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MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139–mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139−/− mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139−/− CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes. Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.

Summary/AbstractWe previously identified the adhesion molecule PSGL-1 as a T cell intrinsic immune checkpoint regulator of T cell exhaustion. Here we show that the ability of PSGL-1 to restrain TCR sginaling correlates with decreased expression of the Zap70 inhibitor Ubash3b (Sts-1) in PSGL-1-deficient T cells. PSGL-1-deficency in T cells supports antitumor responses to a PD-1 blockade resistant melanoma wherein tumor-specific CD8+ T cells sustain an enhanced metabolic state, with an elevated metabolic gene signature that promotes increased glycolysis and glucose uptake to support effector functions. In models of chronic virus infection and cancer, this outcome was associated with CD8+ T cell stemness, as PSGL-1 deficient CD8+ T cells displayed increased TCF-1 and decreased TOX expression, a phenotype shown to be crucial for responsiveness to checkpoint inhibition. Further, we demonstrate that PSGL-1 signaling promotes development of exhaustion in human CD8+ T cells. Finally, pharmacologic blockade of PSGL-1 was sufficient to curtail T cell exhaustion and enhance functionality both with melanoma tumors and chronic LCMV infection, demonstrating that PSGL-1 represents a therapeutic target for immunotherapy for PD-1/PD-L1 resistant tumors.

MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8(+) T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8(+) CTLs compared with naive CD8(+) T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139–mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139(2/2) mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8(+) T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8(+) T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139(2/2) CD8(+) T cells are outcompeted by wild-type CD8(+) T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes. Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2−/− mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2−/− Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2−/− mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy., The ubiquitin ligase Siah2 has been implicated in immune responses. Here, the authors show that Siah2 null immune cells have an increased inflammatory response to inoculated melanoma cells, along with a reduced number of infiltrating immunosuppressive regulatory T cells, resulting in inhibition of tumour growth.

Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in responses to chronic viral infection and melanoma tumors. Subsequently, our studies have focused on investigating the mechanisms by which PSGL-1 regulates T cell activation and exhaustion and evaluating PSGL-1 as a target for cancer immunotherapy. Using the CD4cre system in combination with PSGL-1-floxed mice, we find that T cell intrinsic deletion of PSGL-1 is sufficient to promote enhanced tumor growth control of mesothelioma as well as melanoma tumors. We have found that in vivo PSGL-1 blockade recapitulates the enhanced melanoma tumor growth control observed with PSGL-1-/- CD8+ T cells and limits the development of T cell exhaustion during chronic LCMV Clone 13 viral infection. Conversely, ligation of PSGL-1 in vitro or in vivo drives enhanced upregulation T cell exhaustion markers, most notably PD-1 expression. Phosphoproteomic analysis of early T cell activation revealed the rapid upregulation of more than 30 phosphorylated proteins with significantly enhanced expression following PSGL-1 ligation during T cell activation than T cell activation alone. GSEA and western blot analysis confirmed the increased expression of several regulators of T cell signaling including pAkt1 and pErk in both activated PSGL-1-/- T cells and following PSGL-1 ligation during T cell activation. Single-cell RNA-sequencing of melanoma tumor-specific PSGL-1-/- CD8+ T cells identified differential modulation of genes associated with T cell metabolism and effector responses, including increased Mtor and Hif1a in tumors and Tcf7 in tumor-draining lymph nodes. The Seahorse glycolysis stress test identified that PSGL-1-/- T cells show increased glycolysis after 72 hours of in vitro activation at both sub-optimal and optimal levels of αCD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1-/- CD8+ T cells within two hours of stimulation. Further, 2-NBDG uptake was increased by PSGL-1-/- CD8+ T cells ex vivo from melanoma tumors. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. We hypothesize that PSGL-1 serves as a key regulator of early T cell activation and repeated signaling through PSGL-1 promotes differentiation into exhausted CD8+ T cells by sustaining TCR signaling. Conversely, we hypothesize that the absence of PSGL-1 allows for faster initiation and termination of TCR signaling, inhibiting differentiation into exhausted CD8+ T cells. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile which may drive their enhanced responses to tumors. Citation Format: Jennifer L. Hope, Dennis C. Otero, Eun-ah Bae, Christopher J Stairiker, Ashley B. Palete, Hannah A. Faso, Petrus de Jong, Garth Powis, Linda M. Bradley. PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO014.

