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.     

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.     

Rearrangement and expression of T-cell antigen receptor genes in human T-lymphocyte tumor lines and normal human T-cell clones: evidence for allelic exclusion of Ti beta gene expression and preferential use of a J beta 2 gene segment.

The gene encoding the beta chain of the human T-cell receptor for antigen is composed of variable (V), diversity (D), joining (J), and constant (C) gene segments which undergo specific rearrangements during T-lymphocyte ontogeny. Southern blot analyses of seven human T-cell tumor lines and normal human T-lymphocyte clones revealed that most of these T-cell lines rearrange their Ti beta genes differently. The T-cell tumor line HPB-MLT rearranges and transcribes both of its Ti beta genes. Cloning and sequencing of the Ti beta cDNAs corresponding to these rearrangements revealed that one of the rearranged Ti beta genes is defective, while the other is functional and corresponds to the Ti beta protein expressed on the surface of these cells. Thus, this cell line displays a pattern of allelic exclusion of Ti beta gene expression. A comparison of four C beta 2-containing Ti beta cDNAs from three different cell lines revealed that three of the four utilize the same J beta 2.5 gene segment joined to different D beta and V beta genes, suggesting that there may be preferential use of this J gene during J beta 2 rearrangements. Hybridization analyses with probes for the alpha and beta genes of the T-cell receptor and the T-cell-specific T gamma gene revealed that HPB-MLT cells appear to express approximately equivalent amounts of RNA corresponding to each of the rearranged Ti alpha and Ti beta genes. However, they express a much lower level of T gamma RNA.     

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.     

CD3+4-8- alpha beta T cell population with biased T cell receptor V gene usage. Presence in bone marrow and possible involvement of IL-3 for their extrathymic development.

Analysis of TCR of a series of CD4-8- (double negative; DN) alpha beta T cell lines induced with IL-3 revealed that their V gene usage was biased for V alpha 4 and V beta 2. This has been confirmed in the primary short-term cultures. Thus, IL-3 induced the generation of DN alpha beta T cells with predominant V beta 2 gene expression from the CD4+/CD8+ T cell-depleted spleen or bone marrow (BM) cells of both normal and nude BALB/c mice within 10 days. It was further indicated that the V beta 2+ beta-chain genes contained few junctional N regions in both IL-3-induced primary DN alpha beta T cells and continuous lines. Search for the in vivo counterpart of in vitro IL-3-induced DN alpha beta T cells revealed that BM, but not spleens, of normal BALB/c and B6 mice did contain a significant proportion of DN alpha beta T cells, and that the majority of them expressed V beta 2+ beta-chain genes with few junctional N regions. The presence of V beta 2+ DN alpha beta T cells was similarly observed in the BM of BALB/c nude mice, but their proportion varied markedly among various strains of mice, which was not linked to H-2 haplotypes. The results indicated that V beta 2+ DN alpha beta T cells in the BM represented one of the thymus-independent T cell populations, whose development was under the major histocompatibility Ag complex-unlinked genetic control. TCR of these T cells were shown to be functional as judged by the proliferative response to anti-V beta 2 antibody. Taken together, present results suggested that IL-3 could induce differentiation and/or proliferation of DN alpha beta T cells with uniquely limited repertoire, which existed preferentially in BM in vivo, and implied the possible involvement of extrathymic endogenous ligands as a positive selection force.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

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)     

Amino acid residues in the T cell receptor CDR3 determine the antigenic reactivity patterns of insulin-reactive hybridomas

We examined TCR gene usage in a panel of beef insulin/I-Ad-restricted T cell hybrids obtained from BALB/c mice. These hybrids demonstrated several distinct patterns of reactivity defined by their ability to respond to species variants of insulin. Correlation of TCR-alpha and -beta-gene usage with these patterns of reactivity demonstrated that TCR gene usage was restricted within Ag reactivity groups. In particular, V-J junctional regions (CDR3 equivalent) were restricted with conserved junctional amino acid motifs present in both TCR-alpha- and -beta-chains. Comparison of TCR gene usage in hybrids expressing identical V alpha and V beta gene segments but demonstrating different patterns of reactivity revealed that changes in either J alpha and/or J beta gene segment usage could alter antigenic reactivity. Indeed, single or limited amino acid differences within the CDR3 region were sufficient to markedly alter fine specificity. These data demonstrate the critical role for CDR3 in determining antigenic reactivity in beef insulin-reactive hybrids and are compatible with the current model of TCR/peptide/MHC interaction.     

