Analysis of the susceptibility of CD57 T cells to CD3-mediated apoptosis

After stimulation with anti-CD3 antibody in vitro, CD57(+) T cells showed a greater susceptibility to apoptosis than CD57(-)alphabetaT cell receptor (TCR)(+) T cells (regular alphabeta T cells). The apoptotic fraction of CD57(+) T cells showed an increased production of active caspase-3. An increase in both Fas expression and Fas-ligand (FasL) production was also observed in CD57(+) T cells, whereas the expression of survivin was suppressed in CD57(+) T cells compared to that of regular alphabeta T cells. CD57(+) T cells display a biased expansion of a few Vbeta T cell fractions in individuals, but such Vbeta T cells were not specifically susceptible to CD3-mediated apoptosis. The TCR expression level of CD57(+) T cells was much lower than that of regular T cells and anti-TCR antibody stimulation induced a smaller apoptotic proportion of CD57(+) T cells than did anti-CD3 antibody. Although the CD3epsilon expression levels were similar in both T cell subsets, the CD3zeta level of CD57(+) T cells was significantly higher than that of regular T cells. These results suggest that several apoptotic and anti-apoptotic molecules are involved in the CD3-induced apoptosis of CD57(+) T cells and raise the possibility that the imbalance in expression of the CD3epsilon and CD3zeta chains may also contribute to the susceptibility of CD57(+) T cells to undergo apoptosis.     

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.     

Importance of CD8(+)Vbeta8(+) T cells in IFN-gamma-mediated prevention of toxoplasmic encephalitis in genetically resistant BALB/c mice.

In our attempt to identify a major T cell population(s) that recognizes protective Toxoplasma gondii antigens and produces interferon-gamma (IFN-gamma) for prevention of toxoplasmic encephalitis (TE), we found T cell receptor Vbeta8(+) cells to be the most frequent IFN-gamma-producing population infiltrated into the brain of T. gondii-infected BALB/c mice genetically resistant to the disease. To examine the role of IFN-gamma production by this T cell population for resistance, we transferred Vbeta8(+) immune T cells purified from spleens of infected BALB/c and IFN-gamma(/) mice into infected, sulfadiazine-treated, athymic nude mice. After discontinuation of sulfadiazine treatment, control nude mice that had not received any T cells and animals that had received Vbeta8(+) T cells from IFN-gamma(/) mice all died because of reactivation of infection (TE). In contrast, animals that had received the cells from BALB/c mice survived. Thus, IFN-gamma production by Vbeta8(+) T cells plays an important role in prevention of TE in these animals. When Vbeta8(+) immune T cells were divided into CD4(+) and CD8(+) subsets, a potent protective activity was observed only in the CD8(+) subset, whereas a combination of both subsets provided greater protection than did the CD8(+)Vbeta8(+) population alone. These results indicate that the CD8(+) subset of Vbeta8(+) T cells is a major afferent limb of IFN-gamma-mediated resistance of BALB/c mice against TE, although the CD4(+) subset of the T cell population works additively or synergistically with the CD8(+)Vbeta8(+) population.     

CD8 lymphocytosis in primary cytomegalovirus (CMV) infection of allograft recipients: expansion of an uncommon CD8+ CD57- subset and its progressive replacement by CD8+ CD57+ T cells.

Allograft recipients undergoing cytomegalovirus infection present increased proportions of circulating CD8+ lymphocytes. A longitudinal study of 11 kidney and five liver allograft recipients with primary CMV infection but no other etiological factor of graft dysfunction revealed selective imbalances of peripheral blood CD8+ T cell subsets. Initially, CMV viraemia is associated with elevated CD8+bright T cell numbers and T cell activation. Activation markers fall to normal when viral cultures become negative (before the end of the first month). During the second to sixth month, most (12/16) patients keep up high CD8+ T cell counts (1050-2900 CD8+ cells/mm3), comprising an uncommon CD8+ T cell subset, as 45-73% of CD8+bright lymphocytes were CD3+ and TCR alpha beta+, but were not stained by anti-CD28, CD11b, CD16, CD56, and CD57 antibody. Unexpectedly, CD8+CD57+ T cells, a hallmark of CMV infection, do not appear until the second to sixth month of primary CMV infection, and their numbers increase progressively thereafter. They become the predominant CD8+ T cell subset after 6 months of infection and their persistence for several (up to 4) years is strongly correlated (r = 0.87) with expansion of CD8+ cells. By analysis with MoAbs, there was no bias towards the use of particular TCR-V beta gene families at any time of primary CMV infection. Persistence of CD8 lymphocytosis is thus directly related to the rate of expansion of an uncommon CD8+CD57- subset and its progressive replacement by CD8+CD57+ T cells that are chronically elicited by CMV.     

