Down-regulation of cell surface CXCR6 expression during T cell activation is predominantly mediated by calcineurin

CXCR6, the receptor for the membrane-anchored chemokine, CXCL16, is expressed on a subset of CCR5-bearing memory T cells, and may play a role in recruiting these cells to sites of inflammation. Here, we set out to determine the effect of T cell activation on CXCR6 expression. Highly purified human peripheral blood T cells were cultured for 7-8 days in presence of IL-2 (400 U/ml) to enhance CXCR6 expression. Overnight stimulation with anti-CD3 mAb+anti-CD28 mAb, which resulted in CD69 induction and cytokine (IL-2 and IFN-gamma) production, reduced cell surface expression of CXCR6 by 85% and that of CCR5 by 76%. The Ca(2+) ionophore, ionomycin (125-500 ng/ml), also markedly diminished CXCR6 expression (85%), but without inducing CD69 expression or cytokine production, and reduced CCR5 expression by only 40%. In contrast, the phorbol esters, PdBu or PMA had little effect on CXCR6 expression (23% reduction) but induced CD69 expression and caused a profound down-regulation (92%) of CCR5 expression. Moreover, CCR7, whose expression was low on CXCR6(+) T cells, was little affected by any of these modes of activation. The down-regulation of CXCR6 expression induced by CD3/CD28 activation was blocked by the broad kinase inhibitor, staurosporine, and by the src kinase inhibitor, PP2, but not by the MEK1 inhibitor, U0106. Most interestingly, the calcineurin inhibitor, FK506, consistently inhibited CD3/CD28-induced CXCR6 down-regulation. FK506 also blocked the decrease of CXCR6 expression caused by ionomycin, whereas staurosporine or PP2 had no effect on this decrease. Altogether, these data indicate that CXCR6 expression is down-regulated, independent of CCR5 or CD69 expression and of cytokine induction, by T cell activation signals that involve predominantly the Ca(2+)-dependent calcineurin pathway.     

Modulation of CD40L antigen expression in Jurkat cells: involvement of protein kinase C activity

The CD40L expressed on activated CD4+ T cells delivers contact-dependent proliferative and anti-apoptotic signals to B lymphocytes. Little is known about molecular mechanisms of constitutive expression of CD40L on some non-Hodgkin's lymphomas, especially about involvement of two signal pathways regulating its expression in normal cells; one involving calcineurin, and the other protein kinase C. We analyzed by flow cytometry the effects of 6-hour stimulation of both pathways (stimuli: PMA and ionomycin) and their inhibitors: cyclosporin A and chelerythrine, on CD40L expression. Two Jurkat clones differing in CD40L surface expression: clone 217.6 (CD40L-) and 217.7 (CD40L+) were studied. Our experiments showed that high level of CD40L expression on the surface of 217.7 cells was reduced after stimulation with PMA. The same effect was observed for combination of PMA and chelerythrine or for PKC inhibitor alone. In 217.6 cells, only chelerythrine used alone induced low level of CD40L expression, while PMA and ionomycin were without effect. These results suggest that CD40L surface expression is mainly dependent on protein kinase C activity. By using PepTag Assay we have confirmed that in both Jurkat clones PKC activity is higher than in normal blood lymphocytes.     

Regulation of T lymphocyte apoptosis. Signals for the antagonism between activation- and glucocorticoid-induced death.

Apoptotic death can be induced in T cell hybridomas by glucocorticoids or the stimulation via the TCR/CD3 complex. The two apoptotic processes are mutually antagonistic. We have previously proposed that positive selection of thymocytes for the formation of the T cell repertoire might be based on a similar mechanism. We analyzed the TCR/CD3-mediated signals essential for the regulation of apoptosis in T cell hybridomas. We suggest that both an increase in the intracellular Ca2+ level and an activation of protein kinase C are essential for the TCR/CD3-mediated apoptosis, because we obtained the following results: 1) either reduction of extracellular Ca2+ concentration or addition of a protein kinase inhibitor, 1-(5-isoquinolkinelsulfonyl)-2-methylpiperazine or N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide, inhibited anti-CD3-induced but not dexamethasone-induced DNA fragmentation. 2) The combination of ionomycin and PMA, but neither one alone nor the combination of ionomycin and cyclic nucleotide analogs, induced DNA fragmentation. On the contrary, we suggest that only an increase in the intracellular Ca2+ level is essential for the inhibition of glucocorticoid-induced apoptosis, because ionomycin alone as well as the combination of ionomycin and PMA inhibited dexamethasone- but not anti-CD3-induced DNA fragmentation.     

