Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic γc cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.

Here, we demonstrate that electroporation-enhanced immunization with a rationally designed HPV DNA vaccine (GX-188E), preferentially targeting HPV antigens to dendritic cells, elicits a significant E6/E7-specific IFN-γ-producing T-cell response in all nine cervical intraepithelial neoplasia 3 (CIN3) patients. Importantly, eight out of nine patients exhibit an enhanced polyfunctional HPV-specific CD8 T-cell response as shown by an increase in cytolytic activity, proliferative capacity and secretion of effector molecules. Notably, seven out of nine patients display complete regression of their lesions and viral clearance within 36 weeks of follow up. GX-188E administration does not elicit serious vaccine-associated adverse events at all administered doses. These findings indicate that the magnitude of systemic polyfunctional CD8 T-cell response is the main contributing factor for histological, cytological and virological responses, providing valuable insights into the design of therapeutic vaccines for effectively treating persistent infections and cancers in humans.

Aging is associated with a gradual loss of naïve T cells and a reciprocal increase in the proportion of memory T cells. While reduced thymic output is important, age-dependent changes in factors supporting naïve T cells homeostasis may also be involved. Indeed, we noted a dramatic decrease in the ability of aged mice to support survival and homeostatic proliferation of naïve T cells. The defect was not due to a reduction in IL-7 expression, but from a combination of changes in the secondary lymphoid environment that impaired naïve T cell entry and access to key survival factors. We observed an age-related shift in the expression of homing chemokines and structural deterioration of the stromal network in T cell zones. Treatment with IL-7/mAb complexes can restore naïve T cell homeostatic proliferation in aged mice. Our data suggests that homeostatic mechanisms that support the naïve T cell pool deteriorate with age.

Eosinophils play proinflammatory roles in helminth infections and allergic diseases. Under steady-state conditions, eosinophils are abundantly found in the small intestinal lamina propria, but their physiological function is largely unexplored. In this study, we found that small intestinal eosinophils down-regulate Th17 cells. Th17 cells in the small intestine were markedly increased in the ΔdblGATA-1 mice lacking eosinophils, and an inverse correlation was observed between the number of eosinophils and that of Th17 cells in the small intestine of wild-type mice. In addition, small intestinal eosinophils suppressed the in vitro differentiation of Th17 cells, as well as IL-17 production by small intestinal CD4(+)T cells. Unlike other small intestinal immune cells or circulating eosinophils, we found that small intestinal eosinophils have a unique ability to constitutively secrete high levels of IL-1 receptor antagonist (IL-1Ra), a natural inhibitor of IL-1β. Moreover, small intestinal eosinophils isolated from IL-1Ra-deficient mice failed to suppress Th17 cells. Collectively, our results demonstrate that small intestinal eosinophils play a pivotal role in the maintenance of intestinal homeostasis by regulating Th17 cells via production of IL-1Ra.

Acute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation.

Although murine γδ T cells are largely considered innate immune cells, they have recently been reported to form long-lived memory populations. Much remains unknown about the biology and specificity of memory γδ T cells. Here, we interrogated intestinal memory Vγ4 Vδ1 T cells generated after foodborne Listeria monocytogenes (Lm) infection to uncover an unanticipated complexity in the specificity of these cells. Deep TCR sequencing revealed that a subset of non-canonical Vδ1 clones are selected by Lm infection, consistent with antigen-specific clonal expansion. Ex vivo stimulations and in vivo heterologous challenge infections with diverse pathogenic bacteria revealed that Lm-elicited memory Vγ4 Vδ1 T cells are broadly reactive. The Vγ4 Vδ1 T cell recall response to Lm, Salmonella enterica serovar Typhimurium (STm) and Citrobacter rodentium was largely mediated by the γδTCR as internalizing the γδTCR prevented T cell expansion. Both broadly-reactive canonical and pathogen-selected non-canonical Vδ1 clones contributed to memory responses to Lm and STm. Interestingly, some non-canonical γδ T cell clones selected by Lm infection also responded after STm infection, suggesting some level of cross-reactivity. These findings underscore the promiscuous nature of memory γδ T cells and suggest that pathogen-elicited memory γδ T cells are potential targets for broad-spectrum anti-infective vaccines.

