V beta1+ J beta1.1+/V alpha2+ J alpha49+ CD4+ T cells mediate resistance against infection with Blastomyces dermatitidis

Immunization with a cell wall/membrane (CW/M) and yeast cytosol extract (YCE) crude antigen from Blastomyces dermatitidis confers T-cell-mediated resistance against lethal experimental infection in mice. We isolated and characterized T cells that recognize components of these protective antigens and mediate protection. CD4+ T-cell clones elicited with CW/M antigen adoptively transferred protective immunity when they expressed a V alpha2+ J alpha49+/V beta1+ J beta1.1+ heterodimeric T-cell receptor (TCR) and produced high levels of gamma interferon (IFN-gamma). In contrast, V beta8.1/8.2+ CD4+ T-cell clones that were reactive against CW/M and YCE antigens and produced little or no IFN-gamma either failed to mediate protection or exacerbated the infection depending on the level of interleukin-5 expression. Thus, the outgrowth of protective T-cell clones against immunodominant antigens of B. dermatitidis is biased by a combination of the TCR repertoire and Th1 cytokine production.     

Application of a multiprobe RNase protection assay and junctional sequences to define V beta gene diversity in Sezary syndrome.

Nineteen patients with mycosis fungoides/Sezary syndrome (MF/SZ), a malignancy of the mature helper T-cell phenotype (CD4+TCR alpha beta+), were screened for clonotypic V beta expansions in peripheral blood with a multiprobe RNase protection assay. A different predominant V beta gene was identified in 9 of 14 patients with high peripheral blood CD4/CD8 ratios, whereas 4 of these patients showed T-cell expansions expressing V beta genes other than those included in the assay. In contrast, five patients with few, if any, malignant cells in the circulation had V beta expression levels similar to that in normal peripheral blood. A unique V-D-J sequence was found for each highly expressed V beta gene, thereby documenting monoclonality of the expanded T-cell populations. Polymerase chain reaction (PCR) primers specific for the D-J beta junction accurately identified the corresponding malignant clonotype in peripheral blood. The diverse TCR V beta gene usage found in these MF/SZ patients suggests that T-cell receptor (TCR) specificity has no bearing on this disease.     

T-cell receptor V beta selectivity in T-cell clones alloreactive to HLA-Dw14.

The HLA-DR4 subtypes Dw14 and Dw4 are T-cell-defined allospecificities encoded by the DRB1*0404 and DRB1*0401 genes, respectively. Although these allelic subtypes differ in only two amino acids, allorecognition between Dw14 and Dw4-positive individuals is brisk. This provides an opportunity to analyze T-cell receptor (TCR) usage in a very limited and specifically targeted case, namely the Dw4 anti-Dw14 allogeneic T-cell response. The variable (V), diversity (D), and joining (J) region sequences of the TCR beta chain from two different Dw14-specific alloreactive T-cell clones derived from a Dw4 donor were examined. Clone EMO25 recognized the Dw14.1, Dw14.2, and Dw15 subtypes, which share a DRB1 polymorphism at codon 71 on a DR4 background, while clone EMO36 reacted with only the Dw14.1 subtype associated with polymorphisms at codons 71 and 86. TCR beta cDNA from each clone was amplified using an anchored polymerase chain reaction (PCR) and subsequently expanded with V beta- and C beta-specific primers for asymmetric PCR and direct DNA sequencing. Both clones were found to express the same TCR V beta 8.2 gene segment; however, they have several different residues within the V beta-D beta-J beta junctional regions. V beta 8 usage was also enriched in polyclonal cells obtained from mixed lymphocyte cultures performed between the Dw4 and Dw14 responder-stimulator combination from which EMO25 and EMO36 were derived.     

Evidence for shared recognition of a peptide ligand by a diverse panel of non-obese diabetic mice-derived,islet-specific,diabetogenic T cell clones

MHC class II-restricted autoreactive T cells play a major role in the development of autoimmune diabetes mellitus in both human and mouse. Two of our groups previously established panels of islet-reactive CD4+ T cell clones from prediabetic non-obese diabetic (NOD) mice. These clones express distinct sets of TCR V alpha , V beta , J alpha and J beta , and also differ in the structure of the junctional region of TCR. All of the T cell clones have been shown to cause insulitis and several induce diabetes when transferred to various recipients. The antigen specificities of these T cell clones have not been determined, but they do not react with defined islet cell antigens such as glutamic acid decarboxylase. To identify the peptide ligands recognized by these clones, we examined the reactivity of the T cell clones to peptide mixtures in which anchor residues for H2-A g7 were fixed. Most of the clones showed similar reactivity to the peptide mixtures. To further determine the peptide ligands of the T cell clones, we synthesized several peptides based on the favored amino acid motifs and examined clone reactivity to the synthetic peptides. Some of the peptides, e.g. HLAI-RM and HIPI-RM, could stimulate most of the T cell clones tested, even though the clones expressed different TCR. The results suggest that our islet-reactive T cell clones recognize in islet beta cells a natural ligand that is similar to these peptides.     