Intracellular activation signal requirements for the induction of IL-2 responsiveness in resting T cell subsets in humans

Activation signal requirements for the induction of the IL-2 responsiveness in purified subsets of human resting T cells, T4+ or T8+, have been investigated under the monocyte-depleted conditions. Substantial levels of IL-2 responsiveness were induced in T8+ cells by lectin, Con A, mAb directed against the CD3 Ag, OKT3, Ca2+ ionophore, ionomycin or phorbol ester, PMA. In contrast, none of these stimuli was by itself sufficient for the induction of IL-2 responsiveness in the T4+ subset. The latter cells could, however, be induced to respond to IL-2 by combinations of PMA plus either of Con A, OKT3, or ionomycin (but not any combination of Con A, ionomycin, and OKT3). These data indicate that induction of IL-2 responsiveness in the resting T4+ subset is more complex, possibly requiring two intracellular activation signals, increase in the concentration of intracellular Ca2+ and activation of protein kinase C, whereas either signal may directly trigger IL-2 responsiveness in the resting T8+ cells. The data further suggest that under optimal conditions, growth of both resting T4+ and T8+ subsets may be independent of monocytes.     

Efficient induction of CCR9 on T cells requires coactivation of retinoic acid receptors and retinoid X receptors (RXRs): exaggerated T Cell homing to the intestine by RXR activation with organotins

The active vitamin A metabolite retinoic acid (RA) imprints gut-homing specificity on lymphocytes upon activation by inducing the expression of α4β7 integrin and CCR9. RA receptor (RAR) activation is essential for their expression, whereas retinoid X receptor (RXR) activation is not essential for α4β7 expression. However, it remains unclear whether RXR activation affects the RA-dependent CCR9 expression on T cells and their gut homing. The major physiological RA, all-trans-RA, binds to RAR but not to RXR at physiological concentrations. Cell-surface CCR9 expression was often induced on a limited population of murine naive CD4(+) T cells by all-trans-RA or the RAR agonist Am80 alone upon CD3/CD28-mediated activation in vitro, but it was markedly enhanced by adding the RXR agonist PA024 or the RXR-binding environmental chemicals tributyltin and triphenyltin. Accordingly, CD4(+) T cells treated with the combination of all-trans-RA and tributyltin migrated into the small intestine upon adoptive transfer much more efficiently than did those treated with all-trans-RA alone. Furthermore, naive TCR transgenic CD4(+) T cells transferred into wild-type recipients migrated into the small intestinal lamina propria following i.p. injection of Ag, and the migration was enhanced by i.p. injection of PA024. We also show that PA024 markedly enhanced the all-trans-RA-induced CCR9 expression on naturally occurring naive-like regulatory T cells upon activation, resulting in the expression of high levels of α4β7, CCR9, and Foxp3. These results suggest that RXR activation enhances the RAR-dependent expression of CCR9 on T cells and their homing capacity to the small intestine.     

Athymic nude CD4+8- T cells produce IL-2 but fail to proliferate in response to mitogenic stimuli

A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.     