Targeted deletion of BAFF causes severe deficiency of splenic B cells. BAFF-R is commonly thought to signal to nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase dependent noncanonical NF-κB RelB. However, RelB-deficient mice have normal B-cell numbers. Recent studies showed that BAFF also signals to the canonical NF-κB pathway, and we found that both RelB and cRel are persistently activated, suggesting BAFF signaling coordinates both pathways to ensure robust B-cell development. Indeed, we report now that combined loss of these 2 NF-κB family members leads to impaired BAFF-mediated survival and development in vitro. Although single deletion of RelB and cRel was dispensable for normal B-cell development, double knockout mice displayed an early B-cell developmental blockade and decreased mature B cells. Despite disorganized splenic architecture in Relb(-/-)cRel(-/-) mice, generation of mixed-mouse chimeras established the developmental phenotype to be B-cell intrinsic. Together, our results indicate that BAFF signals coordinate both RelB and cRel activities to ensure survival during peripheral B-cell maturation.

We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the mechanism responsible for PD-1 regulation by PSGL-1 remained to be described. Here we show that, in naïve T cells, PSGL-1 ligation results in enhanced PD-1 expression following activation through the T cell receptor. Furthermore, naïve T cells lacking PSGL-1 fail to upregulate PD-1 to the same extent as wild type T cells following activation. Strikingly we have discovered that T cells lacking PSGL-1 showed reduced calcium signaling. In T cells, the calcium/calcineurin/NFAT pathway is important for induction of many genes including PD-1. Consequently, PSGL-1 deficient T cells also exhibit reduced nuclear translocation of NFAT explaining the reduced PD-1 expression. Conversely, we found that ligating PSGL-1 enhanced NFAT nuclear translocation with TCR signaling thus contributing to upregulation of PD-1. Therefore, by regulating calcium signaling through the T cell receptor, PSGL-1 modulates the extent of PD-1 expression early in activation and subsequently influencing the extent of T cell exhaustion. Further studies aim to elucidate how PSGL-1 regulates calcium signaling through the T cell receptor.

Endoplasmic reticulum (ER) stress is induced by the accumulation of misfolded proteins and other cellular stresses, which lead to the unfolded protein response to restore ER homeostasis. Previous studies have shown that the ER stress response is required for T cell activation and differentiation. However, recent studies demonstrated that inhibition or deletion of ER stress mediators in T cells actually promotes anti-tumor immunity. Therefore, this incongruity as to the role of ER stress response in effector T cell function warrants further investigation. Our lab previously identified that the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), acts as a T cell immune checkpoint regulator. PSGL-1-deficiency enhances T cell function, resulting in the clearance of chronic viral infection and dramatic tumor control. To better understand the contribution of ER stress to T cell function, we assessed T cell phenotype and function together with the ER stress mediators, XBP1s and ATF4, in highly-functional PSGL-1−/− T cells as well as exhausted wild-type T cells in both tumor and virus models. In melanoma models, ER stress was highly induced in activated tumor-infiltrating T cells (TILs). Effector CD8+ TILs producing IFNg displayed elevated XBP1s and ATF4 compared to IFNg− TILs. Upon chronic LCMV Cl13 viral infection in PSGL-1−/− mice, we found increased virus-specific T cells with enhanced multi-functional effector responses and increased memory/stem cell-like T cell populations characterized by the expression of the transcription factor TCF-1, as well as increased ER stress responses. Together, these data suggest that ER stress plays an essential role in effector T cell functions in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in response to chronic viral infection and tumors. Subsequent studies have focused on investigating the role of PSGL-1 signaling in T cell responses, with an emphasis on understanding the mechanisms by which PSGL-1 regulates T cell exhaustion. Single-cell RNA-sequencing of tumor infiltrating PSGL-1−/− CD8+ T cells identified the upregulation and differential modulation of several genes associated with T cell metabolism and enhanced intratumoral responses, including Mtor, Hif1a, and Mki67 in Gzmb/Ifng double-positive cells from tumors and Tcf7 in both tumor draining and non-draining inguinal lymph nodes. These data suggest an important role for PSGL-1 signaling in the development and maintenance of effective anti-tumor T cell responses to melanoma. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. In situ activation of PSGL-1−/− CD8+ T cells demonstrated that PSGL-1−/− CD8+ T cells have increased glycolysis and increased glycolytic capacity at both sub-optimal and optimal levels of α-CD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1−/− CD8+ T cells within two hours of stimulation. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile in response to melanoma tumors.

