Prolonged antitumor NK cell reactivity elicited by CXCL10-expressing dendritic cells licensed by CD40L+ CD4+ memory T cells

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4(+) T (CD4(+) T(EM)) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4(+) T(EM) cells and NK cells. In this interaction, CD4(+) T(EM) cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.     

Autocrine activation-induced cell death of T cells by human peripheral blood monocyte-derived CD4+ dendritic cells

Mature T cells activated by antigen (Ag)-presenting cells are subject to various downmodulatory processes designed to maintain T cell homeostasis. Here we describe experiments in which mature T cells were subjected to apoptosis following stimulation with CD4(+) dendritic cells (DCs) during Ag presentation. The proliferative response of allogeneic T cells was increased by DCs at stimulator to responder (S/R) ratios ranging from 10(-3) to 1, whereas this response was decreased at S/R ratios ranging from 2 to 10. Allogeneic T cells stimulated with DCs at an S/R ratio of 5 underwent apoptosis, whereas this event was not observed in allogeneic T cells stimulated with DCs at an S/R ratio of 0.5. Stimulation of T cells with DCs at an S/R ratio of 5 induced a higher level of expression of CD95 ligand (CD95L) than stimulation of T cells cultured with DCs at an S/R ratio of 0.5, whereas similar levels of expression of CD28 and CD154 were observed in both cells. The abortive proliferation of mature T cells stimulated with DCs was prevented by blocking the CD95-CD95L system. Our results suggest that the CD4(+) DCs play counterregulatory roles in dictating T cell responses during Ag presentation.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Cognate CD4+ help elicited by resting dendritic cells does not impair the induction of peripheral tolerance in CD8+ T cells

Peripheral tolerance is required to prevent autoimmune tissue destruction by self-reactive T cells that escape negative selection in the thymus. One mechanism of peripheral tolerance in CD8(+) T cells is their activation by resting dendritic cells (DC). In contrast, DC can be "licensed" by CD4(+) T cells to induce cytotoxic function in CD8(+) T cells. The question that then arises, whether CD4(+) T cell help could impair peripheral tolerance induction in self-reactive CD8(+) T cells, has not been addressed. In this study we show that CD4(+) T cell activation by resting DC results in helper function that transiently promotes the expansion and differentiation of cognate CD8(+) T cells. However, both the CD4(+) and CD8(+) T cell populations ultimately undergo partial deletion and acquire Ag unresponsiveness, disabling their ability to destroy OVA-expressing pancreatic beta cells and cause diabetes. Thus, effective peripheral tolerance can be induced by resting DC in the presence of CD4(+) and CD8(+) T cells with specificity for the same Ag.     

Modulation of dendritic cell function by naive and regulatory CD4+ T cells

The consequences of interactions between dendric cells (DCs) and either naive CD4+ T cells or regulatory CD4+CD25+ T cells on the expression of proinflammatory IL-6 and anti-inflammatory IL-10 in DC were examined over a period of 12 h, spanning the time frame during which stable T cell-DC interactions shape the development of tolerance and immunity in vivo. We demonstrate that the basal production of IL-6 and IL-10, which is initiated following DC stimulation with LPS, is modified in distinctly different ways by interaction with the two T cell populations. Naive CD4 T cells skew DC cytokine production toward IL-6 and suppress IL-10, whereas CD4+CD25+ T cells have the opposite effect. CD8 T cells or memory CD4 T cells do not influence basal cytokine production by stimulated DC. The effect of CD4+CD25+ T cells is dominant in coculture with naive CD4 T cells as long as inflammatory LPS is absent; the addition of LPS abrogates the suppression of IL-6. However, the modulating influence of CD4+CD25+ T cells remains evident in the enhancement of IL-10 production. Thus, mutual interactions between DC and CD4+ T cell subpopulations following contact with pathogens are likely to influence the strength and quality of incipient immune responses in the local microenvironment.     

Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4+ helper T cell subsets and allogeneic CD8+ T cells.

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th1 or Th2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th1 helper cells in situ. The remaining six cell lines responded to INF-gamma by up-regulating their class II expression and acquiring Th1 antigen-presenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th1-derived INF-gamma. Five of the cell lines provided accessory cell function to Th2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th1 and Th2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8+ T cells occurred in a CD4+ helper cell-independent manner and was unaffected by the addition of INF-gamma. The alloantigen-presenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.     

Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells

Activation of invariant NK T (iNKT) cells with the glycolipid alpha-galactosylceramide promotes CD8(+) cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8(+) T cells occur on the same APCs. We demonstrate that injection of alpha-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8(+) T cell response and abolishes the efficacy of alpha-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8(+) T cells.     

Antigen nonspecific suppression of T cell responses by activated stimulation-refractory CD4+ T cells

Several classes of anergic T cells are capable of suppressing naive T cell proliferation and thereby limiting immune responses. Activated T cells, although not anergic, are transiently refractory to restimulation with Ag. We examine in this study whether activated refractory murine T cells can also suppress naive T cell responses. We find that they can, and that they exhibit many of the suppressive properties of anergic T cells. The activated cells strongly diminish Ag-mediated T cell proliferation, an activity that correlates with their refractory period. Suppression is independent of APC numbers and requires cell contact or proximity. Naive T cells stimulated in the presence of activated refractory cells up-regulate CD25 and CD69, but fail to produce IL-2. The addition of IL-2 to culture medium, however, does not prevent the suppression, which is therefore not solely due to the absence of this growth factor. Persistence of the suppressor cells is also not essential. T cells stimulated in their presence and then isolated from them and cultured do not divide. The suppressive cells, however, do not confer a refractory or anergic state on the target T lymphocytes, which can fully respond to antigenic stimulation if removed from the suppressors. Our results therefore provide evidence that activated T cells act as transient suppressor cells, severely constraining bystander T cell stimulation and thereby restricting their response. These results have potentially broad implications for the development and regulation of immune responses.     

Defective MHC class II presentation by dendritic cells limits CD4 T cell help for antitumor CD8 T cell responses

Cancer immunosurveillance failure is largely attributed to insufficient activation signals and dominant inhibitory stimuli for tumor Ag (TAg)-specific CD8 T cells. CD4 T cells have been shown to license dendritic cells (DC), thereby having the potential for converting CD8 T cell responses from tolerance to activation. To understand the potential cooperation of TAg-specific CD4 and CD8 T cells, we have characterized the responses of naive TCR transgenic CD8 and CD4 T cells to poorly immunogenic murine tumors. We found that whereas CD8 T cells sensed TAg and were tolerized, the CD4 T cells remained ignorant throughout tumor growth and did not provide help. This disparity in responses was due to normal TAg MHC class I cross-presentation by immature CD8alpha+ DC in the draining lymph node, but poor MHC class II presentation on all DC subsets due to selective inhibition by the tumor microenvironment. Thus, these results reveal a novel mechanism of cancer immunosubversion, in which inhibition of MHC-II TAg presentation on DC prevents CD4 T cell priming, thereby blocking any potential for licensing CD8alpha+ DC and helping tolerized CD8 T cells.     

Murine CD4+ T cell responses are inhibited by cytotoxic T cell-mediated killing of dendritic cells and are restored by antigen transfer

Cytotoxic T lymphocytes (CTL) provide protection against pathogens and tumors. In addition, experiments in mouse models have shown that CTL can also kill antigen-presenting dendritic cells (DC), reducing their ability to activate primary and secondary CD8(+) T cell responses. In contrast, the effects of CTL-mediated killing on CD4(+) T cell responses have not been fully investigated. Here we use adoptive transfer of TCR transgenic T cells and DC immunization to show that specific CTL significantly inhibited CD4(+) T cell proliferation induced by DC loaded with peptide or low concentrations of protein antigen. In contrast, CTL had little effect on CD4(+) T cell proliferation induced by DC loaded with high protein concentrations or expressing antigen endogenously, even if these DC were efficiently killed and failed to accumulate in the lymph node (LN). Residual CD4(+) T cell proliferation was due to the transfer of antigen from carrier DC to host APC, and predominantly involved skin DC populations. Importantly, the proliferating CD4(+) T cells also developed into IFN-γ producing memory cells, a property normally requiring direct presentation by activated DC. Thus, CTL-mediated DC killing can inhibit CD4(+) T cell proliferation, with the extent of inhibition being determined by the form and amount of antigen used to load DC. In the presence of high antigen concentrations, antigen transfer to host DC enables the generation of CD4(+) T cell responses regardless of DC killing, and suggests mechanisms whereby CD4(+) T cell responses can be amplified.     