Microbial colonization influences composition and T-cell receptor V beta repertoire of intraepithelial lymphocytes in rat intestine.

Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ-free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T-cell receptor (TCR) alpha/beta + IEL in that the proportions of CD4+ and CD8 alpha beta + TCR alpha/beta + cells were increased, while the double negative (CD4- CD8-) fraction was reduced. microbial colonization also influenced the TCR V beta repertoire of CD8+ IEL in that the proportions of V beta 8.2+ and V beta 10+ cells were increased, whereas V beta 8.5+ and V beta 16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same V beta subsets. Three-colour flow-cytometric analysis demonstrated that skewing of the V beta repertoire was most pronounced in the CD8 alpha alpha + subset, although the numerical increase of IEL mainly included the CD8 alpha beta + subset. In contrast to IEL, the TCR V beta repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCR alpha/beta + IEL subpopulations respond dynamically to the microbial gut flora and suggest that their V beta repertoire can be shaped by luminal microbial antigens.     

Human CD4-8- -Derived Clones Phenotypic and Functional Characteristics and Variation between Donors in Patterns of T-Cell Receptor y Gene Rearrangements

Clones were derived from highly purified human CD4-8- lymphocytes from three different donors and maintained in the presence of interleukin 2 and phytohaemagglutinin. Considerable variation was noted between donors in the phenotype and T-cell receptor (TCR) gamma gene rearrangements of CD4-8- -derived clones. In one donor, most clones remained CD4-8- and all were CD3+WT31- and therefore expressed gamma/delta heterodimers. TCR gamma gene rearrangements almost all involved C gamma 1. In contrast, most clones from a second donor were CD3+WT31+, and therefore expressed alpha/beta heterodimers, and many were positive for CD4 or CD8. Most clones from a third donor were CD3+WT31- with a high proportion of TCR gamma gene rearrangements involving C gamma 2. The V gamma 9JP rearrangement was exclusively confined to CD3+WT31- clones and was present in the majority of clones. Almost all CD3+WT31- clones showed TCR beta as well as gamma gene rearrangements. Most CD3+WT31- clones with at least one chromosome rearranged to C gamma 1 exhibited high non-major histocompatibility complex (MHC)-restricted cytotoxic activity, while most of those with two C gamma 2 rearrangements, and therefore expressing a non-disulphide-linked gamma/delta heterodimer, had low activity. Preincubation of effector cells with anti-CD3 strongly inhibited the cytotoxicity of CD3+WT31- clones while that of CD3+WT31+ clones was enhanced. This implicates the CD3-gamma/delta complex in target cell recognition by cytotoxic gamma/delta-bearing T-cell clones. The results show that there is heterogeneity between donors in the relative proportions of CD4-8- -derived clones expressing alpha/beta heterodimers and the different forms of the gamma/delta heterodimer.     

Mapping of V beta 11+ helper T cell epitopes on mycobacterial antigen in mouse primed with Mycobacterium tuberculosis

Antigenic epitopes for Mycobacterium tuberculosis-reactive T cell immune responses have been mapped using the purified Mycobacterium protein antigen. Lymph node cells from C57BL/6 mice that had been immunized with heat-killed M. tuberculosis were cultured with various Mycobacterium protein antigens and their reactivity was monitored by proliferative response. Usage of the TCR beta chain repertoire was analyzed by flow cytometry. Stimulation of M. tuberculosis-primed lymph node cells with MPT59 (antigen 85B, alpha antigen) induced proliferative response, production of IL-2 and IFN-gamma, and the expansion of V beta 11+ CD4+ T cells in conjunction with antigen- presenting cells in an I-Ab-restricted manner. Lymph node cells from non-primed mice failed to proliferate in response to MPT59. Using peptides covering the complete mature 285 amino acids long MPT59 protein as 15-mer molecules overlapping by five amino acids, we identified the antigenic epitope for MPT59-specific V beta 11+ T cells. The 15-mer peptide, covering amino acid residues 240-254 of MPT59 [peptide-25 (amino acids 240-254)], contains the motif that is conserved for I-Ab and requires processing by antigen-presenting cells to trigger peptide-25-specific V beta 11+ CD4+ T cells. We conclude from these results that MPT59 and peptide-25 (amino acids 240-254) are not superantigens and require antigen processing in order to stimulate V beta 11+ Th1 cells. This experimental system will provide us with a useful tool for delineating the regulation of T cell development in a particular subset of M. tuberculosis infection and for developing antigenic peptides for Th1-dominant immune responses.      