IFN-β is widely used in the treatment of multiple sclerosis, yet the mechanism facilitating its efficacy remains unclear. IL-2 production by activated T cells, including those mediating autoimmunity, and subsequent autocrine stimulation is vital for T cell expansion and function. In this study, we demonstrate that in mouse and human T cells, IFN-β specifically inhibits the production of IL-2 upon TCR engagement without affecting other cytokines or activation markers. Rather than disrupting TCR signaling, IFN-β alters histone modifications in the IL-2 promoter to retain the locus in an inaccessible configuration. This in turn is mediated through the upregulation of the transcriptional suppressor CREM by IFN-β and consequent recruitment of histone deacetylases to the IL-2 promoter. In accordance, ablation of CREM expression or inhibition of histone deacetylases activity eliminates the suppressive effects of IFN-β on IL-2 production. Collectively, these findings provide a molecular basis by which IFN-β limits T cell responses.

P-selectin glycoprotein ligand-1 (PSGL-1) has long been studied as an adhesion molecule involved in immune cell trafficking and is recognized as a regulator of many facets of immune responses by myeloid cells. PSGL-1 also regulates T cell migration during homeostasis and inflammatory settings. However, recent findings indicate that PSGL-1 can also negatively regulate T cell function. Because T cell differentiation is finely tuned by multiple positive and negative regulatory signals that appropriately scale the magnitude of the immune response, PSGL-1 has emerged as an important checkpoint during this process. We summarize what is known regarding PSGL-1 structure and function and highlight how it may act as an immune checkpoint inhibitor in T cells.

We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the function of PSGL-1 in the early activation of naïve T cells has not been fully investigated. We found that, crosslinking PSGL-1 on naïve T cells augments TCR signaling, including enhanced Erk and Akt phosphorylation and Nur77 expression. In addition, T cells expressed more activation markers, including PD-1, proliferated to a greater extent, and secreted more cytokines when PSGL-1 was crosslinked compared to TCR stimulation alone, indicating a role as a costimulatory molecule. Strikingly, we have discovered that PSGL-1 co-localizes with the TCR upon stimulation. CD3 and PSGL-1 can be found in the same micro vesicles following anti-CD3 crosslinking, indicating a close connection between PSGL-1 and the TCR. However, the effects of PSGL-1 ligation are similar to what is seen in T cells that lack PSGL-1 expression, except with respect to PD-1 expression. We propose that decreased induction of PD-1 with PSGL-1 deficiency supports greater effector T cell responses. Further studies will address the function of PSGL-1 in cytoskeletal rearrangement and the formation of the immune synapse. Altogether, these data indicate that the extent of T cell activation is regulated by an association of PSGL-1 with the TCR.

Chronic viruses and cancers thwart immune responses in humans by inducing Tcell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding Tcells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector Tcells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) Tcells inhibited Tcell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which Tcell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved Tcell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate Tcell responses in the tumor microenvironment.

