Non-H-2-linked genetic regulation of cytotoxic responses to hapten-modified syngeneic cells. I. Non-H-2-linked Ir gene defect expressed on T cells is not predetermined at the stage of bone marrow cells

Spleen cells from C3H/He or BALB.K mice immunized to the newly synthesized amino-reactive hapten 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (AED-NH2) were stimulated in vitro with AED-NH2-modified syngeneic cells. After 5 days of culture, effector cells were assayed for their cytotoxic activity against AED-NH2-modified target blast cells. C3H/He and BALB.K mice exhibited the respective high and low anti-AED-NH2 cytotoxic T lymphocyte (CTL) responses. This contrasted with the observation that both of these H-2k strains generated potent CTL responses against aminoreactive haptens, e.g., trinitrophenyl (TNP). Because C3H.SW and BALB.B strains, which are the H-2b counterpart of the above two strains, also represented the respective high and low responders to AED-NH2 hapten, this hapten model enabled us to investigate cellular mechanisms underlying the above non-H-2-associated genetic regulation of CTL responses (C3H vs BALB non-H-2 backgrounds). The results demonstrated that there was no detectable difference between C3H/He and BALB.K strains in the lysability of target cells and the ability of stimulating cells to activate primed spleen cells. Anti-AED-NH2 CTL responses were only marginal when antigen-presenting cells (APC) were eliminated from the primed spleen cells of high responder C3H/He or (C3H/He X BALB.K)F1 mice. The addition of APC to cultures free of APC regained an appreciable CTL response in C3H/He or (C3H/He X BALB.K)F1 mice, irrespective of whether APC were derived from high (C3H/He) or low (BALB.K) responders. We have also demonstrated that allogeneic radiation bone marrow chimera (BALB.K----C3H/He) exhibited a CTL response comparable to that induced by C3H/He mice, whereas the reverse direction of allogeneic chimera (C3H/He----BALB.K) induced a marginal CTL response. These results indicate that this non-H-2-associated Ir gene defect is expressed on T cells (CTL precursors and/or helper T cells) rather than APC, and that this T cell defect is not predetermined at the level of bone marrow cells. The results are discussed in the light of the genetic and cellular mechanisms underlying non-H-2-linked Ir gene control.     

Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses

Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study investigates the role of haptenic structure and hapten-cell surface reaction patterns in influencing the generation of the T cell specificity as well as the H-2-linked genetic control. CTL and helper T cell responses were generated against two structurally related haptens, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene-diamine (SH-reactive AEDANS; AED-SH) and 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (NH2-reactive form of AEDANS; AED-NH2) by immunizing C57BL/6N (H-2b) mice with these hapten-modified syngeneic spleen cells. Spleen cells from primed C57BL/6N mice generated strong CTL and helper T cell activities upon in vitro restimulation with the respective hapten-modified self. The generation of potent anti-AED-NH2 CTL and helper T cell responses in C57BL/6N mice sharply contrasted with the failure of NH2-reactive haptens studied thus far to generate strong anti-hapten cytotoxic responses in H-2b mice. Antibodies induced against the above two haptens exhibited extensive cross-reactivity detected by hemagglutination, whereas CTL and helper T cells clearly discriminated the structural difference between AED-NH2 and AED-SH haptens. The hapten specificity in T cell recognition was also observed between AED-NH2 and trinitrophenyl (TNP) haptens, which were demonstrated to functionally modify similar cell surface sites. These results indicate that hapten epitope structure and hapten-cell membrane conjugation patterns influence the generation of H-2-linked genetic control and T cell specificity in anti-hapten self cytotoxic as well as helper T cell responses.     

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.     

Analysis of Ir gene control of cytotoxic response to hapten-modified self: helper T cells specific for a sulfhydryl hapten can substitute for an anti-TNP-H-2b self helper cell defect

