Superantigen-induced anergy of V beta 8+ CD4+ T cells induces functional but non-proliferative T cells in vivo.

The response profile of staphylococcal enterotoxin B (SEB)-primed murine V beta 8+ CD4+ and V beta 8+ CD8+ T cells was analysed upon rechallenge in vitro. While in vitro responses to secondary stimulation with SEB were reduced to background levels, the in vivo reactivity after rechallenge with SEB was retained, in that SEB-primed mice succumbed to lethal T-cell shock, lymphokines [interleukin-1 (IL-1), IL-2, Il-4, IL-6, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)], and lymphokine-specific mRNA accumulation could be detected in V beta 8+ CD4+ and V beta 8+ CD8+ T cells. However, V beta 8+ CD4+ T cells failed to enter the cell cycle. While the phenotype of V beta 8+ CD8+ T cells was indistinguishable from that of their counterparts from naive mice, V beta 8+ CD4+ T cells exhibited in vivo an unusual phenotype as non-proliferative but functional T cells. We conclude that in vitro-defined anergy does not disclose the functional abilities of ligand-reactive V beta 8+ T cells in vivo, and that priming with superantigen (SAg) induces in vivo a differentiation of SEB-reactive V beta 8+ CD4+ T cells into a non-proliferative but functional phenotype.     

H-2 class I Ld antigen positively selects different TCR V beta gene products in CD8+ and CD4+ T cells.

Expression of CD4+ or CD8+ determinants and ten different TCR V beta genes was analysed by two-colour flow cytometry in lymph node cells (LNC) of BALB/c and dm2 (a BALB/c Ld loss mutant) mice. TCR V beta 14 expression of CD8+ T cells and TCR V beta 7 and 14 of CD4+ T Cells were significantly more frequent in BALB/c than in dm2 LNC, whereas no differences were observed in the frequency distribution of TCR V beta+ CD4-CD8- double negative LNC from BALB/c versus dm2 mice. These results represent the first published evidence for positive selection of particular TCR V beta genes by individual MHC class I molecules in non-manipulated mice.     

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.      

Human double-negative (CD4-CD8-) T cells bearing alpha beta T cell receptor possess both helper and cytotoxic activities.

Expression of CD4 or CD8 on the cell surface is an important guide for discriminating the immunological functions of T cells. However, a minor T cell subset, which lacks both CD4 and CD8 molecules but bears the usual form of T cell receptor (TCR) alpha beta (CD4-CD8-TCR alpha beta+ T cells), has recently been found not only in mice but also in humans, and its role in immune response is now of considerable interest. In order to clarify the characteristics of this newly defined T cell subpopulation, we established five IL-2-dependent CD4-CD8-TCR alpha beta+ T cell clones from the peripheral blood of a healthy individual, and examined their various biological functions. It was found that all clones not only helped B cells in immunoglobulin production, but also exerted major histocompatibility complex-unrestricted cytotoxicity. Although their CD3/TCR complexes were functionally competent, the cytotoxicity seemed to be mediated via unknown molecules other than the CD3/TCR complex, as evidenced by the failure of CD3 MoAb to inhibit the cytotoxic activity. Our present findings showed that CD4-CD8-TCR alpha beta+ T cells possess potential bifunction, i.e. helper and cytotoxic activities. Their roles in the pathogenesis of immunodeficiency are discussed.     

Definition of unique traits of human CD4-CD8- alpha beta T cells.

We have studied the nature of human CD4-CD8- (double negative) alpha beta T cells to determine whether they possess unique characteristics which could further differentiate them from conventional CD4+ or CD8+ (single positive) T cells. We observed that double negative TCR alpha beta+ T cells differ from single positive T cells in the following respects: (i) their T cell receptor (TCR) repertoire is different, as revealed by the analysis of 47 clones derived from three individuals and by analysis of peripheral blood lymphocytes (PBL) prior to in vitro manipulation; (ii) their in vivo CD3:TCR expression is lower before in vitro manipulation and expansion; (iii) their direct proliferative response to IL-3, which is not mediated by secondary release of other T cell growth factors. These characteristics have also been recently ascribed to murine double negative alpha beta T cells, which develop extrathymically and are considered to be a distinct T cell lineage. Our data suggest that, like their murine counterparts, human double negative alpha beta T cells may represent a distinct T cell lineage which might develop extrathymically.     