We recently demonstrated that PSGL-1 (P-selectin glycoprotein-1) could act as a T cell-intrinsic checkpoint inhibitor during chronic viral infection. As such, PSGL-1 may be a viable target to control T cell responses in the setting of autoimmune disease. In this study, we investigated the role of PSGL-1 on the activation of T cells and in mouse models of autoimmune disease. Here we find that the lack of PSGL-1 on T cells results in clinically worse disease in both EAE and adoptive transfer colitis, which supports its function as a negative regulator of T cell activation. However, in vivo treatment with an anti-PSGL-1 antibody known to block binding of the ligand, P-selectin, from the time of disease initiation had little effect on disease severity in the transfer colitis model and led to exacerbated disease in the EAE model. Crosslinking PSGL-1 on naïve T cells in vitro in the presence of TCR signals results in up-regulation of PD-1 but also up-regulation of other activation markers, suggesting that PSGL-1 exerts regulatory effects during the early activation program in T cells. Surprisingly, however, in an in vitro assay, crosslinking PSGL-1 on effector T cells resulted in resistance to regulatory T cell suppression. Therefore, anti-PSGL-1 treatment results in enhanced T cell activation and resistance to Treg suppression, thereby leading to more severe autoimmune disease by altering/blocking PSGL-1 signaling to phenocopy PSGL-1-deficiency.

Expression of B cell-activating factor (BAFF), a critical B cell survival factor, is elevated in autoimmune and lymphoproliferative disorders. Mice overproducing BAFF develop systemic lupus erythematosus (SLE)-like disease and exhibit B cell activation of classical and alternative NF-kappaB-signaling pathways. We used a genetic approach and found that both NF-kappaB-signaling pathways contributed to disease development but act through distinct mechanisms. Whereas BAFF enhanced long-term B cell survival primarily through the alternative, but not the classical, NF-kappaB pathway, it promoted immunoglobulin class switching and generation of pathogenic antibodies through the classical pathway. Activation of the alternative NF-kappaB pathway resulted in integrin upregulation, thereby retaining autoreactive B cells in the splenic marginal zone, a compartment that contributes to their survival. Thus, both classical and alternative NF-kappaB signaling are important for development of lupus-like disease associated with BAFF overproduction. The same mechanisms may be involved in the pathogenesis of human SLE.

IkappaB Kinase (IKK)alpha is required for activation of an alternative NF-kappaB signaling pathway based on processing of the NF-kappaB2/p100 precursor protein, which associates with RelB in the cytoplasm. This pathway, which activates RelB:p52 dimers, is required for induction of several chemokine genes needed for organization of secondary lymphoid organs. We investigated the basis for the IKKalpha dependence of the induction of these genes in response to engagement of the lymphotoxin beta receptor (LTbetaR). Using chromatin immunoprecipitation, we found that the promoters of organogenic chemokine genes are recognized by RelB:p52 dimers and not by RelA:p50 dimers, the ubiquitous target for the classical NF-kappaB signaling pathway. We identified in the IKKalpha-dependent promoters a novel type of NF-kappaB-binding site that is preferentially recognized by RelB:p52 dimers. This site links induction of organogenic chemokines and other important regulatory molecules to activation of the alternative pathway.

Proliferative expansion of pro-B cells is an IL-7-dependent process that allows for the rearrangement of H chain genes and the expression of the pre-B cell receptor (pre-BCR). Further B cell differentiation is dependent upon signals elicited through the pre-BCR, which are thought to be responsible for allelic exclusion, induced L chain gene rearrangement, and continued proliferation. CD19 promotes the proliferation and survival of mature B cells, but its role in early B cell development is less well understood. Here we identify and characterize impairments in early B cell development in CD19−/− mice. Following sublethal irradiation, we found decreased numbers of autoreconstituted early B cells, which was first evident in the large cycling pre-B cell fraction. Reduced cell progression due to a defect in proliferation was made evident from cell cycle analysis and bromodeoxyuridine labeling of bone marrow cells from CD19−/− and wild-type mice. Studies of IL-7-dependent pre-B cell cultures derived from wild-type and CD19−/− mouse bone marrow suggested that CD19 has little affect on IL-7 signaling. By contrast, signaling through the pre-BCR was impaired in the absence of CD19, as demonstrated by reduced activation of Bruton’s tyrosine kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase. Thus, in addition to promoting mature B cell homeostasis and Ag-induced responses, the early onset of CD19 expression acts to enhance B cell generation.