The pigeon cytochrome c-specific T cell response of low responder mice. I. Identification of antigenic determinants on fragment 1 to 65

An examination of the proliferative response to pigeon cytochrome c fragments 1 to 65 and 1 to 80 by T cells from mice that are low responders to the native molecule revealed that some of the strains could respond to antigenic determinants on these fragments. T cell clones derived from B10.A(3R) and B10.A(4R) mice were used to characterize the antigenic determinants on fragment 1 to 65. All of the clones recognized syngeneic A beta:A alpha Ia molecules as their restriction element. Three B10.A(3R) clones and six B10.A(4R) clones recognized fragment 39 to 65. Another four B10.A(4R) clones responded to fragment 1 to 38. By stimulating with a series of cytochrome c fragments from different species, as well as a synthetic peptide, it was possible to localize the antigenic determinant(s) recognized by the B10.A(3R) clones to residues 45 to 58. Each clone showed a unique pattern of responsiveness to the various fragments, suggesting a diversity of T cell receptors specific for the same peptide. One B10.A(3R) clone could be stimulated by many of the 1 to 65 fragments in association with allogeneic B10.SM presenting cells and by tuna fragment 1 to 65 in association with B10.M presenting cells, although the rank order of potency for several of the fragments was different than that observed with syngeneic antigen-presenting cells. In addition, the clone was poorly reactive to a synthetic peptide containing a conservative substitution, serine for threonine, at position 49. The implications of these results for subsite dissection (agretope and epitope) of the antigenic determinant recognized by this clone are discussed.     

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.     

Comparison of the T cell receptors on insulin-specific hybridomas from insulin transgenic and nontransgenic mice. Loss of a subpopulation of self-reactive clones.

Transgenic mice expressing the human insulin gene do not produce insulin-specific antibody after injection of human insulin. Nevertheless, they have some peripheral T cells that proliferate to human insulin in vitro. To investigate the nature of these T cells, human insulin-specific T cell hybridomas were produced from transgenic and nontransgenic mice. Transgenic hybridomas required more insulin to achieve maximum responses and they produced lower levels of lymphokines than nontransgenic hybridomas. The majority of nontransgenic hybridomas recognized only human and pork insulin whereas transgenic hybridomas recognized beef, sheep, and/or horse insulin in addition to human and pork insulin. The TCR expressed by transgenic and nontransgenic hybridomas were determined by Northern analysis. Both types of hybridomas used several different V alpha and V beta gene families and no favored association between V alpha and V beta gene usage was detected in either type. V beta 1 was used by 7 of 16 nontransgenic hybridomas but only by 1 of 16 transgenic hybridomas. V beta 6 receptors were predominantly expressed by the transgenic hybridomas and all V beta 6-bearing hybridomas recognized beef as well as human insulin. The differences in Ag reactivity and TCR gene usage suggest that V beta 1-bearing human insulin-reactive T cells were clonally deleted or inactivated in the transgenic animal. Other clones, representing a minor subpopulation in nontransgenic mice, were recovered from transgenic mice.     

Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes

The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up- regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL, the infiltrating T cells could be stained with mAbs to alpha 3 or beta 1 and were localized in close proximity to the epiligrin-containing basement membrane. Infiltrating lymphocytes in malignant cutaneous B disease (CBCL) did not express alpha 3 beta 1 by immunohistochemical techniques and did not associate with the epidermal basement membrane. The present findings clearly define a function for alpha 3 beta 1 in T cells and strongly suggest that alpha 3 beta 1 interaction with epiligrin may be involved in the pathogenesis of cutaneous inflammation.     

T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments.

Both cDNA and genomic clones of the T cell receptor (TCR) alpha- and beta-chain genes of the alloreactive cytotoxic T lymphocyte (CTL) clone F3 were examined. Two distinct rearrangement events, one functional and one non-functional, were found for both the alpha and beta loci. Thus only a single functional TCR alpha beta heterodimer could be defined, consistent with allelic exclusion in the TCR genes. The V alpha gene employed by F3 is part of a six-member V alpha subfamily. Genomic clones containing each member of this subfamily were isolated and the V alpha nucleotide sequences determined. Five of these six genes are functional; these genes differ from each other by 7-14% at the amino acid level. A single dominant hypervariable region was defined within this subfamily, in contrast to the pattern of variability seen between V alpha genes in general.     

Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.     

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.     

