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Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD. [ABSTRACT FROM AUTHOR]

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-KB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (BcllO) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. BcllO-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimu-lation induced BcllO association with IKB kinase ß, a key component of the canonical NF-KB pathway. Consistently, BcllO-deficient B cells were impaired in BAFF-induced IKIta phosphorylation and formation of nuclear p50/c-Rel complexes. BcllO-deficient B cells also displayed reduced expression of NF-KB2/pl00, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, BcllO-deficient B cells failed to express Bcl-xL, a BAFF-induced NF-KB target gene. Taken together, these data demonstrate that BcllO controls BAFF-induced canonical NF-KB activation directly and non-canonical NF-KB activation indirectly. The BAFF-R/BC110/NF-KB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells. [ABSTRACT FROM AUTHOR]

One of the important signaling events following TCR engagement is activation of phospholipase Cγ (PLCγ). PLCγ has two isoforms, PLCγI and PLCγ2. It is known that PLCγI is important for TCR signaling and TCR-mediated T cell selection and functions, whereas PLCγ2 is critical for BCR signal transduction and BCR-mediated B cell maturation and functions. In this study, we report that PLCγ2 was expressed in primary T cells, and became associated with linker for activated T cells and Src homology 2-domain containing leukocyte protein of 76 kDa and activated upon TCR stimulation. PLCγI/PLCγ2 double-deficient T cells displayed further block from CD4 and CD8 double-positive to single-positive transition compared with PLCγI single-deficient T cells. TCR-mediated proliferation was further impaired in PLCγI/PLCγ2 double-deficient T cells compared with PLCγI single-deficient T cells. TCR-mediated signal transduction, including Ca2+ mobilization and Erk activation, was further impaired in PLCγI/PLCγ2 double-deficient relative to PLCγI single-deficient T cells. In addition, in HY TCR transgenic mouse model, thymic positive and negative selections were reduced in PLCγI heterozygous- and PLCγ2 homozygous-deficient (PLCγl+/- PLCγ2-/-) relative to wild-type, PLCγ2 single-deficient (PLCγ2-/-), or PLCγI heterozygous-deficient (PLCγ1+/-) mice. Taken together, these data demonstrate that PLCγ2 participates in TCR signal transduction and plays a role in T cell selection. [ABSTRACT FROM AUTHOR]

The caspase recruitment domain family member (CARD)11-Bcl10-Malt1 signalosome controls TGF-β-activated kinase 1 (TAK1) activation and regulates BCR-induced NF-κB activation. In this study, we discovered that CARD19 interacted with TAK1 and inhibited TAB2-mediated TAK1 ubiquitination and activation. Although CARD19 deficiency in mice did not affect B cell development, it enhanced clonal deletion, receptor editing, and anergy of self-reactive B cells, and it reduced autoantibody production. Mechanistically, CARD19 deficiency increased BCR/TAK1-mediated NF-κB activation, leading to increased expression of transcription factors Egr2/3, as well as the E3 ubiquitin ligases c-Cbl/Cbl-b, which are known inducers of B cell tolerance in self-reactive B cells. RNA sequencing analysis revealed that although CARD19 deficiency did not affect the overall Ag-induced gene expression in naive B cells, it suppressed BCR signaling and increased hyporesponsiveness of self-reactive B cells. As a result, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus. In summary, CARD19 negatively regulates BCR/TAK1-induced NF-κB activation and its deficiency increases Egr2/3 and c-Cbl/Cbl-b expression in self-reactive B cells, thereby enhancing B cell tolerance., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Heparin-induced thrombocytopenia is a relatively common drug-induced immune disorder that can have life-threatening consequences for affected patients. Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive Abs are central to the pathogenesis of heparin-induced thrombocytopenia. Regulatory T (Treg) cells are a subpopulation of CD4 T cells that play a key role in regulating immune responses, but their role in controlling PF4/heparin-specific Ab production is unknown. In the studies described in this article, we found that Foxp3-deficient mice lacking functional Treg cells spontaneously produced PF4/heparin-specific Abs. Following transplantation with bone marrow cells from Foxp3-deficient but not wild-type mice, Rag1-deficient recipients also produced PF4/heparin-specific Abs spontaneously. Adoptively transferred Treg cells prevented spontaneous production of PF4/heparin-specific Abs in Foxp3-deficient mice and inhibited PF4/heparin complex-induced production of PF4/heparin-specific IgGs in wild-type mice. Treg cells suppress immune responses mainly through releasing anti-inflammatory cytokines, such as IL-10. IL-10-deficient mice spontaneously produced PF4/heparin-specific Abs. Moreover, bone marrow chimeric mice with CD4 T cell-specific deletion of IL-10 increased PF4/heparin-specific IgG production upon PF4/heparin complex challenge. Short-term IL-10 administration suppresses PF4/heparin-specific IgG production in wild-type mice. Taken together, these findings demonstrate that Treg cells play an important role in suppressing PF4/heparin-specific Ab production., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The three major Ras members, Kras, Hras, and Nras, are highly homologous and individual Ras genes can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological functions of this Ras family member. In this study, we generated and examined mice with hematopoietic-specific deletion of Kras and bone marrow (BM) chimeric mice with B cell-specific targeted deletion of Kras. Hematopoietic-specific deletion of Kras impaired early B cell development at the pre-B cell stage and late B cell maturation, resulting in the reduction of BM pre-, immature, and mature B cells and peripheral follicular, marginal zone, and B1 mature B cells. In contrast, Kras deficiency did not affect T cell development. Studies of BM chimeric mice with B cell-specific deletion of Kras demonstrated that Kras deficiency intrinsically impaired B cell development. Kras deficiency reduced BCR-induced B cell proliferation and survival. Furthermore, Kras deficiency specifically impaired pre-BCR- and BCR-induced activation of the Raf-1/MEK/ERK pathway in pre-B and mature B cells, respectively. Thus, Kras is the unique Ras family member that plays a critical role in early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in approximately 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R alpha-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.