Effective T cell responses depend on adhesion mechanisms that guide T cells to sites of infection and inflammation. P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to impact T cell migration; however, its role in the differentiation of responding T cells has not been investigated. Since PSGL-1 is highly expressed on naïve, effector, and memory T cells with varying degrees of glycosylation that impact ligand binding, we hypothesized that PSGL-1 may regulate T cell fates. Using mice deficient in PSGL-1 and three murine models we found that PSGL-1 limits the magnitude of the ensuing T cell response. PSGL-1-deficient mice had an increased accumulation of anti-viral CD4+ and CD8+ T cells after both acute and chronic LCMV infection. Virus-specific PSGL-1-deficient T cells had enhanced survival and were not hyper proliferative. Furthermore, PSGL-1-deficient T cells had superior effector function, downregulation of PD-1 and other inhibitory receptors, and enhanced viral control. The enhanced T cell response resulted in significant mortality of PSGL-1-deficient mice infected with chronic LCMV, whereas all PSGL-1-deficient mice survived the acute LCMV infection. After viral clearance, more CD4+ and CD8+ PSGL-1-deficient T cells survived to form memory cells. Interestingly, resting memory T cells from PSGL-1-deficient mice sustained PD-1 downregulation implying that PSGL-1 may regulate inhibitory receptors in memory T cells. We found that PSGL-1-deficient T cells had superior function in a tumor model and were more effective in controlling melanoma tumors. Our findings in three separate models show a previously unrecognized role for PSGL-1 in negatively regulating T cell responses and may have high therapeutic potential.

SUMMARY BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences., Graphical abstract

The contributions of IFN regulatory factor (IRF) 3/7 and the type I IFNs IFN-α/β to the innate host defense have been extensively investigated; however, their role in thymic development is less clear. In this study, we show that mice lacking the type I IFN receptor IFN-α/β receptor (IFNAR) or the downstream transcription factor STAT1 harbor a significant reduction in self-Ag–presenting, autoimmune regulator (AIRE)+ medullary thymic epithelial cells (mTECs). Constitutive IFNAR signaling occurs in the thymic medulla in the absence of infection or inflammation. Receptor activator for NF-κB (RANK) ligand stimulation results in IFN-β upregulation, which in turn inhibits RANK signaling and facilitates AIRE expression in mTECs. Finally, we find that IRF7 is required for thymic IFN-β induction, maintenance of thymic architecture, and mTEC differentiation. We conclude that spatially and temporally coordinated cross talks between the RANK ligand/RANK and IRF7/IFN-β/IFNAR/STAT1 pathways are essential for differentiation of AIRE+ mTECs.

STAT1 is an essential part of interferon signaling, and STAT1-deficiency results in heightened susceptibility to infections or autoimmunity in both mice and humans. Here we report that mice lacking the IFNα/β-receptor (IFNAR1) or STAT1 display impaired deletion of autoreactive CD4(+)CD8(+)-T-cells. Strikingly, co-existence of WT T cells restored thymic elimination of self-reactive STAT1-deficient CD4(+)CD8(+)-T cells. Analysis of STAT1-deficient thymocytes further revealed reduced Bim expression, which was restored in the presence of WT T cells. These results indicate that type I interferons and STAT1 play an important role in the survival of MHC class I-restricted T cells in a T cell intrinsic and non-cell intrinsic manner that involves regulation of Bim expression through feedback provided by mature STAT1-competent T cells.

