory cell interaction required for TH cell activation, and an H-2K/D-restricted pCTL-stimulator cell interaction required for pCTL stimulation. The interaction between activated TH cells and stimulated pCTL is mediated, at least in part, by nonspecific soluble helper factors.