Simultaneous expression of allogenic class II MHC and B7.1 (CD80) molecules in A20 B-lymphoma cell line enhances tumor immunogenicity

We expressed the allogenic class II MHC antigen and B7.1 (CD80) co-stimulatory molecule in A20 beta-lymphoma cells in order to test their efficacy as immuno-stimulating adjuvant agents in inducing tumor-specific immunity. The transduction of the allogenic I-Ab alpha and beta chain genes into A20 cell resulted in a surface expression of the allogenic class II MHC molecules. The expression of the allogenic class II MHC antigen (I-Ab) in A20 cells enhanced the proliferation of T cells in a mixed lymphocyte tumor culture and in vitro cytotoxic T lymphocyte (CTL) generation against parental cells. The B7.1 gene, which is known to be a potent co-stimulatory molecule, was also transduced and expressed in A20 cells, either alone or in combination with I-Ab. The B7.1 transduction alone leads to a similar in vitro immune enhancing effect as I-Ab. When both the I-Ab and B7.1 genes were transduced, the in vitro immunostimulating capacity was further enhanced. Finally, we also tested the A20 cells that were transduced with I-Ab and/or B7.1 for their efficacy as preventive tumor vaccines in vivo. The results indicate that the A20 cells that express both the I-Ab and B7.1 have more potent vaccinating potential, compared to the cells that express only one of the molecules.     

Structure-function analysis of the Abm12 beta mutation using site-directed mutagenesis and DNA-mediated gene transfer

The B6.C-H-2bm12 (bm12) mouse possesses a naturally occurring mutation in its class II MHC A beta gene. The three amino acid substitutions at positions 67, 70, and 71 that comprise this mutation lead to changes in both Ia expression and immune recognition of the resultant A beta A alpha molecule. The experiments reported here utilize a combination of oligonucleotide-mediated site-directed mutagenesis and DNA-mediated gene transfer to explore the roles played by each of the three mutant residues in these various phenotypic changes. A beta genes comprising all permutations of the residues distinguishing Ab beta from Abm12 beta were created and were individually co-transfected with Ab beta into mouse L cells. Sublines expressing high levels of membrane Ia were selected by preparative flow cytometry and were studied for reactivity with a panel of monoclonal anti-Ia antibodies, or for their ability to act as antigen-presenting cells (APC) for the stimulation of T cell hybridomas. During the generation of these transfectant lines, it was noted that expression of a high level of Abm12 beta Ab alpha was more difficult to achieve than a similar level of Ab beta Ab alpha. Northern blot analysis of specific A beta and A alpha mRNA levels in these various lines indicated that more class II mRNA, and presumably more A beta and A alpha chains, were required to achieve expression of Abm12 beta Ab alpha equal to that of Ab beta Ab alpha, suggesting that the previously noted reduction of Ia expression on cells from bm12 mice reflects a decreased ability of Abm12 beta Ab alpha chains to pair, or to reach the membrane. Staining of the panel of transfectants with monoclonal antibodies revealed that antibodies which did not distinguish Ab beta Ab alpha from Abm12 beta Ab alpha also reacted equally well with all molecules involving in vitro mutant A beta chains. Monoclonal antibodies reactive with Ab beta Ab alpha but not Abm12 beta Ab alpha were specific for an epitope primarily determined by the presence or absence of Arg 70 in Ab beta. In striking contrast, all three mutant positions were found to play crucial roles in T cell recognition, because all substitutions led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complex regulation of class II gene expression: analysis with class II mutant cell lines