The growth of A-1 fibroblasts depends on exogenous amyloid beta/A4 protein precursor (APP), providing a simple bioassay to study the function of APP. Our preliminary study, testing the activity of a series of fragments derived from the secreted form of APP-695 (sAPP-695) on this bioassay, has shown that at least one of the active sites of sAPP-695 was localized within a 40-mer sequence (APP296-335, Kang sequence; Roch, J.-M., I. P. Shapiro, M. P. Sundsmo, D. A. C. Otero, L. M. Refolo, N. K. Robakis, and T. Saitoh. 1992. J. Biol. Chem. 267:2214-2221). In the present study, to further characterize the growth-promoting activity of sAPP-695 on fibroblasts, we applied a battery of synthetic peptides on this bioassay and found that: (a) the sequence of five amino acids, RERMS (APP328-332), was uniquely required for the growth-promoting activity of sAPP-695; (b) the activity was sequence-specific because the reverse-sequence peptide of the active domain had no activity; and (c) the four-amino-acid peptide RMSQ (APP330-333), which partially overlaps the COOH-terminal side of the active sequence RERMS, could antagonize the activity of sAPP-695. Furthermore, a recombinant protein which lacks this active domain (APP20-591 without 306-335) did not promote fibroblast cell growth, suggesting that this domain is the only site of sAPP-695 involved in the growth stimulation. The availability of these biologically active, short peptides and their antagonists should prove to be an essential step for the elucidation of APP involvement in regulation of cellular homeostasis.

The secreted form of Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in cell growth regulation (Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T., and Schenk, D.B. (1989) Cell 58, 615-622). Using a strong prokaryotic expression system, we expressed, in Escherichia coli, peptide fragments covering different regions of the secreted form of APP-695. The longest of these fragments (KB75, 572 amino acids from Val-20 to Ile-591), which contained neither the Kunitz-type protease inhibitor (KPI) domain nor the amyloid beta/A4-protein domain, was purified and shown to be biologically active in terms of growth regulation. Two other APP fragments (KB48, 316 amino acids from Val-20 to Met-335; and RB17, 150 amino acids from Thr-296 to Pro-445), overlapping by only 40 amino acids at a close site C-terminal to the KPI insertion site, were also active. Furthermore, a chemically synthesized 40-residue peptide corresponding to this region of overlap also stimulated the growth of A-1 fibroblasts. These results establish the presence of growth-promoting activity in the secreted form of APP-695 and suggest that the site of this activity of APP-695 lies within a 40-amino acid domain next to the KPI insertion site.

Mice lacking activity of the kinase MEKK1 ('Map3k1(deltaKD)' mice) have defective activation of the kinase Jnk and increased production of T helper type 2 cytokines after T cell receptor ligation. Here we show that Map3k1(deltaKD) mice had defective germinal center formation and diminished production of antibodies recognizing thymus-dependent antigens. Those defects were B cell intrinsic, as MEKK1 was necessary for CD40-mediated activation of the kinases Jnk and p38 and transcription factor c-Jun, as well as for expression of cyclin D2 and activation-induced deaminase. MEKK1 was recruited to CD40 and adaptor molecule TRAF2 after CD40 ligation, and Map3k1(deltaKD) B cells were hypoproliferative after CD40 stimulation. Our data emphasize that MEKK1 is an essential component of signaling cascades needed for thymus-dependent antigen-induced B cell proliferation and antibody production.

The importance of lymphotoxin (LT) βR (LTβR) as a regulator of lymphoid organogenesis is well established, but its role in host defense has yet to be fully defined. In this study, we report that mice deficient in LTβR signaling were highly susceptible to infection with murine CMV (MCMV) and early during infection exhibited a catastrophic loss of T and B lymphocytes, although the majority of lymphocytes were themselves not directly infected. Moreover, bone marrow chimeras revealed that lymphocyte survival required LTα expression by hemopoietic cells, independent of developmental defects in lymphoid tissue, whereas LTβR expression by both stromal and hemopoietic cells was needed to prevent apoptosis. The induction of IFN-β was also severely impaired in MCMV-infected LTα−/− mice, but immunotherapy with an agonist LTβR Ab restored IFN-β levels, prevented lymphocyte death, and enhanced the survival of these mice. IFN-αβR−/− mice were also found to exhibit profound lymphocyte death during MCMV infection, thus providing a potential mechanistic link between type 1 IFN induction and lymphocyte survival through a LTαβ-dependent pathway important for MCMV host defense.

