Characterization of antigen-specific, Ia-restricted, L3T4+ cytolytic T lymphocytes and assessment of thymic influence on their self specificity

The goals of the present study were: (a) to generate antigen-specific L3T4+ cytolytic T lymphocytes (CTL), (b) to determine their major histocompatibility complex (MHC) restriction specificity, and (c) to assess the influence of thymic MHC determinants on their self specificity. We found that L3T4+ CTL specific for either trinitrophenyl (TNP)-modified self determinants or minor histocompatibility antigens could be generated from Lyt-2- responder T cells provided that the response cultures were supplemented with supernatants rich in helper factors. Such antigen-specific L3T4+ CTL were Ia-restricted by the criteria that they lysed only Ia+ target cells and that their lysis of Ia+ target cells was specifically inhibited by anti-Ia monoclonal antibodies. The relative frequency of L3T4+ pCTL was found to be only 5-10% of the total anti-TNP pCTL present in the spleens of normal mice. Finally, we utilized radiation bone marrow chimeras to assess the influence of the thymic haplotype on the self-Ia specificity of L3T4+ CTL. Both bulk culture and limiting dilution experiments revealed that the self-Ia specificity of L3T4+ anti-TNP CTL from F1----parent and A----B allogeneic chimeras was not markedly skewed toward the haplotype of the chimeric thymus. These results contrast with those obtained previously for L3T4+ anti-TNP Th cells and demonstrate that in the radiation bone marrow chimera model of T cell differentiation, the self specificity of Th cells but not pCTL is markedly influenced by the haplotype of the chimeric thymus.     

Self-recognition specificity expressed by T cells from nude mice. Absence of detectable Ia-restricted T cells in nude mice that do exhibit self-K/D-restricted T cell responses

The presence in athymic nude mice of precursor T cells with self-recognition specificity for either H-2 K/D or H-2 I region determinants was investigated. Chimeras were constructed of lethally irradiated parental mice receiving a mixture of F1 nude mouse (6-8 wk old) spleen and bone marrow cells. The donor inoculum was deliberately not subjected to any T cell depletion procedure, so that any potential major histocompatibility complex-committed precursor T cells were allowed to differentiate and expand in the normal parental recipients. 3 mo after reconstitution, the chimeras were immunized with several protein antigens in complete Freund's adjuvant in the footpads and their purified draining lymph node T cells tested 10 d later for ability to recognize antigen on antigen-presenting cells of either parental haplotype. Also, their spleen and lymph node cells were tested for ability to generate a cytotoxic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified stimulator cells of either parental haplotype. It was demonstrated that T cell proliferative responses of these F1(nude)----parent chimeras were restricted solely to recognizing parental host I region determinants as self and expressed the Ir gene phenotype of the host. In contrast, CTL responses could be generated (in the presence of interleukin 2) to TNP-modified stimulator cells of either parental haplotype. Thus these results indicate that nude mice which do have CTL with self-specificity for K/D region determinants lack proliferating T cells with self-specificity for I region determinants. These results provide evidence for the concepts that development of the I region-restricted T cell repertoire is strictly an intrathymically determined event and that young nude mice lack the unique thymic elements responsible for education of I region-restricted T cells.     

Role of self carriers in the immune response and tolerance. V. Reversal of trinitrophenyl-modified self suppression of the B-cell response by blocking of H-2 antigens

Trinitrophenylated syngeneic spleen cells (TNP-SC) are potent tolerogens of the anti-TNP plaque-forming cell (PFC) response in vivo and in vitro. This unresponsive state requires T cells for both its induction and maintenance. Because H-2K/D-restricted cytotoxic T cells are also induced by exposure to TNP-SC, we determined the role(s) of histocompatibility antigens (K, I, and D) in the suppression of the PFC response by TNP-SC. We treated syngeneic TNP-modified stimulator cells with antiserum directed at K-, I-, or D-region determinants and found that blocking of H-2K or D antigens on TNP-SC transformed these tolerogens into immunogens capable of eliciting an anti-TNP PFC response in the absence of extrinsic immunogens like TNP-polymerized flagellin. In H-2k or H-2a(k/d) mice, only H-2Kk needs to be blocked on the stimulator cells, whereas H-2K or D recognition was apparent in B10.A(4R) mice. These observations indicate that suppression of the PFC response by TNP-SC shows the same restriction in recognition as does the cytotoxic T-cell response. Furthermore, our results suggest that TNP-I-A is recognized by the helper cells in this system as the intrinsic antigen. When both TNP-K and TNP-I-A are present and available on the same stimulator cell, suppression (via modified K recognition) is dominant over help.     