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.     

Interleukin-18 regulates T helper 1 or 2 immune responses of human cord blood CD4+ V alpha 24+V beta 11+ natural killer T cells

Natural killer T (NKT) cells, exhibiting both T-cell and NK-cell markers, are known to regulate immune responses by secreting T-helper (Th) 1 and Th2 cytokines. We analyzed NKT cells in cord blood (CB) for phenotypical and functional characteristics and regulatory mechanisms that control Th1 and Th2 determination. Human CB V alpha 24+V beta 11+ NKT cells were predominantly the CD4+ single positive (SP) phenotype (approximately 96%), in contrast to adult peripheral blood V alpha 24+V beta 11+ NKT cells which are composed of a dominant population of the CD4-CD8-double negative (DN) phenotype and a minor population of the CD4+ SP phenotype. The CB CD4+ V alpha 24+ NKT cells, following stimulation with the primary culture, gained the capacity to secrete interferon (IFN)-gamma, a Th1 cytokine, and interleukin (IL)-13, a Th2 cytokine. The combination of IL-18 and IL-12 induced IFN-gamma production in CB CD4+ V alpha 24+ NKT cells, while IL-18 in combination with IL-2 induced IL-13 production in these cells. Thus, IL-18 regulates the determination of the Th1 or Th2 immune response by human CD4+ V alpha 24+ NKT cells through different cytokine combinations.     

Intrathymic selection of murine TCR alpha beta+CD4-CD8- thymocytes

The CD4-CD8- thymocyte population contains the precursors of all other thymocytes. However, it also contains a significant proportion of cells which express surface TCR alpha beta, and have little or no precursor activity. Like peripheral T cells, but unlike most other thymocytes, these TCR alpha beta+CD4-CD8- thymocytes do not express heat stable antigen. Both the origin and developmental status of these cells are unclear, and are the subject of this report. We have measured the proportion of V beta 8.1+ cells amongst TCR+HSA-CD4-CD8- thymocytes in MIs-1a versus MIs-1b mice, in order to determine whether they have undergone negative selection. The proportions were similar in both strains, in contrast to mature T cells, indicating that neither they nor their precursors had undergone clonal deletion. We also measured the accumulation of these cells over the early life of the animal and found that it was extremely slow. Our data also show that although TCR-V beta 8.1+ cells are reactive to MIs-1a in association with MHC class II, most mature TCR-V beta 8.1+ cells in MIs-1b mice are CD8+, suggesting an additional reactivity with MHC class I. We raise the possibility that TCR-V beta 8.1+CD4-CD8- thymocytes are derived from TCR-V beta 8.1+CD4+CD8+ thymocytes, and that the reactivity of TCR-V beta 8.1 with both MHC classes I and II has resulted in the down-regulation of both CD4 and CD8.     

Neonatal mouse CD4+ mature thymocytes show responsiveness to interleukin 2 and interleukin 7: growth in vitro of negatively selected V beta 6- and V beta 11-expressing CD4+ cells from (C57BL/6 x DBA/2)F1 mice

By three colour flow microfluorimetry, we have recently shown that neonatal mouse CD4 single positive thymocytes are a population of proliferating cells. Furthermore, analysis of CD4+ thymocytes from (C57BL/6 x DBA/2)F1 mice showed that they proliferate regardless of whether they express particular V beta-encoded TCR molecules (V beta 6 and V beta 11) that are undergoing intrathymic deletion. In this report, cell culture experiments demonstrate that unstimulated neonatal CD4+ thymocytes from such mice proliferate in vitro in response to a combination of r-IL-2 and r-IL-7. Simultaneous three colour analysis of V beta TCR, CD4 expression, and DNA content of these cultured cells shows that V beta 6+, -8+, and -11+ cells grow equally well. Experiments where cells were cultured overnight in unsupplemented medium did not reveal preferential loss of negatively selected (V beta 6+ and V beta 11+) subpopulations of CD4+ cells. Taken together, these results suggest that IL-2 and IL-7 play a role in the intrathymic proliferation of developing mature T cells.     

Positive selection of V beta 2+ CD8+ T cells.