CD40-CD40 ligand interaction between dendritic cells and CD8+ T cells is needed to stimulate maximal T cell responses in the absence of CD4+ T cell help

Stimulation of CD40 on APCs through CD40L expressed on helper CD4+ T cells activates and "licenses" the APCs to prime CD8+ T cell responses. Although other stimuli, such as TLR agonists, can also activate APCs, it is unclear to what extent they can replace the signals provided by CD40-CD40L interactions. In this study, we used an adoptive transfer system to re-examine the role of CD40 in the priming of naive CD8+ T cells. We find an approximately 50% reduction in expansion and cytokine production in TCR-transgenic T cells in the absence of CD40 on all APCs, and on dendritic cells in particular. Moreover, CD40-deficient and CD40L-deficient mice fail to develop endogenous CTL responses after immunization. Surprisingly, the role for CD40 and CD40L are observed even in the absence of CD4+ T cells; in this situation, the CD8+ T cell itself provides CD40L. Furthermore, we show that although TLR stimulation improves T cell responses, it cannot fully substitute for CD40. Altogether, these results reveal a direct and unique role for CD40L on CD8+ T cells interacting with CD40 on APCs that affects the magnitude and quality of CD8+ T cell responses.     

NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells

Modification in the function of dendritic cells (DC), such as that achieved by microbial stimuli or T cell help, plays a critical role in determining the quality and size of adaptive responses to Ag. NKT cells bearing an invariant TCR (iNKT cells) restricted by nonpolymorphic CD1d molecules may constitute a readily available source of help for DC. We therefore examined T cell responses to i.v. injection of soluble Ag in the presence or the absence of iNKT cell stimulation with the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GalCer). Considerably enhanced CD4(+) and CD8(+) T cell responses were observed when alpha-GalCer was administered at the same time as or close to OVA injection. This enhancement was dependent on the involvement of iNKT cells and CD1d molecules and required CD40 signaling. Studies in IFN-gammaR(-/-) mice indicated that IFN-gamma was not required for the adjuvant effect of alpha-GalCer. Consistent with this result, enhanced T cell responses were observed using OCH, an analog of alpha-GalCer with a truncated sphingosine chain and a reduced capacity to induce IFN-gamma. Splenic DC from alpha-GalCer-treated animals expressed high levels of costimulatory molecules, suggesting maturation in response to iNKT cell activation. Furthermore, studies with cultured DC indicated that potentiation of T cell responses required presentation of specific peptide and alpha-GalCer by the same DC, implying conditioning of DC by iNKT cells. The iNKT-enhanced T cell responses resisted challenge with OVA-expressing tumors, whereas responses induced in the absence of iNKT stimulation did not. Thus, iNKT cells exert a significant influence on the efficacy of immune responses to soluble Ag by modulating DC function.     

Memory functions and death proneness in three CD4+CD45RO+ human T cell subsets

We propose a classification of human CD4(+)CD45RO(+) memory T cells into three new subsets based on cell surface expression levels of CD43. The first subset consists of cells whose CD43 expression is relatively high; this subset also contains the highest proportion of recall Ag-reactive precursors, and its constituent cells respond far more strongly than cells in either of the other subsets to immobilized CD3 Ab in addition to secreting substantially more IFN-gamma and IL-4. Cells of the second subset express similar levels of CD43 to naive cells, and they also respond weakly to TCR-mediated stimuli as judged by either their ability to proliferate or capacity for cytokine production. The third subsets consists of cells whose CD43 expression levels are clearly down-regulated; its cells appear to be anergic to TCR-mediated stimuli, and when examined ex vivo many of them appear to be undergoing either spontaneous apoptosis via a caspase-independent pathway or Fas-mediated apoptosis via a caspase-dependent pathway, even in the resting state. An analysis of telomere lengths revealed that the typical telomere of a cell in the second subset was significantly longer than the typical telomere in the first or third subset. Taken together, these results appear to indicate that CD4(+)CD45RO(+) T cells fall into three functionally differing subsets, one being a subset of cells with fully matured memory phenotype, a second being a less mature subset of cells that retain longer telomeres and whose memory functionality is marginal, and a third consisting of anergic cells that give every appearance of being death-prone and/or in the process of dying.     