Aberrant T-cell receptor signalling of interferon-gamma- and tumour necrosis factor-alpha-producing cytotoxic CD8+ Vdelta1/Vbeta16 T cells in a patient with chronic neutropenia

We previously found that the peripheral blood (PB) mononuclear cells (MCs) (PBMCs) of a patient with chronic neutropenia contained an expanded population of cytotoxic CD8+ T cells using a variable (V) region delta1 gene product in the T-cell receptor-alpha (TCR-alpha) polypeptide [Vdelta1-constant(C)alpha+ T cells]. Sequencing of polymerase chain reaction (PCR) amplification products have now revealed a productive Vdelta1/joining (J)alphaIGRJa03/Calpha rearrangement of the TCR-alpha gene, predominantly associated with a Vbeta16/Dbeta2.1/Jbeta2.1/Cbeta2 TCR-beta gene, in these cells. Furthermore, we detected a markedly deficient proliferative response of the patient PBMCs to triggering with monoclonal antibodies (MoAbs) to the CD3 molecule, contrasting with a substantial response to the Vbeta3, 12, 14, 15, 17 and 20-specific staphylococcal enterotoxin B (SEB) superantigen, suggesting defective TCR-mediated activation of the Vdelta1+/Vbeta16+ clone. Moreover, whereas triggering of Vdelta1- T cells cultured with interleukin-2 (IL-2) by MoAb to the CD3 molecule enhanced proliferation, Vdelta1-Calpha+ T cells were inhibited by MoAbs to either CD3 or Vdelta1. Vdelta1-Calpha+ T-cell clones spontaneously secrete interferon-gamma (IFN-gamma) and were further induced to release tumour necrosis factor (TNF-alpha) when triggered by anti-CD3 plus phorbol ester. Aberrant signalling by the clonotypic TCR together with the functional properties of the CD8+ Vdelta1+/Vbeta16+ clone may thus contribute to the immunohaematological abnormalities observed in this patient.     

Monoclonal antibody against murine T cell receptor V alpha 14 cross-reacts with human CD3 epsilon and detects disulfide-linked dimeric form.

A monoclonal antibody against V14+ alpha-chain of murine T cell receptor (TCR) was established by fusing spleen cells from a rat immunized with a soluble chimeric TCR/IgG3 protein containing murine TCR V alpha 14J alpha 281 in place of the VHDHJH of an IgG3. lambda 1, and subjected to screening on a human transfectant (Jurkat variant) expressing the murine V14J281 alpha-chain. The anti-mouse V alpha 14 antibody precipitated TCR alpha beta molecules from Triton X-100-solubilized extracts of 125I-labeled murine thymocytes and spleen cells. Unexpectedly, the antibody showed cross-reactivity to the human CD3 epsilon molecule and detected a disulfide-linked 20 kDa dimeric form of human CD3 epsilon, which is a novel family component of the CD3 complex and is associated closely with the CD3 zeta-zeta homodimer as well as TCR alpha beta or TCR gamma delta.     

Dominant TCR V alpha usage by virus and tumor-reactive T cells with wide affinity ranges for their specific antigens

We have studied the TCR features and functional responses of three sets of human cytolytic T cell (CTL) clones, recognizing antigenic peptides presented by HLA-A2 and derived from the Epstein-Barr virus proteins BMLF1 and BRLF1 and from the melanoma protein Melan-A/MART-1. Within each set, a majority of clones used a recurrent V alpha region, even though they expressed highly diverse TCR beta chains and V(D)J junctional sequences. Functional assays and peptide/MHC multimer binding studies indicated that this restricted V alpha usage was not associated with the affinity/avidity of the CTL clones. The V alpha dominance, which may be a frequent feature of antigen-specific T cells, likely reflects a restricted geometry of TCR/peptide/MHC complexes, primarily determined by V alpha CDR.     

Restricted alpha/beta receptor gene usage of idiotype-specific major histocompatibility complex-restricted T cells: selection for CDR3-related sequences.