Several Ia-negative variants of a homozygous Iad-expressing antigen-presenting B lymphoma cell line, M12, have been obtained by repeated cycles of negative immunoselection after mutagenesis with ethylmethane sulfonate or gamma-irradiation. Two such Iad-negative cell lines, selected with a mixture of alpha I-Ad and alpha I-Ed monoclonal antibodies, failed to present antigen to all cloned Iad-restricted T cells tested, whereas the third cell line, selected with alpha I-Ad reagents only, stimulated I-Ed but not I-Ad-restricted T cells. The mutations in all three cell lines resulted in the absence of RNA specific for the A beta d gene. In addition, two-dimensional gel electrophoresis of immunoprecipitates from one of the I-Ed-negative cell lines demonstrated the presence of intracytoplasmic Ed polypeptides that exhibited significantly decreased amounts of oligosaccharide-induced heterogeneity. The introduction of class II A beta b and A alpha b genes by DNA-mediated transfection resulted in the serologic and functional expression of a class II I-Ab molecule but not the reexpression of the endogenous class II molecules; thus a transacting regulatory element is unlikely to be the target of the mutagenic event. The analysis of these and other Ia variant cell lines may prove useful in understanding the molecular mechanisms that control the expression of class II molecules in B cells.     

Delineation of Ia:nominal antigen complementary determinants recognized by T cells in studies of gene complementation in response to insulin

The immune response to beef insulin in mice is controlled by genes in the IA subregion. We have previously shown that B6.C-H-2bm12 (bm12) mice, an A beta gene mutation of B6, have a selective loss of responsiveness to beef insulin, whereas other IAb controlled responses such as (TG)AL and collagen are unchanged. F1 hybrid mice between two nonresponder genotypes Ik and Ibm12 were found to be good responders to beef insulin suggesting functional complementation. In this report, we define the cellular and molecular basis of this complementation by investigating the determinants on Ia molecules and nominal antigen that are recognized by (B10.A X bm12)F1 proliferating T cells. Genetic analyses demonstrated that the Ik region was the only nonresponder genotype that complemented Ibm12, thus restoring responsiveness to beef insulin. More precisely an IAk and not an IEk gene product was found to be responsible for this complementation. Antibody blocking studies furthermore showed that the A alpha b:A beta k hybrid Ia mediated the response to beef insulin in (B10.A X bm12)F1 mice. Clonal analyses of the response to beef insulin in these F1 mice confirmed these conclusions, because the insulin-specific response in all 21 F1-T cell clones studied thus far was found to be dependent upon presentation via the A alpha b:A beta k hybrid Ia molecule. Dissection of the antigenic specificity of the F1-T cell clones demonstrated recognition of at least two insulin determinants, one A-loop (A8-A10) associated and the other non-loop (A4 or B chain) associated. Therefore these studies identify the molecular and antigenic basis of the Ir gene complementation seen in the response to beef insulin of (B10.A X bm12)F1 hybrids.     

Activation of murine T cells by streptococcal pyrogenic exotoxin type A. Requirement for MHC class II molecules on accessory cells and identification of V beta elements in T cell receptor of toxin-reactive T cells

We investigated the mechanisms of murine T cell activation by streptococcal pyrogenic exotoxin type A (SPE A), focusing on the role of MHC class II molecules on accessory cells (AC) and V beta usage in alpha beta TCR of SPE A-reactive T cells in comparison with staphylococcal enterotoxin B-reactive T cells. L cells transfected with I-Ab genes functioned as effective AC for SPE A-induced responses by C57BL/6 T cells, proliferation, and IL-2 production, but control L cells were not effective AC. Anti-I-Ab mAb inhibited the SPE A-induced responses. Staphylococcal enterotoxin B-induced C57BL/6 T cell blasts were composed of cells bearing V beta 3, members of the V beta 8 family, and V beta 11. Most of the SPE A-induced T cell blasts (about 80%) bore V beta 8.2. mAb reactive to V beta 8.2 markedly inhibited SPE A-induced T cell responses. Apparently, SPE A activates mainly T cells bearing V beta 8.2 in physical association with MHC class II molecules expressed on AC. We also discuss the pathogenic activities of SPE A in relation to toxic shock syndrome.     

Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.     

