Evidence for monoclonal expansion of synovial T cells bearing V alpha 2.1/V beta 5.5 gene segments and recognizing a synthetic peptide that shares homology with a number of putative autoantigens.

A peptide of 15 amino acids derived from the cereal glycine-rich cell wall protein (GRP), sharing a significant homology with Epstein-Barr virus nuclear antigen-1 (EBNA-1), fibrillar and procollagen, stimulated synovial fluid (SF) T cells from juvenile (JRA) and adult (RA) rheumatoid arthritis patients. An overexpression of the V alpha 2 gene family was found in the SF from patients who responded significantly to the peptide. To investigate in more detail the SF T-cell responses to the GRP peptide, we established peptide-specific T-cell lines and clones from a DR8+ positive JRA patient with pauciarticular form. The T-cell clones were phenotyped as T-cell receptor (TCR)alpha beta+/CD4+ and their clonality was investigated by polymerase chain reaction (PCR) and flow cytometric analysis. TCR sequences from different clones demonstrated that the clones were identical and used the V alpha 2.1/J alpha 6 combined with V beta 5.5/J beta 2.7 gene segments. Interestingly, direct sequencing of the V alpha 2 family PCR product obtained from cDNA prepared from freshly isolated SF mononuclear cells identified the same TCR sequence as that used by the clones, suggesting the monoclonality of SF CD4+ T cells bearing V alpha 2.1/J alpha 6 gene products. The present data suggest a recruitment and expansion of a SF T-cell subpopulation, and also support the hypothesis that autoimmune diseases can be triggered by protein epitopes with crucial amino acids homologous to self-proteins.     

Cloning, functional activities and in vivo tissue distribution of rat NKR-P1+ TCR alpha beta + cells

We have successfully cloned nine NKR-P1+ TCR alpha beta + cells from PVG rat spleens, utilizing murine macrophage inflammatory protein-1 alpha (MIP-1 alpha) and IL-2. These clones are either double negative (DN, CD4-CD8-), which included clones 3.31, 3.71, 4.19, 4.59 and 4.65, or single positive (SP, CD4+CD8-), which included clones 1.64, 3.8, 3.76 and 3.78. No CD8+ clone was recovered. All nine clones are restricted in terms of their expression of the V beta antigens, since they express V beta 8.2 but not V beta 8.5, V beta 10 or V beta 16. These clones are agranular and they fall to generate NK or LAK activity upon incubation with IL-2, IL-12 or their combination. On the basis of their production of intracellular cytokines they can be divided into three categories: (I) SP clones (1.64, 3.8, 3.76 and 3.78) do not produce IL-2 or IL-4, but produce IFN-gamma and IL-12, and they vary in their production of IL-1, RANTES or tumor necrosis factor (TNF)-alpha; (II) DN clones 4.59 and 4.65 produce IL-1 alpha and IFN-gamma only, and fall to produce other cytokines; and (III) DN clones 3.31, 3.71 and 4.19 produce IL-1 alpha, IL-1 beta, IL-2, IL-12, IFN-gamma, RANTES and TNF-alpha. From all the clones examined only DN clones 3.31 and to a lesser degree 4.19 produce IL-4. In vivo tissue localization of clones 3.8, 3.31 and 4.59 shows that these cells distribute into the liver and bone marrow 24 h post i.v. administration. Their accumulation in the liver and bone marrow along with their ability to secrete various cytokines suggest that these cells may influence the generation, differentiation or apoptosis of immune or hematopoietic cells.      

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2- type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross- linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.      

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.      

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.     

'Natural' T cells responsive to malaria: evidence implicating immunological cross-reactivity in the maintenance of TCR alpha beta+ malaria-specific responses from non-exposed donors.