We have sequenced the T cell receptor (TcR) V alpha and V beta genes of seven independent BALB/c CD4+ T cell clones specific for the immunoglobulin lambda 2 light chain produced by the MOPC 315 myeloma (lambda 2(315)). All the clones recognize a peptide of residues 91-101 of lambda 2(315) and are restricted by the major histocompatibility complex (MHC) molecule I-E(d). The results indicate that in BALB/c mice, this anti-idiotypic response uses a very limited number of TcR. The four clones which cross-react between Phe94 and Tyr94 peptide analogues use very similar receptors (V alpha 3, J alpha 1, V beta 6, J beta 1.1). The V alpha 3 gene used by all of these clones is identical and has not been previously described. Although the four clones differ in nucleotide sequence in the V/J borders, two had identical receptors at the amino acid level. One of the cross-reactive clones exhibits a heteroclitic response to the Tyr94 peptide variant resulting from a single amino acid exchange in the V/J junction of the alpha chain. The three remaining clones which recognize only the Phe94 and not the Tyr94 peptide have somewhat more diverse TcR, however, two of these three clones use V beta 6. One of these non-crossreacting clones is alloreactive, the specificity of which can be attributed to differences in the N-D-J sequences. Taken together these data indicate that this T cell response to an immunoglobulin idiotope is very restricted in terms of the TcR used. These data in conjunction with recently published results indicate that, although there can be strong preference for individual V alpha or V beta gene segments, certain V alpha/V beta combinations are preferentially selected for interacting with a given peptide/MHC combination, and that the CDR3-related regions are crucial for antigen fine specificity and alloreactivity.     

Limited heterogeneity of biased T-cell receptor V beta gene usage in lung but not blood T cells in active pulmonary sarcoidosis.

Sarcoidosis is a multisystem disorder characterized by non-caseating granulomas and the accumulation of CD4+ T cells in involved tissues such as the lung. To evaluate the diversity of the CD4+ T-cell repertoire in this disorder, a detailed clonal analysis was performed in five individuals with active sarcoidosis who demonstrated preferential accumulation of T cells expressing the T-cell receptor variable gene family V beta 8 in either the lung or blood. In three individuals, analysis of unselected samples of nucleotide sequences derived from V beta 8+ lung T cells demonstrated degrees of clonality ranging from 11% to 46%, indicating the expansion of limited numbers of V beta 8+ T-cell clones in the lung. Analysis of the corresponding deduced amino acid sequences demonstrated common VDJ junctional amino acid residues in the dominant V beta 8+ T-cell clones derived from two oligoclonal V beta 8+ lung T-cell populations, consistent with an antigen-specific T-cell response. In contrast, analysis of V beta 8+ CD4+ T cells from the blood of an individual with a marked bias for peripheral blood V beta 8+ T cells demonstrated no evidence of oligoclonality, suggesting that the stimulus for circulating biased V beta-specific T cells in sarcoidosis may derive from a different, perhaps superantigenic, origin. Clinical improvement in the disease either in response to treatment with corticosteroids or as a result of spontaneous resolution was associated with a decrease in the proportion of V beta 8-specific T cells in the biased lung and/or blood T-cell compartments. Together, these observations are consistent with a role for this T-cell subset in the clinical manifestations of active granulomatous disease.     

A persistent T cell expansion in the peripheral blood of a normal adult male: a new clinical entity?

A dramatic and persistent T cell expansion in a healthy adult male was initially identified, using anti-T cell receptor for antigen (TCR)-specific MoAbs. The expanded T cells were found to be expressing TCR containing V alpha 12.1 and V beta 5.2, and they composed approximately one third of all the CD8+ T cells. The cells were shown to be not only non-activated (HLA-DR-, IL-2R-) but also of 'virgin' cell type (CD45RA+/CD45RO-) and they persisted over the observation period of more than one and a half years. Various T and B cell markers, and all other laboratory and physical parameters analysed, were normal. The expanded CD8+ T cells were further characterized by polymerase chain reaction (PCR) amplification, using V beta- and C beta-specific primers, followed by hybridization with J beta-specific probes. Close to 90% of the V alpha 12.1+ V beta 5.2+ T cells were found to utilize the J beta 2.5 gene segment, thus strongly suggesting the expanded T cells to be monoclonal. The condition may constitute a T cell counterpart to 'monoclonal gammopathy of undetermined significance' (MGUS), and by analogy we suggest it should be designated 'monoclonal T cell expansion of undetermined significance' (MTUS).     

