Lnk prevents inflammatory CD8+ T‐cell proliferation and contributes to intestinal homeostasis

The intracellular adaptor Lnk (also known as SH2B3) regulates cytokine signals that control lymphohematopoiesis, and Lnk^?/? mice have expanded B‐cell, megakaryocyte, and hematopoietic stem‐cell populations. Moreover, mutations in the LNK gene are found in patients with myeloproliferative disease, whereas LNK polymorphisms have recently been associated with inflammatory and autoimmune diseases, including celiac disease. Here, we describe a previously unrecognized function of Lnk in the control of inflammatory CD8⁺ T‐cell proliferation and in intestinal homeostasis. Mature T cells from newly generated Lnk–Venus reporter mice had low but substantial expression of Lnk, whereas Lnk expression was downregulated during homeostatic T‐cell proliferation under lymphopenic conditions. The numbers of CD44^hiIFN‐γ⁺CD8⁺ effector or memory T cells were found to be increased in Lnk^?/? mice, which also exhibited shortening of villi in the small intestine. Lnk^?/? CD8⁺ T cells survived longer in response to stimulation with IL‐15 and proliferated even in nonlymphopenic hosts. Transfer of Lnk^?/? CD8⁺ T cells together with WT CD4⁺ T cells into Rag2‐deficient mice recapitulated a sign of villous abnormality. Our results reveal a link between Lnk and immune cell‐mediated intestinal tissue destruction.     

T‐bet modulates the antibody response and immune protection during murine malaria

CD4⁺ T‐cell subtypes govern the synthesis of different Ab isotypes and other immune functions. The influence of CD4⁺ T‐cell differentiation programs on isotype switching and other aspects of host immunological networks during malaria infection are currently poorly understood. Here, we used Tbx21^?/? mice deficient for T‐bet, a regulator of Th1 CD4⁺ T‐cell differentiation, to examine the effect of Th1 CD4⁺ T cells on the immune protection to nonlethal murine malaria Plasmodium yoelii 17XNL. We found that Tbx21^?/? mice exhibited significantly lower parasite burden that correlated with elevated levels of IgG1, indicating that T‐bet‐dependent Ab isotype switching may be responsible for lower parasite burden. Absence of T‐bet was also associated with a transient but significant loss of T cells during the infection, suggesting that T‐bet may suppress malaria‐induced apoptosis or induce proliferation of T cells. However, Tbx21^?/? mice produced greater numbers of Foxp3⁺CD25⁺ regulatory CD4⁺ T cells, which may contribute to the early contraction of T cells. Lastly, Tbx21^?/? mice exhibited unimpaired production of IFN‐γ by a diverse repertoire of immune cell subsets and a selective expansion of IFN‐γ‐producing T cells. These observations may have implications in malaria vaccine design.     

Antigen presentation by B cells guides programing of memory CD4+ T‐cell responses to a TLR4‐agonist containing vaccine in mice

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T‐helper 1 (T_H1) CD4⁺ T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B‐cell deficient mice (μMT^?/?), tetramer‐positive CD4⁺ T cells, markers of memory “precursor” effector cells (MPECs), and T‐cell adoptive transfers and demonstrated that the early antigen‐specific cytokine‐producing T_H1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory T_H1 response in μMT^?/? mice. We further show that antigen‐presentation by B cells is necessary for their role in MPEC generation using B‐cell adoptive transfers from wt or MHC class II knock‐out mice into μMT^?/? mice. Our study challenges the view that B‐cell deficiency exclusively alters the T_H1 response at memory time‐points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring T_H1 cell‐mediated immunity.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