Physiological induction of regulatory Qa-1-restricted CD8+ T cells triggered by endogenous CD4+ T cell responses

T cell-dependent autoimmune diseases are characterized by the expansion of T cell clones that recognize immunodominant epitopes on the target antigen. As a consequence, for a given autoimmune disorder, pathogenic T cell clones express T cell receptors with a limited number of variable regions that define antigenic specificity. Qa-1, a MHC class I-like molecule, presents peptides from the variable region of TCRs to Qa-1-restricted CD8+ T cells. The induction of Vß-specific CD8+ T cells has been harnessed in an immunotherapeutic strategy known as the “T cell vaccination” (TCV) that comprises the injection of activated and attenuated CD4+ T cell clones so as to induce protective CD8+ T cells. We hypothesized that Qa-1-restricted CD8+ regulatory T cells could also constitute a physiologic regulatory arm of lymphocyte responses upon expansion of endogenous CD4+ T cells, in the absence of deliberate exogenous T cell vaccination. We immunized mice with two types of antigenic challenges in order to sequentially expand antigen-specific endogenous CD4+ T cells with distinct antigenic specificities but characterized by a common Vß chain in their TCR. The first immunization was performed with a non-self antigen while the second challenge was performed with a myelin-derived peptide known to drive experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We show that regulatory Vß-specific Qa-1-restricted CD8+ T cells induced during the first endogenous CD4+ T cell responses are able to control the expansion of subsequently mobilized pathogenic autoreactive CD4+ T cells. In conclusion, apart from the immunotherapeutic TCV, Qa-1-restricted specialized CD8+ regulatory T cells can also be induced during endogenous CD4+ T cell responses. At variance with other regulatory T cell subsets, the action of these Qa-1-restricted T cells seems to be restricted to the immediate re-activation of CD4+ T cells.     

Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.     

Multiple dendritic cell populations activate CD4+ T cells after viral stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+) T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+) T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+) and CD8(+) T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+) and CD4(+) T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+) T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+) T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+) T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.     

Increasing the survival of dendritic cells in vivo does not replace the requirement for CD4+ T cell help during primary CD8+ T cell responses

The survival of dendritic cells (DC) in vivo determines the duration of Ag presentation and is critical in determining the strength and magnitude of the resulting T cell response. We used a mouse model to show that Ag-loaded C57BL/6 DC (MHC class II(+/+) (MHC II(+/+))) that reach the lymph node survived longer than Ag-loaded MHC II(-/-) DC, with the numbers of C57BL/6 DC being approximately 2.5-fold the number of the MHC II(-/-) DC by day 4 and approximately 5-fold by day 7. The differential survival of DC in vivo was not affected by low doses of LPS, but in vitro pretreatment with CD40L or with high doses of LPS increased the numbers of MHC II(-/-) DC to levels approaching those of C57BL/6 DC. Regardless of their numbers and relative survival in lymph nodes, MHC II(-/-) DC were profoundly defective in their ability to induce CTL responses against the gp33 peptide epitope, and were unable to induce expansion and optimal cytotoxic activity of CD8(+) T cells specific for the male Ag UTY. We conclude that CD4(+) T cell help for CD8(+) responses involves mechanisms other than the increased survival of Ag-presenting DC in the lymph node.     

CD4+ CD25+ regulatory T cells impair HIV-1-specific CD4 T cell responses by upregulating interleukin-10 production in monocytes

T cell dysfunction in the presence of ongoing antigen exposure is a cardinal feature of chronic viral infections with persistent high viremia, including HIV-1. Although interleukin-10 (IL-10) has been implicated as an important mediator of this T cell dysfunction, the regulation of IL-10 production in chronic HIV-1 infection remains poorly understood. We demonstrated that IL-10 is elevated in the plasma of individuals with chronic HIV-1 infection and that blockade of IL-10 signaling results in a restoration of HIV-1-specific CD4 T cell proliferation, gamma interferon (IFN-γ) secretion, and, to a lesser extent, IL-2 production. Whereas IL-10 blockade leads to restoration of IFN-γ secretion by HIV-1-specific CD4 T cells in all categories of subjects investigated, significant enhancement of IL-2 production and improved proliferation of CD4 T helper cells are restricted to viremic individuals. In peripheral blood mononuclear cells (PBMCs), this IL-10 is produced primarily by CD14(+) monocytes, but its production is tightly controlled by regulatory T cells (Tregs), which produce little IL-10 directly. When Tregs are depleted from PBMCs of viremic individuals, the effect of the IL-10 signaling blockade is abolished and IL-10 production by monocytes decreases, while the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), increases. The regulation of IL-10 by Tregs appears to be mediated primarily by contact or paracrine-dependent mechanisms which involve IL-27. This work describes a novel mechanism by which regulatory T cells control IL-10 production and contribute to dysfunctional HIV-1-specific CD4 T cell help in chronic HIV-1 infection and provides a unique mechanistic insight into the role of regulatory T cells in immune exhaustion.