Although the B-cell antigen receptor (BCR) factors most prominently in the maintenance and differentiation of mature B cells, it is now appreciated that co-receptor molecules can positively or negatively modulate signals through the BCR. Co-receptors are functionally defined as modifiers of BCR engagement and signal transduction, and are distinct from other accessory molecules that act independently to regulate B-cell growth. The co-receptor CD19 functions to augment signals by the pre-BCR/BCR and in doing so can modulate B-cell fate decisions at multiple stages of development. In mature B cells, CD19 also associates with complement receptor 2 (CR2/CD21) and is pivotal for transducing signals induced by co-recognition of complement C3d-fixed antigens by the BCR and CD21. In this article, we focus on recent progress in the understanding of CD19 function through the characterization of mouse models that relate in vivo function to biochemical properties of CD19.

Loss of membrane-bound Ig results in the rapid onset of apoptosis in recirculating B cells. This observation implies that a competent B cell receptor (BCR) is not only required for Ag-dependent differentiation, but also for continued survival in the peripheral immune system. Expression of the B cell coreceptor, CD19, is likewise essential for key B cell differentiative events including the formation of B-1, germinal center, and marginal zone (MZ) B cells. In this study, we report that CD19 also exerts a role before Ag encounter by promoting the survival of naive recirculating B cells. This aspect of CD19 signaling was first suggested by the analysis of mixed bone marrow chimeras, wherein CD19−/− B cells fail to effectively compete with wild-type B cells to reconstitute the peripheral B cell compartment. Consistent with this observation, Bromodeoxyuridine- and CFSE-labeling studies reveal a shorter in vivo life span for CD19−/− B cells vs their wild-type counterparts. Moreover, we find that CD19 is necessary for propagation of BCR-induced survival signals and thus may contribute to homeostatic mechanisms of tonic signaling. To determine whether provision of a constitutive survival signal could compensate for the loss of CD19 in vivo, Bcl-2-transgenic mice were bred onto the CD19−/− background. Here, we observe an increase in follicular B cell numbers and selective recovery of the MZ B cell compartment. Together these findings suggest that maintenance of the follicular and MZ B cell compartments require CD19-dependent survival signals.

CD19 is rapidly phosphorylated upon B-cell antigen receptor (BCR) cross-linking, leading to the recruitment of downstream signaling intermediates. A prominent feature of CD19 signaling is the binding and activation of phosphoinositide 3-kinase (P13K), which accounts for the majority of PI3K activity induced by BCR ligation. Recent findings have implicated activation of the serine/threonine kinase Akt as imparting survival signals in a PI3K-dependent fashion. Using CD19-deficient B-lymphoma cells and mouse splenic B-cells, we show that CD19 is necessary for efficient activation of Akt following cross-linking of surface immunoglobulin or Igbeta. In the absence of CD19, Akt kinase activity is reduced and transient. In addition, coligation of CD19 with surface immunoglobulin leads to augmented Akt activity in a dose-dependent manner. Thus, CD19 is a key regulator of Akt activity in B-cells; as such it may contribute to pre-BCR or BCR-mediated cell survival in vivo.