Helper T cells specific for N-iodoacetyl-N'-(5-sulfonic 1-naphthyl) ethylene diamine (I-AED) were generated in (C56BL/6 X C3H/He)F1 mice by immunization with I-AED-modified syngeneic cells (AED-self). The requirements for activation of hapten-induced helper cells were investigated. The results demonstrated that activation of AED and trinitrophenyl- (TNP) helper cells was strictly hapten specific. In addition, F1 AEd-helpers could be activated efficiently by either I-AED-modified H-2b or H-2k self components to enhance the anti-AED self-CTL responses. This contrasts with the previous findings demonstrating the failure of TNP-H-2b self to activate F1 TNP-helper cells. After AED-helpers were activated, they were capable of augmenting sensitization of cytotoxic T cells (CTL) against TNP-self. These results indicate that although the activation of hapten-reactive helper cells is antigen (hapten)-specific, the subsequent helper activity, as determined by augmentation of CTL responses against another hapten, is antigen nonspecific. Since helper function was antigen nonspecific, F1 AED-helper cells activated by AED-H-2b or AED-H-2k self were tested for their ability to augment the F1 and anti-TNP-H-2b CTL response. The results indicate that the Ir gene defect in the ability of F1 spleen cells to respond to TNP-H-2b self could not be corrected by these helper cells. These results are discussed in the light of Ir gene controlled differences in the activation of AED and TNP-helper cells and possible models for augmenting CTL responses against various antigens in strains that generate marginal helper activity to TNP-self.     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

Production and characterization of T-cell clones specific for trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self)

Using serial antigenic challenge as the method of selection and stimulation, continuous lines of cytotoxic T-lymphocytes (CTL) directed against TNBS-modified syngeneic spleen cells (TNP-self) have been generated. Spleen cells from C3H/HeJ (H-2k) mice were primed in vitro with autologous spleen cells modified with TNBS, and subsequently cloned by limiting dilution and in soft agar in the presence of IL2. These CTL clones grew continuously in medium supplemented with IL2 and in the presence of antigen. They are antigen specific and H-2 restricted in their target cell recognition. They all express Thyl and Lyt2 surface markers. None of the clones exhibit natural killer (NK) cell activity. All CTL clones tested so far are restricted in their target cell recognition to H-2Kk-TNP and none were found to be restricted to H-2Dk-TNP. These findings demonstrate at the clonal level the H-2K/D restriction of TNP-self specific CTL. These clones provide tools that may facilitate an understanding of the development and regulation of antigen specific CTL. They may also serve as models useful towards an understanding of the mechanism of lysis by CTL.     

Cell-mediated lympholysis responses against autologous cells modified with haptenic sulfhydryl reagents. IV. Self-determinants recognized by wild-type anti-H-2Kb and H-2Db-restricted cytotoxic T cells specific for sulfhydryl and amino-reactive haptens are absent in certain H-2 mutant strains

Virus-specific H-2-restricted cytotoxic T cells (CTL) have been found to discriminate between wild-type and mutant class I molecules. The only results reported concerning a hapten-self model, however, indicate that TNP-specific CTL do not discriminate between wild-type and mutant self determinants (7). In the present study, hapten-specific CTL generated against N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine-modified syngeneic cells (AED-self) were used to determine whether a hapten that is known to react with different cell surface sites than TNP can induce CTL that distinguish mutant H-2K and D molecules from those of wild type. The findings of this study indicate that H-2Kb-AED-self cytotoxic effector cells can discriminate between self-determinants of H-2Kb wild-type and the H-2bm1 and H-2bm11 mutants, but not between wild-type and the H-2bm6 and H-2bm9 mutants. H-2Db-AED-self effector cells were also found to discriminate between self-determinants of H-2Db wild-type and the H-2bm13 and H-2bm14 mutants. Furthermore, cold target competition experiments indicated that the bm1 and bm11 Kb products also lack some determinants recognized by anti-wild-type Kb TNP-specific CTL. These findings provide the first demonstration that hapten-self-specific effectors can detect alterations in H-2 mutant class I molecules. The results in the present report also support the hypothesis that haptens do not have to derivatize H-2 molecules in order to form antigens recognized by H-2-restricted CTL. These findings are discussed with respect to the involvement of self-determinants on MHC and non-MHC cell surface molecules.     

In vivo development of cytolytic T lymphocytes (CTL) to hapten-altered self: MIs-disparate cells facilitate the response by neutralizing IL 2 inhibitor