RELT negatively regulates the early phase of the T‐cell response in mice

RELT (tumor necrosis factor receptor superfamily member 19‐like, TNFRSF19L) is a TNFR superfamily member that is primarily expressed in immune cells and lymphoid tissues, but whose immunological function is not well‐defined. Here, we show that RELT is expressed by naive T cells and DCs, and their activation or maturation decreases RELT expression. Using RELT knockout (RELT^?/?) mice, we demonstrate that RELT deficiency selectively promotes the homeostatic proliferation of CD4⁺ T cells but not CD8⁺ T cells, and enhances anti‐tumor CD8⁺ T‐cell responses. We also demonstrate, using an adoptive transfer model in which RELT is knocked‐out in either the transferred transgenic CD8⁺ T cells or the recipient melanoma‐bearing mice, that RELT on multiple immune cells limits the hyper‐response of tumor‐specific CD8⁺ T cells. Hyper‐responsiveness of RELT‐deficient T cells was induced by promoting their proliferation. Taken together, our findings suggest that RELT acts as a negative regulator that controls the early phase of T‐cell activation probably by promoting T‐cell apoptosis.     

Differential requirements for IRF4 in the clonal expansion and homeostatic proliferation of naive and memory murine CD8+ T cells

Interferon regulatory factor 4 (IRF4) has critical roles in immune cell differentiation and function and is indispensable for clonal expansion and effector function in T cells. Here, we demonstrate that the AKT pathway is impaired in murine CD8⁺ T cells lacking IRF4. The expression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT pathway, was elevated in Irf4^?/? CD8⁺ T cells. Inhibition of PTEN partially rescued downstream events, suggesting that PTEN constitutes a checkpoint in the IRF4‐mediated regulation of cell signaling. Despite the clonal expansion defect, in the absence of IRF4, memory‐like CD8⁺ T cells could be generated and maintained, although unable to expand in recall responses. The homeostatic proliferation of naïve Irf4^?/? CD8⁺ T cells was impaired, whereas their number eventually reached a level similar to that of wild‐type CD8⁺ T cells. Conversely, memory‐like Irf4^?/? CD8⁺ T cells underwent homeostatic proliferation in a manner similar to that of wild‐type memory CD8⁺ T cells. These results suggest that IRF4 regulates the clonal expansion of CD8⁺ T cells at least in part via the AKT signaling pathway. Moreover, IRF4 regulates the homeostatic proliferation of naïve CD8⁺ T cells, whereas the maintenance of memory CD8⁺ T cells is IRF4‐independent.     

CD40L expression by CD4+ but not CD8+ T cells regulates antiviral immune responses in acute LCMV infection in mice

CD40‐CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T‐cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8⁺ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus‐specific CD8⁺ T‐cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L^?/? mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV‐specific effector and memory T cells consists of CD40L⁺ CD8⁺ T cells. However, deficiency of CD40L in CD8⁺ T cells did influence neither the quantity nor the quality of primary T‐cell responses in LCMV infection. Virus‐specific CD8⁺ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8⁺ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8⁺ T‐cell responses also under inflammatory conditions, CD40L expression by CD8⁺ T cells themselves is dispensable in acute LCMV infection.     

IL‐2 is positively involved in the development of colitogenic CD4+ IL‐7Rαhigh memory T cells in chronic colitis

IL‐2 and IL‐7 share a common γ‐chain receptor and are critical for T‐cell homeostasis. We aimed to clarify the reciprocal roles of IL‐2 and IL‐7 in the development and persistence of chronic colitis. We performed a series of adoptive transfers of IL‐2^?/? CD4⁺CD45RB^high T cells into RAG‐2^?/? mice and assessed the role of IL‐2 in the induction of IL‐7Rα on colitogenic CD4⁺ T cells and the development of chronic colitis. RAG‐2^?/? mice transferred with WT but not with IL‐2^?/? CD4⁺CD45RB^high T cells developed Th1/Th17‐mediated colitis. Consistently, re‐expression of IL‐7Rα was severely impaired on IL‐2^?/? but not on WT CD4⁺ T cells from the transferred mice. To exclude a contribution of the preclinical autoimmunity of IL‐2^?/?mice, WT Ly5.1⁺ or IL‐2^?/? Ly5.2⁺ CD4⁺CD45RB^high T cells from GFP mice previously transplanted with the same number of WT and IL‐2^?/? BM cells were transferred into RAG‐2^?/? mice. RAG‐2^?/? mice transferred with IL‐2^?/?‐derived CD4⁺CD45RB^high T cells did not develop colitis, but their splenic CD4⁺ T cells changed from effector‐memory to central‐memory type. These results show that IL‐2 is critically involved in the establishment and maintenance of IL‐7‐dependent colitogenic memory CD4⁺IL‐7Rα^high T cells.     