Functional and phenotypic characterization of CD57+CD4+ T cells and their association with HIV-1-induced T cell dysfunction

HIV-1 replication is associated with reduced or absent HIV-1-specific CD4+ T cell proliferation and skewing of HIV-1-specific CD4+ T cells toward an IFN-gamma-producing, CCR7- phenotype. The CCR7- T cell population is heterogeneous and can be subdivided based on the expression of CD57. Although CD57 expression on CD8+ T cells is associated with proliferation incompetence and replicative senescence, less is known about the function of CD57-expressing CD4+ T cells. In this study, the frequency, phenotype, and function of CD57+CD4+ T cells were evaluated in 25 HIV-1-infected subjects and 10 seronegative controls. CD57+CD4+ T cells were found to be proliferation incompetent, even after strong mitogen stimulation. Percentages of CD4+ T cells that expressed CD57 were significantly higher in untreated HIV-1-infected subjects than in HIV-1-seronegative donors, and CD57 expression did not normalize in subjects receiving at least 6 mo of effective antiretroviral therapy. CD57 was predominately expressed on the CCR7- fraction of the CD4+ T cell compartment and accounted for the majority of cells in the CCR7-CD45RA+ population from untreated HIV-1-infected subjects. HIV-1-specific CD4+ T cells producing only IFN-gamma had the highest expression of CD57, whereas few cells producing IL-2 alone expressed CD57. These findings further define a novel population of proliferation-incompetent CD4+ T cells that are generated in the presence of chronic Ag exposure. A better understanding of the generation and persistence of CD57+ T cells in HIV-1 infection could provide important insights into the immunopathogenesis of this disease.     

Expression of IL-17 in human memory CD45RO+ T lymphocytes and its regulation by protein kinase A pathway.

In the present study, the authors compared the interleukin 17 (IL-17 expression of human naive and phenotypically defined memory T cells as well as its regulation by cAMP pathway. Our data showed that IL-17 mRNA was highly expressed in memory human peripheral CD8(+)45RO+T cells and CD4(+)45RO+T cells when peripheral blood mononuclear cells were first stimulated with ionomycin/PMA. IL-17 expression in memory CD8(+)T cells required accessory signals since culture of ionomycin/PMA-activated CD8(+)45RO+T cells alone did not result to IL-17 expression. In contrast, memory CD4(+)T cell population seems to be more independent. IL-17 and interferon gamma(IFN-gamma) mRNA were both inhibited in the presence of PGE2 or the cAMP analogue (dibutyryl-cAMP), while the anti-inflammatory cytokine IL-10 was highly increased. In contrast, naive CD45RA+T cells were unable to express IL-17 whatever the culture conditions. Naive CD4(+)and CD8(+)T cells were sensitive to the PKA regulatory pathway since they represent a significant source of IL-10 when PBMC were first cultured with ionomycin/PMA in the presence of either PGE2 or db-cAMP. The authors showed that naive cells are highly dependent to their microenvironment, since culture of ionomycin/PMA-activated CD45RA+T cells alone did not result in detectable levels of cytokines even in the presence of PGE2. Results also showed that PGE2 induced quite the same levels of intracellular cAMP in naive and memory cells suggesting that these cell populations are equally sensitive to PGE2. However, we suggest that PGE2 may be more efficient in blocking both IL-17 and IFN-gamma expression in already primed memory T cells, rather than in suppressing naive T cells that could represent a significant source of IL-10. Data suggest that PKA activation pathway plays a critical role in the regulation of cytokine profiles and consequently the functional properties of both human naive and memory CD4(+) and CD8(+)T cells during the immune and inflammatory processes.     

Analysis of protein phosphorylation patterns reveals unanticipated complexity in T lymphocyte activation pathways.

