Interleukin-7 enhances proliferation responses to T-cell receptor stimulation in naïve CD4+ T cells from human immunodeficiency virus-infected persons

Proliferation responses of naïve CD4⁺ T cells to T-cell receptor and interleukin-7 (IL-7) stimulation were evaluated by using cells from human immunodeficiency virus-positive (HIV⁺) donors. IL-7 enhanced responses to T-cell receptor stimulation, and the magnitude of this enhancement was similar in cells from healthy controls and from HIV⁺ subjects. The overall response to T-cell receptor stimulation alone or in combination with IL-7, however, was diminished among viremic HIV⁺ donors and occurred independent of antigen-presenting cells. Frequencies of CD127⁺ cells were related to the magnitudes of proliferation enhancement that were mediated by IL-7. Thus, IL-7 enhances but does not fully restore the function of naïve CD4⁺ T cells from HIV-infected persons.Interleukin-7 (IL-7) plays an important role in T-cell homeostasis by modulating thymic output (1, 16, 22) and by enhancing the peripheral expansion and survival of both naïve and memory T-cell subsets (12, 18, 20, 25, 26, 31, 32). Under normal circumstances, the homeostatic maintenance of naïve CD4⁺ T cells is regulated by at least two types of signals that include T-cell receptor (TCR) engagement and IL-7 (10, 26, 30). In addition, IL-7 may play an important role in the conversion of effector T cells into long-term memory cells (12, 14).Homeostasis of T cells is dysregulated in human immunodeficiency virus (HIV) infection such that there is a marked depletion of CD4⁺ cells and a progressive loss of naïve CD4 and CD8⁺ T cells (24). Although the mechanisms for these deficiencies are not fully understood, it is possible that impairments in T-cell proliferation and responsiveness to immunomodulatory cytokines could play a role. In HIV disease, IL-7 is increased in plasma (2, 5, 11, 15, 19, 21, 23) and the alpha chain of the IL-7 receptor, CD127, is less frequently expressed among T lymphocytes (2, 5, 11, 21, 23). The ability of patient T cells to respond to IL-7 stimulation may be diminished in HIV disease but may not be related to the density of CD127 expression as it is in T cells from healthy controls (4). Moreover, the responsiveness of T cells, including naïve CD4⁺ lymphocytes, to TCR stimulation is diminished in HIV disease (27-29). Thus, defects in responsiveness to cytokines or TCR stimulation could contribute to the perturbations in T-cell proliferation and survival in HIV disease.In these studies, we examined the responsiveness of naïve CD4⁺ T cells from viremic HIV-positive (HIV⁺) donors (median plasma HIV RNA level, 25,200 copies/ml [range, 1,015 to 1,000,000 copies/ml]; median CD4 cell count, 429 cells/μl [range, 41 to 950 cells/μl]; median age, 38 years [range, 22 to 64 years]; n = 25) and aviremic, highly active antiretroviral therapy (HAART)-treated HIV⁺ donors (plasma HIV RNA level, <400 copies/ml; median CD4 cell count, 309 cells/μl [range, 74 to 918 cells/μl]; median age, 48 years [range, 37 to 55 years]; n = 12) to the combined stimulus of recombinant IL-7 (Cytheris) plus agonistic anti-CD3 antibody. Peripheral blood mononuclear cells (PBMC) were depleted of CD45RO⁺ cells by magnetic bead depletion (>90% purity) and were incubated in medium alone or were stimulated with anti-CD3 antibody, IL-7, or anti-CD3 antibody plus IL-7. CD4⁺CD45RO⁻CD28⁺CD27⁺ cells were assessed for the expression of Ki67 2 days poststimulation by flow cytometric analyses. The addition of IL-7 to anti-CD3 antibody enhanced the induction of Ki67 expression in cells from both HIV⁺ and HIV-negative (HIV⁻) donors (Fig. ​(Fig.11 and Fig. ​Fig.2).2). A diminished response to anti-CD3 antibody was observed among naïve CD4⁺ T cells from viremic HIV⁺ donors. In contrast, cells from aviremic HIV⁺ donors (all receiving antiretroviral therapy) had normal responses to anti-CD3 antibody compared to cells from healthy donors (Fig. ​(Fig.2).2). Importantly, the addition of IL-7 to the cultures significantly improved the responses to above those observed with anti-CD3 alone in HIV⁻ and HIV⁺ donors, regardless of viremia (Wilcoxon signed