CXCR3 regulates leukocyte trafficking, maturation, and various pathophysiological conditions. Alternative splicing generates three CXCR3 isoforms in humans. Previous studies investigated the roles of CXCR3 isoforms, and some biochemical data are not correlated with biological relevance analyses. RT-PCR analyses indicate that most cells express all three splicing variants, suggesting that they may mutually affect the chemokine binding and cellular responses of other splicing variants. Here, we performed an integrative analysis of the functional relations among CXCR3 splicing variants and their chemokine-dependent signaling using NanoBiT live cell protein interaction assays. The results indicated that the CXCR3 N-terminal region affected cell surface expression levels and ligand-dependent activation. CXCR3A was efficiently expressed in the plasma membrane and responded to I-TAC, IP-10, and MIG chemokines. By contrast, CXCR3B had low plasma membrane expression and mediated I-TAC-stimulated cellular responses. CXCR3Alt was rarely expressed on the cell surface and did not mediate any cell responses to the tested chemokines; however, CXCR3Alt negatively affected the plasma membrane expression of CXCR3A and CXCR3B and their chemokine-stimulated cellular responses. Jurkat cells express endogenous CXCR3, and exogenous CXCR3A expression enhanced chemotactic activity in response to I-TAC, IP-10, and MIG. By contrast, exogenous expression of CXCR3B and CXCR3Alt eliminated or reduced the CXCR3A-induced chemotactic activity. The PF-4 chemokine did not activate any CXCR3-mediated cellular responses. NanoBiT technology are useful to integrative studies of CXCR3-mediated cell signaling, and expand our knowledge of the cellular responses mediated by molecular interactions among the splicing variants, including cell surface expression, ligand-dependent receptor activation, and chemotaxis.

C-X-C motif chemokine ligand 12(CXCL12) is an essential chemokine for organ development and homeostasis in multiple tissues. Its receptor, C-X-C chemokine receptor type 4(CXCR4), is expressed on the surface of target cells. The chemokine and receptor are expressed almost ubiquitously in human tissues and cells throughout life, and abnormal expression of CXCL12 and CXCR4 is observed in pathological conditions, such as inflammation and cancer. CXCR4 is reportedly translated into five splicing variants of different lengths, which each have different amino acids in the N-terminus. As the N-terminus is the first recognition site for chemokines, CXCR4 variants may respond differently to CXCL12. Despite these differences, the molecular and functional properties of CXCR4 variants have not been thoroughly described or compared. Here, we explored the expression of CXCR4 variants in cell lines and analyzed their roles in cellular responses using biochemical approaches. RT-PCR revealed that most cell lines express more than one CXCR4 variant. When expressed in HEK293 cells, the CXCR4 variants differed in protein expression efficiency and cell surface localization. Although variant 2 demonstrated the strongest expression and cell surface localization, variants 1, 3, and 5 also mediated chemokine signaling and induced cellular responses. Our results demonstrate that the N-terminal sequences of each CXCR4 variant determine the expression of the receptor and affect ligand recognition. Functional analyses revealed that CXCR4 variants may also affect each other or interact during CXCL12-stimulated cellular responses. Altogether, our results suggest that CXCR4 variants may have distinct functional roles that warrant additional investigation and could contribute to future development of novel drug interventions.

Examining the rate of in vivo T cell turnover (proliferation) in aged mice revealed a marked reduction in turnover at the level of memory-phenotype CD44(hi) CD8(+) cells relative to young mice. Based on adoptive transfer experiments, the reduced turnover of aged CD44(hi) CD8(+) cells reflected an inhibitory influence of the aged host environment. Aged CD44(hi) CD8(+) cells also showed poor in vivo responses to IL-15 and IL-15-inducing agents, but responded well to IL-15 in vitro. Two mechanisms could account for the reduced turnover of aged CD44(hi) CD8(+) cells in vivo. First, aging was associated with a prominent and selective increase in Bcl-2 expression in CD44(hi) CD8(+) cells. Hence, the reduced turnover of aged CD44(hi) CD8(+) cells may in part reflect the antiproliferative effect of enhanced Bcl-2 expression. Second, the impaired in vivo response of aged CD44(hi) CD8(+) cells to IL-15 correlated with increased serum levels of type I interferons (IFN-I) and was largely reversed by injection of anti-IFN-I antibody. Hence the selective reduction in the turnover of aged CD44(hi) CD8(+) cells in vivo may reflect the combined inhibitory effects of enhanced Bcl-2 expression and high IFN-I levels.