Helper T cells for cytotoxic T lymphocytes need not be I region restricted

We investigated the antigenic requirements for restimulation of H-2- restricted cytolytic T lymphocytes (CTL) in vitro to determine whether H-2 I region-restricted helper T cells are required in these responses. In one set of experiments, we studied the in vitro response of (responder x nonresponder)F(1) female T cells to the male antigen H-Y. We chose to examine this response because it has been suggested that the defect in nonresponder strains is a failure of helper T cells to recognize H-Y in association with nonresponder I region determinants. However, we find that nonresponder male stimulator cells are as effective as F(1) male stimulator cells at inducing H-Y-specific CTL responses. This finding calls into question reports that secondary CTL responses to H-Y are dependent upon the activation of H-Y- specific helper T cells restricted to responder type I region determinants. In a second set of experiments, we examined the requirements for restimulation of H-2-restricted T cells specific for minor-histocompatibility antigens from long-term mixed lymphocyte cultures. These cultures were established by repeatedly restimulating cultures of specific T cells with H- 2-matched stimulator cells expressing foreign minor histocompatibility antigens. We found that H-2D-restricted T ceils, including CTL, could be restimulated with cells that were matched with the responding cells at only the D region genes. This response did not appear to result from positive allogeneic effects or from antigen processing and “representation” by responder type APC that might contaminate the cultures. Thus, we find no evidence for a requirement for I region-restricted helper T cells in these CTL responses. However, helper T cells are required because we find that CTL lines derived by limit-dilution cloning from these long-term MLC are absolutely dependent upon exogenous helper factors for growth. The most simple interpretation of these results is that the helper cells are restricted to H-2 antigens other than I region antigens or to antigens that code outside of the H-2 complex. Finally, we show that factor-dependent CTL lines must recognize their specific antigen to proliferate, even in the presence of exogenous factors. The requirement of activated CTL for antigen to proliferate provides an explanation for how specific CTL can be selectively enriched in MLC by specific antigen stimulation. Furthermore, it is at variance with reports that memory CTL or activated CTL require only interleukin 2 for restimulation.     

Cytotoxic T lymphocytes induced against allogeneic I-region determinants react with Ia molecules on trinitrophenyl-conjugated syngeneic target cells

The major histocompatibility complex codes for determinants which are recognized by and serve as targets for cytolytic T lymphocytes (CTL) (1). Antigens coded for by the K and D loci of the H-2 complex can activate xenogeneic or allogeneic CTL (2,3). In addition, the H-2K or H-2D gene products function as those molecules against which syngeneic CTL responses specific for chemical, viral, and minor H antigens are directed (4-8). It has recently been shown that Ia determinants can also serve as target antigens for distinct but weaker CTL responses (9-13). Those clones which recognize Ia antigens see them independently of K- or D- coded antigens as shown in genetic studies and by antisera-blocking experiments (12,13). We have proposed that the existence of clones of CTL specific for I-region-coded determinants is not fortuitous; rather these clones specifically recognize Ia determinants and may have an immunoregulatory role. These CTL may affect those immune functions which are at least partially dependent on or controlled by I-region-coded molecules. Two predictions can be made and tested concerning the role of Ia determinants in cytolytic systems and the role, if any, of I-region- specific CTL in regulating the immune response: (a) that if as we and others have shown, certain Ia specificities can serve as a third series of major histocompatibility antigens, then Ia antigens should be susceptible to the same types of antigenic modifications as H-2K- or H-2D-coded structures and thus serve as targets for CTL directed against modified-self in selected systems; and (b) that allogeneically induced I-region-specific CTL should demonstrate cross-reactivity with targets bearing modified syngeneic I-region-coded determinants. Data will be present which demonstrates that trinitrophenyl (TNP)-modified syngeneic I-region determinants can serve as targets for CTL induced by allogeneic Ia antigens.     