Production of interferon-gamma and tumour necrosis factor-alpha by human T-cell clones expressing different forms of the gamma delta receptor.

Panels of human T-cell clones bearing the gamma delta T-cell receptor (TcR) were obtained from peripheral blood and decidual tissue and maintained in the presence of interleukin-2 (IL-2). TcR V gamma and V delta gene expression was determined in 40 TcR delta 1+ clones using the gamma delta T-cell subset markers Ti gamma A and delta TCS1, in conjunction with Southern blot analysis using TcR J gamma and J delta probes. gamma delta T-cell clones, together with control alpha beta T-cell clones derived from the same lymphocyte populations, were stimulated with phytohaemagglutinin (PHA) and their ability to produce interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) tested using specific ELISA. Many clones representative of the major peripheral V gamma 9/V delta 2J1 subset produced high amounts of both cytokines and mean levels were not significantly different from those produced by alpha beta T-cell clones. Panels of clones expressing V gamma 9 and V delta 2J1 produced significantly higher levels of TNF-alpha than clones not expressing V delta 2J1 and those expressing V delta 1J1. There was no relationship between levels of IFN-gamma and TNF-alpha produced by individual gamma delta T-cell clones and also no relationship between their non-major histocompatibility complex (MHC)-restricted cytotoxic activity and levels of either cytokine. There was a significant tendency for gamma delta T-cell clones to produce more TNF-alpha than IFN-gamma in comparison to alpha beta T-cell clones. The significance of these findings is discussed in the light of the reported differences in distribution in vivo of V delta 1J1+ and V delta 2J1+ cells.     

Control of TCR V alpha-mediated positive repertoire selection and alloreactivity by differential J alpha usage and CDR3 alpha composition

In rats expressing the f allele of the rat MHC (RT1f), CD8 T cells utilizing the V alpha 8.2 segment are 10-fold overselected during thymic development, resulting in V alpha 8.2 expression by 14% of mature CD8 T cells as compared to 1-2% in MHC congenic strains. In the alloreactive responses of CD8 T cells from RT1f-negative rats against RT1f, V alpha 8.2+ CD8 T cells are also preferentially expanded. Neither overselection nor alloreactivity of V alpha 8.2+ TCR require selective V beta pairing. However, RT1f alloreactive V alpha 8.2+ TCR preferentially use a related set of J alpha segments which contribute short homogeneous CDR3 alpha loops, with features suggesting peptide promiscuity, and little N additions. In contrast, only few overselected V alpha 8.2+ CD8 T cells showed an imprint of positive selection on J usage or CDR3 composition. The results demonstrate that a single V alpha segment can promote both MHC allele-specific positive selection and alloreactivity, and that the latter is more dependent on an additional contribution of CDR3 alpha, possibly by promoting reactivity with a diverse set of MHC-bound peptides or by providing additional MHC contacts.      

Single cell analysis of T cells infiltrating labial salivary glands from patients with Sj?gren's syndrome.

Sj?gren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into the lacrimal and salivary glands leading to symptomatic dry eyes and mouth. To analyze the function of T cells infiltrating the labial salivary glands, we analyzed T cell receptor (TCR) beta and alpha chains, the expression of various cytokine mRNAs, and apoptosis associated genes in predominant TCR BV2+ T cells in the labial salivary glands of patients with SS at the single cell level. TCR BV2+ T cells in the labial salivary glands were sorted as single cells by flow-cytometry, and then examined by a single cell polymerase chain reaction (PCR). We isolated 18 TCR BV2+ T cell clones from three patients with SS. In six clones, there were highly conserved amino acid motifs (RDxG, GNT, QGxxQETQ) in the CDR3 region of the TCR beta chain. Three of the six clones showed conserved amino acids (EDxTG, or ExxTG) in the CDR3 region of the TCR alpha chain, suggesting restricted T cell epitopes. All TCR BV2+ clones expressed IL-2 mRNA, and six clones were able to produce IL-4, indicating that the cells were Th0 type T cells. All TCR BV2+ clones in the labial salivary glands were CD4+ T cells, and ten clones overexpressed Fas antigen at the mRNA level. In contrast, only one clone expressed Fas-ligand (Fas-L) mRNA, and neither perforin nor granzyme A/B was expressed. In conclusion, these findings support the notion that TCR BV2+ T cells that infiltrate labial salivary glands recognize restricted epitopes and function as CD4+ Th0 type T cells in the induction phase of autoimmunity.