Previous work has shown that memory-phenotype CD44(hi) CD8(+) cells are controlled by a cytokine, interleukin (IL)-15. However, the dependency of CD44(hi) CD8(+) cells on IL-15 is partial rather than complete. Here, evidence is presented that CD44(hi) CD8(+) cells comprise a mixed population of IL-15-dependent and IL-15-independent cells. The major subset of CD122(hi) CD44(hi) CD8(+) cells is heavily dependent on IL-15 by three different parameters, namely (1) "bystander" proliferation induced via IFN-induced stimulation of the innate immune system, (2) normal "background" proliferation, and (3) T cell survival; IL-15 dependency is most extreme for the Ly49(+) subset of CD122(hi) CD44(hi) CD8(+) cells. In contrast to CD122(hi) cells, the CD122(lo) subset of CD44(hi) CD8(+) cells is IL-15 independent; likewise, being CD122(lo), CD44(hi) CD4(+) cells are IL-15 independent. Thus, subsets of memory-phenotype T cells differ radically in their sensitivity to IL-15.

Allergic contact hypersensitivity (CHS) is an inflammatory skin disease mediated by allergen specific T cells. In this study, we investigated the role of transcription factor NFAT1 in the pathogenesis of contact hypersensitivity. NFAT1 knock out (KO) mice spontaneously developed CHS-like skin inflammation in old age. Healthy young NFAT1 KO mice displayed enhanced susceptibility to hapten-induced CHS. Both CD4(+) and CD8(+) T cells from NFAT1 KO mice displayed hyper-activated properties and produced significantly enhanced levels of inflammatory T helper 1(Th1)/Th17 type cytokines. NFAT1 KO T cells were more resistant to activation induced cell death (AICD), and regulatory T cells derived from these mice showed a partial defect in their suppressor activity. NFAT1 KO T cells displayed a reduced expression of apoptosis associated BCL-2/BH3 family members. Ectopic expression of NFAT1 restored the AICD defect in NFAT1 KO T cells and increased AICD in normal T cells. Recipient Rag2(-/-) mice transferred with NFAT1 KO T cells showed more severe CHS sensitivity due to a defect in activation induced hapten-reactive T cell apoptosis. Collectively, our results suggest the NFAT1 plays a pivotal role as a genetic switch in CD4(+)/CD8(+) T cell tolerance by regulating AICD process in the T cell mediated skin inflammation.

Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8+ T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4+CD25+ regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8+ T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The gammaC cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of gammaC cytokine-responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8+ T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells.

Mice that are deficient in suppressor of cytokine signaling-1 (SOCS-1) succumb to neonatal mortality that is associated with extensive cellular infiltration of many tissues. T cells seem to be necessary for disease, which can be alleviated largely by neutralizing interferon-gamma. Examining T cell receptor (TCR) specificity shows that even monospecific T cells can mediate disease in SOCS-1-deficient mice, although disease onset is substantially faster with a polyclonal T cell repertoire. A major phenotype of SOCS-1-/- mice is the accumulation of CD44(high)CD8+ peripheral T cells. We show that SOCS-1-deficient CD8, but not CD4, T cells proliferate when transferred into normal (T cell-sufficient) mice, and that this is dependent on two signals: interleukin (IL)-15 and self-ligands that are usually only capable of stimulating homeostatic expansion in T cell-deficient mice. Our findings reveal that SOCS-1 normally down-regulates the capacity of IL-15 to drive activation and proliferation of naive CD8 T cells receiving TCR survival signals from self-ligands. We show that such dysregulated proliferation impairs the deletion of a highly autoreactive subset of CD8 T cells, and increases their potential for autoimmunity. Therefore, impaired deletion of highly autoreactive CD8 T cells, together with uncontrolled activation of naive CD8 T cells by homeostatic survival ligands, may provide a basis for the T cell-mediated disease of SOCS-1-/- mice.

