Synthetic peptides as antigens and competitors in recognition by H-2-restricted cytolytic T cells specific for HLA

The specificity of peptide recognition by a number of Kd-restricted CTL clones specific for HLA-CW3 or HLA-A24 was investigated. The CTL clones were derived from DBA/2 (H-2d) mice immunized with syngeneic P815 mouse cells transfected with genes encoding HLA-CW3 or HLA-A24 class I molecules. We had previously shown that CTL clones that lysed P815-CW3 transfectant target cells could lyse P815 (HLA-) target cells incubated with synthetic CW3 peptides corresponding to the COOH-terminal end of the alpha 2 domain. In the present study, we found that Kd-restricted CTL clones that lysed P815-A24 transfectant target cells recognized a synthetic peptide from the same region (residues 170-182) of the A24 molecule. CW3 and A24 differ by only one amino acid within this region. Recognition of CW3 or A24 peptides corresponded exactly with lysis of P815-HLA transfectants both for clones that mutually exclusively lysed CW3 or A24 transfectant target cells and for CW3/A24 crossreactive CTL clones. The latter CTL clones that lysed both CW3 and A24 transfectant target cells showed a clear preference for the peptide corresponding to the immunizing HLA allele. The homologous CW3 and A24 peptides could compete with each other for recognition, in contrast to a peptide from the same region of HLA-B7. Peptides from the corresponding region of the endogenous Kd and Dd/Ld molecules could also inhibit recognition of CW3 and A24 peptides. Competition with peptides apparently occurred at the level of the target cell. These results are consistent with a model whereby MHC class I molecules position protein fragments or peptides for specific recognition by T cells.     

H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.     

On the role of the transmembrane anchor sequence of influenza hemagglutinin in target cell recognition by class I MHC-restricted, hemagglutinin-specific cytolytic T lymphocytes

We have examined the requirement for the transmembrane hydrophobic anchor sequence of the influenza hemagglutinin (HA) in the formation of the antigenic moiety on the surface of target cells recognized by class I MHC-restricted murine CTL. For this analysis we have used a line of CV-1 monkey epithelial cells that express the transfected murine H-2Kd gene product as target cells and have used recombinant SV40-based late replacement vectors to achieve expression of genes encoding wild-type and mutant forms of HA. We have found that the majority of Kd-restricted HA-specific CTL clones recognize target cells that express a secreted HA molecule that lacks the transmembrane and cytoplasmic domains of the parent glycoprotein. Several Kd-restricted CTL clones that recognize subtype-specific and crossreactive epitopes on HA fail to recognize the anchor-negative, secreted HA or chimeric HA molecules containing the transmembrane and cytoplasmic domains of unrelated glycoproteins. These CTL clones appear to be directed to antigenic epitopes located within the transmembrane domain of HA, as defined by their capacity to recognize target cells sensitized with a synthetic 23-amino-acid peptide corresponding to sequences within this domain. The implications of these results for class I MHC-restricted CTL recognition are discussed.     

Thymic selection and adaptability of cytotoxic T lymphocyte responses in transgenic mice expressing a viral protein in the thymus

Upon primary challenge with lymphocytic choriomeningitis virus (LCMV), H-2d (BALB/cByJ) mice mount a cytotoxic T lymphocyte (CTL) response to a single immunodominant domain of the viral nucleoprotein (NP) but no detectable response to the viral glycoprotein (GP). To manipulate this CTL response, the viral NP gene was expressed in the thymus and peripheral T lymphocytes using the murine Thy1.2 promoter. As a result, such Thy1.2-NP (H-2d) transgenic (tg) mice deleted their high-affinity anti-LCMV-NP CTL, but generated equal numbers of lower-affinity NP CTL. Further, they made an alternative anti-LCMV-GP CTL response that is not normally found in non-tg mice indicating a hierarchial control of the CTL response. Unlike the H-2d mice, H-2b (C57Bl/6J) mice normally mount a CTL response to both LCMV-GP and -NP. When the LCMV-NP was expressed using the Thy1.2 promoter in these H-2b mice, the LCMV-NP-specific CTL response was completely aborted and no CTL to new, alternative viral epitopes were generated. Dilutions of H-2b or H-2d NP peptides indicated that 3-4 logs less H-2b NP peptide was required to sensitize syngeneic target cells for CTL-specific lysis, suggesting that the differing affinities of H-2b and H-2d major histocompatibility complex molecules for their peptides likely account for the total removal of NP CTL in the H-2b mice but only partial removal in H-2d mice made to express thymic NP. Thymic grafting experiments done with thymi from newborn Thy1.2-NP tg mice show that selection processes studied in this model are of central (thymic) origin and are not caused by Thy1.2- positive LCMV-NP-expressing T lymphocytes in the periphery.     