The molecular basis of alloreactivity in antigen-specific, major histocompatibility complex-restricted T cell clones

We have studied the relationship between major histocompatibility complex (MHC)-restricted antigen recognition and alloreactivity by examining T cell receptor (TCR) alpha and beta gene expression in cytochrome c-specific, Ek alpha:Ek beta (Ek)-restricted helper T cell clones derived from B10.A mice. The clones could be segregated on the basis of four distinct alloreactivity patterns. Clones cross-reactive for three different allogeneic la molecules (As alpha:As beta [As], Ab alpha:Ab beta [Ab], Ek alpha: Eb beta [Eb]) expressed the same V alpha and V beta gene segments, generating the distinct alloreactive specificities via unique V alpha-J alpha and V beta-D beta-J beta joining events. Ek alpha:Es beta (Es)-alloreactive B10.A clones expressed the same V alpha, J alpha, and V beta segments as an Es-restricted, Ek-alloreactive, cytochrome c-specific, H-2-congenic B10.S(9R) clone. This homology between TCRs mediating allorecognition of la molecules and recognition of the same la molecules as restriction elements associated with nominal antigen suggests that MHC-restricted recognition and allorecognition represent differences in the affinity of the TCR-MHC molecule interaction.     

Generation and characterization of cloned T helper cell lines for anti-DNA responses in NZB.H-2bm12 mice.

We have previously demonstrated that the introduction of the bm12 mutation into NZB mice results in animals that spontaneously produce high titer IgG autoantibodies to dsDNA. The observation that NZB.H-2bm12 develop lupus although NZB.H-2b control mice do not, provides a unique system to study the role of Th cells in the production of antibodies to dsDNA. We have isolated, in the absence of a known stimulating autoantigen, a series of seven autoreactive T cell clones that provide help in vitro for the production of IgG anti-dsDNA antibodies by syngeneic B cells. The data on these seven cloned T cell lines was compared to two cloned T cell lines specific for keyhole limpet hemocyanin. The seven cloned T cell lines, coined clones 19D, 23G, 410F, 410H, C1, C15, and C52 all show significant help in vitro for production of IgM and IgG antibodies to ssDNA and dsDNA; antibody levels increased 7- to 30-fold compared to cultures without T cells. Clones C1, C15, and C52 were furthered studied and were shown to provide help for IgM antihistone and anti-OVA responses but provided significantly less help for IgG antibodies. In contrast, keyhole limpet hemocyanin-specific cloned T cell lines TK2 and TK5 provided help for IgM antibodies to ssDNA, dsDNA, and histone, but failed to significantly increase IgG antibodies to ssDNA, dsDNA, or histone. The cloned T cell lines were restricted to H-2bm12 and proliferated only in response to APC from NZB.H-2bm12 and B6.C-H-2bm12 but not NZB.H-2b or NZB.H-2d mice; their in vitro helper activity was inhibited by antibodies to class II. All cloned T cell lines expressed Thy-1, CD5, and TCR-alpha/beta. Three of the seven clones used TCR-V beta 4. However, the V beta expression of the four remaining autoreactive T cell clones could not be determined. All of the autoreactive cloned T cell lines produce significant IL-4 but no detectable IL-2 or IFN-gamma. We believe that HPLC-purified peptides eluted from I-Abm12 molecules from APC can potentially provide insight on the putative autoantigen.     

Molecular analysis of antigen recognition by insulin-specific T-cell hybridomas from B6 wild-type and bm12 mutant mice.

Molecular analysis of the heterodimeric T-cell antigen receptor of insulin-specific class II-restricted T-cell hybridomas (THys) derived from C57BL/6 (B6) wild-type and B6.C-H-2bm12 (bm12) mutant mice revealed that such T cells use a diverse V gene repertoire. Analysis of three THys that use related V genes, however, showed a number of novel features. Two THys that share major histocompatibility complex restriction use V alpha genes that are 98.6% homologous. Two THys sharing the same antigen fine specificity use a particular germ line V beta D beta J beta combination. A 21-base-pair deletion in the 5' segment of the J beta gene occurs in one THy, suggesting a novel mechanism for generating diversity in T-cell antigen receptor beta genes. The first amino acid encoded by N sequences at the V-D junction is conserved in a pair of T cells which recognize identical antigenic epitopes. The implications of these findings for the structural mechanisms underlying major histocompatibility complex-restricted antigen-specific T-cell recognition are discussed.     