Inability to develop CTL in vivo to hapten-altered self can be attributed in part to an inhibitor of interleukin 2 (IL 2) that is present in the serum of normal mice. We have shown earlier that hapten-specific CTL can be generated in C3H mice (H-2k, MIsc) provided CBA/J (H-2k MIsd) spleen cells are injected simultaneously with hapten-modified syngeneic spleen cells into the hind foot paws. In efforts to determine whether serum levels of the inhibitor of IL 2 are altered as a consequence of this successful immunization method, we have compared the activity of the inhibitor in serum at intervals after the injection of syngeneic spleen cells, CBA spleen cells, or TNP-C3H spleen cells alone or together with CBA spleen cells, by using a murine IL 2-dependent, cloned cytotoxic T cell line, CT-6. The results indicate that inhibitor was neutralized optimally 48 to 72 hr after injection of TNP-C3H spleen cells mixed with CBA/J spleen cells. The order in which neutralization occurred was as follows: TNP-C3H cells + CBA/J cells greater than CBA cells greater than TNP-C3H cells greater than normal C3H spleen cells. Furthermore, supernatants from cultures of C3H lymph node cells stimulated in vivo with CBA/J cells also contained IL 2 activity. Thus, injection of CBA/J cells with TNP-modified syngeneic spleen cells produces IL 2 in vivo in sufficient quantity to neutralize the activity of the inhibitor as well as to facilitate the maturation of pre-CTL into hapten-altered self-specific CTL.     

Regulation of the hapten-specific T cell response. I. Preferential induction of hyporesponsiveness to the D-end of the major histocompatibility complex in the hapten-specific cytotoxic T cell response

In this paper we have examined the phenomenon of hapten-specific tolerance in the cytolytic T lymphocyte (CTL), using the trinitrophenyl (TNP) and azobenzenearsonate haptens. We found that the H-2 K and H-2 D-end restricted CTL in H-2a mice are differentiable in the ease with which they are tolerized to the TNP hapten. With TNP modified syngeneic spleen cells (TNP-SC), or low amounts of trinitrobenzylsulfonic acid as tolerogen, preferential hyporesponsiveness of D-end restricted CTL can be observed. Larger doses of hapten, e.g. a higher amount of trinitrobenzylsulfonic acid, will tolerize both K- and D-end restricted TNP-specific CTL in H-2a mice. The phenomenon of preferential D-end restricted CTL hyporesponsiveness is not observed in H-2d, H-2k, or H-2b mice, nor is it observed in H-2a mice with respect to the azobenzenearsonate hapten. We have also shown that the clones of CTL responsible for lysis of TNP-modified allogeneic targets (cross-reactive lysis) in H-2a mice probably overlap with the D-end restricted TNP-specific CTL since D-end restricted hyporesponsiveness induced by intravenous injection of TNP spleen cells also results in the elimination of cross-reactive lysis of TNP-modified allogeneic targets. The possible mechanisms of preferential D-end hyporesponsiveness to the TNP hapten in the H-2a mice as well as its significance and relationship to previous work in this area are discussed in this paper.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Multiple cytolytic T-cell clones with distinct cross-reactivity patterns coexist in anti-self + hapten cell lines

C57BL/10 T cells sensitized with TNBS-treated syngeneic cells and maintained in culture by repeated stimulations exhibit high cytolytic activity toward syngeneic TNBS-treated target cells with marked cross-reactivity on TNBS-treated target cells from mice of independent H-2 haplotypes (ten tested). The analysis of the reactivities of 48 T-cell clones derived by limiting dilution from such T-cell populations revealed three types of cytotoxic T-cell clones: (1) clones restricted by H-2K^b + TNP without cross-reaction on TNBS-treated or untreated target cells of other tested mouse haplotypes; (2) clones that lysed H-2^b + TNP and also TNBS-treated target cells from not more than one, two or three different H-2 haplotypes; (3) clones that lysed untreated H-2^k target cells. No T-cell clone was found to exhibit the wide pattern of cross-reactivity on any TNBS-treated mouse cell, characteristic of the original T-cell populations, indicating that these were composed of individual T-cell clones specific for TNP + private or for TNP + distinct public H-2 determinants. Correlation with described serological public H-2 specificities was possible for some cytotoxic T-cell clone reactivity, but not for others. The general pattern of T-cell reactivity as revealed by clonal analysis in this study, as well as in published work, includes cross-reactions between self H-2^a + X and allogeneic H-2ⁿ + X, between self H-2^a + X and unmodified allogeneic H-2ⁿ, or between allogeneic H-2ⁿ and allogeneic H-2^m + X, and is consistent with the hypothesis that MHC-class restriction is the main rule in T-cell recognition.Abbreviations used in this paper CTL cytotoxic T lymphocyte - TNBS trinitrobenzene sulfonate - TNP trinitrophenyl - TNCB trinitrochlorobenzene - H-2 mouse major histocompatibility complex - IL-2 interleukin 2 - Con A concanavalin A - FCS fetal calf serum - SCA IL-2 containing supernatant - LPS E. coli lipopolysaccharide - X a non-H-2 conventional antigen     