Antitumor vaccination using a major histocompatibility complex (MHC) class I-restricted pseudopeptide with reduced peptide bond

Synthetic peptides have raised a considerable interest in the fields of vaccines and immunotherapy. The authors previously introduced modifications into the peptide backbone of the H-2Kd-restricted epitope CW3. One of these pseudopeptides, C7, bound to Kd with an affinity identical to the parent peptide and was recognized by T cells specific for the parent peptide. The authors now show that this analog has an increased resistance to trypsin and displays an extended half-life in serum. The authors further tested its immunogenicity both in vitro and in vivo and found that cytotoxic T lymphocytes (CTL) induced against the peptide analog recognize the parent peptide. Moreover, analysis of T-cell receptor rearrangements by Immunoscope software revealed that C7-induced CTL display the hallmarks of the response against the parental epitope CW3. Administration of the pseudopeptide into DBA/2 mice induces a protective immune response against a lethal challenge with tumor cells expressing the parent peptide. Therefore, modifications in the backbone of antigenic peptides can decrease protease susceptibility while preserving immunogenicity. Such peptide analogues may therefore prove useful for the development of new therapeutic tools aimed at eradicating pathogens or tumors.     

Introduction of H-2Dd determinants into the H-2Ld antigen by site-directed mutagenesis

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.     

A single amino acid substitution in the alpha 3 domain of an H-2 class I molecule abrogates reactivity with CTL

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.     

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.     

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.     

T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire

We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all.     

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.     

Use of DNA-mediated gene transfer to analyze the role of H-2Ld in controlling the specificity of anti-vesicular stomatitis virus cytotoxic T cells

Mouse thymidine kinase (tk-) C3H L (H-2k) cells transformed by the technique of DNA-mediated gene transfer with the herpes simplex virus tk gene together with the BALB/c H-2Ld gene express H-2Ld molecules indistinguishable from their counterparts on spleen cells. An established cloned cell line (8-5) was used to assess the function of the H-2Ld antigen in determining the specificity of alloreactive as well as anti-vesicular stomatitis virus (VSV) cytotoxic T cells (CTL). Both anti-H-2d and anti-H-2Ld CTL displayed a cytotoxic effect against 8-5 cells but not a control cell line transformed with the tk gene only (tk+ cells). Further evidence that 8-5 cells express H-2Ld was provided by the finding that monoclonal anti-H-2Ld but not H-2Dd antibodies blocked target cell lysis by the effector cells. Both BALB/c (H-2d) and DBA/2 (H-2d) animals generated anti-VSV CTL that lysed infected 8-5 but not tk+ cells. To further establish that H-2Ld controlled the specificity of the effector cells, a monoclonal antibody directed against H-2Ld was shown to inhibit lysis of infected 8-5 target cells. To determine whether other H-2d-encoded gene products could serve as restricting antigens for anti-VSV CTL in BALB/c animals, unlabeled VSV infected 8-5 cells were tested for their ability to block lysis of 51chromium-labeled P815 (H-2d)-infected target cells. The 8-5-VSV inhibitor cells inhibited lysis to a slightly lesser extent than unlabeled P815-VSV cells, indicating that H-2Ld plays a major if not exclusive role in restricting anti-VSV CTL in H-2d animals.     

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.     

An immunodominant epitope present in multiple class I MHC molecules and recognized by cytotoxic T lymphocytes

