Selective antigen‐specific CD4+ T‐cell,but not CD8+ T‐ or B‐cell,tolerance corrupts cancer immunotherapy

Self‐tolerance, presumably through lineage‐unbiased elimination of self‐antigen‐specific lymphocytes (CD4⁺ T, CD8⁺ T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self‐tolerance reflects selective elimination of antigen‐specific CD4⁺ T cells, but preservation of CD8⁺ T‐ and B‐cell populations. In mice, antigen‐specific CD4⁺ T‐cell tolerance restricted CD8⁺ T‐ and B‐cell responses targeting the endogenous self‐antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4⁺ T‐cell tolerance blocked GUCY2C‐specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self‐antigen‐independent CD4⁺ T‐cell help. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4⁺ T‐cell help, revealing the latent functional capacity of GUCY2C‐specific CD8⁺ T‐ and B‐cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines targeting self‐antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4⁺ T‐cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self‐antigens characterized by selective CD4⁺ T‐cell tolerance and abrogation of such tolerance through self‐antigen‐independent T‐cell help is essential for future immunotherapeutics.     

Efficient and versatile manipulation of the peripheral CD4+ T‐cell compartment by antigen targeting to DNGR‐1/CLEC9A

DC NK lectin group receptor‐1 (DNGR‐1, also known as CLEC9A) is a C‐type lectin receptor expressed by mouse CD8α⁺ DC and by their putative equivalents in human. DNGR‐1 senses necrosis and regulates CD8⁺ T‐cell cross‐priming to dead‐cell‐associated antigens. In addition, DNGR‐1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8⁺ T‐cell and Ab responses. In this study, we evaluated whether DNGR‐1 targeting can be additionally used to manipulate antigen‐specific CD4⁺ T lymphocytes. Injection of small amounts of antigen‐coupled anti‐DNGR‐1 mAb into mice promoted MHC class II antigen presentation selectively by CD8α⁺ DC. In the steady state, this was sufficient to induce proliferation of antigen‐specific naïve CD4⁺ T cells and to drive their differentiation into Foxp3⁺ regulatory lymphocytes. Co‐administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4⁺ T‐cell behavior: poly I:C induced a strong IL‐12‐independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR‐1 is a versatile approach for inducing functionally distinct CD4⁺ T‐cell responses. Given the restricted pattern of expression of DNGR‐1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.     

DC‐induced CD8+ T‐cell response is inhibited by MHC class II‐dependent DX5+CD4+ Treg

CD4⁺ T cells are important for CD8⁺ T‐cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4⁺ T cells to influence CD8⁺ T‐cell responses induced by activated DC. Surprisingly, mice depleted for CD4⁺ cells were able to generate stronger antigen‐specific CD8⁺ T‐cell responses after DC vaccination than non‐depleted mice. The same observation was made when mice were vaccinated with MHC class II^?/? DC, indicating the presence of a MHC class II‐dependent CD4⁺ T‐cell population inhibiting CD8⁺ T‐cell responses. Recently we described the expansion of DX5⁺CD4⁺ T cells, a T‐cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8⁺ T‐cell induction and expansion of DX5⁺CD4⁺ T cells as the latter cells did not expand after vaccination with MHC class II^?/? DC. In vitro, DX5⁺CD4⁺ T cells were able to limit proliferation, modulate cytokine production and induce Foxp3⁺ expression in OVA‐specific CD8⁺ T cells. Together, our data show an inhibitory role of CD4⁺ T cells on the induction of CD8⁺ T‐cell responses by activated DC and indicate the involvement of DX5⁺CD4⁺, but not CD4⁺CD25⁺, T cells in this process.     

H2‐M3‐restricted CD8+ T cells augment CD4+ T‐cell responses by promoting DC maturation

Infection with Listeria monocytogenes triggers the activation and expansion of nonconventional CD8⁺ T cells restricted by the MHC class Ib molecule, H2‐M3. H2‐M3‐restricted CD8⁺ T cells exhibit a memory phenotype, rapidly produce cytokines, and reach peak frequencies sooner than conventional MHC class Ia‐restricted CD8⁺ T cells. In this study, we found that simultaneous in vivo priming of H2‐M3‐restricted T cells and adoptively transferred OT‐II CD4⁺ T cells on the same DC enhances the survival of OT‐II cells. Stimulation of H2‐M3‐restricted T cells were found to induce DC maturation resulting in costimulatory molecule upregulation and production of T_H1‐type cytokines, which was dependent on both cell‐to‐cell contact and soluble factors, particularly TNF‐α, produced by activated H2‐M3‐restricted T cells. Interestingly, H2‐M3‐restricted T cells were more efficient than activated NK cells in inducing DC maturation. Furthermore, we found that OVA_323–339‐coated DC matured by coculturing with peptide‐stimulated H2‐M3‐restricted T cells were more efficient in stimulating the proliferation of Ag‐activated OT‐II cells. This study indicates that H2‐M3‐restricted T cells promote immune responses by CD4⁺ T cells by inducing DC maturation and suggests novel mechanisms for vaccine development.     