ranks test; for each comparison, P was <0.04), and the magnitude of that enhancement, although slightly diminished in cells from HIV⁺ donors, was not significantly different between groups of subjects when measured as either the enhancement (n-fold; not shown) or as the change in percent Ki67⁺ cells above the background observed for cells stimulated with anti-CD3 alone (Fig. ​(Fig.3).3). Although IL-7 enhanced responses to TCR stimulation in HIV⁻ subjects, the overall magnitude of the responses among cells from HIV viremic subjects did not reach the levels seen with cells from healthy donors, even in the presence of IL-7 (Fig. ​(Fig.2).2). It should be noted, however, that these functional readouts were not related to clinical indices of plasma HIV RNA level, CD4 cell count, or age when considered as continuous variables, suggesting that the functional perturbations in naïve CD4⁺ T cells are probably undermined by complexities extending beyond HIV replication (not shown). Together, these results suggest that TCR responsiveness is diminished in naïve CD4⁺ T cells from viremic HIV⁺ subjects, whereas responsiveness to IL-7 stimulation is relatively preserved.Open in a separate windowFIG. 1.IL-7 enhances the induction of Ki67 expression in naïve CD4⁺ T cells from healthy controls and HIV⁺ donors. CD45RO-depleted PBMC were incubated with anti-CD3 antibody (100 ng/ml), IL-7 (50 ng/ml), anti-CD3 antibody plus IL-7, or medium alone (RPMI with 10% fetal bovine serum). Cells were gated on CD4⁺CD27⁺CD28⁺ lymphocytes and examined for Ki67 expression by intracellular flow cytometry.Open in a separate windowFIG. 2.IL-7 responsiveness in cells from viremic and aviremic HIV⁺ donors. Plotted values represent the percentages of CD4⁺CD27⁺CD28⁺CD45RO⁻ T cells that expressed Ki67 after a 2-day incubation with anti-CD3 or with anti-CD3 plus IL-7. Percentages of Ki67⁺ cells in cultures without stimulation or with IL-7 only were subtracted from the values shown. Responses of cells from healthy controls (n = 9), HIV⁺ subjects with plasma HIV RNA levels of >400 copies/ml (n = 25), and HIV⁺ subjects on HAART with suppressed viral replication (<400 copies/ml; n = 12) are shown. Statistically significant differences between cells from controls and HIV⁺ donors are indicated. Analyses included Kruskal-Wallis test (P = 0.002) for multigroup comparisons and Mann-Whitney U test for comparison of two groups (*, P < 0.05).Open in a separate windowFIG. 3.IL-7 enhances responses to anti-CD3 antibody stimulation to a similar degree in cells from HIV⁺ and HIV⁻ donors. Naïve CD4⁺ T cells were incubated with IL-7, anti-CD3, anti-CD3 plus IL-7, or medium alone for 2 days. Background division (percent Ki67⁺ cells) in medium alone or IL-7 alone was first subtracted from the responses observed with cells stimulated with anti-CD3 alone or with anti-CD3 plus IL-7, respectively. The magnitude of IL-7 enhancement was then calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7. n = 9, 25, and 12 for healthy controls, viremic subjects, and aviremic subjects, respectively.Previous studies indicate that the frequency of CD127⁺ T cells, particularly memory T-cell subsets, is reduced in patients with HIV disease (5, 11, 21, 23). This could, in part, result from the modulation of receptor expression through increased exposure to IL-7 in vivo and also may reflect accumulation of CD127⁻ effector memory cells (21). We assessed the expression of CD127 in naïve CD4⁺CD45RA⁺CD28⁺CD27⁺ and memory CD4⁺CD45RO⁺ T cells in a subset of patients and asked if the frequencies of CD127⁺ cells were related to the induction of Ki67 expression by anti-CD3 or by anti-CD3 plus IL-7 among naïve CD4⁺ T cells. We reasoned that the ability of IL-7 to enhance responses to TCR stimulation might be limited if CD127 expression was diminished among naïve CD4⁺ T cells from HIV⁺ donors. Alternatively, a defect in functional responses also could be related to increased exposure to IL-7 in vivo, as may be reflected by the absence of CD127 receptor expression on memory T-cell subsets.In agreement with previous studies, our results suggest that CD127 expression is relatively preserved in naïve CD4⁺ T