Helper cells in the autologous mixed lymphocyte reaction (AMLR): IV. H-2 restriction and specificity of cytotoxic cells induced by AMLR helper factor

Supernatants from Day 3 cultures of T cells stimulated with syngeneic non-T cells in the autologous mixed lymphocyte reaction (AMLR) contained a T-cell factor distinct from interleukin 2 (IL-2). The AMLR factor could mediate the cytotoxic response of T cells to hapten-modified nonstimulatory syngeneic cells. The activity of this helper factor was H-2 unrestricted. However, the cytotoxic cells which developed in the presence of this factor were H-2 restricted and hapten specific.     

Cloned auto-Ia-reactive T cells elicit lichen planus-like lesion in the skin of syngeneic mice

To explore the physiologic or pathologic roles of autoreactive T cells, we examined immunological functions of several autoreactive mouse T cell clones in vitro and in vivo. All of the T cell clones were Lyt-2-, L3T4+ and showed self-I region-restricted proliferative responses (one clone was self-I-E restricted, the other clones were self-I-A restricted). One clone derived from C57BL/6 mouse and reactive to the self-I-Ab product (clone bb1-2) showed cross-reactivity to the I-Ak product. Among four such auto-Ia-reactive T cell clones examined, one clone produced fairly large amounts of interleukin 2 (IL 2) in response to syngeneic stimulator cells, and mediated help for the in vitro cytotoxic T cell (CTL) responses of syngeneic thymocytes, whereas this clone did not mediate in vitro antibody responses of syngeneic B cells. The other three clones were producers of small amounts of IL 2 and did not mediate the in vitro CTL responses. Among the three clones, clone bb1-2 showed strong regulatory function, and clone kk-1 (B10.BR origin and self-I-Ak reactive) showed weak regulatory function in vitro antibody responses of syngeneic B cells. The physiologic or pathologic roles of autoreactive T cells in vivo were explored by injecting subcutaneously clone kk-1 T cells or clone bb1-2 T cells into the footpads of the respective syngeneic mice. Clone kk-1 T cells injected into syngeneic mice elicited swelling of the footpad and marked accumulation of mononuclear cells in the dermis, leaving the epidermis intact, as in the delayed-type hypersensitivity reaction. As a notable finding, clone bb1-2 T cells injected into syngeneic mice elicited marked swelling of the footpad and lichen planus-like skin lesions, i.e., infiltration of lymphocytes in the epidermis and epidermal cell damage. The lymphocytes infiltrating in the epidermis were evaluated, as were the injected clone bb1-2 T cells expressing the Lyt-1.2 phenotype, by examination of the skin lesions elicited in C3H/He mice (H-2k, Lyt-1.1, 2.1) by the clone T cells. Clone bb1-2 T cells exerted in vitro cytotoxicity against H-2b and H-2k target cells, whereas clone kk-1 T cells did not show any cytotoxic activity, indicating a correlation between the cytotoxic activity of clone bb1-2 T cells and their ability to elicit lichen planus-like lesions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unresponsiveness in hapten-specific cytotoxic lymphocytes. III. Influence of H-2 and non-H-2 gene loci on the in vitro trinitrophenyl-specific CTL response following either acute or chronic in vivo treatment with trinitrobenzene sulfonic acid

The regulation of the in vitro generation of cytotoxic T lymphocytes (CTLs) directed against hapten-modified syngeneic cells has been investigated. The results indicate that acute intravenous pretreatment with water-soluble hapten, trinitrobenzene sulfonic acid (TNBS), can either positively or negatively affect the in vitro generation of trinitrophenyl (TNP)-specific CTLs. In general, mice bearing the H-2d haplotype are most likely to develop a reduced in vitro response pattern following a single acute in vivo TNBS treatment, wheras mice bearing the H-2k or H-2b haplotypes display either unchanged or augmented in vitro response patterns. We have shown that, in addition to the influences of H-2 genes, non-H-2 genes can also influence the in vitro hapten-specific CTL response following intravenous pretreatment with water-soluble hapten. Further, in two (H-2k X H-2d) F1 combinations between an H-2k strain displaying an unchanged in vitro response pattern following acute in vivo TNBS treatment and an H-2d strain displaying a reduced in vitro response pattern following similar treatment, it was observed that a single in vivo TNBS pretreatment did not induce the unresponsive state when F1-TNP stimulator cells were used. These results suggest that the mechanisms responsible for the reduced in vitro response pattern are not dominant within the F1 environment. However, when TNP-modified parental stimulators are used, a split-response pattern is observed in cells from TNBS-treated F1 mice which reflect the response patterns of the respective parents. These latter results again emphasize the influence of gene loci on the in vitro response patterns following acute TNBS treatment. In contrast to the significant influence of H-2 and non-H-2 genes on the in vitro TNP-specific response following acute in vivo TNBS treatment, these genes do not appear to significantly influence the in vitro TNP-specific response pattern following chronic TNBS treatment. Chronic TNBS treatment renders all strains tested specifically unresponsive.     