T cells bearing V beta 4, V beta 6, V beta 10, V beta 14, and V beta 17a are positively selected by MHC class I and/or class II molecules with poorly elucidated mechanisms. In this paper levels of V beta 2+ CD4+ and V beta 2+ CD8+ T cells from 33 inbred, five F1 hybrid, and 48 [(C58 x DBA/2)F1 x DBA/2] backcross mice have been examined. The results show that (i) V beta 2+ CD8+ T cells are positively selected by MHC class I H-2k molecules, (ii) this positive selection might be mediated by a non-H-2 ligand(s) in association with the Kk molecule, and (iii) inbred strains of mice, so far examined, do not have endogenous superantigens for deletion of V beta 2+ T cells.     

Down syndrome (DS) peripheral blood contains phenotypically mature CD3+TCR alpha, beta+ cells but abnormal proportions of TCR alpha, beta+, TCR gamma, delta+, and CD4+ CD45RA+ cells: evidence for an inefficient release of mature T cells by the DS thymus.

Down syndrome (DS) thymocytes have a markedly diminished proportion of cells expressing high levels of the alpha, beta T cell receptor (TCR alpha, beta) and the associated CD3 molecule. Thus, we examined the surface expression of TCR alpha, beta and CD3 as well as TCR gamma, delta, CD4, CD8, CD16, and CD45RA on peripheral blood lymphocytes (PBL) from 13 noninstitutionalized subjects with DS and 13 closely age-matched sibling controls using immunofluorescence and flow cytometry. DS PBL expressed high surface levels of TCR alpha, beta and CD3, but, as compared to controls, they had a lower proportion of cells expressing TCR alpha, beta (61% vs. 68%, respectively; P less than or equal to 0.05). Moreover, the absolute number of TCR alpha, beta+ cells was considerably lower for DS subjects than for controls (1634 +/- 229 vs. 2763 +/- 530, respectively; P less than or equal to 0.05). DS subjects had a markedly higher proportion of cells expressing TCR gamma, delta than did the controls (12% vs. 7%, respectively; P less than or equal to 0.02). In addition, DS subjects had a lower proportion of CD4+CD45RA+ cells than controls (22% vs. 35%, respectively; P less than or equal to 0.02), representing naive T cells which have recently emigrated from the thymus. The imbalance in the proportions of T cell subpopulations we have observed in DS PBL may contribute to the increased susceptibility to infection associated with DS and may represent a diminished efficiency in the production of newly differentiated T cells by the DS thymus.     

Comparison of activation marker and TCR V beta gene product expression by CD4+ and CD8+ T cells in peripheral blood and lymph nodes from HIV-infected patients.

Since lymphoid organs constitute the site of active and progressive HIV disease, analysis of their lymphocytes may provide more accurate information on T cell abnormalities than that obtained from studying peripheral blood lymphocytes. The objective of this study was to compare the expressions of activation markers and T cell receptor (TCR) V beta gene products by CD4+ and CD8+ T cells in lymph nodes (LN) and peripheral blood (PB) from healthy individuals and asymptomatic HIV-infected patients to determine whether anomalies that could be identified at the HIV replication site could support the hypothesis of T cell activation by HIV-encoded antigens or superantigens. CD4+ and CD8+ T cells in paired LN and PB obtained from six healthy controls and five asymptomatic HIV-infected individuals were analysed by flow cytometry, using anti-CD38, anti-HLA-DR and 13 anti-V beta MoAbs that cover, approximately, 45% of the T cell repertoire. Analysis of T cell activation marker expression indicated that the percentages of CD4+ and CD8+ T cells bearing CD38 or CD38 and HLA-DR molecules were higher in patients than in controls and, in patients, higher in LN than in PB. Comparison between the V beta repertoires of CD4+ and CD8+ T cells in LN and PB showed that, in each healthy individual, a limited number of V beta families expressed by CD4+ or CD8+ T cells had different repartition in LN and PB, whereas in each HIV+ patient, more V beta families exhibited different distributions and these differences recurred among certain V beta segments, such as V beta 5.3 and V beta 21 in the CD4+ T cell population and V beta 5.2/5.3, V beta 12 and V beta 21 in the CD8+ T cell population. Taken together, these data argue for a skewed TCR repertoire in HIV infection and sustained activation of T cells by HIV-encoded antigens at the site of HIV replication, and further demonstrate that a high proportion of CD4+ T cells are in an activation state that may, indirectly, participate in their functional abnormalities.     