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

Marked differences in the efficiency of expression of distinct alpha beta T cell receptor heterodimers

Ag recognition by most T lymphocytes is mediated by clonally distributed alpha beta heterodimeric receptors. A major fraction of TCR diversity is believed to be due to the random coexpression in individual T cells of the products of independently rearranging alpha- and beta-genes (combinatorial diversity). However, analysis of cell surface receptors on transfected T hybridoma cells synthesizing various sets of alpha- and beta-chains revealed marked differences in the efficiency of expression of certain alpha beta-pairs. Specifically, using the functionally rearranged gene products of the 2B4 cytochrome c specific T hybridoma (V beta 3, V alpha 11.2) and BW5147 parent lymphoma (V beta 1, V alpha BW), a hierarchy of expression efficiency relative to indirectly measured precursor chain levels in the cell was shown to be 2B4 alpha-BW beta greater than 2B4 alpha - 2B4 beta greater than BW alpha - BW beta greater than BW alpha - 2B4 beta. The estimated difference between the best expressed and worst expressed pairs is on the order of 50-fold. For the beta-chain, the primary determinant of expression efficiency with a given alpha-chain appears to be the V segment, as a second V beta 1-chain with distinct D and J regions from BW beta was expressed with the same pattern. These data imply that alpha- and beta-chains do not form well-expressed TCR in a random manner and that limitations on the useful combinatorial association of these chains may significantly affect the functional T cell repertoire.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

Separation of the immune response genes for LDH-B and MOPC-173. II. Description of an immune response defect in B10.ASR7

By using the intra-I region recombinant mouse strain B10.ASR7 (H-2as3), 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 H-2s and H-2b strains respond to LDH-B and MOPC-173 whereas H-2a and H-2k strains failed to respond due to haplotype-specific suppression of I-Ak molecule-activated T helper cells by I-Ek molecule-activated T suppressor cells. In the experiments reported here, B10.ASR7 mice, which lack I-Ek expression, mounted a significant T cell proliferative response to LDH-B but not to MOPC-173. Separation of the Ia determinants used in restricting these two antigen responses was further confirmed when pretreatment of B10.S(9R) (A beta sA alpha sE beta sE alpha k) macrophages with A.TL anti-B10.HTT serum (anti-As beta Es beta Js) adsorbed with B10.ASR7 spleen cells blocked the MOPC-173 response but not the LDH-B response. Unadsorbed serum blocked both antigen responses. The B10.ASR7 E beta allele was determined to be s due to the ability of (A.TL X B10.ASR7)F1 hybrids to mount a T cell proliferative response to the terpolymer GLPhe. Monoclonal antibody blocking of the B10.ASR7 T cell proliferative response to LDH-B demonstrated that the Ia.2 and Ia.17, and not the Ia.15 epitopes are spatially related to the Ia epitopes involved in the restriction of the B10.ASR7 LDH-B T cell proliferative response. In addition, B10.ASR7 helper T cells generated in response to LDH-B were suppressed in a haplotype-specific manner by I-Ek molecule-restricted suppressor T cells in that (A.TL X B10.ASR7)F1 hybrids failed to respond to LDH-B. This nonresponsiveness was eliminated by treatment with monoclonal antibodies directed against the I-Ek molecule. These results suggest the possibility that the immune response defect in B10.ASR7 could be related to the site of recombination.     

Phenotypic characterization of murine tumor-infiltrating T lymphocytes.

Tumor infiltrating lymphocytes (TIL) can be isolated from solid tumors and selectively expanded in long term culture with IL-2 and autologous irradiated tumor. Such long term cultured cells express anti-tumor activity in vitro, mediate the regression of established tumor in murine models of cancer, and have been used for the treatment of cancer in humans. We have characterized freshly isolated mouse Thy-1+ TIL populations, as well as long term TIL cultures, from several different C57BL/6 (B6) tumors. Freshly isolated Thy-1+ TIL include both CD4+ and CD8+ cells, as well as cells bearing NK markers. These cells are predominantly TCR alpha beta+, with a smaller population of TCR gamma delta+ cells. The TCR alpha beta+ cells expressed a broad distribution of V beta phenotypes that was statistically different from that expressed in normal B6 splenic Thy-1+ cells or CD8+ cells, presumably reflecting in vivo selection in the host anti-tumor response. NK cells are present in these tumors at a greater frequency than noted in splenic T cells. Cultured TIL populations rapidly became exclusively Thy-1+/CD8+/CD4- and TCR alpha beta+/gamma delta-. Individual long term TIL populations initially expressed multiple V beta products, but rapidly restricted their V beta expression, frequently expressing a single dominant V beta. The identity of this dominant V beta varied among different TIL lines, but the overall representation of V beta phenotypes in these cultures was statistically different from that seen in Thy-1+ or CD8+ splenocytes. No statistical difference was noted between lines derived from antigenically distinct tumors. The selection of tumor specific T cells in vitro is therefore not reflected in any simple predominance of V beta usage. The complexity of TCR usage in the anti-tumor response may result from the involvement of multiple alpha- and beta-chain regions in the response to a single antigenic determinant, or may reflect multiple antigenic determinants expressed on a single syngeneic tumor.