Most memory phenotype (MP) CD44(hi) CD8(+) cells are resting interleukin (IL)-15-dependent cells characterized by high expression of the IL-2/IL-15 receptor beta (CD122). However, some MP CD8(+) cells have a CD122(lo) phenotype and are IL-15 independent. Here, evidence is presented that the CD122(lo) subset of MP CD8(+) cells is controlled largely by major histocompatibility complex (MHC) class I molecules. Many of these cells display surface markers typical of recently activated T cells (CD62L(lo), CD69(hi), CD43(hi), and CD127(lo)) and show a high rate of background proliferation. Cells with this phenotype are highly enriched in common gamma chain-deficient mice and absent from MHC-I(-/-) mice. Unlike CD122(hi) CD8(+) cells, CD122(lo) MP CD8(+) cells survive poorly after transfer to MHC-I(-/-) hosts and cease to proliferate. Although distinctly different from typical antigen-specific memory cells, CD122(lo) MP CD8(+) cells closely resemble the antigen-dependent memory CD8(+) cells found in chronic viral infections.

CD4 T cells differentiate into RORγt/IL-17A-expressing cells in the small intestine following colonization by segmented filamentous bacteria (SFB). However, it remains unclear whether SFB-specific CD4 T cells can differentiate directly from naïve precursors, and whether their effector differentiation is solely directed towards the Th17 lineage. In this study, we used adoptive T cell transfer experiments and showed that naïve CD4 T cells can migrate to the small intestinal lamina propria (sLP) and differentiate into effector T cells that synthesize IL-17A in response to SFB colonization. Using single cell RT-PCR analysis, we showed that the progenies of SFB responding T cells are not uniform but composed of transcriptionally divergent populations including Th1, Th17 and follicular helper T cells. We further confirmed this finding using in vitro culture of SFB specific intestinal CD4 T cells in the presence of cognate antigens, which also generated heterogeneous population with similar features. Collectively, these findings indicate that a single species of intestinal bacteria can generate a divergent population of antigen-specific effector CD4 T cells, rather than it provides a cytokine milieu for the development of a particular effector T cell subset.

The fast and intense proliferative responses have been well documented for naïve T cells adoptively transferred into chronic lymphopenic hosts. This response known as spontaneous proliferation (SP), unlike antigen-independent lymphopenia-induced proliferation (LIP), is driven in a manner dependent on antigens derived from commensal microbiota. However, the precise nature of the SP response and its impact on homeostasis and function for T cells rapidly responding under this lymphopenic condition are still unclear. Here we demonstrate that, when naïve T cells were adoptively transferred into specific pathogen-free (SPF) but not germ-free (GF) RAG-/- hosts, the SP response of these cells substantially affects the intensity and tempo of the responding T cells undergoing LIP. Therefore, the resulting response of these cells in SPF RAG-/- hosts was faster and stronger than the typical LIP response observed in irradiated B6 hosts. Although the intensity and tempo of such augmented LIP in SPF RAG-/- hosts were analogous to those of antigen-dependent SP, the former was independent of antigenic stimulation but most importantly, dependent on IL-2. Similar observations were also apparent in other acute lymphopenic settings where antigen-dependent T cell activation can strongly occur and induce sufficient levels of IL-2 production. Consequently, the resulting T cells undergoing IL-2-driven strong proliferative responses showed the ability to differentiate into functional effector and memory cells that can control infectious pathogens. These findings therefore reveal previously unappreciated role of IL-2 in driving the intense form of T cell proliferative responses in chronic lymphopenic hosts.