Identification of the immunodominant H-2K(k)-restricted cytotoxic T-cell epitope in the Borna disease virus nucleoprotein

Borna disease virus (BDV)-induced immunopathology in mice is most prominent in strains carrying the major histocompatibility complex H-2k allele and is mediated by CD8(+) T cells that are directed against the viral nucleoprotein p40. We now identified the highly conserved octamer peptide TELEISSI, located between amino acid residues 129 and 136 of BDV p40, as a potent H-2K(k)-restricted cytotoxic T-cell (CTL) epitope. When added to the culture medium of L929 target cells, TELEISSI conferred sensitivity to lysis by CTLs isolated from brains of BDV-infected MRL mice with acute neurological disease. Vaccinia virus-mediated expression of a p40 variant with mutations in the two K(k)-specific anchor residues of the TELEISSI peptide (p40(E130K,I136T)) did not sensitize L929 target cells for lysis by BDV-specific CTLs, whereas expression of wild-type p40 did. Furthermore, unlike vaccination with wild-type p40, vaccination of persistently infected symptomless B10.BR mice with p40(E130K,I136T) did not result in central nervous system inflammation and neurological disease. These results demonstrate that TELEISSI is the immunodominant CTL epitope of BDV p40 in H-2k mice.     

Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL.

T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell-dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc-IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN-gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro.     

Ongoing hapten-specific delayed-type hypersensitivity prevents generation of cytolytic T lymphocytes to the same hapten

Cytolytic T lymphocytes (CTL) specific for trinitrophenyl (TNP)-altered self antigens can be generated in vivo through the simultaneous injection of TNP-modified syngeneic spleen cells and H-2-compatible, minor histocompatibility locus (Mls)-disparate auxiliary spleen cells into the footpads of mice. The latter stimulates host helper cells to produce differentiative and proliferative signals required for the generation of CTL. Advent of this protocol allowed investigation of the initiation of two different cell-mediated immune responses, delayed-type hypersensitivity (DTH) and the generation of CTL, in the same experimental animal. Mice presensitized for CTL were able to develop DTH as well as normal controls. However, when mice were first sensitized for DTH, they were thereafter incapable of generating CTL. This effect was hapten specific, relatively long lasting, and preventable by treating mice with cyclophosphamide before sensitizing for DTH. Adoptive transfer of lymphoid cells from DTH-immune mice conferred DTH reactivity upon naive recipients but not a suppressed CTL response. Therefore, cells mediating DTH were not responsible for suppression of CTL. The mechanism for suppression has been discussed from the viewpoint of the suppressor-T-cell circuits that are known to be generated when animals are sensitized for DTH and which are susceptible to treatment with cyclophosphamide.     

Characterization of a progressive tumor from C3H fibroblasts transformed in vitro with SV40 virus. Immunoresistance in vivo correlates with phenotypic loss of H-2Kk

Cultured SV40-transformed fibroblasts from C3H mice (SV-C3H) were "adapted" to in vivo growth by serial passage through sublethally irradiated, syngeneic recipients. After four in vivo passages, a population of cells was obtained (V4) that was weakly oncogenic in nonirradiated mice. Cells isolated from large V4 tumors (V5) were found to be highly oncogenic, producing lethal tumors at doses of less than 10(3) cells. V5 is insensitive to SV40-specific transplantation immunity in syngeneic animals but can be rejected completely by H-2 allogeneic mice. In vitro studies revealed that although V4 and the parent SV-C3H cells can induce SV40-specific cytotoxic T cells (CTL) in vitro and are lysed by these CTL, V5 does neither. The failure of V5 to interact with CTL was traced to the loss of H-2Kk antigen expression on these cells. The correlation between H-2Kk loss and immunoresistance in vivo suggests a central role for the cytotoxic T cell in in vivo tumor elimination in this system.