Activating receptors such as NKG2D and Ly49D mediate a multitude of effector functions including cytotoxicity and cytokine generation in NK cells. However, specific signaling events that are responsible for the divergence of distinct effector functions have yet to be determined. In this study, we show that lack of caspase recruitment domain-containing protein Bcl10 significantly affected receptor-mediated cytokine and chemokine generation, but not cytotoxicity against tumor cells representing "missing-self" or "induced-self." Lack of Bcl10 completely abrogated the generation of GM-CSF and chemokines and it significantly reduced the generation of IFN-gamma (>75%) in NK cells. Commitment, development, and terminal maturation of NK cells were largely unaffected in the absence of Bcl10. Although IL-2-activated NK cells could mediate cytotoxicity to the full extent, the ability of the freshly isolated NK cells to mediate cytotoxicity was somewhat reduced. Therefore, we conclude that the Carma1-Bcl10-Malt1 signaling axis is critical for cytokine and chemokine generation, although it is dispensable for cytotoxic granule release depending on the activation state of NK cells. These results indicate that Bcl10 represents an exclusive "molecular switch" that links the upstream receptor-mediated signaling to cytokine and chemokine generations.

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.

The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B(-/-) fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3(-/-) mice. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B(-/-) fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.

Krüppel-like transcription factor (KLF)13, previously shown to regulate RANTES expression in vitro, is a member of the Krüppel- like family of transcription factors that controls many growth and developmental processes. To ascertain the function of KLF13 in vivo, Klf13-deficient mice were generated by gene targeting. As expected, activated T lymphocytes from Klf13(-/-) mice show decreased RANTES expression. However, these mice also exhibit enlarged thymi and spleens. TUNEL, as well as spontaneous and activation-induced death assays, demonstrated that prolonged survival of Klf13(-/-) thymocytes was due to decreased apoptosis. Microarray analysis suggests that protection from apoptosis-inducing stimuli in Klf13(-/-) thymocytes is due in part to increased expression of BCL-X(L), a potent antiapoptotic factor. This finding was confirmed in splenocytes and total thymocytes by real-time quantitative PCR and Western blot as well as in CD4+CD8- single-positive thymocytes by real-time quantitative PCR. Furthermore, EMSA and luciferase reporter assays demonstrated that KLF13 binds to multiple sites within the Bcl-X(L) promoter and results in decreased Bcl-X(L) promoter activity, making KLF13 a negative regulator of BCL-X(L).