Ia specificities on parental and hybrid cells of an I-A mutant mouse strain

Splenic B cells and B cell blasts from the I-A mutant mouse strain B6.C-H-2bm12 were tested by serology with a series of new monoclonal anti-Iab antibodies. Four out of 5 of those monoclonal antibody-defined specificities that are determined by wild-type I-Ab antigens were undetectable on B6.C-H-2bm12 cells. Specificities both present and absent on mutant cells appear to be determinants on the same wild-type molecule, as indicated by sequential precipitation experiments with soluble H-2b antigens. The lack of expression of certain Ia specificities on mutant cells was found not to be the result of disparate control by the Xid gene, which was previously shown to control the expression of Ia.W39, another specificity absent in B6.C-H-2bm12 mice. Serologic testing of Ia specificities on cells and blasts from F1-hybrid mice suggested that the Iabm12 antigens are codominantly expressed, indicating a failure to detect trans regulation or complementation of the mutant phenotype. Another monoclonal antibody-defined Ia specificity dependent on the expression of the E beta polypeptide was normally expressed in B6.C-H-2bm12 mice. These data thus suggest that the lesion of these mutant mice occurred in the A alpha and/or A beta structural gene, resulting in the loss of several Ia specificities.     

Cytotoxic T lymphocyte response to minor H-42a alloantigen in H-42b mice: clonal inactivation of the precursor cytotoxic T lymphocytes by veto-like spleen cells that express the H-42a antigen

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by "veto cells" contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

Tolerance induction of allo-class II H-2 antigen-reactive L3T4+ helper T cells and prolonged survival of the corresponding class II H-2-disparate skin graft

The present study investigates the effects of i.v. presensitization with class II H-2-disparate allogeneic cells on various L3T4+ T cell functions including the capability of rejecting the corresponding allogeneic skin graft. C57BL/6 (B6) mice were i.v. presensitized with class II H-2 disparate B6-C-H-2bm12 (bm12) spleen cells. Such presensitization did not affect the bm12-specific L3T4+ T cell-mediated proliferative and interleukin 2 (IL-2)-producing capacities. A single cell suspension of (B6 x bm12)F1 spleen cells was depleted of APC by two round-passages over Sephadex G-10 columns. This APC-depleted fraction of (B6 x bm12)F1 cells failed to stimulate B6 responding cells in mixed lymphocyte reactions (MLR). The addition of recombinant IL-1 to the MLR restored anti-bm12 MLR responses, indicating that APC-depleted (B6 x bm12)F1 cells bear bm12 alloantigens but are unable to stimulate B6 anti-bm12 L3T4+ T cells. A single i.v. administration of APC-depleted (B6 x bm12)F1 cells into B6 mice resulted in almost complete abrogation of the capacity of recipient B6 lymphoid cells to give anti-bm12 MLR and IL2 production. This suppression was bm12 alloantigen-specific and attributed to the elimination or functional impairment of anti-bm12 T cell clones rather than the induction of suppressor cells. The tolerance was also observed in graft-rejection responses. The strikingly prolonged survival of bm12 skin grafts was produced when grafts were implanted into B6 mice which had been presensitized with APC-depleted, but not with untreated (B6 x bm12)F1 spleen cells. These results indicate that allo-class II H-2 antigen-reactive L3T4+ T cells are rendered tolerant by i.v. presensitization with APC-depleted fraction of the corresponding allogeneic cells.     