Response of V beta 8.1+ T cell clones to self Mls-1a: implications for the origin of autoreactive T cells.

Clonal deletion and anergy are two major mechanisms of self-tolerance. However, the molecular mechanisms underlying clonal deletion and anergy, as well as the threshold of TCR affinity/avidity required for these processes, are not known. Expression of the V beta 8.1 TCR correlates with the reactivity of the T cells to the minor lymphocyte stimulating locus-1a (Mls-1a) and T cells expressing this TCR are deleted in the thymus of Mls-1a mice. Similarly, in TCR V beta 8.1 transgenic mice, the number of CD4+CD8-T cells is reduced in Mls-1a mice. However, small numbers of CD4+CD8-T cells remain in the periphery of adult Mls-1a transgenic mice. We have generated T cell clones from TCR V beta 8.1 transgenic mice by stimulation of lymph node T cells with C57BL/6 alloantigens. Interestingly, CD4+CD8-V beta 8.1+ clones isolated from the transgenic mice of Mls-1a background responded to the self-antigen Mls-1a, to which they did not respond in primary assay. Reactive patterns of the clones were compared with clones derived from Mls-1b mice. Proliferation and cytokine production of the clones from Mls-1a mice to the self-antigen Mls-1a were generally reduced when compared with clones from Mls-1b mice. More importantly, T cell clones from Mls-1a mice required more Mls-1a antigen for their activation, and were more susceptible to the inhibitory effects of anti-CD4 antibody on the proliferative responses to Mls-1a than those from Mls-1b mice. These results suggest that the T cell receptor on clones derived from Mls-1a mice have functional but reduced affinity/avidity for self-antigen Mls-1a.     

Analysis of J beta gene segment usage by CD4+ and CD8+ human peripheral blood T lymphocytes.

Certain T cell antigen receptor V gene products in man have been shown by us and others to display a reproducible bias for preferential expression in CD4+ or CD8+ T cell subsets. In order to investigate whether such a skewed representation of V gene segments is also present at the J gene segment level, we tested the relative J beta gene usage by V beta 5.1 + T cells, as this V beta gene is biased towards CD4+ T cell expression in virtually all individuals. To analyze the usage of the 13 J beta gene segments, we developed a new approach using V beta 5.1 and C beta specific oligonucleotides as 5' and 3' primers respectively for polymerase chain reaction (PCR) amplification of cDNA derived from CD4+ or CD8+ peripheral blood lymphocyte (PBL) T cells. The PCR products were visualized for reactivity with individual J beta 1.1-1.6 and J beta 2.1-2.7 32P-labelled oligonucleotide probes using autoradiography and quantitative gel-scanning. Eleven normal blood donors provided the PBL T cells. The results showed that in every individual's V beta 5.1+ T cell populations (CD4 and CD8), all V beta/J beta combinations were used although at varying but reproducible levels for each J beta gene. Thus, no discernible disallowance of combinations existed. Moreover, we could show that six of 13 J beta genes were unequally expressed when compared in pairs with regard to expression in CD4+ and CD8+ T cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human T cell antigen receptor repertoire: skewed use of V beta gene families by CD8+ T cells.

The TCR repertoire of human CD8+ peripheral blood lymphocytes has been determined using MoAbs to the V beta 2, 3, 5.1, 5.2/5.3, 6.7, 8, 12 and 19(17)V beta gene families. The CD8T cell repertoire for V beta 2 and V beta 3 is shown to be skewed, with an excess of individuals having higher values than are consistent with a normal distribution. A significant majority of these individuals are over the age of 40. High values of V beta CD8+ cells were found for each V beta family studied except for 6.7a. Individual high values are stable for at least 12 months. In addition, the total percentage of CD4 and CD8 cells reacting with this panel of reagents was determined. There is a significant excess of V beta + CD4+ cells (33%) over CD8+V beta + cells (21.9%). Thus the human CD8 V beta repertoire differs from the human CD4 repertoire in a number of important ways.     

Cytotoxic activity of V beta 8+ T cells in Crohn's disease: the role of bacterial superantigens.