CTL derived from (C3H x B6.K1)F1 animals were sensitized against L cells that express the transfected gene product Q10d/Ld. These CTL were highly crossreactive against three other class I molecules, H-2Kbm1, H-2Ld, and H-2Kd. In an attempt to define this crossreactive epitope it was noted that between 25 and 39% of amino acids in the alpha helices and central beta strands of these three molecules vary from Q10d. These amino acids represent residues that have been proposed to potentially interact with a peptide antigen or TCR (21). However, all four molecules share the amino acid tyrosine at positions 155 and 156. Additionally, Q10d, H-2Kbm1, and H-2Ld share alanine at position 152, while H-2Kd has an aspartic acid. We showed that these residues were important in controlling this epitope by the finding that anti-Q10d CTL did not recognize H-2Kbm1 revertant molecules that had either the position 152 alanine changed back to the wild-type H-2Kb residue (glutamic acid) or position 155 and 156 tyrosines changed back to wild-type residues arginine and leucine. Further evidence that these molecules share a crossreactive epitope was noted by the failure of (C3H x H-2Kbm1)F1 animals to generate CTL that recognized H-2Ld or H-2Kd, and the inability of (C3H x BALB/c)F1 animals to generate CTL reactive against H-2Kbm1. CTL from these mice were still able to recognize Q10d/Ld indicating that other epitopes could be detected if natural tolerance prevented recognition of the crossreactive epitope. To further define the epitope, CTL clones were generated against Q10d/Ld and maintained on either H-2Kbm1 or BALB/c feeder cells. In addition to testing these clones on the target cells described above, mutant molecules derived from H-2Ld, which have amino acid substitutions in their alpha 1 domain, were analyzed. It was noted that some anti-Q10 clones that did not crossreact on H-2Ld did react against H-2Ld mutant antigens that had H-2Dd amino acid substitutions in the alpha 1 domain at positions 63, 65, 66, and 70. Other clones had differential reactivities on these H-2Ld mutants further substantiating that alpha 1 domain amino acids play a role in controlling the expression of the crossreactive epitope. Thus, four class I molecules with multiple amino acid differences in their alpha 1 and alpha 2 domains share a crossreactive epitope readily recognized by alloreactive CTL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haplotype-specific suppression of cytotoxic T cell induction by antigen inappropriately presented on T cells

To detect a strong cytotoxic T lymphocyte (CTL) response to minor histocompatibility (H) antigens in a 5-d mixed lymphocyte culture, it is necessary to use a responder that has been primed in vivo with antigen-bearing cells. It has previously been shown that minor-H- specific CTL can be primed in vivo both directly by foreign spleen cells and by presentation of foreign minor H antigens on host antigen- presenting cells. This latter route is evident in the phenomenon of cross-priming, in which H-2 heterozygous (A x B)F1 mice injected 2 wk previously with minor H-different H-2A (A') spleen cells generate both H-2A- and H-2B-restricted minor-H-specific CTL. In a study of the kinetics of direct- vs. cross-priming to minors in F1 mice, we have found that minor H-different T cells actually suppress the induction of virgin CTL capable of recognizing them. CTL activity measured from F1 mice 3-6 d after injection with viable A' spleen cells is largely H-2B restricted. The H-2A-restricted response recovers such that roughly equal A- and B-restricted activity is detected in mice as early as 8-10 d postinjection. This temporary hyporeactivity does not result from generalized immunosuppression--it is specific for those CTL that recognize the foreign minor H antigen in the context of the H-2 antigens on the injected spleen cells. The injected spleen cells that mediate this suppression are radiosensitive T cells; Lyt-2+ T cells are highly efficient at suppressing the induction of CTL in vivo. No graft vs. host reaction by the injected T cells appears to be required, as suppression of direct primed CTL can be mediated by spleen cells that are wholly tolerant of both host H-2 and minor H antigens. Suppression cannot be demonstrated by in vitro mixing experiments. Several possible mechanisms for haplotype-specific suppression are discussed, including inactivation of responding CTL by veto cells and in vivo sequestration of responding CTL by the injected spleen cells.     

Does B7-1 expression confer antigen-presenting cell capacity to tumors in vivo?

Tumors engineered to express the costimulatory molecule B7-1 can elicit CD8+ cytotoxic T lymphocyte (CTL)-dependent antitumor responses in immunocompetent mice. It has been postulated that this result reflects direct priming of CTL by the modified tumor in vivo. Previous studies of the immune response to a B7-1- murine colon carcinoma expressing influenza nucleoprotein (NP) as a model tumor antigen have demonstrated the crucial role of bone marrow-derived antigen-presenting cells (APCs) in the priming of NP-specific CTL in vivo. In this system, no evidence of direct CTL priming by tumor was detected. We have performed a similar analysis to determine if B7-1 transfectant of this tumor results in the direct priming of CTL, and to compare this response to that primed by host APCs. When H-2b-->H-2bxd bone marrow chimeras were immunized with a single injection of CT26/NP/B7-1 (H-2d), NP-specific CTL were detected that were restricted to the bone marrow haplotype (H- 2b), but not to the tumor haplotype. In contrast, CTL recognizing the NP antigenic epitope in the context of the tumor's major histocompatibility complex were detectable only after multiple immunizations. These results suggest that whereas B7-1+ tumor vaccines result in some degree of direct presentation to CD8+ T cells, the dominant mechanism of CTL priming is through the uptake and presentation of tumor antigens by bone marrow-deprived APCs. However, repeated immunization with B7-1+ tumor cells can efficiently expand the directly primed CD8+ CTL population.     