Origin of CD57+ T cells which increase at tumour sites in patients with colorectal cancer.

Human T cells carrying natural killer (NK) markers, CD57 or CD56 antigens, appear to be distinguishable from other T cell subsets in terms of their granular lymphocyte morphology and their numerical increase in patients with AIDS and in recipients of bone marrow transplantation. At the beginning of this study, we observed that CD57+ T cells as well as CD56+ T cells were abundant at tumour sites in many patients with colorectal cancer. Since all these findings for CD57+ T cells are quite similar to those of extrathymic T cells seen in mice, we investigated how CD57+ T cells are distributed to various immune organs in humans. They were found to be present mainly in the bone marrow and liver, but to be completely absent in the thymus. Similar to the case of extrathymic T cells in mice, they were observed to consist of double-negative CD4-8- subsets as well as single-positive subsets (preponderance of CD8+ cells), and to contain a considerable proportion of gamma delta T cells. These features are striking when compared with those of CD57- T cells, which are characterized by an abundance of CD4+ subsets and alpha beta T cells. Not only at tumour sites but also in the peripheral blood, some patients with colorectal cancer displayed elevated levels of CD57+ cells. These results suggest that CD57+ T cells may be of extrathymic origin, possibly originating in the bone marrow and liver, and may be associated with tumour immunity, similar to another extrathymic population of CD56+ T cells in humans.     

Intrathyroidal lymphocyte subsets, including unusual CD4+ CD8+ cells and CD3loTCR alpha beta lo/-CD4-CD8- cells, in autoimmune thyroid disease.

Intrathyroidal lymphocyte subsets were analysed in 13 euthyroid patients with autoimmune thyroid disease by two-colour flow cytometry and compared with subsets in peripheral blood. In both Graves' and Hashimoto's diseases, proportions of intrathyroidal CD5- B cells were higher than in peripheral blood. The numbers of such cells were correlated with serum levels of anti-thyroid microsomal antibodies. Proportions of T cells bearing alpha beta chains of T cell receptors (TCR alpha beta+ T; T alpha beta) and CD16+CD57+ natural killer (NK) cells were lower in the thyroid, but proportions of CD3hiTCR alpha beta-TCR gamma delta+ (T gamma delta) cells were not different. Proportions of CD4+Leu-8- helper T cells and CD4+CD57+ germinal centre T cells were higher and proportions of CD4+Leu-8+ suppressor-inducer T cells and CD8+CD57+ or CD8+CD11b+ suppressor T cells were lower than in the blood in both diseases. Proportions of CD5+ B cells were high in Graves' disease, and proportions of CD8+CD11b- cytotoxic T cells were high in Hashimoto's disease. Unexpectedly, CD4+CD8+ cells and CD3loTCR alpha beta lo/-CD4-CD8- cells were present in thyroid tissues of both diseases. These findings suggest that: (i) an imbalance in the numbers of regulatory T cells and of NK cells that had appeared in the thyroid resulted in the proliferation of CD5- B cells, which were related to thyroid autoantibody production; (ii) CD5+ B cells and cytotoxic T cells are important for the different pathological features in Graves' and Hashimoto's diseases, respectively; and (iii) intrathyroidal CD4+CD8+ cells and CD3loTCR alpha beta lo/-CD4-CD8- cells may be related to the pathogenesis of autoimmune thyroid disease.     

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.     