Functional analysis of T cells expressing Ia antigens. I. Demonstration of helper T-cell heterogeneity

We have examined the expression of I-region antigens on functional subpopulations of murine T cells. A.TH anti-A.TL (anti-Ik, Sk, Gk) alloantiserum was raised by immunization of recipients with concanavalin A (Con A) stimulated thymic and peripheral T-cell blasts. In contrast to similar antisera made by conventional methods, the anti-Ia blast serum was highly cytotoxic for purified T lymphocytes. Moreover, it reacted in a specific fashion with T cells having particular functions. Treatment of keyhole limpet hemocyanin (KLH)-primed B10.A (H-2 alpha) T cells with this antiserum plus complement resulted in the elimination of helper activity for B-cell responses to trinitrophenyl-KLH. Inhibition was shown to be a result of the selective killing of one type of helper T cell whose activity could be replaced by a factor(s) found in the supernate of Con A-activated spleen cells. A second type of helper cell required for responses to protein-bound antigens appeared to be Ia-. By absorption and analysis on H-2 recombinants, at least two specificities were detectable on helper T cells; one mapping in the I-A subregion and a second in a region(s) to the right of I-J. In addition, the helper T cell(s) involved in the generation of alloreactive cytotoxic lymphocytes was shown to be Ia+, whereas cytotoxic effector cells and their precursors were Ia- with this antiserum. These results provide strong evidence for the selective expression of I-region determinants on T-cell subsets and suggest that T-cell-associated Ia antigens may play an important role in T-lymphocyte function.     

T cells from fully H-2 allogeneic (A replaced by B) radiation bone marrow chimeras are functionally competent and host restricted but are alloreactive against hybrid Ia determinants expressed on (A x B)F1 cells

In this communication it is demonstrated that T cells from fully allogeneic A replaced by B radiation bone marrow chimeras are alloreactive against the hybrid Ia molecules expressed on the surface of heterozygous A X B cells. These results suggested that previous failures to generate cytotoxic T lymphocyte (CTL) responses from fully allogeneic chimeras by sensitizing the chimeric T cells to antigen in an (A X B)F1-priming environment might have been confounded by an ongoing alloreaction against determinants created by hybrid Ia molecules expressed on F1 cells. Consequently, the ability to generate CTL responses from fully allogeneic chimeras was re-examined by sensitizing the chimeric T cells to antigen presented by homozygous rather that F1 stimulator cells. It was found that T cells of donor bone marrow origin that mediate cytotoxic responses to trinitrophenyl- modified self determinants do differentiate into functional competence in an H-2-incompatible host environment and are restricted to the host H-2 haplotype.     

H-2-restricted helper factor secreted by clone hybridoma cells

Biological and serological characteristics of a helper factor secreted by cloned hybridoma cells was described. The factor is carrier specific and contains determinants shared with immunoglobulin VH bu does not react with V kappa- or V lambda-specific antibodies. Presence of four H-2I-controlled antigenic specificities, Ia.ml, Ia.m2, Ia.17, and Ia.m7, was detected. Hence, it is possible that both A beta and E alpha loci may be involved in its control. Helper effect could be obtained only toward B cell sources that shared the H-2K and I-A antigens with the hybridoma cells. Similarly, the factor was absorbed only by spleen cells syngeneic in I-A. Previous studies have demonstrated that this clone binds antigen in an H-2-restricted manner. It follows that H-2-restricted helper cells produce H-2-restricted helper factors. Hence, they support the view that specific T cell factors may represent secreted T cell receptors.     

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.     

Helper activity is required for the in vivo generation of cytotoxic T lymphocytes

B6.T1a(a) (Qa-1(a)) mice that are primed in vivo and restimulated in vitro with Qa-1 congenic spleen cells from B6 (Qa-1(b)) animals are unable to generate anti-Qa-1(b) cytotoxic T lymphocytes (CTL). This nonresponsive pattern was observed regardless of the route of immunization or the time of testing in vitro. Although B6.T1a(a) mice are nonresponders to Qa-1(b) when presented on B6 cells, these mice can generate anti-Qa-1(b) CTL when primed in vivo with Qa-1 and H-Y alloantigens (females primed with B6 male cells) or Qa-1 and minor-H- alloantigens (primed with sex-matched A.BY cells). Therefore, the inability to generate anti-Qa-1(b) CTL is due to a lack of helper or accessory antigens on B6 immunizing cells obligatory during in vivo priming, rather than an absence of anti-Qa-1(b) CTL precursors (CTL-P). Demonstration that the additional determinants required during in vivo priming actually function as carrier or helper determinants was shown by the requirement for linked recognition of Qa-1 and the helper determinants (H-Y) in vivo, and the fact that H-Y was not present on susceptible target ceils. Animals primed in vivo with H-Y only could not generate anti-Qa-1 CTL activity when challenged in vitro with both Qa-1 and H-Y, indicating that recognition of the helper determinant causes in vivo priming of CTL-P rather than generating helper activity that might activate unprimed CTL-P in vitro. Whereas unprimed peripheral CTL-P require the presence of both Qa-1 (CTL) and H-Y (helper) determinants for successful in vivo priming, helper determinants were not required in vitro because primed CTL-P from B6.T1a(a) mice could be driven to CTL in vitro using sex-matched B6 stimulator cells. The generation of anti-Qa-1(b) CTL is under immune response (Ir) gene control because F(1) mice, obtained by crossing responder A/J with nonresponder B6.T1a(a) animals, generated CTL to the Qa-1(b) alloantigen when presented on B6 spleen cells. Progeny testing of backcross mice further demonstrated that the Ir gene(s) is linked to the H-2 complex. These data indicate that an H-2-linked Ir gene controls the recognition of helper determinants required for CTL priming in vivo. These helper determinants can be distinguished from CTL determinants and both must be recognized together for successful priming of CTL-P.     