cells from HIV⁺ donors (representative histograms in Fig. ​Fig.4)4) (mean percentage of CD127⁺ cells, 87 and 83 for HIV⁻ donors [n = 5] and HIV⁺ donors [n = 17], respectively; P = 0.96) but is diminished in memory CD4⁺ T cells from HIV⁺ donors (mean percentage of CD127⁺ cells, 83 and 59 for HIV⁻ and HIV⁺ donors, respectively; P = 0.01). The frequencies of CD127⁺ naïve T cells were directly related to the frequencies of CD127⁺ memory T cells (Spearman''s correlations; r = 0.711, P = 0.001; n = 18) in HIV⁺ subjects. This result suggests that a similar mechanism modulates the expression of CD127 in these T-cell subsets, even though the loss of CD127 expression is clearly greater among the memory T cells in HIV disease. Neither CD127 expression among naïve CD4⁺ T cells nor CD127 expression among memory CD4⁺ T cells was related to the functional response of naïve CD4⁺ T cells to anti-CD3 (r = 0.238 and P = 0.36 for naïve CD127 expression; r = 0.293 and P = 0.25 for memory CD127 expression) or to anti-CD3 plus IL-7 (r = 0.32 and P = 0.21 for naïve CD127 expression; r = 0.31 and P = 0.22 for memory CD127 expression). There was a relationship between the percentage of CD127⁺ naïve T cells and the delta Ki67 expression that resulted from the addition of IL-7 to anti-CD3-treated cultures (percentage of Ki67⁺ cells in cultures treated with anti-CD3 plus IL-7 minus the percentage of Ki67⁺ cells in cultures treated with anti-CD3 alone) (Fig. ​(Fig.4).4). This relationship was statistically significant by Pearson''s correlation (r = 0.5, P = 0.041), the use of which was justified based on the normal distribution of the data. Spearman''s analysis, which is independent of data distribution, indicated a similar trend that was not statistically significant (r = 0.41, P = 0.1). The mean fluorescence intensity of CD127 expression on CD4⁺CD45RA⁺CD27⁺CD28⁺ T cells was not significantly related to the delta Ki67 expression induced by IL-7 but also suggested a trend consistent with a direct relationship between these indices (r = 0.45 and P = 0.07 by Pearson''s correlation; r = 0.34 and P = 0.18 by Spearman''s correlation). Despite the relative preservation of IL-7 receptor in naïve CD4⁺ T cells from HIV⁺ donors, the association between the frequencies of CD127⁺ cells and CD4⁺ T-cell proliferation responses to TCR plus IL-7 suggests that subtle IL-7 receptor perturbations might contribute to functional defects of naïve CD4⁺ T cells in HIV-infected persons.Open in a separate windowFIG. 4.CD127 receptor expression is related to enhancement of proliferation by IL-7. (A) Whole blood from a healthy control and an HIV-infected person was examined by flow cytometry for expression of CD127 on CD4⁺CD45RA⁺CD27⁺CD28⁺ (naïve) T cells. The gating strategy for identifying naïve cells involved an initial gate for lymphocyte forward and side scatter (SSC) characteristics (not shown) and then sequential gates for CD4 positive, CD45RA positive and, finally, CD28⁺CD27⁺ double-positive cells. (B) Plotted values indicating the relationship between the delta Ki67 expression in naïve CD4⁺ T cells and the percentage of CD127⁺ naïve T cells that was determined by using freshly isolated whole blood. The delta Ki67 expression was calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7.To consider the possibility that antigen-presenting cells could contribute to the diminished response of T-cells to stimulation with TCR plus IL-7, we next asked if defects in TCR-plus-IL-7 stimulation could be detected in purified naïve CD4⁺ T-cell populations. CD4⁺CD45RO⁻ cells were negatively selected by magnetic bead depletion, achieving a purity of >90% as determined by flow cytometric analyses. Purified naïve CD4⁺ T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) tracking dye and incubated with IL-7, anti-CD3 antibody that was immobilized on a plate, anti-CD3 plus IL-7, or medium alone. The induction of proliferation was measured 7 days later by the dilution of CFSE tracking dye among CD4⁺CD27⁺ cells by calculating the division index (average number of cell divisions of all CD4⁺CD27⁺ cells) and the proliferation index (average number of divisions of CD4⁺CD27⁺ cells