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.     

Friend virus-specific cytotoxic T lymphocytes recognize both gag and env gene-encoded specificities

We have constructed a series of "synthetic" target cell lines for an analysis of the specificity of anti-Friend virus (FV) CTL. Our results show that murine H-2 genes and individual retroviral genes can be stable expressed in Fisher rat embryo (FRE) cells, and that their products have the potential to form target structures recognized by mouse CTL. Cells expressing H-2Db and either the env or gag genes of one component of FV, helper Friend murine leukemia virus (FMuLV), were lysed by anti-FV CTL and by CTL generated against FMuLV alone. Experiments with Db-transfected FRE clones infected only with the replication-defective spleen focus-forming virus (SFFV) component of FV indicate that the SFFV genome also provides specificities recognized by both anti-FV and anti-FMuLV CTL, thus demonstrating the existence of a crossreactive CTL population. An unexpected finding was that anti-FMuLV CTL, but not anti-FV CTL were also able to lyse FRE clones that expressed H-2Kb in either the presence or absence of FV. The use of heterologous cell lines for the construction of synthetic target cells thus offers a useful approach for the analysis of T cell specificity.     

Allospecific cytolytic T lymphocytes recognize conformational determinants on hybrid mouse transplantation antigens

Alloreactive cytolytic T cell (CTL) lines and clones have been used to identify the sites of polymorphism of antigens of the major histocompatibility complex (MHC). Specific CTL were generated against wild-type H-2b products by cells from H-2b mutant mice that had one or a few amino acid changes in either the alpha 1 or alpha 2 domains of the Kb or Db class I molecules. These CTL populations, which might be expected to react with determinants expressed on single MHC domains, were examined for lytic activity on L cells expressing newly constructed hybrid class I molecules. Transformed cell lines expressing native class I molecules or hybrid class I molecules in which the alpha 1 and alpha 2 domains of H-2Kb had been substituted by those domains of H-2Db were lysed by H-2Db-specific CTL. Similarly, all H-2Kb-specific CTL recognized hybrid molecules in which the alpha 1 and alpha 2 domains of H-2Kb were inserted into the H-2Db molecule. In contrast, exchange of the alpha 1 domains of H-2Kb and H-2Db resulted in a total loss of recognition by Kb and Db-specific CTL. These results suggest that the allodeterminants recognized by H-2 mutant CTL are influenced by interactions between the alpha 1 and alpha 2 domains, findings similar to those seen using conventional alloreactive T cells (11). These results were compared to the binding of alloreactive mAbs, including 5 new mAbs specific for the Kb molecules. Finally, it was shown that primary and secondary CTL responses could be generated by direct sensitization against hybrid class I molecules, demonstrating that these molecules express neoantigenic determinants recognized by alloreactive CTL.     

Heterogeneity and specificity of cloned lines of influenza-virus specific cytotoxic T lymphocytes

Continuous lines of murine cytotoxic T lymphocytes (CTL) directed to type A influenza viruses have been generated in vitro by stimulation of individual CTL precursors in the presence of T cell-growth factor TCGF and syngeneic virus-infected stimulator cells. The cloned CTL lines are H-2 restricted in their target cell recognition and exhibit distinct patterns of influenza virus recognition. All CTL lines appear to be restricted in target cell recognition to either the H-2K or the H-2D end of the appropriate H-2 haplotype. Likewise, CTL lines of F1 origin are restricted in recognition exclusively to one of the parental haplotypes. All CTL lines examined express the Thy-1.2 and the Lyt-2- surface antigen markers. 4 of 11 cytotoxic lines examined also expressed detectable levels of the Lyt-1- surface antigen. These findings confirm at the clonal level previous observations on the H- 2K/D restriction of virus-specific CTL and also demonstrate heterogeneity among H-2 restricted CTL both from the standpoint of viral antigen recognition and cell surface phenotype.