Gadd45b and Gadd45g are important for anti‐tumor immune responses

An effective Th1 type cell‐mediated immune response against cancer cells is critical in limiting cancer progression. Gadd45b, a signaling molecule highly up‐regulated during Th1 type responses, is studied for its role in limiting tumor growth. Mouse B16 melanoma cells implanted into Gadd45b^?/? mice grew faster than those in WT or Gadd45b^+/? littermate controls. The defect of Gadd45b^?/? mice in tumor immunosurveillance was attributed to the reduced expression of IFN‐γ, granzyme B, and CCR5 in Gadd45b^?/? CD8⁺ T cells at the tumor site. Activation of p38 MAP kinase, but not ERK or JNK, by either TCR‐stimuli or IL‐12 and IL‐18 is diminished in Gadd45b^?/? CD8⁺ T cells, resulting in reduced production of IFN‐γ. In addition, mRNA of T‐bet and Eomes were reduced in Gadd45b^?/? CD8⁺ T cells, supporting a critical role of Gadd45b in shaping the Th1 fate. More importantly, the tumor vaccination, which is effective in WT mice, failed in Gadd45b/Gadd45g doubly deficient mice. Collectively, these data demonstrate that members of the Gadd45 gene family are important for anti‐tumor immune responses.     

Postarrest stalling rather than crawling favors CD8+ over CD4+ T‐cell migration across the blood–brain barrier under flow in vitro

Although CD8⁺ T cells have been implied in the pathogenesis of multiple sclerosis (MS), the molecular mechanisms mediating CD8⁺ T‐cell migration across the blood–brain barrier (BBB) into the central nervous system (CNS) are ill defined. Using in vitro live cell imaging, we directly compared the multistep extravasation of activated CD4⁺ and CD8⁺ T cells across primary mouse brain microvascular endothelial cells (pMBMECs) as a model for the BBB under physiological flow. Significantly higher numbers of CD8⁺ than CD4⁺ T cells arrested on pMBMECs under noninflammatory and inflammatory conditions. While CD4⁺ T cells polarized and crawled prior to their diapedesis, the majority of CD8⁺ T cells stalled and readily crossed the pMBMEC monolayer preferentially via a transcellular route. T‐cell arrest and crawling were independent of G‐protein‐coupled receptor signaling. Rather, absence of endothelial ICAM‐1 and ICAM‐2 abolished increased arrest of CD8⁺ over CD4⁺ T cells and abrogated T‐cell crawling, leading to the efficient reduction of CD4⁺, but to a lesser degree of CD8⁺, T‐cell diapedesis across ICAM‐1^null/ICAM‐2^?/? pMBMECs. Thus, cellular and molecular mechanisms mediating the multistep extravasation of activated CD8⁺ T cells across the BBB are distinguishable from those involved for CD4⁺ T cells.     

Competition between colitogenic Th1 and Th17 cells contributes to the amelioration of colitis

Th17 cells and Th1 cells coordinate to play a critical role in the formation of inflammatory bowel diseases. To examine how Th17 and Th1 cells are regulated at inflammatory sites, we used Th1‐dominant CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? and Th1/Th17‐mixed IL‐10^?/? mice. Interestingly, not only did colitic RAG‐2^?/? mice that were parabiosed with WT mice show significant amelioration of colitis, but amelioration of disease was also observed in those parabiosed with colitic IL‐10^?/? mice. To assess the interference between Th1 and Th17 colitogenic T cells, we co‐transferred colitogenic CD4⁺ T cells from the lamina propria (LP) of CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? mice and IL‐10^?/? mice into RAG‐2^?/? mice. Surprisingly, the co‐transferred RAG‐2^?/? mice showed a vast cellular infiltration of LP CD4⁺ T cells similar to that seen in RAG‐2^?/? mice re‐transferred with the cells from colitic RAG‐2^?/? mice alone, but the co‐transferred RAG‐2^?/? mice did not have the wasting symptoms, which are also absent in RAG‐2^?/? mice transferred with cells from colitic IL‐10^?/? mice alone. Furthermore, the percentages of Th1 and Th17 cells originating from IL‐10^?/? mice and those of Th1 cells originating from colitic RAG‐2^?/? mice were all significantly decreased in the co‐transferred mice as compared with the singly‐transferred paired RAG‐2^?/? mice, suggesting that Th1 and Th17 cells are in competition, and that their orchestration results in a merged clinical phenotype of the two types of murine colitis.     