Protein kinases are considered likely to play important roles in the still dimly understood process by which mitogens induce resting T lymphocytes to enter the cell cycle. Using two-dimensional electrophoretic analysis of lysates from orthophosphate-labeled cells, we have compared patterns of phosphorylation in freshly isolated murine splenic T cells exposed to three mitogenic agents: antibody to the epsilon-chain of the TCR CD3 complex, the plant lectin Con A, and a mixture of PMA and ionomycin, which together bypass the signal transduction apparatus to activate intracellular pathways. Of 14 phosphoproteins found whose level of phosphorylation was increased (at least fivefold) by anti-CD3 epsilon antibody, 13 also responded to the mixture of PMA and ionomycin. Surprisingly, however, only 5 of these 14 also responded strongly to Con A exposure. We also identified two substrates that were phosphorylated in response to Con A but not to anti-CD3. Phosphorylation patterns were also studied in T cells exposed to either PMA or ionomycin alone, to gain further insight into the role of protein kinase C and calcium-dependent events in the activation process. Of 16 phosphoproteins that responded to mixtures of PMA and ionomycin, 4 were shown to require the ionomycin signal, 2 to require the PMA signal, and 3 others to respond only when both activators were present; the other 7 responded to either agonist added alone. In addition, we found two PMA-sensitive phosphoproteins in which phosphorylation was inhibited by ionomycin induced calcium signals. Finally, we identified several phosphoproteins which show differential responsiveness in CD4+ and CD8+ T cells. Classification of kinase substrates based on their differential susceptibility to these stimuli should provide new insights into the mode of action of agents and diseases that affect T cell activation.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

Stepwise differentiation of CD4 memory T cells defined by expression of CCR7 and CD27

To study the steps in the differentiation of human memory CD4 T cells, we characterized the functional and lineage relationships of three distinct memory CD4 subpopulations distinguished by their expression of the cysteine chemokine receptor CCR7 and the TNFR family member CD27. Using the combination of these phenotypic markers, three populations were defined: the CCR7+CD27+, the CCR7-CD27+, and the CCR7-CD27- population. In vitro stimulation led to a stepwise differentiation from naive to CCR7+CD27+ to CCR7-CD27+ to CCR7-CD27-. Telomere length in these subsets differed significantly (CCR7+CD27+ > CCR7-CD27+ > CCR7-CD27-), suggesting that these subsets constituted a differentiative pathway with progressive telomere shortening reflecting antecedent in vivo proliferation. The in vitro proliferative response of these populations declined, and their susceptibility to apoptosis increased progressively along this differentiation pathway. Cytokine secretion showed a differential functional capacity of these subsets. High production of IL-10 was only observed in CCR7+CD27+, whereas IFN-gamma was produced by CCR7-CD27+ and to a slightly lesser extent by CCR7-CD27- T cells. IL-4 secretion was predominantly conducted by CCR7-CD27- memory CD4 T cells. Thus, by using both CCR7 and CD27, distinct maturational stages of CD4 memory T cells with different functional activities were defined.     

Functional expression of the chemokine receptor CCR5 on virus epitope-specific memory and effector CD8+ T cells

Because the chemokine receptor CCR5 is expressed on Th1 CD4(+) cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8(+) T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+-->CCR5+CCR7+-->CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.     

Retinoic acids inhibit activation-induced apoptosis in T cell hybridomas and thymocytes.

Apoptosis is induced in immature thymocytes and T cell hybridomas upon stimulation via the TCR/CD3 complex. This phenomenon appears to be related to negative selection of T cell clones in the thymus. In T cell hybridomas, it has been shown that glucocorticoids inhibit TCR/CD3-mediated apoptosis, whereas glucocorticoids alone induce apoptosis. All-trans-retinoic acid (RA) at 0.1 to 10 microM also inhibited TCR/CD3-mediated apoptosis assessed by DNA fragmentation and cytolysis, but RA alone hardly induced apoptosis. RA enhanced the effects of glucocorticoids to induce apoptosis and to inhibit TCR/CD3-mediated apoptosis. TCR/CD3-mediated stimulation can be mimicked by the combination of ionomycin, a calcium ionophore, and PMA, an activator of protein kinase C, and the combination-induced DNA fragmentation was also inhibited by RA. RA, however, failed to inhibit the combination-induced increase in intracellular Ca2+ concentration or the combination-induced translocation of protein kinase C from the cytosolic fraction to the particulate fraction. Time course studies of RA addition into the culture indicated that a 3- to 6-h delay in the addition of RA did not reduce its inhibitory effect on anti-CD3-induced DNA fragmentation. These results suggest that RA interferes with the apoptotic process at some point after its initiation stage. It has been suggested that negative selection involves not only TCR/CD3-mediated signals but also LFA-1-mediated signals. RA at 0.01 to 1 microM significantly inhibited the induction of thymocyte apoptosis by co-immobilized mAb to CD3 and LFA-1 molecules. RA by itself hardly induced apoptosis, but enhanced glucocorticoid-induced apoptosis. The results suggest that thymic selection might be influenced by RA at near-physiologic concentrations. The receptors of glucocorticoids and RA belong to the erbA oncogene-related steroid hormone receptor superfamily. Thyroid hormones and 1 alpha,25-dihydroxy vitamin D3, whose receptors also belong to the superfamily, failed to modulate apoptosis in both T cell hybridomas and thymocytes.     