In Crohn's disease, disease-related stimuli could alter the T cell receptor (TCR) repertoire. To examine the possibility that changes in function may occur in T cell subsets without obvious changes in expression of TCR, we analysed the TCR repertoire of cytotoxic T lymphocytes in Crohn's disease peripheral blood. Furthermore, we examined the effect of bacterial superantigens, staphylococcal enterotoxin B (SEB) and E (SEE) on the cytotoxic function of T cell subsets bearing different TCR V genes using MoAbs specific for CD3 and TCR V gene products in a redirected cytotoxicity assay. There was no difference between patients and controls in the cytotoxicity measured in concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 or with six of seven anti-TCR V gene MoAbs. However, the cytotoxicity of V beta 8 T cells was decreased in Crohn's disease patients. This was not due to a decrease in total or CD8+ T cells expressing V beta 8. Furthermore, in normal subjects, PBMC stimulation with SEE and SEB selectively expanded and increased the cytotoxicity of V beta 8 and V beta 12 T cells, respectively. In Crohn's disease, although SEB stimulation increased the number and cytolytic function of the V beta 12 subset, SEE stimulation failed to increase cytolytic activity of V beta 8+ T cells in spite of the expansion of V beta 8+ T cells. These results suggest that the changes in cytotoxic function observed in V beta 8 T cells in Crohn's patients may reflect previous exposure to a V beta 8-selective superantigen.     

Decreases in alpha beta T cell receptor negative T cells and CD8 cells, and an increase in CD4+ CD8+ cells in active Hashimoto''s disease and subacute thyroiditis.

We examined peripheral lymphocyte subsets in patients with autoimmune thyroid disease, or subacute thyroiditis, in the active stage when possible. During destructive thyrotoxicosis arising from alpha beta T cell receptor (TCR) negative T (WT31-CD3+) cells and CD8 (CD4-CD8+) cells decreased and those of CD4+CD8+ cells increased slightly, resulting in proportional increases in CD4 (CD4+CD8-) cells, non-T, non-B (CD5-CD19-) cells, and the CD4/CD8 cell ratio. Changes were similar in active subacute thyroiditis. During stimulative thyrotoxicosis in active Graves' disease, the numbers of such T lymphocyte subsets were not changed, but only the number of CD5+ B (CD5+CD19+) cells increased markedly, resulting in proportional decreases in total T (CD3+) cells, alpha beta+ TCR T (WT31+CD3+) cells, CD8 cells, and non-T, non-B cells. A serial study of some of the patients showed opposite changes in alpha beta TCR- T cells, the CD4/CD8 cell ratio, and CD5+ B cells between the active stages of Graves' and Hashimoto's diseases. alpha beta TCR- T cells were mostly gamma delta TCR+ T (IIF2+ CD3+) cells in these patients. These data suggest that alpha beta TCR-T (gamma delta TCR+ T), CD8, and CD4+ CD8+ cells are important in thyroid destruction in Hashimoto's disease and subacute thyroiditis, and that CD5+ B cells are important in thyroid stimulation in Graves' disease.     

Spontaneous production of various cytokines except IL-4 from CD4+ T cells in the affected organs of sarcoidosis patients.

We investigated surface antigens and spontaneous cytokine production of T cells from bronchoalveolar lavage fluid (BALF) and aqueous humor (AH) from pulmonary sarcoidosis patients for a better understanding of the role of T cells in granuloma formation. The levels of CD3, CD11b, and CD28 antigen expression on freshly isolated T cells in the BALF of patients were significantly lower than those in peripheral blood lymphocytes (PBL) of either sarcoidosis patients or healthy donors (HD). In contrast, the levels of CD80 (B7/B7-1) and CD86 (B70/B7-2) antigen expression were significantly higher on these T cells and alveolar macrophages in the BALF of patients. Fifty-three T cell clones (TCC) established from the BALF and AH of the three sarcoidosis patients displayed primarily either CD4+ CD11b+ CD28+ or CD4+ CD11b- CD28- phenotypes. Most (61-90%) of these TCC spontaneously produced greater amounts of IL-1 alpha, IL-10, tumour necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than did TCC from the PBL from sarcoidosis patients or HD (P < 0.05). Interferon-gamma (IFN-gamma), IL-6, and IL-2, but not IL-4, were also produced by 40-48% of these TCC. These results suggest that CD4+ T cells of the affected organs of sarcoidosis patients are activated and involved in the immunopathogenesis of sarcoidosis through production of various cytokines.