Characterization of CD4+ CD8alphaalpha+ and CD4-CD8alphaalpha+ intestinal intraepithelial lymphocytes in rats

Intestinal intraepithelial lymphocytes (i-IEL) of aged rats comprise CD4+CD8alphaalpha+ and CD4-CD8alphaalpha+ T cells expressing TCR alphabeta. In the present study, we compared characteristics between CD4+CD8alphaalpha+ and CD4-CD8alphaalpha+ i-IEL, which were purified by a cell sorter from the i-IEL of 6-month-old Lewis rats. Most of the CD4+CD8alphaalpha+ i-IEL were of the CD44(hlgh) phenotype, while CD4-CD8alphabeta+ i-IEL were CD44(low). Vbeta usage in the CD4-CD8alphaalpha+ i-IEL was much diversified, while CD4+CD8alphaalpha+ i-IEL showed a skewed Vbeta repertoire. The CD4+CD8alphaalpha+ i-IEL but not the CD4-CD8alphaalpha+ i-IEL proliferated in response to syngeneic spleen cells, which was partially inhibited by addition of anti-MHC class I mAb. The CD4+CD8alphaalpha+ i-IEL produced IFN-gamma and IL-2 but no IL-4 or transforming growth factor (TGF)-beta in response to syngeneic spleen cells, while CD4-CD8alphaalpha+ i-IEL produced abundant levels of TGF-beta but no IL-2, IFN-gamma or IL-4. CD4+CD8alphaalpha+ i-IEL proliferated in response to exogenous IL-2 but not to IL-15, while CD4-CD8alphaalpha+ i-IEL could respond to IL-15 as well as IL-2. These results suggest that a significant fraction of CD4+CD8alphaalpha+ i-IEL belongs to Th1-type T cells capable of responding to self-MHC class I, while CD4-CD8alphaalpha+ i-IEL are a unique population with a diversified Vbeta repertoire that respond to IL-15 in rats.     

Changes in Natural Killer Cells, the CD57CD8 Subset, and Related Cytokines in Healthy Aging

Aging has been shown to be accompanied by various changes in the lymphocyte subset distribution in the elderly. We have investigated more fully, and in a large number of subjects, age-related changes within several subpopulations bearing natural killer (NK) cell-associated surface antigens and changes in several cytokines involved in NK cell expansion. A total of 229 healthy subjects from all decades of life from 20 to 98 years of age was included in this cross-sectional study. A significant increase with age was found in both the absolute counts and the proportions of CD3–CD(16+56)+, CD3+CD(16+56)+, CD57+CD8+, CD57+CD8_(low)+, and CD57+CD8– cells, whereas the CD57+CD8_(high)+ subset, which may represent the cytolytic T cell population more precisely, showed less change with age. Some evidence is also provided to suggest that these expanded NK cell populations are in an activated state. Soluble IL-2 receptor levels were also found to increase significantly with age and correlated with certain NK cell subsets. Although the functions of some of these subsets remain to be elucidated, their expansion in the elderly may represent a remodeling of the immune system with increasing age, with an increase in non-MHC-restricted cells perhaps compensating for the previously reported decline in T and B cells in the elderly. Alternatively, increased numbers of these cells may be a direct result of cytokine dysregulation or increased antigenic or neoplastic cell challenge.     

Contribution of NK, NK T, gamma delta T, and alpha beta T cells to the gamma interferon response required for liver protection against Trypanosoma cruzi

In the present work, we show that intracellular Trypanosoma cruzi is rarely found in the livers of acutely infected mice, but inflammation is commonly observed. The presence of numerous intrahepatic amastigotes in infected gamma interferon (IFN-gamma)-deficient mice corroborates the notion that the liver is protected by an efficient local immunity. The contribution of different cell populations was suggested by data showing that CD4- and CD8-deficient mice were able to restrain liver parasite growth. Therefore, we have characterized the liver-infiltrating lymphocytes and determined the sources of IFN-gamma during acute T. cruzi infection. We observed that natural killer (NK) cells increased by day 7, while T and B cells increased by day 14. Among CD3+ cells, CD4+, CD8+, and CD4- CD8- cell populations were greatly expanded. A large fraction of CD3+ cells were positive for PanNK, a beta1 integrin expressed by NK and NK T cells. However, these lymphocytes were not classic NK T cells because they did not express NK1.1 and showed no preferential usage of Vbeta8. Otherwise, liver NK T (CD3+ NK1.1+) cells were not increased in acutely infected mice. The majority of PanNK+ CD4+ and PanNK+ CD8+ cells expressed T-cell receptor alphabeta (TCRalphabeta), whereas PanNK+ CD4- CD8- cells were positive for TCRgammadelta. In fact, gammadelta T cells showed the most remarkable increase (40- to 100-fold) among liver lymphocytes. Most importantly, intracellular analysis revealed high levels of IFN-gamma production at day 7 by NK cells and at day 14 by CD4+, CD8+, and CD4- CD8- TCRgammadelta+ cells. We concluded that NK cells are a precocious source of IFN-gamma in the livers of acutely infected mice, and, as the disease progresses, conventional CD4+ and CD8+ T cells and gammadelta T cells, but not classic NK-T cells, may provide the IFN-gamma required for liver protection against T. cruzi.     