Cytotoxic T lymphocytes specific for I region determinants do not require interactions with H-2K or D gene products

Gene products coded for by the major hisocompatibility complex (MHC) can serve as target antigens for cytotoxic T lymphocytes (CTL) (1). A variety of test systems are available which have yielded information consistently reinforcing the importance of this complex of genes in the generation and effector phases of the cytotoxic immune response. Originally, it was shown that allogeneically-induced CTL had specificity primarily for the products of the K and D loci of the mouse H-2 complex (2). More recently this has also been found to be the case for xenogeneic immunizations (3,4). Additional examples of T cell-mediated lysis have been reported involving viral-infected or chemically- modified syngeneic stimulating and target cells in which homology at H-2K or H-2D was required between the responding and target cells for appreciable lysis to occur (5-7). Moreover, CTL specific for minor histocompatability antigens are able to lyse only target cells bearing these membrane antigens and sharing a common H-2K or H2-D gene product with the effector (8,9). Two hypotheses have been proposed to explain the requirement for H-2 identity between effector and targets in these systems. CTL may recognize new antigenic determinants created by the interaction of the modifier with syngeneic K and D gene products. Alternately, a dual recognition system my exist, requiring an antigen-specific receptor as well as a second receptor with specificity for homologous H-2K or H-2D determinants (5). Neither model can be excluded at this time. The I region also contains genes coding for histocompatibility loci since animals differing at the I-A or I-C regions of the H-2 complex reject skin grafts (10-12), though less rapidly than mice differing at the H-2K or H-2D regions, Also CTL can be generated to I region determinants but less efficiently than CTL specific for H-2K or H-2D gene products (12-14). The question can therefore be raised, whether the I region minor histocompatibility loci function independently from the H-2K or H-2D loci or whether I region-specific cytolysis requires the participation of H-2K or H-2D gene products of the target cell. This communication illustrates the generation of CTL showing specificity for I region determinants in primary mixed lymphocyte cultures. Further, we demonstrate by genetic analysis and byt eh use of speficit alloantisera that CTL directed to Ia determinants (a) do not see these antigens as modifications of H-2K or H-2D gene products but as independent gene products coded for by the I region, and (b) they do not require interaction with target cells bearing the same H-2K or H-2D gene product as the effect CTL.     

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.     

Inhibition of T-lymphocyte-mediated tumor-specific lysis by alloantisera directed against the H-2 serological specificities of the tumor

After appropriate in vivo or in vitro immunization, cytotoxic T lymphocytes (CTL) are generated which efficiently kill cells bearing particular membrane antigens in common with the immunizing cell (reviewed in reference 1). Such CTL have been most thoroughly studied in mice, employing alloimmunization with cells differing at the major histocompatibility locus, H-2. in such cases, the predominant cell surface antigens recognized by the CTL appear to be the molecules carrying the serologically defined H-2 specificities, coded for by the K and D regions of the H-2 complex (2). In other syngeneic models of cell-mediated specific cytolysis, involving lymphocyte chariomeningitis (LCM) virus- or ectromelia virus-infected cells or TNP-modified lymphoid cells, thymus-derived cells also constitute the main effector cell type. The CTL generated in these latter systems function most efficiently when virus-infected or TNP-modified target cells share identitites at the H-2K or H-2D loci with the effector CTL and stimulator cells (3-5). Another set of experimental systems in which CTL are generated and play a significant biological role is that of immunity to tumor-associated antigens (TAA) (6). The nature of the TAA which the CTL recognize is only beginning to be understood. Several recent reports indicated the existence of physiochemical and/or antigenic relationships between TAA and H-2 antigens (7,8). These relationships, together with the genetic restrictions cited above in the generation of CTL involving products of the H-2K or H-2D loci suggested the possibility that in certain tumor systems, the TAA which are able to most effectively stimulate CTL responses might be structurally similar to, or linked with, the H-2K or H- 2D molecules on the tumor surface. It has been previously demonstrated in allogenic models that antisera specific for the appropriate H-2K or H-2D products present on a target cell could specifically block CTL-mediated lysis (1,9). This report demonstrates that certain anti-H-2 alloantisera specific for the target tumor cells can block lysis of those target cells mediated by syngeneic tumor-specific CTL effector cells.     