It is now generally accepted that peripheral blood of humans not exposed previously to malaria contains T cells which proliferate vigorously in response to malaria parasites and antigens. Although it has been claimed that these cells express a memory phenotype, their origin is uncertain. We have examined the phenotype and immunological responses of such cells. We confirm that these cells do express the 'memory phenotype', CD45Ro, in that depletion of such cells, but not of CD45Ra (virgin) cells, abrogates the immune response to malaria parasites. In an effort to define the genesis of these responses, numerous malaria-specific T cell clones have been generated from non-exposed individuals. These were tested for reactivity to a large panel of common bacterial, viral, and fungal pathogenic and non-pathogenic organisms. Most clones proliferated vigorously in response to one or more such organisms, while many clones demonstrated smaller but significant degrees of proliferation in response to many different organisms. Our data offers insights into the maintenance of immunological memory. All clones examined were CD3+, CD4+, CD8-, TCR alpha beta+, and TCR delta-. The ratio of TCR alpha beta+ to TCR delta+ cells among peripheral blood lymphocytes increased during polyclonal culture in the presence of parasite. The high frequency of such cells in peripheral blood (1/800-1/9000), and their response to a wide range of geographically different Plasmodium falciparum isolates and clones by both proliferation and lymphokine secretion (predominantly IFN-gamma) with a high degree of sensitivity (less than 1 parasite/microliters blood in some cases) suggests that these cells must be quickly activated following malaria infection. Their contribution to the outcome of the disease (protection/immunopathology) may be significant.     

Protection against tuberculosis by passive transfer with T-cell clones recognizing mycobacterial heat-shock protein 65.

We have previously shown that mice vaccinated by injection with J774 macrophage-like tumour cells that expressed Mycobacterium leprae heat-shock protein (hsp) 65 as a transgene had acquired a remarkably high degree of protection against subsequent challenge with virulent M. tuberculosis. We show here that antigen-specific T cells cloned from spleens of such vaccinated animals can transfer a high level of protection to non-vaccinated recipients. The most efficient cells were of T-cell receptor (TCR) alpha beta+ and CD4- CD8+ type and specifically lysed mycobacteria-infected macrophages. These findings are consistent with the importance for protective immunity of engaging the endogenous antigen-presenting pathway to bias the immune response towards a cytolytic action against a mycobacterial antigen that is expressed at the surface of infected macrophages. TCR gamma delta+ and TCR alpha beta+ cells interacted synergistically.     

Various membrane proteins of Francisella tularensis induce interferon-gamma production in both CD4+ and CD8+ T cells of primed humans.

Tularaemia is an intracellular infection, which is controlled by the host as a result of an immunospecific T-cell response. A crucial product of the responding T cells is interferon-gamma (IFN-gamma), which acts by enhancing the microbicidal activity of macrophages. T cells of tularaemia-vaccinated individuals respond in vitro to a multitude of protein antigens of the vaccine strain Francisella tularensis LVS. In the present study, the responses to four of these antigens were shown to be confined mostly to the CD45RO+ memory T-cell subset. To characterize further the phenotype of the responding cells, purified CD4+ and CD8+ T cells were stimulated with the antigens. CD4+ T cells, but not CD8+ T cells, proliferated and produced IFN-gamma. However, when CD8+ T cells were isolated from bulk cultures of lymphocytes, which had been stimulated with antigen for 3 days, they responded to an extent similar to that of CD4+ T cells. Purified CD8+ T cells also responded when they were supplemented with interleukin-2 (IL-2). There was a direct quantitative correlation between the proliferative response of CD4+ and CD8+ T cells and their production of IFN-gamma. IL-2 was produced in the cultures, the amounts being higher in the cultures of CD4+ than in those of CD8+ cells. IL-4 was not detected in the culture medium of any of the T-cell subsets. Seventeen human alpha beta + CD4+ CD8- CD3+ T-cell clones, specific to antigens of F. tularensis, were raised. When proliferating, these clones did invariably produce IL-2 and IFN-gamma but no IL-4. In conclusion, both CD4+ and CD8+ T cells of tularaemia-vaccinated individuals respond with proliferation to various protein antigens of F. tularensis, and the proliferative response is strictly associated with IFN-gamma production. The CD8+ T-cell response seems to depend on cytokines supplied by proliferating CD4+ T cells.     

Development of CD8 alpha/beta + TCR alpha beta intestinal intraepithelial lymphocytes in athymic nu/nu mice and participation in regional immune responses.