Proliferation and interleukin 5 production by CD8hi CD57+ T cells

CD8hi CD57+ T cells have previously been described as effector memory T cells with minimal expansion capacity and high susceptibility to activation-induced cell death. In contrast, we demonstrate here that CD8hi CD57+ T cells are capable of rapid expansion using multiple techniques including [(3)H]thymidine uptake, flow cytometric bead-based enumeration and standard haemocytometer counting. Previous reports can be explained by marked inhibition of activation-induced expansion and increased 7-amino-actinomycin D uptake by CD8hi CD57+ T cells following treatment with CFSE, a dye previously used to measure their proliferation, combined with specific media requirements for the growth of this cell subset. The ability of CD8hi CD57+ T cells to further differentiate is highlighted by a distinct cytokine profile late after activation that includes the unexpected release of high levels of interleukin 5. These data indicate that CD8hi CD57+ T cells should not be considered as "end-stage" effector T cells incapable of proliferation, but represent a highly differentiated subset capable of rapid division and exhibiting novel functions separate from their previously described cytotoxic and IFN-gamma responses.     

The CD39 molecule defines distinct cytotoxic subsets within alloactivated human CD8-positive cells.

Lymphocyte activation induces or increases the expression of several surface structures, none of which is characteristic of an activated cell subset. In particular, structures such as CD45RO, CD25, CD26, CD49b, CD54, CD71 are expressed by the vast majority of lymphocytes at various times following in vitro activation. CD39 molecules were originally identified on activated B lymphocytes and have recently been described on activated T cell clones. In the present report, we have characterized phenotypically and functionally defined cell subsets generated during an in vitro allostimulation. Results indicated that the percentage of CD39+ cells reached a maximum at day 6 and remained stable thereafter. We demonstrate that CD39 expression allows the identification within the allosensitized CD8+ cytotoxic cells of distinct subsets of cells mediating allo cytotoxic T lymphocyte or natural killer (NK)-like reactivity. More precisely, CD8+CD39+ alloactivated cells mainly mediate specific killer activity, whereas CD8+CD39- alloactivated cells predominantly exhibit NK-like reactivity. Further, we show a high functional correlation associated with the lack of CD39 expression on NK-like alloactivated CD8+ cells, while there is no association with CD56 or CD57 NK-associated structures.     

T cell receptor vbeta repertoire in bronchoalveolar lavage in Wegener's granulomatosis and sarcoidosis.

BACKGROUND: Lymphocytic alveolitis is a common feature of Wegener's granulomatosis (WG) and sarcoidosis. In pulmonary sarcoidosis analysis of the T cell receptor (TCR) configuration disclosed a biased TCR repertoire in cells obtained by bronchoalveolar lavage (BAL), suggesting that the pulmonary T cell population contains an oligoclonal, probably antigenically selected component. We examined whether this is also a feature of WG. METHODS: The distribution of 16 TCR Vbeta phenotypes on BAL cells was compared with the distribution on autologous blood cells in 8 WG patients with lymphocytic alveolitis. A control group was composed of 5 patients with active pulmonary sarcoidosis. The two groups were comparable with respect to the lymphocyte count in BAL and the distribution of the CD4+ and CD8+ T cell subsets in BAL and the blood. Overutilization of individual Vbeta phenotypes on BAL cells was considered to be significant when the percentage of positive cells in BAL doubled than in the blood and the frequency in BAL was at least 10% of total BAL T cells. RESULTS: All 5 sarcoidosis patients showed substantial overutilization of individual Vbeta families in BAL as compared with the blood. By contrast, only 2 of the 8 WG patients showed overutilization of individual Vbeta phenotypes in BAL. CONCLUSIONS: These findings do not suggest that the T cell population in BAL of WG patients contains a prominent oligoclonal component. While BAL is a convenient approach to obtain T cells from a site of active disease, BAL cells do not appear to be good material for the isolation of clonally selected T cells in WG.     

Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll-like receptor 9 ligand

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 T cells in culture. The CD8+ DC showed a reduced capacity to stimulate T cell proliferation compared to either of the CD8- DC subsets, regardless of antigen and DC dose. In contrast to CD8- DC, the quiescent CD8+ DC did not induce IFN-gamma production from CD4 T cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC class II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll-like receptor 9 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 T cell proliferation and IFN-gamma production. In contrast, similar activation of CD8- DC produced a more modest increase in capacity to stimulate CD4 T cell proliferation and no increase in capacity to stimulate IFN-gamma production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8- DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.     

Factors controlling levels of CD8+ T-cell lymphocytosis associated with murine gamma-herpesvirus infection.

Intranasal infection of mice with murine gamma-herpesvirus 68 (MHV-68) elicits a striking CD8+ T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vbeta4+ CD8+ T cells. The Vbeta4+ CD8+ T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vbeta4+ CD8+ T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8+ T cell activation and Vbeta4+ CD8+ T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vbeta4+ CD8+ T-cell response correlates with the degree of CD8+ T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vbeta4+ CD8+ T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.     

Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells

The pathogenesis of pulmonary sarcoidosis has been related to an increased production of Th1-like cytokines. However, cytokine expression in sarcoidosis has not been systematically studied at a single-cell level. We therefore investigated the expression of IL-2, IL-4, IL-13, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) intracellularly in bronchoalveolar lavage (BAL) and peripheral blood CD3+ T lymphocytes from patients with pulmonary sarcoidosis (radiologic stage II-III, n = 8) and normal controls (n = 9) by flow cytometry. In contrast to IL-4 and IL-13, the percentage of T lymphocytes expressing intracellular IL-2 (49.3 +/- 21.3% versus 14.5 +/- 15.6%), IFN-gamma (75.5 +/- 14.9% versus 32.6 +/- 18.7%) and TNF-alpha (68.3 +/- 18.7% versus 36.8 +/- 20.8%) was significantly higher in patients with sarcoidosis than in normal controls (each P < 0.005). In contrast to BAL lymphocytes, expression of these cytokines in peripheral blood lymphocytes did not differ between patients with sarcoidosis and normal controls. Close correlations were observed between the percentages of BAL lymphocytes expressing intracellular IL-2, IFN-gamma and TNF-alpha, but not for IL-4 or IL-13. Analysis of the expression of these cytokines in T lymphocyte subsets revealed IL-2, IFN-gamma, and TNF-alpha in CD4+ as well as CD8+ T lymphocytes, suggesting a contribution of TC1 cells to the production of proinflammatory cytokines in sarcoidosis. We conclude that a Th1-like cytokine pattern can be observed in CD4+ as well as in CD8+ BAL T lymphocytes in patients with pulmonary sarcoidosis.     

TH1/TH2 and TC1/TC2 profiles in peripheral blood and bronchoalveolar lavage fluid cells in pulmonary sarcoidosis

BACKGROUND: Sarcoidosis is thought to be a type-1 cytokine-mediated disorder. However, few data are available on the profiles of cytokine expression by TH cells at the single-cell level, as assessed by intracellular cytokine flow cytometry. Additionally, it remains to be determined whether the balance of TC1 and TC2 cells can be altered in sarcoidosis. OBJECTIVE: The aim of this study was to evaluate the TH1/TH2 and TC1/TC2 balances in sarcoidosis. METHODS: Using triple-color flow cytometry and phorbol 12-myristate acetate/ionomycin stimulation, we measured the production of the intracellular cytokines IFN-gamma and IL-4 in CD4+ and CD8+ T cells separately, which were obtained from peripheral blood and bronchoalveolar lavage fluid (BALF) of 20 patients with sarcoidosis, and compared their cytokine expressions with those of 10 normal subjects. RESULTS: Under unstimulated conditions, there were no significant differences in the proportion of cytokine-producing CD4+ or CD8+ T cells in peripheral blood or BALF between patients with sarcoidosis and normal control subjects. On stimulation with phorbol 12-myristate acetate/ionomycin for 4 hours, in BALF of the patients, but not in peripheral blood, we found a significant increase in the percentage of IFN-gamma-producing CD4+ T cells and a decrease in the percentage of IL-4-producing CD4+ T cells, resulting in a 3.5-fold higher ratio of IFN-gamma/IL-4-producing CD4+ T cells compared with that found in normal subjects. In contrast, no difference was found in the proportions of cytokine-producing CD8+ T cells or the ratio of IFN-gamma/IL-4-producing CD8+ T cells in either the peripheral blood or BALF between the patients and normal subjects. CONCLUSIONS: These findings suggest that the prominent shift toward a type-1 phenotype may occur in CD4+ T-cell populations but not in CD8+ T-cell populations in the affected organs of sarcoidosis.     