that had diluted tracking dye; Flow-Jo analysis software). These purified CD4⁺ T cells proliferated poorly in response to anti-CD3 antibody stimulation alone, providing functional evidence that the samples were free of antigen-presenting cell contamination (Fig. ​(Fig.5A).5A). The combined treatment of anti-CD3 and IL-7 induced cellular expansion, whereas alone, neither stimulus induced cellular proliferation during the 7-day period (Fig. ​(Fig.5A).5A). Responses of cells from HIV⁺ donors were reduced compared to those of cells from healthy donors, confirming that the defects in naïve CD4⁺ T-cell expansion are independent of antigen-presenting cells and not fully corrected by IL-7 (Fig. ​(Fig.5B5B).Open in a separate windowFIG. 5.Diminished responses to TCR plus IL-7 in purified naïve CD4⁺ T cells from HIV⁺ donors. CD4⁺CD45RO⁻ cells were purified from PBMC by negative selection. Cells from HIV⁺ donors (n = 7) and healthy controls (n = 7) were labeled with CSFE and incubated with anti-CD3 immobilized on a plate (5 μg/ml, overnight at 4°C) plus IL-7 (10 ng/ml). CFSE dye dilution was measured among the CD4⁺CD27⁺ cells. (A) Representative histograms showing the dilution of CFSE and CD27 expression among cells incubated with anti-CD3 antibody alone, IL-7 alone, or the combination of anti-CD3 plus IL-7. Placements of quadrant gates were based on an isotype control antibody stain (for CD27 expression) and on cells that had been incubated in medium alone (for CFSE dye dilution). (B) Division indices (average number of cell divisions among CD4⁺CD27⁺ cells) and proliferation indices (average number of cell divisions among CD4⁺CD27⁺ cells that had diluted tracking dye) are shown.IL-7 is a promising candidate for therapeutic and vaccine adjuvant applications in HIV disease. This cytokine may be especially beneficial in circumstances of immune reconstitution, since it normally plays an essential role in T-cell proliferation and survival. Here, we demonstrate that IL-7 efficiently enhances TCR-triggered naïve CD4⁺ T-cell expansion in cells from healthy individuals and from HIV⁺ donors. The mechanism of IL-7 activity is not discerned in these experiments but may involve effects on survival, such as the induction of Bcl-2 (9), or may involve the enhancement of IL-2 or IL-2 receptor expression (6, 8). In any case, our studies provide evidence that IL-7 should provide an effective therapy for the regulation of naïve CD4⁺ T-cell homeostasis and may be useful for vaccine adjuvant applications in HIV disease. The potential of this approach has been illustrated by recent human trials of IL-7 that demonstrated the expansion of naïve T cells in vivo after IL-7 administration to HIV-infected persons (13) and by animal studies, wherein IL-7 administration enhanced T-cell responses to immunization in mice (17).Notably, the depletion studies and purification methods employed here did not necessarily eliminate terminally differentiated effector memory CD4⁺ T cells from our cultures; however, studies of CMV-specific terminally differentiated cells suggested that these cells are primarily CD27⁻ (3), and the use of three markers to identify naïve CD4⁺ T cells, including the ones used here (CD27, CD28, and CD45RO) is estimated to provide 98% assurance that the cells being examined are truly naïve (7). Thus, it is likely that terminally differentiated cells were largely removed from our analyses.Our observations provide confirmation of a significant defect in the responses of naïve CD4⁺ T cells to TCR triggering in HIV disease, and this defect is not fully corrected by IL-7, as shown here, or by IL-2, as we demonstrated previously (27). These deficiencies are reproduced even among naïve CD4⁺ T cells that are purified from professional antigen-presenting cells, indicating that the defects are intrinsic to the T cells and not a consequence of dysfunctional antigen-presenting cells. We propose that functional defects in naïve CD4⁺ T cells from HIV⁺ donors stem primarily from deficiencies in TCR signaling. Further studies that define the nature of naïve CD4⁺ T-cell defects in HIV disease will be required to address the underlying mechanisms.     

Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells

γδ T cells are considered to be innate lymphocytes that play an important role in host defense against tumors and infections. We recently reported that IL-18 markedly amplified γδ T cell responses to zoledronate (ZOL)/IL-2. In an extension of this finding, we analyzed the mechanism underlying the IL-18-mediated expansion of γδ T cells. After incubation of PBMCs with ZOL/IL-2/IL-18, the majority of the cells expressed γδ TCR, and the rest mostly exhibited CD56(bright)CD11c(+) under the conditions used in this study. CD56(bright)CD11c(+) cells were derived from a culture of CD56(int)CD11c(+) cells and CD14(+) cells in the presence of IL-2 and IL-18 without the addition of ZOL. They expressed IL-18Rs, HLA-DR, CD25, CD80, CD83, CD86, and CD11a/CD18. In addition, they produced IFN-γ, TNF-α, but not IL-12, when treated with IL-2/IL-18, and they exerted cytotoxicity against K562 cells, thus exhibiting characteristics of both NK cells and dendritic cells. Incubation of purified γδ T cells with CD56(bright)CD11c(+) cells in the presence of ZOL/IL-2/IL-18 resulted in the formation of massive cell clusters and led to the marked expansion of γδ T cells. However, both conventional CD56(-/int)CD11c(high) dendritic cells induced by GM-CSF/IL-4 and CD56(+)CD11c(-) NK cells failed to support the expansion of γδ T cells. These results strongly suggest that CD56(bright)CD11c(+) cells play a key role in the IL-18-mediated proliferation of γδ T cells.     

Selective proliferation of human γδ T cells in vitro

The effect of monoethylphosphate (MEP,commercial available or synthesized) together with IL-2 on the selective proliferation of human γδT cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated.The γδT cells were stimulated by MEP to proliferate in a dose-dependent manner.The effect of synthesized MEP was 10 times greater than that of commercial MEP.When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP,the total cell number increased about 1000-3000 fold;and the ratio of γδT cells reached to 70-80%.The selective expansion of γδT cells depended on the synergic action of MEP and IL-2.The bulk cultured γδT cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5,K562 and Daudi) and autologous tumor cells.The culture system described here not only offers a simple method for obtaining a large number of γδT cells which may become a new effector in the adoptive immunotherapy,but also provides a useful model for the further studies of the structure and function of γδT cells in vitro.     

Praziquantel facilitates IFN-γ-producing CD8+ T cells (Tc1) and IL-17-producing CD8+ T cells (Tc17) responses to DNA vaccination in mice

Background

CD8⁺ cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.

Methodology/Principal Findings

Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8⁺ T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8⁺ T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8⁺ T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.

Conclusions/Significance

Our results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8⁺ T cell-based immunotherapy that breaks tolerance to HBsAg.     

Activated murine endothelial cells have reduced immunogenicity for CD8+ T cells: a mechanism of immunoregulation?