DNGR‐1 is dispensable for CD8+ T‐cell priming during respiratory syncytial virus infection

During respiratory syncytial virus (RSV) infection CD8⁺ T cells both assist in viral clearance and contribute to immunopathology. CD8⁺ T cells recognize viral peptides presented by dendritic cells (DCs), which can directly present viral antigens when infected or, alternatively, “cross‐present” antigens after endocytosis of dead or dying infected cells. Mouse CD8α⁺ and CD103⁺ DCs excel at cross‐presentation, in part because they express the receptor DNGR‐1 that detects dead cells by binding to exposed F‐actin and routes internalized cell debris into the cross‐presentation pathway. As RSV causes death in infected epithelial cells, we tested whether cross‐presentation via DNGR‐1 is necessary for CD8⁺ T‐cell responses to the virus. DNGR‐1‐deficient or wild‐type mice were intranasally inoculated with RSV and the magnitude of RSV‐specific CD8⁺ T‐cell induction was measured. We found that during live RSV infection, cross‐presentation via DNGR‐1 did not have a major role in the generation of RSV–specific CD8⁺ T‐cell responses. However, after intranasal immunization with dead cells infected with RSV, a dependence on DNGR‐1 for RSV‐specific CD8⁺ T‐cell responses was observed, confirming the ascribed role of the receptor. Thus, direct presentation by DCs may be the major pathway initiating CD8⁺ T‐cell responses to RSV, while DNGR‐1‐dependent cross‐presentation has no detectable role.     

Antigen‐specific Treg impair CD8+ T‐cell priming by blocking early T‐cell expansion

Foxp3⁺ Treg are crucial for the maintenance of self‐tolerance and have been shown to control CD8⁺ T‐cell effector functions. In addition, Treg are thought to control the priming of CD8⁺ T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA‐specific Treg suppress antigen‐linked CTL responses through early blockade of CD8⁺ T‐cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8⁺ T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA‐specific CD8⁺ T‐cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA‐specific Treg are less effective but still reduce the frequency of newly primed HA‐specific CD8⁺ T cells and the ensuing frequency of memory CD8⁺ T cells. Our results demonstrate that Treg control CTL priming in an antigen‐specific manner at the level of T‐cell expansion, highlighting how self‐reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T‐cell proliferation.     

Steady‐state antigen‐expressing dendritic cells terminate CD4+ memory T‐cell responses

CD4⁺ T cells are important effectors of inflammation and tissue destruction in many diseases of immune dysregulation. As memory T cells develop early during the preclinical stages of autoimmune and inflammatory diseases, immunotherapeutic approaches to treatment of these diseases, once established, must include the means to terminate memory T‐cell responses. Traditionally, it has been considered that, due to their terminally differentiated nature, memory T cells are resistant to tolerance induction, although emerging evidence indicates that some immunotherapeutic approaches can terminate memory T‐cell responses. Here, we demonstrate that CD4⁺ memory T‐cell responses can be terminated when cognate antigen is transgenically expressed in steady‐state DC. Transfer of in‐vitro‐generated CD4⁺ memory T cells establishes, in nontransgenic recipients, a stable and readily recalled memory response to cognate antigen. In contrast, upon transfer to mice expressing cognate antigen targeted to DC, memory CD4⁺ T cells undergo a phase of limited proliferation followed by substantial deletion, and recall responses are effectively silenced. This finding is important in understanding how to effectively apply immunotherapy to ongoing T‐cell‐mediated autoimmune and inflammatory diseases.     

Antigens expressed by myelinating glia cells induce peripheral cross‐tolerance of endogenous CD8+ T cells

Auto‐reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self‐antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self‐antigens is not robust. To investigate the fate of nervous system‐specific CD8⁺ T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus‐derived CD8⁺ T‐cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8⁺ T‐cell epitopes induced robust tolerance of endogenous auto‐reactive T cells, which proved thymus‐independent and was mediated by cross‐presenting bone‐marrow‐derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia‐derived antigens in DC, suggesting that peripheral tolerance of CD8⁺ T cells results from uptake and presentation by steady state DC.     