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.     

Specificity of cytotoxic T cells from athymic mice

In normal mice, self-H-2 antigens in the thymus have a profound influence on T cell specificity. We have therefore investigated the properties of cytotoxic T lymphocyte (CTL) precursors from athymic nude mice (5) with the notion that they may provide a model system for the study of T cells whose receptro specificity is closer to the germ-line- encoded repertoire. It was found that the precursors of nude CTL are, themselves, THy-1+ cells. The possibility that these nude t cells were derived from the phenotypically normal mother by placental transfer was ruled out. In the presence of T cell growth factor, nude CTL can be induced by polyclonal activation with concanavalin A or by stimulation with allogeneic or trinitrophenyl (TNP)-modified syngeneic stimulator cells, but not by stimulation with minor H antigens in the context of self-H-2. Alloreactive, nude CTL--like those from normal mice-- recognize H-2K- and H-2D-region-encoded antigens in killer-target cell interactions, but, unlike normal CTL, did not cross-react on third- party target cells. Whereas the anti-TNP response of nude mice is H-2 restricted, it does not seem to be influenced by self-H-2 antigens in the same manner as in normal mice. This is suggested by the finding that the immunodominance of H-2k over H-2d in the anti-TNP-self response of normal (H-2d X H-2b)F1 mice is absent in (H-2d X H-2k)F1 nude mice. These observations are discussed in relation to the role of the thymus in the generation of the normal mature T cell receptor repertoire.     

Protection against graft vs. host-associated immunosuppression in F1 mice. I. Activation of F1 regulatory cells by host-specific anti-major histocompatibility complex antibodies

Injection of parental spleen cells into unirradiated F1 hybrid mice results in suppression of the potential to generate cytotoxic T lymphocyte (CTL) responses in vitro. In an attempt to protect the F1 mice from immunosuppression, the recipients were injected with antibodies specific for major histocompatibility complex (MHC)-encoded antigens of the F1 mice 24 h before inoculation of the parental spleen cells. 8-14 d later, the generation of CTL responses in vitro against H- 2 alloantigens was tested. Alloantiserum directed against either parental haplotype of the F1 strain markedly diminished the suppression of CTL activity. Furthermore, monoclonal antibodies recognizing H-2 or Ia antigens protected the F2 mice from parental spleen cell-induced suppression. Although this study has been limited to reagents that recognize host H-2 determinants, these findings do not necessarily imply that protection against graft vs. host (GvH) can be achieved only with anti-MHC antibodies. However, protection was observed only by antibodies reactive with F1 antigens, and small amounts of the alloantibodies were sufficient to diminish CTL suppression. Adoptive transfer of spleen cells from syngeneic F1 mice treated with anti-h-2a alloantiserum 24 h previously provided protection equal to that of injection of the recipients with alloantibodies. The cells necessary for this effect were shown to be T cells and to be radiosensitive to 2000 rad. This cell population is induced by antisera against F1 cell surface antigens and effectively counteracts GvH-associated immuno- suppression.     

Differences in antigen presentation to MHC class I-and class II-restricted influenza virus-specific cytolytic T lymphocyte clones

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA-specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II-restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets.     