The immunogenic properties of primary cultures of murine lung microvascular endothelial cells (EC) were analyzed. Resting endothelial cells were found to constitutively express low levels of MHC class I and CD80 molecules. IFN-gamma treatment of EC resulted in a marked up-regulation of MHC class I, but no change was observed in the level of CD80 expression. No CD86 molecules were detectable under either condition. The ability of peptide-pulsed EC to induce the proliferation of either the HY-specific, H2-K(k)-restricted CD8(+) T cell clone (C6) or C6 TCR-transgenic naive CD8(+) T cells was analyzed. Resting T cells were stimulated to divide by quiescent peptide-prepulsed EC, while peptide-pulsed, cytokine-activated EC lost the ability to induce T cell division. Furthermore, Ag presentation by cytokine-activated EC induced CD8(+) T cell hyporesponsiveness. The immunogenicity of activated EC could be restored by adding nonsaturating concentrations of anti-H2-K(k) Ab in the presence of an optimal concentration of cognate peptide. This is consistent with the suggestion that the ratio of TCR engagement to costimulation determines the outcome of T cell recognition. In contrast, activated peptide-pulsed EC were killed more efficiently by fully differentiated effector CD8(+) T cells. Finally, evidence is provided that Ag recognition of EC can profoundly affect the transendothelial migration of CD8(+) T cells. Taken together, these results suggest that EC immunogenicity is regulated in a manner that contributes to peripheral tolerance.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Age-associated changes in interferon-γ and interleukin-4 secretion by purified human CD4+ and CD8+ T cells

Aging is associated with a decline in immune function. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), two important immune deviation-related cytokines, are mainly produced by type 1 and type 2 T cells, respectively. To investigate the age-associated changes in the secretion of these two cytokines, 20 elderly and 20 young subjects fulfilling the SENIEUR protocol were enrolled. The ratios of CD4+ to CD8+ T cells were not different between the two age groups. The CD4+ and CD8+ T cells were purified by a magnetic cell sorting system, and then activated by concurrent anti-CD3 and anti-CD28 stimulation. The released cytokines were determined by ELISA. Both the CD4+ and the CD8+ T cells of the elderly individuals secreted a significantly larger amount of IFN-gamma after activation. Profound IL-4 production by CD8+ T cells was observed in the older subjects compared with that of the young subjects. These data suggested that age-associated decrease in immunity may be related to an imbalance in the secretion of immune deviation cytokines. The number of IL-4-secreting CD8+ T cells (T cytotoxic 2) rose significantly in the older individuals. Our design also provided a useful way to differentiate the T cell subsets secreting the same cytokine, such as IFN-gamma-producing T helper 1 and T cytotoxic 1 cells.     

NLRC5 potentiates anti-tumor CD8+ T cells responses by activating interferon-β in endometrial cancer

ObjectivesNLR family CARD domain containing 5 (NLRC5) could promote major histocompatibility complex class I (MHC-I)-dependent CD8⁺ T cell-mediated anticancer immunity. In this study, the immunosurveillance role and underlying mechanisms of NLRC5 in endometrial cancer (EC) were characterized.MethodsCD8⁺ T cells were separated from healthy women's peripheral blood by using magnetic beads. The effect of NLRC5 and interferon-β (IFN-β) on immunosurveillance of EC were examined through a mouse tumor model and a CD8⁺ T cell-EC cell coculture system after NLRC5 overexpression and IFN-β overexpression or depletion. The effect of NLRC5 on IFN-β expression was examined with gain- and loss-of-function experiments.ResultsNLRC5 overexpression in the EC cell and CD8⁺ T cell coculture system inhibited EC cell proliferation and migration and promoted EC cell apoptosis and CD8⁺ T cell proliferation. In vivo, NLRC5 overexpression increased the proportion of CD8⁺ T cells and inhibited EC progression. Furthermore, IFN-β overexpression in the EC cell and CD8⁺ T cell coculture system activated CD8⁺ T cell proliferation; however, genetic depletion of IFN-β exerted the opposite effects. In addition, NLRC5 could negatively regulate IFN-β expression in EC cells. Mechanistically, NLRC5 potentiated the antitumor responses of CD8⁺ T cells to EC by activating IFN-β.ConclusionsTaken together, our findings demonstrated that NLRC5 potentiates anti-tumor CD8⁺ T cells responses by activating interferon-β in EC, suggesting that genetically escalated NLRC5 and IFN-β may act as potential candidates for the clinical translation of adjuvant immunotherapies to patients with EC.     