Pro‐inflammatory self‐reactive T cells are found within murine TCR‐αβ+CD4−CD8−PD‐1+ cells

TCR‐αβ⁺ double negative (DN) T cells (CD3⁺TCR‐αβ⁺CD4^?CD8^?NK1.1^?CD49b^?) represent a minor heterogeneous population in healthy humans and mice. These cells have been ascribed pro‐inflammatory and regulatory capacities and are known to expand during the course of several autoimmune diseases. Importantly, previous studies have shown that self‐reactive CD8⁺ T cells become DN after activation by self‐antigens, suggesting that self‐reactive T cells may exist within the DN T‐cell population. Here, we demonstrate that programmed cell death 1 (PD‐1) expression in unmanipulated mice identifies a subset of DN T cells with expression of activation‐associated markers and a phenotype that strongly suggests they are derived from self‐reactive CD8⁺ cells. We also found that, within DN T cells, the PD‐1⁺ subset generates the majority of pro‐inflammatory cytokines. Finally, using a TCR‐activation reporter mouse (Nur77‐GFP), we confirmed that in the steady‐state PD‐1⁺ DN T cells engage endogenous antigens in healthy mice. In conclusion, we provide evidence that indicates that the PD‐1⁺ fraction of DN T cells represents self‐reactive cells.     

DC within the pregnant mouse uterus influence growth and functional properties of uterine NK cells

The vascular addressins mucosal addressin cell adhesion molecule‐1, P‐selectin and ICAM‐1 permit α₄β₇‐integrin‐expressing DC, especially those of the myeloid lineage (CD11c⁺CD11b⁺ DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule‐1 in P‐selectin^?/? mice or experimental approaches with β7‐integrin^?/? or ICAM‐1^?/? mice revealed that limited access or absence of CD11c⁺CD11b⁺ DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into β7‐integrin^?/? mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c⁺CD11b⁺ DC are characterized by decreased IL‐15 and IL‐12 mRNA and/or protein levels. Chronic administration of IL‐15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL‐12 positively affects the IFN‐γ expression of uNK cells. Real‐time RT‐PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL‐15 and IL‐12 in the pregnant mouse uterus.     

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4⁺ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4⁺ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4⁺ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4⁺ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4⁺ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2^?/? recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam₃Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4⁺ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.     

Micro‐RNA‐155‐mediated control of heme oxygenase 1 (HO‐1) is required for restoring adaptively tolerant CD4+ T‐cell function in rodents

T cells chronically stimulated by a persistent antigen often become dysfunctional and lose effector functions and proliferative capacity. To identify the importance of micro‐RNA‐155 (miR‐155) in this phenomenon, we analyzed mouse miR‐155‐deficient CD4⁺ T cells in a model where the chronic exposure to a systemic antigen led to T‐cell functional unresponsiveness. We found that miR‐155 was required for restoring function of T cells after programmed death receptor 1 blockade. Heme oxygenase 1 (HO‐1) was identified as a specific target of miR‐155 and inhibition of HO‐1 activity restored the expansion and tissue migration capacity of miR‐155^?/? CD4⁺ T cells. Moreover, miR‐155‐mediated control of HO‐1 expression in CD4⁺ T cells was shown to sustain in vivo antigen‐specific expansion and IL‐2 production. Thus, our data identify HO‐1 regulation as a mechanism by which miR‐155 promotes T‐cell‐driven inflammation.