Vaccine molecules targeting Xcr1 on cross‐presenting DCs induce protective CD8+ T‐cell responses against influenza virus

Targeting antigens to cross‐presenting dendritic cells (DCs) is a promising method for enhancing CD8⁺ T‐cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross‐presenting murine CD8α⁺ DCs, and that the expression is conserved on homologous DC subsets in humans (CD141⁺ DCs), sheep (CD26⁺ DCs), and macaques (CADM1⁺ DCs). We therefore tested if targeting antigens to Xcr1 on cross‐presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α⁺ DCs and increased proliferation of antigen‐specific T cells. DNA vaccines encoding dimeric Xcl1‐hemagglutinin (HA) fusion proteins induced cytotoxic CD8⁺ T‐cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8⁺ T‐cell responses, targeting of antigen to Xcr1 induced CD4⁺ Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α⁺ DCs using Xcl1 represents a novel and promising method for induction of protective CD8⁺ T‐cell responses.     

T‐cell help dependence of memory CD8+ T‐cell expansion upon vaccinia virus challenge relies on CD40 signaling

Due to their capacity to differentiate into long‐lived memory cells, CD8⁺ T cells are able to resolve subsequent infections faster than during the primary response. Among other factors, CD4⁺ T cells play a crucial role during primary and secondary CD8⁺ T‐cell responses. However, the timing and mechanisms by which they influence CD8⁺ T cells may differ in primary and secondary responses. Here, we demonstrate that during both primary and secondary vaccinia virus infection, CD4⁺ T cells are necessary to promote CD8⁺ T‐cell responses. While CD4⁺ T cells contributed to memory CD8⁺ T‐cell development, they were even more important during memory recall responses during challenge, as absence of CD4⁺ T cells during challenge resulted in markedly decreased proliferation and increased apoptosis. T‐cell help during primary and secondary responses was mediated via CD40 signaling, with DCs being an integral part of that pathway. As opposed to primary CD8⁺ T‐cell responses where only a combination of agonistic CD40 signaling and provision of IL‐2 could substitute for T‐cell help, agonistic CD40 triggering alone was sufficient to rescue memory CD8⁺ T‐cell responses in absence of T‐cell help in the context of vaccinia virus infection.     

CD8+ T‐cell immunosurveillance constrains lymphoid premetastatic myeloid cell accumulation

Increasing evidence suggests that premetastatic niches, consisting mainly of myeloid cells, provide microenvironment critical for cancer cell recruitment and survival to facilitate metastasis. While CD8⁺ T cells exert immunosurveillance in primary human tumors, whether they can exert similar effects on myeloid cells in the premetastatic environment is unknown. Here, we show that CD8⁺ T cells are capable of constraining premetastatic myeloid cell accumulation by inducing myeloid cell apoptosis in C57BL/6 mice. Ag‐specific CD8⁺ T‐cell cytotoxicity against myeloid cells in premetastatic lymph nodes is compromised by Stat3. We demonstrate here that Stat3 ablation in myeloid cells leads to CD8⁺ T‐cell activation and increased levels of IFN‐γ and granzyme B in the premetastatic environment. Furthermore, Stat3 negatively regulates soluble Ag cross‐presentation by myeloid cells to CD8⁺ T cells in the premetastatic niche. Importantly, in tumor‐free lymph nodes of melanoma patients, infiltration of activated CD8⁺ T cells inversely correlates with STAT3 activity, which is associated with a decrease in number of myeloid cells. Our study suggested a novel role for CD8⁺ T cells in constraining myeloid cell activity through direct killing in the premetastatic environment, and the therapeutic potential by targeting Stat3 in myeloid cells to improve CD8⁺ T‐cell immunosurveillance against metastasis.     

A CD40/CD40L feedback loop drives the breakdown of CD8+ T‐cell tolerance following depletion of suppressive CD4+ T cells

Dendritic cells (DCs) are the key APCs not only for the priming of naïve T cells, but also for the induction and maintenance of peripheral T‐cell tolerance. We have recently shown that cognate interactions between Foxp3⁺ Tregs and steady‐state DCs are crucial to maintain the tolerogenic potential of DCs. Using DIETER mice, which allow the induction of antigen presentation selectively on DCs without altering their maturation status, we show here that breakdown of CD8⁺ T‐cell tolerance, which ensues after depletion of suppressive CD4⁺ T cells, is driven by a positive feedback loop in which autoreactive CD8⁺ T cells activate DCs via CD40. These data identify ligation of CD40 on DCs as a stimulus that promotes autoreactive T‐cell priming when regulatory T‐cell suppression fails and suggest that feedback from autoreactive T cells to DCs may contribute to the well‐documented involvement of CD40 in many autoimmune diseases.     