CD4 T cells play important roles in maintaining IL-17-producing γδ T-cell subsets in naive animals

A proportional balance between αβ and γδ T-cell subsets in the periphery is exceedingly well maintained by a homeostatic mechanism. However, a cellular mechanism underlying the regulation remains undefined. We recently reported that a subset of developing γδ T cells spontaneously acquires interleukin (IL)-17-producing capacity even within naive animals through a transforming growth factor (TGF)β1-dependent mechanism, thus considered 'innate' IL-17-producing cells. Here, we report that γδ T cells generated within αβ T cell (or CD4 T cell)-deficient environments displayed altered cytokine profiles; particularly, 'innate' IL-17 expression was significantly impaired compared with those in wild-type mice. Impaired IL-17 production in γδ T cells was directly related to CD4 T-cell deficiency, because depletion of CD4 T cells in wild-type mice diminished and adoptive CD4 T-cell transfer into T-cell receptor β-/- mice restored IL-17 expression in γδ T cells. CD4 T cell-mediated IL-17 expression required TGFβ1. Moreover, Th17 but not Th1 or Th2 effector CD4 T cells were highly efficient in enhancing γδ T-cell IL-17 expression. Taken together, our results highlight a novel CD4 T cell-dependent mechanism that shapes the generation of IL-17+ γδ T cells in naive settings.     

Cutting edge: Generation of colitogenic Th17 CD4 T cells is enhanced by IL-17+ γδ T cells

Th 17 cells have been implicated in the pathogenesis of colitis; however, a cellular mechanism by which colitogenic Th17 immunity arises in vivo remains unclear. In this study, we report that a subset of IL-17(+) γδ T cells plays a crucial role in enhancing in vivo Th17 differentiation and T cell-mediated colitis. TCRβ(-/-) mice were highly susceptible to T cell-mediated colitis, whereas TCRβδ(-/-) mice were resistant to the disease. Importantly, cotransfer of IL-17(+) but not of IL-17(-) γδ T cells with CD4 T cells was sufficient to enhance Th17 differentiation and induce full-blown colitis in TCRβδ(-/-) recipients. Collectively, our results provide a novel function of IL-17(+) γδ T cell subsets in supporting in vivo Th17 differentiation and possibly in fostering the development of intestinal inflammation.     

Circulating CD8+CD28− suppressor T cells tied to poorer prognosis among metastatic breast cancer patients receiving adoptive T-cell therapy: A cohort study

Background aims

This study aimed to determine the prognostic value of circulating CD8⁺CD28^? T lymphocytes among breast cancer patients treated with adoptive T-lymphocyte immunotherapy after chemotherapy.

Ectopically expressed human tumor biomarker MutS homologue 2 is a novel endogenous ligand that is recognized by human γδ T cells to induce innate anti-tumor/virus immunity

Human (h) MutS homologue 2, a nuclear protein, is a critical element of the DNA mismatch repair system. Our previous studies suggest that hMSH2 might be a protein ligand for TCRγδ. Here, we show that hMSH2 is ectopically expressed on a large panel of epithelial tumor cells. We found that hMSH2 interacts with both TCRγδ and NKG2D and contributes to Vδ2 T cell-mediated cytolysis of tumor cells. Moreover, recombinant human MSH2 protein stimulates the proliferation and IFN-γ secretion of Vδ2 T cells in vitro. Finally, hMSH2 expression is induced on the cell surface of Epstein-Barr virus-transformed lymphoblastoid cell lines, and the induction increases the sensitivity of these lymphoblastoid cell lines to γδ T cell-mediated cytolysis. Our data suggest that hMSH2 functions as a tumor-associated or virus infection-related antigen recognized by both Vδ2 TCR and NKG2D, and it plays a role in eliciting the immune responses of γδ T cells against tumor- and virus-infected cells. The recognition of ectopic surface-expressing endogenous antigen by TCRγδ and NKG2D may be an important mechanism of innate immune response to carcinogenesis and viral infection.     