The adaptor protein B cell lymphoma 10 (Bcl10) plays an essential role in the functions of the AgRs in T and B cells. In this study, we report that Bcl10 also plays an important role in mast cells. Bcl10 is expressed in mast cells. Although Bcl10-deficient mast cells undergo normal development, we demonstrate that Bcl10 is essential for specific functions of FcepsilonR. Although Bcl10-deficient mast cells have normal de novo synthesis and release of the lipid mediator arachidonic acid, the mutant cells possess impaired FcepsilonR-mediated degranulation, indicated by decreased serotonin release, and impaired cytokine production, measured by release of IL-6. In addition, Bcl10-deficient mice display impaired IgE-mediated passive cutaneous anaphylaxis. Moreover, although Bcl10-deficient mast cells have normal FcepsilonR-mediated Ca(2+) flux, activation of PI3K, and activation of the three types of MAPKs (ERKs, JNK, and p38), the mutant cells have markedly diminished FcepsilonR-mediated activation of NF-kappaB and decreased activation of AP-1. Thus, Bcl10 is essential for FcepsilonR-induced activation of AP-1, NF-kappaB, degranulation, and cytokine production in mast cells.

NK cells play a central role in mediating innate immune responses. Activation of NK cells results in cytotoxicity, cytokine, and chemokine secretions. In this study, we show that in mice with targeted deletion of phospholipase Cgamma (PLCgamma)2, one of the key signal transducers, there are profound effects on the development and terminal maturation of NK cells. Lack of PLCgamma2 significantly impaired the ability of lineage-committed NK precursor cells to acquire subset-specific Ly49 receptors and thereby terminal maturation of NK cells. Overexpression of isozyme, PLCgamma1, in PLCgamma2-deficient NK cells resulted in the successful Ly49 acquisition and terminal maturation of the NK cells; however, it could only partially rescue NKG2D-mediated cytotoxicity with no cytokine production. Furthermore, PLCgamma2-deficient NK cells failed to mediate antitumor cytotoxicity and inflammatory cytokine production, displaying a generalized hyporesponsiveness. Our results strongly demonstrate that PLCgamma1 and PLCgamma2 play nonredundant and obligatory roles in NK cell ontogeny and in its effector functions.

Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton's tyrosine kinase and phospholipase C-gamma2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes.

The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

Both type-2 CD4(+) Th cells (CD4(+)Th2) and type-2 innate effector cells play critical roles in generating type-2 immunity that can either be protective against parasitic infection or cause tissue damage in allergy and asthma. How innate effector cells acquire the capacity to produce Th2 cytokines is not entirely known. We previously showed that IL-4 induced differentiation of Th2 cytokine-producing eosinophils. To determine whether other Th2 cytokines can also induce Th2 cytokine-producing capacity in innate effector cells, we cultured bone marrow progenitor cells in the presence of various Th2 cytokines. IL-5, but not IL-13 or IL-25, primed bone marrow progenitor cells to differentiate into robust IL-4-producing cells. The majority of IL-4-producing cells induced by IL-5 were eosinophils. Importantly, IL-5 completely depended on STAT5 to promote IL-4-producing capacity in eosinophils. Thus, our study demonstrates that IL-5 functions as a potent factor that drives bone marrow progenitor cells into IL-4-producing eosinophils.

Phospholipase Cgamma2 (PLCgamma2) plays a critical role in the functions of the B cell receptor in B cells and of the FcRgamma chain-containing collagen receptor in platelets. Here we report that PLCgamma2 is also expressed in mast cells and monocytes/macrophages and is activated by cross-linking of Fc(epsilon)R and Fc(gamma)R. Although PLCgamma2-deficient mice have normal development and numbers of mast cells and monocytes/macrophages, we demonstrate that PLCgamma2 is essential for specific functions of Fc(epsilon)R and Fc(gamma)R. While PLCgamma2-deficient mast cells have normal mitogen-activated protein kinase activation and cytokine production at mRNA levels, the mutant cells have impaired Fc(epsilon)R-mediated Ca(2+) flux and inositol 1,4,5-trisphosphate production, degranulation, and cytokine secretion. As a physiological consequence of the effect of PLCgamma2 deficiency, the mutant mice are resistant to IgE-mediated cutaneous inflammatory skin reaction. Macrophages from PLCgamma2-deficient mice have no detectable Fc(gamma)R-mediated Ca(2+) flux; however, the mutant cells have normal Fc(gamma)R-mediated phagocytosis. Moreover, PLCgamma2 plays a nonredundant role in Fc(gamma)R-mediated inflammatory skin reaction.