Functional and phenotypic evidence for presentation of E alpha 52-68 structurally related self-peptide(s) in I-E alpha-deficient mice

The Y-Ae mAb and the 1H3.1 TCR-alpha beta (V alpha 1/V beta 6) are two immune receptors specific for I-Ab MHC class II molecules complexed to the 52-68 fragment of the alpha-chain of I-E class II molecules (the E alpha 52-68 peptide). A profound intrathymic negative selection occurs in 1H3.1 TCR transgenic mice in the presence of an I-E alpha transgene. The administration of mAbs to 1H3.1/I-E alpha double-transgenic newborn mice reveals that Y-Ae, but not the isotype-matched anti-I-E Y17 mAb, rescues a significant number of mature (V beta 6highCD4+CD8-) thymocytes and allows the detection of E alpha 52-68-reactive T cells in the periphery. These observations indicate that deletion of autoreactive T cells can be specifically inhibited in vivo by an mAb specific for the deleting self-peptide:self-MHC class II complex. Similar inhibition experiments indicate that C57BL/6 (I-Ab+/I-E alpha-) mice constitutively express an E alpha-independent, Y-Ae-recognizable epitope(s). This finding is confirmed by the phenotypic analysis of mature (MHC class II high) C57BL/6 bone marrow-derived dendritic cells. Collectively, these observations further illustrate the peptide specificity of negative selection and demonstrate that MHC class II-positive cells from unmanipulated C57BL/6 mice that lack a functional I-E alpha gene can assemble one or more self-peptide:I-Ab complexes recognizable by the E alpha 52-68:I-Ab complex-specific Y-Ae mAb.     

Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules.

To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site.     

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.     

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.     

Separation of the immune response genes for LDH-B and MOPC-173. I. Description of an immune response defect in B10.BASR1

By using the intra-I-region recombinant mouse strain, B10.BASR1 (H-2as4), the immune response (Ir) genes for LDH-B and MOPC-173 were genetically and serologically separated, as assayed by T cell proliferation. Previous work demonstrated that the H-2s and H-2b strains respond to LDH-B and MOPC-173, whereas the H-2a and H-2k strains failed to respond due to haplotype-specific suppression of I-Ak-activated T helper cells by I-Ek-activated T suppressor cells. In the experiments reported here, B10.BASR1 mice, which lack I-Ek expression, mounted a significant T cell proliferative response to MOPC-173 but not to LDH-B. Separation of the Ia determinants used in restricting these two antigen responses was further confirmed when pretreatment of B10.S(9R) (A alpha sA beta sE beta sJk) macrophages with A.TL anti-B10.HTT (anti-A beta sE beta sJs) serum absorbed with B10.BASR1 spleen cells blocked the LDH-B response but not the MOPC-173 response. Unabsorbed serum blocked both antigen responses. The primary immunogenic determinant recognized by LDH-B or MOPC-173 immune T cells was not present on both antigens, as MOPC-173-primed T cells and LDH-B-primed T cells responded only to the priming antigen. Lastly, by using the A beta mutant strain, B6CH-2bm12, it was shown that the Ir gene and Ia determinants affected by this mutation had no effect on the LDH-B and MOPC-173 proliferative responses. These results suggest the possibility of an intragenic recombinatorial event in either the A alpha or A beta chain resulting in the separation of these two immune response gene functions.     

Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

T cell clones specific for IgG2a of the a allotype: direct evidence for presentation of endogenous antigen

T cell clones specific for IgG2a of the alpha allotype have been isolated from C57BL/6J mice. Antigenic determinants recognized by these clones were localized by using a panel of hybrid IgG2b-IgG2a myeloma proteins. These experiments provide evidence for two distinct antigenic sites, one located in a segment encompassing the hinge region and most of the CH2 domain, and the other in a segment spanning the CH3 domain and the C-terminal eight residues of the CH2 domain. As judged by their failure to respond in the presence of B6.C-H-2bm12 spleen cells, all the clones recognize determinants created, in part, by the I-A beta chain. A strong proliferative response was observed in the presence of spleen cells from several H-2b strains, including C3H.SW, A.BY, D1.LP, and BALB.B. Experiments testing reactivity directed toward spleen cells from appropriate allotype-congenic mouse strains demonstrated that this response was controlled by Igh-linked genes. These results clearly indicated 1) that Igh-1a-specific T cell clones are stimulated by endogenously synthesized IgG2a, and 2) these T cells recognize shared determinants expressed on IgG2a molecules of various strains. These experiments thus provide strong evidence for presentation of self antigens under normal physiological conditions.