On the basis of the CD8 coreceptor expression, T-cell receptor (TCR)alpha beta-bearing intestinal intraepithelial lymphocytes (i-IEL) segregate into two populations. The CD8 alpha alpha + TCR alpha beta i-IEL develop thymus independently, whereas the CD8 alpha beta + TCR alpha beta i-IEL are generally considered to be thymus dependent. Flow cytometry analysis revealed a distinct population of CD8 alpha beta + TCR alpha beta i-IEL in individual athymic nu/nu mice. The i-IEL encompassing CD8 alpha beta + TCR alpha beta cells expressed potent cytolytic and interferon-gamma-producing activities. These findings demonstrate that CD8 alpha beta + TCR alpha beta i-IEL can develop in nu/nu mice independently from a functional thymus and suggest that these cells, directly or indirectly, perform biological functions in the gut.     

Interleukin-12 primes CD4+ T cells for interferon-γ production and protective immunity during Mycobacterium avium infection

Interleukin-12 (IL-12) is a crucial cytokine for the generation of a protective immune response against Mycobacterium avium infection. In contrast to infected control mice, IL-12-deficient mice were unable to control bacterial proliferation and their spleen T cells were almost unresponsive in vitro to specific antigens of M. avium. Susceptibility of mice deficient in IL-12 was similar to that of interferon-gamma (IFN-gamma)-deficient mice. These data indicate a crucial role of IL-12 in the development of a T-cell population able to produce IFN-gamma and to mediate protection against M. avium infection. Treatment of M. avium-infected mice with IL-12 induced CD4+ T cells with enhanced capacity to produce IFN-gamma as well as to confer increased protection against M. avium.     

Protective role for heat shock protein-reactive alpha beta T cells in murine yersiniosis.

To investigate the role of heat shock proteins (HSP) of Yersinia enterocolitica for the host immune response against this pathogen, we cloned and expressed a 60-kDa HSP of Y. enterocolitica serotype O8. A fragment of Y. enterocolitica O8 HSP60 encoded by amino acids 90 to 286 was sequenced and showed more than 90% homology with HSP60 of Y. enterocolitica O3 and GroEL of Escherichia coli and 59% homology with HSP65 of Mycobacterium bovis. The arthritogenic T-cell epitope of mycobacterial HSP65 (amino acid residues 180 to 188) was not found on Yersinia HSP60. To determine whether Yersinia HSP60 is an immunodominant antigen, the immune responses of Yersinia-infected C57BL/6 mice were analyzed. Yersinia-infected mice evolved a significant serum antibody and splenic T-cell response against Yersinia HSP60. CD4+ alpha beta T-cell clones which were generated from splenic T cells isolated from either Yersinia-infected or Yersinia HSP60-immunized mice, recognized both heat-killed Yersinia serotypes O3 and O8 as well as recombinant Yersinia HSP60 but not heat-killed Yersinia pseudotuberculosis, Salmonella typhimurium, or recombinant HSP65 of Mycobacterium bovis. The adoptive transfer of HSP60-reactive T-cell clones mediated significant protection against a lethal infection with Y. enterocolitica O8. These results indicate that HSP60 of Y. enterocolitica is an immunodominant antigen which is recognized by both antibodies and CD4+ alpha beta T cells. Moreover, this is the first report providing direct evidence that microbial HSP may elicit a protective immune response which is not associated with autoimmunity.     

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.     

Human leucocyte antigen-A2 restricted and Mycobacterium tuberculosis 19-kDa antigen-specific CD8+ T-cell responses are oligoclonal and exhibit a T-cell cytotoxic type 2 response cytokine-secretion pattern