Amyloid β/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line B103 which synthesizes no detectable levels of APP. When B103 cells were plated at a low density in a defined serum-free medium, neurite outgrowth was promoted by the conditioned medium of APP-695 over-producing cells and by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328–332) within this stretch of sequence, was the shortest active peptide. Binding assay using 125l-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites which are extractable with detergent. These data indicate that sAPP induces neurite extension through a cell surface binding and that the RERMS domain represents the active site responsible for this function. The RERMS-containing APP peptide also exhibited neuronal survival activity on rat primary cortical neuronal culture. In addition, the infusion of the trophic APP peptide into rat brain ventricles increased the synaptic density in the frontoparietal cortex and enhanced the memory retention. These results firmly establish the neurotrophic properties of sAPP and raise the possibility that small APP peptides or non-peptidic compounds mimicking their activity may be used therapeutically for the improvement of memory performance.

Diffuse large B-cell lymphoma (DLBCL) accounts for approximately 40% of non-Hodgkin lymphomas, with an overall 3-year relapse-free survival of 53%. DLBCL consists of a germinal center B-cell (GCB-like) subgroup and an activated B-cell (ABC-like) subgroup exist, associated with good and poor prognosis, respectively. In addition to differential expression of coding genes, ABC-type lymphoma cells express high levels of the non-coding microRNA, miR-155, an onco-miR whose ectopic expression in the mouse has been shown to give rise to B-cell malignancies. DLBCL feature a dysregulated PI3 kinase pathway, which can promote proliferation and survival. PI3K is regulated by phosphatases PTEN (phosphatase and tensin homolog deleted on chromosome ten) and SHIP (SH2 domain-containing inositol 5-phosphatase). PTEN is a known tumor suppressor and is mutated in human cancer with a frequency approaching that of p53. cDNA array analyses of DLBCL patient specimens indicate that low mRNA expression level of SHIP can be used as a negative prognostic marker for survival. We have now identified SHIP as the first bona-fide target of miR-155. RT-PCR analysis of micro-dissected lymphoma samples confirmed high levels of miR-155 and low levels of SHIP in poor-prognosis ABC-cells as compared to GCB-cells. We further demonstrate that elevated levels of miR-155, and consequent abrogation of SHIP expression, is not due to point mutations or promoter hypermethylation of SHIP in primary DLBCL cells, but rather the result of a TNFa-mediated autocrine loop, a pro-inflammatory cytokine whose serum levels are elevated in DLBCL patients. Consequently, anti-TNFa regimens (Etenercept (Enbrel®), Infliximab (Remicade®)) are sufficient to reduce miR-155 levels, restore expression of SHIP, decrease p-Akt levels, and slow tumor cell growth of ABC-sub-type DLBCL cell lines. Furthermore, we observe a highly significant decrease in tumor growth when DLBCL-xenograft mice are treated with Etenercept, suggesting a novel approach for restoring tumor suppressor activity and inducing growth inhibition of these aggressive tumors. Our findings offer the first direct evidence that specific miRs can function as a the link between inflammation and cancer. Finally, our hypothesis, that TNFa promotes survival of DLBCL via miR-155, SHIP and pAkt, opens up a novel and immediately accessible (co)treatment for poor prognosis DLBCL.

SummaryMice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2−/− brown preadipocytes. Furthermore, Tyk2−/− mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

Radiation-induced apoptosis (RiA) is used therapeutically for tumor cell ablation as well as a tool to characterize hemopoietic cell lineages. We report that the peritoneal B-1 B cell subset is selectively resistant to RiA. Inherent radioresistance is not shared by splenic B-2 or B-1 cells. However, it is conferred upon B-2 cells by BCR crosslinking in the presence of IL-6 or IL-10. In vivo experiments with gene-targeted mice confirm that IL-6 and, to a lesser extent, IL-10 are the relevant stimuli that combine with BCR ligands to promote B-1 cell radioresistance. STAT3 promotes cell survival in response to selected growth factors, and is activated by combined BCR crosslinking and IL-6 (IL-10). Importantly, STAT3−/− B-1 cells become susceptible to irradiation, indicating that STAT3 activation by the BCR in the presence of IL costimuli account for the inherent radioresistance of peritoneal B-1 B cells.