Role of the H-2 complex in the induction of T cell tolerance to self minor histocompatibility antigens

The present study has utilized cytotoxic T lymphocyte (CTL) responses specific for minor histocompatibility (minor H) antigens as an experimental approach to determining whether recognition of self MHC determinants is involved in the induction of T cell tolerance to self antigens. It was observed that C3H.SW splenic T cells from C3H.SW leads to B10 X B10.BR radiation bone marrow chimeras contained CTL precursors (pCTL) reactive against self C3H minor H antigens + H-2k but were tolerant to self C3H minor H antigens + H-2b. Precursor CTL with the reciprocal reactivity pattern were observed for C3H leads to B10 X B10.BR chimeras. In addition, it was observed that C3H.SW thymocytes from C3H.SW leads to B10 X B10.BR chimeras could generate minor H- specific CTL responses and were reactive against self C3H minor H antigens + H-2k, but were tolerant to self C3H minor H antigens + H-2b. Thus, the present study demonstrates that for peripheral and intrathymic T cell populations at least a component of T cell tolerance to self antigens is restricted by products of the MHC.     

Thymus dictates major histocompatibility complex (MHC) specificity and immune response gene phenotype of class II MHC-restricted T cells but not of class I MHC-restricted T cells

Athymic H-2b nude mice received grafts from C57BL/6 (Sendai virus and H-Y antigen cytotoxic T lymphocyte [CTL] responder type), bm1 (H-2Kb mutant, Sendai CTL nonresponder type), or bm12 (H-21-A mutant, H-Y CTL nonresponder type) neonates. In observations of the CTL response to H-Y, both recipients and thymus donors were female. All types of thymus engraftment resulted in mature H-2b splenic T lymphocyte surface phenotype in nude hosts. T cell immunocompetence (as measured by major histocompatibility complex [MHC] CTL responses to allogeneic cells) was restored, and induced nonresponsiveness to the MHC determinants of the engrafted thymus in the nude host. The CTL reaction to Sendai virus in both responder type C57BL/6 and nonresponder type bm1 neonatal thymuses allowed maturation of Sendai-specific, H-2Kb-restricted CTL. For the CTL reaction to H-Y, only responder type C57BL/6 thymuses restored the CTL response, whereas this was not achieved with thymuses from nonresponder type bm12 neonatal females. Results of double thymus (B6 and bm12) engraftment excluded the possibility that this latter effect was caused by suppression. In addition, athymic bm1 mice were engrafted with thymuses from either B6 (Sendai CTL responder type) or syngeneic bm1 neonates (Sendai CTL nonresponder type). Again, both types of neonate thymuses restored T cell competence as measured by MHC/CTL responses to allogeneic cells. However, neither responder B6 nor nonresponder bm1 neonate thymus grafts allowed maturation of Sendai-specific CTL. In conclusion, the thymus dictates MHC specificity and immune response gene phenotype of T cells restricted to class II MHC molecules but not of T cells restricted to class I MHC molecules.     

On the thymus in the differentiation of "H-2 self-recognition" by T cells: evidence for dual recognition?

In the thymus, precursor T cells differentiate recognition structures for self that are specific for the H-2K, D, and I markers expressed by the thymic epithelium. Thus recognition of self-H-2 differentiates independently of the T cells H-2 type and independently of recognition of nonself antigen X. This is readily compatible with dual recognition by T cells but does not formally exclude a single recognition model. These conclusions derive from experiments with bone marrow and thymic chimeras. Irradiated mice reconstituted with bone marrow to form chimeras of (A X B)F1 leads to A type generate virus-specific cytotoxic T cells for infected targets A only. Therefore, the H-2 type of the host determines the H-2-restricted activity of killer T cells alone. In contrast, chimeras made by reconstituting irradiated A mice with adult spleen cells of (A X B)F1 origin generate virus-specific cytotoxic activity for infected A and B targets, suggesting that mature T cells do not change their self-specificity readily. (A X B)F1 leads to (A X C)F1 and (KAIA/DC) leads to (KAIA/DB) irradiation bone marrow chimeras responded against infected A but not B or C targets. This suggests that cytotoxicity is not generated against DC because it is abscent from the host's thymus epithelium and not against DB because it is not expressed by the reconstituting lymphoreticular system. (KBIB/DA) leads to (KCIC/DA) K, I incompatible, or completely H-2 incompatible A leads to B chimeras fail to generate any measurable virus specific cytotoxicity, indicating the necessity for I-specific helper T cells for the generation of killer T cells. Finally adult thymectomized, irradiated and bone marrow reconstituted (A X B)F1 mice, transplanted with an irradiated thymus of A origin, generate virus-specific cytotoxic T cells specific for infected A targets but not for B targets; this result formally demonstrates the crucial role of thymic epithelial cells in the differentiation of anti-self-H-2 specificities of T cells.