IL‐10 promotes homeostatic proliferation of human CD8+ memory T cells and,when produced by CD1c+ DCs,shapes naive CD8+ T‐cell priming

IL‐10 is an anti‐inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8⁺ CTL, IL‐10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL‐10 produced by human APC subsets in T‐cell responses. IL‐10 production was restricted to CD1c⁺ DCs and CD14⁺ monocytes. Interestingly, it was differentially regulated, since R848 induced IL‐10 in DCs, but inhibited IL‐10 in monocytes. Autocrine IL‐10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high‐affinity peptides. Nevertheless, it completely blocked cross‐priming and priming with low‐affinity peptides of a self/tumor‐antigen. IL‐10 also inhibited CD1c⁺ DC‐induced CD4⁺ T‐cell priming and enhanced Foxp3 induction, but was insufficient to induce T‐cell IL‐10 production. CD1c⁺ DC‐derived IL‐10 had also no effect on DC‐induced secondary expansions of memory CTL. However, IL‐15‐driven, TCR‐independent proliferation of memory CTL was enhanced by IL‐10. We conclude that DC‐derived IL‐10 selects high‐affinity CTL upon priming. Moreover, IL‐10 preserves established CTL memory by enhancing IL‐15‐dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL‐10 promotes CTL responses in humans.     

Optimal stimulation for CD70 induction on human monocyte‐derived dendritic cells and the importance of CD70 in naive CD4+ T‐cell differentiation

Studies in mice have shown that CD70 on dendritic cells (DCs) is sufficient to convert T‐cell tolerance into immunity and hence induce anti‐tumour immune responses. Therefore, it is important to investigate (i) optimal stimuli to induce CD70 on human monocyte‐derived DCs (MoDCs), which are widely used for tumour immunotherapy, and (ii) the role of CD70 in functional differentiation of naive CD4⁺ and CD8⁺ T cells stimulated with MoDCs. We show that interferon‐α (IFN‐α) is a key cytokine to differentiate monocytes into DCs with the capacity to express CD70 upon maturation. CD70 expression on IFN‐α‐induced MoDCs was elicited by different categories of maturation‐inducing factors (Toll‐like receptor ligands, CD40 ligand and pro‐inflammatory mediators), among which prostaglandin E₂ was most effective. Naive T cells stimulated with MoDCs also expressed CD70. Stimulation with MoDCs promoted naive CD4⁺ T cells to acquire the ability to produce T helper type 1 and 2 cytokines in a CD70‐dependent manner. In contrast, the CD70–CD27 interaction diminished the production of an immunoregulatory cytokine IL‐10. The CD27 signal did not play a dominant role in the induction of effector molecules in naive CD8⁺ T cells during the stimulation with MoDCs. This study adds a novel function to the versatile cytokines, type I IFNs, that is, the induction of CD70 on MoDCs. CD70 promotes naive CD4⁺ T cells to acquire immunostimulatory activity through the DC–T‐cell and T‐cell–T‐cell interactions during the stimulation with MoDCs. Hence, the CD70–CD27 interaction may play an important role in inducing effective immune responses in DC‐based immunotherapy.     

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8⁺ T‐cell precursor frequency (PF) precludes the requirement of CD4⁺ helper T (Th) cells for primary CD8⁺ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DC_OVA) derived from C57BL/6, CD40 knockout (CD40^?/?) or CD40 ligand knockout (CD40L^?/?) mice were used to immunize C57BL/6, Ia^b?/?, CD40^?/? or CD40L^?/? mice, whose PF was previously increased with transfer of 1 × 10⁶ CD8⁺ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40^?/?) or OTI(CD40L^?/?) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4⁺ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4⁺ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4⁺ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4⁺ T‐cell functions.     

Analysis of the functional WT1‐specific T‐cell repertoire in healthy donors reveals a discrepancy between CD4+ and CD8+ memory formation