The primary induction sites for intestinal IgA are the gut-associated lymphoid tissues (GALT), such as Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). The commensal microbiota is known to contribute to IgA production in the gut; however, the role of dietary antigens in IgA production is poorly understood. To understand the effect of dietary antigens on IgA production, post-weaning mice were maintained on an elemental diet without any large immunogenic molecules. We found that dietary antigens contribute to IgA production in PPs through induction of follicular helper T cells and germinal center B cells. The role of dietary antigens in the PP responses was further confirmed by adding bovine serum albumin (BSA) into the elemental diet. Although dietary antigens are important for PP responses, they have fewer effects than the microbiota on the development and maturation of ILFs. Furthermore, we demonstrated that dietary antigens are essential for a normal antigen-specific IgA response to Salmonella typhi serovar Typhimurium infection. These results provide new insights into the role of dietary antigens in the regulation of mucosal immune responses.

Immune tolerance in the lung is important for preventing hypersensitivity, such as allergic asthma. Maintenance of tolerance in the lung is established by coordinated activities of poorly understood cellular and molecular mechanisms, including participation of dendritic cells (DCs). We have previously identified DC expression of the signaling molecule TRAF6 as a non-redundant requirement for the maintenance of immune tolerance in the small intestine of mice. Because mucosal tissues share similarities in how they interact with exogenous antigens, we examined the role of DC-expressed TRAF6 in the lung. As with the intestine, we found that the absence TRAF6 expression by DCs led to spontaneous generation of Th2-associated immune responses and increased susceptibility to model antigen-induced asthma. To examine the role of commensal microbiota, mice deficient in TRAF6 in DCs were treated with broad-spectrum antibiotics and/or re-derived on a germ-free (GF) background. Interestingly, we found that antibiotics-treated specific pathogen-free, but not GF, mice showed restored immune tolerance in the absence of DC-expressed TRAF6. We further found that antibiotics mediate microbiota-independent effects on lung T cells to promote immune tolerance in the lung. This work provides both a novel tool for studying immune tolerance in the lung and an advance in our conceptual understanding of potentially common molecular mechanisms of immune tolerance in both the intestine and the lung.

C-C motif chemokine receptor 2 (CCR2), the main receptor for monocyte chemoattractant protein-1 (MCP-1), is expressed on immune cells, including monocytes, macrophages, and activated T cells, and mediates cell migration toward MCP-1 in inflammation-related diseases. The CCR2 gene encodes two isoforms: CCR2A and CCR2B. The CCR2B open reading frame is localized in a single exon, similar to other chemokine receptors, and CCR2A and CCR2B feature different amino acid sequences in their C-terminal intracellular loops due to alternative splicing. Most biochemical studies on CCR2-related cellular responses in the immune system have focused on CCR2B, with few reports focused on CCR2A. Understanding the functional properties of CCR2A in cellular responses may elucidate the roles played by MCP-1 and CCR2 in pathophysiological responses.

High doses of interleukin-2 (IL-2) have been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy, with a ~15% response rate. Remarkably, 7%-9% of patients achieve complete or long-lasting responses. Many patients treated with IL-2 experienced an expansion of regulatory T cells (Tregs), specifically the expansion of ICOS+ highly suppressive Tregs, which correlate with worse clinical outcomes. This partial efficacy together with the high toxicity associated with the therapy has limited the use of IL-2-based therapy. Taking into account the understanding of IL-2 structure, signaling, and in vivo functions, some efforts to improve the cytokine properties are currently under study. In previous work, we described an IL-2 mutein with higher antitumor activity and less toxicity than wtIL-2. Mutein was in silico designed for losing the binding capacity to CD25 and for preferential stimulation of effector cells CD8+ and NK cells but not Tregs. Mutein induces a higher anti-metastatic effect than wtIL-2, but the extent of the in vivo antitumor activity was still unexplored. In this work, it is shown that mutein induces a strong antitumor effect on four primary tumor models, being effective even in those models where wtIL-2 does not work. Furthermore, mutein can change the in vivo balance between Tregs and T CD8+ memory/activated cells toward immune activation, in both healthy and tumor-bearing mice. This change reaches the tumor microenvironment and seems to be the major explanation for mutein efficacy in vivo.