CD8+ T cells can be grouped into two different types of secretory T lymphocytes, based on the cytokine-secretion pattern upon antigen exposure: those with a T-cell cytotoxic type 1 response (Tc1), which secrete interferon-gamma (IFN-gamma), or those with a T-cell cytotoxic type 2 response, which secrete interleukin (IL)-4 and IL-10. We examined the CD8+ T-cell response directed against an immunodominant human leucocyte antigen (HLA)-A2-presented peptide derived from a 19-kDa Mycobacterium tuberculosis-associated antigen. T cells were examined by functional analysis and by T-cell receptor (TCR) complementarity-determining region 3 (CDR3)-spectratyping, which defines the complexity of a T-cell response. T-cell stimulation with the immunodominant VLTDGNPPEV epitope yielded a Tc2 (IL-4) cytokine-secretion pattern and resulted in oligoclonal expansion of TCR-variable beta chain (VB) families, which differed from patient to patient. Generation of T-cell clones corroborated the notion that the CD8+ T-cell response directed against the HLA-A2-presented VLTDGNPPEV epitope leads to a Tc2 cytokine-secretion pattern in CD8+ T cells, as defined by IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. Characterization of the cytokine-secretion profile in HLA-A2/VLTDGNPPEV-tetramer sorted T cells from patients with active tuberculosis supported this observation: peptide-specific T cells from three of three patients secreted IL-4 and only one of three patients produced IFN-gamma in response to the nominal target epitope. Permutation of this T-cell epitope may aid to elicit a qualitatively different CD8+ T-cell response in patients with M. tuberculosis infection.     

Decreases in interleukin-4 secretion by invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells in peripheral blood of patientswith relapsing-remitting multiple sclerosis

The cytokine profile of invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells from patients with multiple sclerosis (MS) was compared with that of healthy controls. CD4(-)CD8(-)V alpha 24(+) T cells from the peripheral blood of 12 patients with relapsing-remitting MS (RR-MS), 5 patients with progressive MS (CP-MS), and 9 control individuals were directly sorted into single wells and expanded in vitro for analysis of IL-4 and IFN-gamma secretion; 315 V alpha 24J alpha Q T cell clones were generated and their T cell receptor (TCR) sequenced. T cell functionality was determined by examining cytokine secretion upon TCR cross-linking. RR-MS patients exhibited lower frequencies of IL-4 secreting CD4(-)CD8(-)V alpha 24J alpha Q T cell clones than patients with CP-MS and controls. No differences in IFN-gamma secretion were observed between the groups. An IL-4 positive cytokine profile could be correlated to the cloning efficiency of the V alpha 24J alpha Q T cells. We conclude that alterations in cytokine secretion patterns of CD4(-)CD8(-)V alpha 24J alpha Q T cells may influence the immune system and thus contribute to relapsing-remitting MS.     

WI-1, a novel 120-kilodalton surface protein on Blastomyces dermatitidis yeast cells, is a target antigen of cell-mediated immunity in human blastomycosis.

A large body of experimental data has demonstrated the central role of T cells in acquired resistance to the dimorphic fungus Blastomyces dermatitidis. We examined the human T-cell response to WI-1, a 120-kDa B. dermatitidis yeast cell surface protein recently shown to be an immunodominant antigen of the B-cell response in infected humans. Peripheral blood lymphocytes from 10 blastomycosis patients studied proliferated in response to WI-1 (mean, 19,431 cpm) and to the standard, crude cell wall antigen, Blastomyces alkali- and water-soluble antigen (B-ASWS) (mean, 19,131 cpm); lymphocytes from 10 histoplasmosis patients and 10 normal control subjects did not respond to WI-1. WI-1 stimulation of patient lymphocytes and rechallenge with WI-1 or B-ASWS showed that the antigens share immunodominant epitopes. Of 100 WI-1-responsive T-cell clones derived from peripheral blood, 10 were studied in detail to assess the phenotype, function, and ligands recognized. The clones exhibit the CD3+ CD4+ phenotype of helper T cells; 2 of 10 clones (and 21% of antigen-stimulated peripheral blood lymphocytes) use the V beta 8 T-cell receptor gene element to respond to WI-1. All the clones proliferate in response to both WI-1 and B-ASWS but not other fungal antigens, and some mediate potent cytolytic effects on WI-1- and B-ASWS-labeled targets. WI-1 recognition requires antigen processing and presentation of epitopes in association with HLA-DR (to noncytolytic clones) and HLA-DP (to cytolytic clones). From these findings, we conclude that CD4+ T cells with regulatory and cytolytic properties are involved in the development of acquired resistance of B. dermatitidis, that the cells are directed against WI-1, and that the manner of display of WI-1 peptide epitopes in conjunction with major histocompatibility complex class II may influence the profile of the immune response.     

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.     

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.