Perforin expression in human peripheral blood mononuclear cells. Definition of an IL-2-independent pathway of perforin induction in CD8+ T cells.

Perforin gene expression upon in vitro stimulation was studied at the mRNA level in normal human PBMC and in subpopulations. Freshly isolated PBMC express low levels of perforin mRNA. Increased perforin expression is rapidly induced by the calcium ionophore A23187 and by rIL-2. Phorbolesters (PMA), by comparison, are poor inducers of perforin RNA. Perforin induction by Ca-ionophore, unlike granzyme 2 and IL-2 induction, did not synergize with phorbolesters in PBMC or in purified T cells. Instead, perforin mRNA induction by A23187 in purified T cells requires the presence of adherent cells. Ca-ionophore plus adherent cell-induced perforin occurred in CD8+ T cells and was abolished by depletion of CD8+ T cells but not by depletion of CD4+ T cells. Adherent cells alone did not express perforin under any condition. Perforin mRNA induction by both A23187 and by rIL-2 is independent of de novo protein synthesis. The half-life of perforin mRNA induced by either stimulus is approximately 100 min. Cyclosporin A completely abrogates perforin induction by A23187 but only slightly inhibits the effect of rIL-2 on perforin mRNA expression. These data show that A23187 activates perforin gene expression in CD8+ cells by an IL-2-independent pathway and that the molecular mechanism of perforin expression may be different from the one induced by IL-2. Granzyme 2 (human leukocyte protease-HLP, homologous to murine granzyme B) mRNA expression was studied in comparison to perforin. Granzyme 2 in contrast to perforin responds to the synergistic action of phorbolester and Ca-ionophore in PBMC. In addition, the kinetics of the induction of granzyme and perforin mRNA, by various signals are different. Our data suggest that situations in vivo may exist that allow perforin expression in CD8+ cells in the absence of cytokines by a combination of Ca signals and accessory receptor ligation. The same signals may not be sufficient for granzyme 2 expression in any T cell subpopulation.     

Lineage-specific telomere shortening and unaltered capacity for telomerase expression in human T and B lymphocytes with age

Age effects on telomere length and telomerase expression in peripheral blood lymphocytes were analyzed from 121 normal individuals age newborn to 94 years and revealed several new findings. 1) Telomere shortening was observed in CD4+ and CD8+ T and B cells with age. However, the rate of telomere loss was significantly different in these populations, 35 +/- 8, 26 +/- 7, and 19 +/- 7 bp/year for CD4+ and CD8+ T and B cells, respectively. In addition, CD4+ T cells had the longest average telomeres at all ages, followed by B cells, with CD8+ T cell telomeres the shortest, suggesting that these lymphocyte populations may have different replicative histories in vivo. 2) Telomerase activity in freshly isolated T and B cells was indistinguishably low to undetectable at all ages but was markedly increased after Ag and costimulatory receptors mediated stimulation in vitro. Furthermore, age did not alter the magnitude of telomerase activity induced after stimulation of T or B lymphocytes through Ag and costimulatory receptors or in response to PMA plus ionomycin treatment. 3) The levels of telomerase activity induced by in vitro stimulation varied among individual donors but were highly correlated with the outcome of telomere length change in CD4+ T cells after Ag receptor-mediated activation. Together, these results indicate that rates of age-associated loss of telomere length in vivo in peripheral blood lymphocytes is specific to T and B cell subsets and that age does not significantly alter the capacity for telomerase induction in lymphocytes.