KLRG1+NKG2A+ CD8 T cells mediate protection and participate in memory responses during γ-herpesvirus infection

Functional CD8 T cell effector and memory responses are generated and maintained during murine γ-herpesvirus 68 (γHV68) persistent infection despite continuous presentation of viral lytic Ags. However, the identity of the CD8 T cell subpopulations that mediate effective recall responses and that can participate in the generation of protective memory to a γ-herpesvirus infection remains unknown. During γHV68 persistence, ～75% of γHV68-specific CD8 T cells coexpress the NK receptors killer cell lectin-like receptor G1 (KLRG1) and NKG2A. In this study, we take advantage of this unique phenotype to analyze the capacity of CD8 T cells expressing or not expressing KLRG1 and NKG2A to mediate effector and memory responses. Our results show that γHV68-specific KLRG1(+)NKG2A(+) CD8 T cells have an effector memory phenotype as well as characteristics of polyfunctional effector cells such us IFN-γ and TNF-α production, killing capacity, and are more efficient at protecting against a γHV68 challenge than their NKG2A(-)KLRG1(-) counterparts. Nevertheless, γHV68-specific NKG2A(+)KLRG1(+) CD8 T cells express IL-7 and IL-15 receptors, can survive long-term without cognate Ag, and subsequently mount a protective response during antigenic recall. These results highlight the plasticity of the immune system to generate protective effector and proliferative memory responses during virus persistence from a pool of KLRG1(+)NKG2A(+) effector memory CD8 T cells.     

Cardiolipin binds to CD1d and stimulates CD1d-restricted γδ T cells in the normal murine repertoire

Cardiolipin (CL), a major phospholipid in bacterial cell walls, is sequestered from the immune system in mammalian mitochondria and is, therefore, a potential danger signal. Based on growing evidence that phospholipids constitute natural ligands for CD1 and that CD1d-restricted T cells recognize phospholipids, we hypothesized that CD1d binds and presents CL and that T cells in the normal immune repertoire respond to CL in a CD1d-restricted manner. We determined the murine CD1d-CL crystal structure at 2.3 ? resolution and established through additional lipid loading experiments that CL, a tetra-acylated phospholipid, binds to murine CD1d with two alkyl chains buried inside the CD1d binding groove and the remaining two exposed into the solvent. We furthermore demonstrate the functional stimulatory activity of CL, showing that splenic and hepatic γδ T cells from healthy mice proliferate in vitro in response to mammalian or bacterial CL in a dose-dependent and CD1d-restricted manner, rapidly secreting the cytokines IFN-γ and RANTES. Finally, we show that hepatic γδ T cells are activated in vivo by CD1d-bearing dendritic cells that have been pulsed with CL, but not phosphatidylcholine. Together, these findings demonstrate that CD1d is able to bind and present CL to a subset of CL-responsive γδ T cells that exist in the spleen and liver of healthy mice and suggest that these cells could play a role in host responses to bacterial lipids and, potentially, self-CL. We propose that CL-responsive γδ T cells play a role in immune surveillance during infection and tissue injury.     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

Antigen-specific inhibition of CD4+ T-cell responses to β-lactoglobulin by its single amino acid-substituted mutant form through T-cell receptor antagonism

T cell responses can be antagonized by some single amino acid-substituted analogs of a peptide ligand for T-cell receptors (TCR), and these are called TCR antagonists. In this study, we addressed the question of whether TCR antagonism can be elicited by a whole protein antigen carrying a mutated T-cell determinant region corresponding to a TCR antagonist peptide. To clarify this, we examined the ability of a single amino acid-substituted mutant form of bovine β-lactoglobulin (β-Lg) to inhibit three CD4⁺ T-cell clones recognizing a peptide corresponding to an immunodominant determinant region 119-133 of β-Lg (p119-133). First, we identified pD129A, an analog of p119-133 with a substitution of Ala for ¹²⁹Asp, as an antagonist which can inhibit the response of two of the three T-cell clones. Then, using a yeast expression system, we prepared a mutant β-Lg (mutD129A) with the same substitution of Ala for ¹²⁹Asp as that in pD129A. This mutant protein could inhibit the proliferation of the two T-cell clones in a manner similar to the effect of pD129A. From these results we can demonstrate that TCR antagonism can be elicited by peptides naturally processed from a single-substituted mutant protein as well as by the corresponding peptides added exogenously. This revised version was published online in June 2006 with corrections to the Cover Date.