CD4-CD8- T cells bearing invariant Valpha24JalphaQ TCR alpha-chain are decreased in patients with atopic diseases

Atopic disorders are caused by disregulated activation of T helper 2 (Th2) cells that produce IL-4 and IL-5. Because the presence of IL-4 potently augments the differentiation of naive T cells into Th2 cells, it is important to seek the cell population which provides IL-4 for naive T cells. Recently, a unique subpopulation of T cells, natural killer (NK) T cells, has been shown to produce a large amount of IL-4 upon activation, suggesting their regulatory role in initiation of Th2 cell differentiation. To determine whether NK T cells play a regulatory role in human Th2 cell-mediated atopic diseases, we analysed the frequency of invariant Valpha24JalphaQ CD4-CD8- double-negative (DN) T cells, human NK T cells, in patients with atopic asthma and atopic dermatitis. We also studied cytokine production from Valpha24+ Vbeta11+ DN T cells, which comprise most of Valpha24JalphaQ DN T cells. We found that the invariant Valpha24JalphaQ DN T cells were greatly diminished in patients with asthma and atopic dermatitis. On the other hand, there was no significant difference in Valpha24+ CD4+ T cells possessing invariant Valpha24JalphaQ TCR between healthy subjects and atopic patients. We also found that Valpha24+ Vbeta11+ DN T cells from healthy subjects predominantly produced interferon-gamma (IFN-gamma) but not IL-4 upon activation. These results suggest that NK T cells may not be essential for human atopic disease and that the disappearance of NK T cells, most of which produce IFN-gamma, may be involved in the pathogenesis of atopic diseases.     

Vbeta spectratype analysis reveals heterogeneity of CD4+ T-cell responses to minor histocompatibility antigens involved in graft-versus-host disease: correlations with epithelial tissue infiltrate.

Lethal graft-versus-host disease (GVHD) can be induced after hematopoietic stem cell transplantation between major histocompatibility complex-matched murine strains expressing multiple minor histocompatibility antigen (miHA) differences. In the C57BL/6By (B6)-->C.B10-H2b/LiMcdJ (BALB.B) irradiation model, both CD4+ and CD8+ donor T cells can mediate lethal GVHD, whereas in the B6-->CXB-2/By (CXBE) model, in which the recipient expresses a subset of BALB.B miHA, only the CD8+ T cells are lethal. Phenotypic analysis of CD4+ T cells collected from the thoracic duct lymphocyte pool of recipient mice had indicated expansion of the donor T-cell receptor Vbeta6-9 families in BALB.B recipients, and only Vbeta7 and Vbeta9 populations in CXBE mice. CDR3-size spectratyping, used to further analyze these responses, revealed overlapping oligoclonal expansion of Vbeta4, Vbeta6-10, and Vbeta12-14 families in both BALB.B and CXBE recipients injected with host-presensitized B6 T cells. In addition, the B6-->BALB.B CD4+ T-cell response appeared to involve the recognition of unique BALB.B-specific miHA, indicated by additional skewing of Vbeta2 and Vbeta11 families. On the other hand, the B6-->CXBE strain combination exhibited unique skewing of the Vbeta16 and Vbeta18 families. Immunohistochemical staining of lingual epithelial sections from BALB.B recipients of naive B6 CD4+ T cells correlated with the involvement of several of the spectratype-skewed Vbeta families in GVHD lesions. Furthermore, magnetic cell separation techniques were used to positively select the spectratype-skewed Vbeta families from the donor B6 CD4+ T cells; the former were found to have significant GVHD potential upon transplantation into lethally irradiated BALB.B recipients. In contrast, mice that received transplants from the unskewed Vbeta families all survived with minimal symptoms of GVHD. Taken together, these results demonstrate that the expansion of particular Vbeta families, as identified by spectratype analysis, correlates with the induction and pathogenesis of lethal GVHD.