The Wilms’ tumour‐1 (WT1) protein is considered a prime target for cancer immunotherapy based on its presumptive immunogenicity and widespread expression across a variety of malignancies. However, little is known about the naturally occurring WT1‐specific T‐cell repertoire because self‐derived antigens typically elicit low frequency responses that challenge the sensitivity limits of current detection techniques. In this study, we used highly efficient cell enrichment procedures based on CD137, CD154, and pHLA class I tetramer staining to conduct a detailed analysis of WT1‐specific T cells from the peripheral blood. Remarkably, we detected WT1‐specific CD4⁺ and CD8⁺ T‐cell populations in the vast majority of healthy individuals. Memory responses specific for WT1 were commonly present in the CD4⁺ T‐cell compartment, whereas WT1‐specific CD8⁺ T cells almost universally displayed a naive phenotype. Moreover, memory CD4⁺ and naive CD8⁺ T cells with specificity for WT1 were found to coexist in some individuals. Collectively, these findings suggest a natural discrepancy between the CD4⁺ and CD8⁺ T‐cell lineages with respect to memory formation in response to a self‐derived antigen. Nonetheless, WT1‐specific T cells from both lineages were readily activated ex vivo and expanded in vitro, supporting the use of strategies designed to exploit this expansive reservoir of self‐reactive T cells for immunotherapeutic purposes.     

Antigen presenting cell‐targeted proinsulin expression converts insulin‐specific CD8+ T‐cell priming to tolerance in autoimmune‐prone NOD mice

Type 1 diabetes (T1D) results from autoimmune destruction of insulin‐producing pancreatic β cells. Therapies need to incorporate strategies to overcome the genetic defects that impair induction or maintenance of peripheral T‐cell tolerance and contribute to disease development. We tested whether the enforced expression of an islet autoantigen in antigen‐presenting cells (APC) counteracted peripheral T‐cell tolerance defects in autoimmune‐prone NOD mice. We observed that insulin‐specific CD8⁺ T cells transferred to mice in which proinsulin was transgenically expressed in APCs underwent several rounds of division and the majority were deleted. Residual insulin‐specific CD8⁺ T cells were rendered unresponsive and this was associated with TCR downregulation, loss of tetramer binding and expression of a range of co‐inhibitory molecules. Notably, accumulation and effector differentiation of insulin‐specific CD8⁺ T cells in pancreatic lymph nodes was prominent in non‐transgenic recipients but blocked by transgenic proinsulin expression. This shift from T‐cell priming to T‐cell tolerance exemplifies the tolerogenic capacity of autoantigen expression by APC and the capacity to overcome genetic tolerance defects.     

CD70 and IFN‐1 selectively induce eomesodermin or T‐bet and synergize to promote CD8+ T‐cell responses

CD70‐mediated stimulation of CD27 is an important cofactor of CD4⁺ T‐cell licensed dendritic cells (DCs). However, it is unclear how CD70‐mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type‐1 interferon (IFN‐1) and IL‐12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin^hiT‐bet^lo CD8⁺ T‐cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8⁺ T‐cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN‐γ production and the proportion of the population with characteristics of short‐lived effector cells, yet also promotes the ability to form long‐lived memory. Notably, while IFN‐1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN‐1's effect directly on CD8⁺ T cells, and is associated with the increased expression of T‐bet in T cells. Surprisingly, we find that IL‐12 fails to synergize with CD27 stimulation to promote CD8⁺ T‐cell expansion, despite its capacity to drive effector CD8⁺ T‐cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8⁺ T‐cell responses.     

DX5+CD4+ T cells modulate CD4+ T‐cell response via inhibition of IL‐12 production by DCs

DX5⁺CD4⁺ T cells have been shown to dampen collagen‐induced arthritis and delayed‐type hypersensitivity reactions in mice. These cells are also potent modulators of T‐helper cell responses through direct effects on CD4⁺ T cells in an IL‐4 dependent manner. To further characterize this T‐cell population, we studied their effect on DCs and the potential consequences on T‐cell activation. Here, we show that mouse DX5⁺CD4⁺ T cells modulate DCs by robustly inhibiting IL‐12 production. This modulation is IL‐10 dependent and does not require cell contact. Furthermore, DX5⁺CD4⁺ T cells modulate the surface phenotype of LPS‐matured DCs. DCs modulated by DX5⁺CD4⁺ T‐cell supernatant express high levels of the co‐inhibitor molecules PDL‐1 and PDL‐2. OVA‐specific CD4⁺ T cells primed with DCs exposed to DX5⁺CD4⁺ T‐cell supernatant produce less IFN‐γ than CD4⁺ T cells primed by DCs exposed to either medium or DX5^?CD4⁺ T‐cell supernatant. The addition of IL‐12 to the co‐culture with DX5⁺ DCs restores IFN‐γ production. When IL‐10 present in the DX5⁺CD4⁺ T‐cell supernatant is blocked, DCs re‐establish their ability to produce IL‐12 and to efficiently prime CD4⁺ T cells. These data show that DX5⁺CD4⁺ T cells can indirectly affect the outcome of the T‐cell response by inducing DCs that have poor Th1 stimulatory function.