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Short-chain fatty acids, such as butyrate, are major gut microbial metabolites that are beneficial for gastrointestinal health. Clostridium butyricum MIYAIRI588 (CBM588) is a bacterium that produces a robust amount of butyrate and therefore has been used as a live biotherapeutic probiotic in clinical settings. Clostridioides difficile causes life-threatening diarrhea and colitis. The gut resident microbiota plays a critical role in the prevention of C. difficile infection (CDI), as the disruption of the healthy microbiota by antibiotics greatly increases the risk for CDI. We report that CBM588 treatment in mice significantly improved clinical symptoms associated with CDI and increased the number of neutrophils and Th1 and Th17 cells in the colonic lamina propria in the early phase of CDI. The protective effect of CBM588 was abolished when neutrophils, IFN-γ, or IL-17A were depleted, suggesting that induction of the immune reactants is required to elicit the protective effect of the probiotic. The administration of tributyrin, which elevates the concentration of butyrate in the colon, also increased the number of neutrophils in the colonic lamina propria, indicating that butyrate is a potent booster of neutrophil activity during infection. However, GPR43 and GPR109a, two G protein–coupled receptors activated by butyrate, were dispensable for the protective effect of CBM588. These results indicate that CBM588 and butyrate suppress CDI, in part by boosting antimicrobial innate and cytokine-mediated immunity.

Invariant NKT (iNKT) cells provide rapid innate T cell responses to glycolipid Ags from host cells and microbes. The numbers of CD1d-restricted iNKT cells are tightly controlled in mucosal tissues, but the mechanisms have been largely unclear. We found that vitamin A is a dominant factor that controls the population size of mucosal iNKT cells in mice. This negative regulation is mediated by the induction of the purinergic receptor P2X7 on iNKT cells. The expression of P2X7 is particularly high on intestinal iNKT cells, making iNKT cells highly susceptible to P2X7-mediated cell death. In vitamin A deficiency, iNKT cells fail to express P2X7 and are, therefore, resistant to P2X7-mediated cell death, leading to iNKT cell overpopulation. This phenomenon is most prominent in the intestine. We found that iNKT cells are divided into CD69+ sphingosine-1-phosphate receptor 1 (S1P1)− tissue resident and CD69− S1P1+ nonresident iNKT cells. The CD69+ S1P1− tissue-resident iNKT cells highly express P2X7 and are effectively controlled by the P2X7 pathway. The regulation of iNKT cells by vitamin A by the P2X7 pathway is important to prevent aberrant expansion of effector cytokine-producing iNKT cells. Our findings identify a novel role of vitamin A in regulating iNKT cell homeostasis in many tissues throughout the body.

Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and profoundly affect host health and disease. SCFAs generate IL-10+ regulatory T cells, which may promote immune tolerance. However, SCFAs can also induce Th1 and Th17 cells upon immunological challenges and, therefore, also have the potential to induce inflammatory responses. Because of the seemingly paradoxical SCFA activities in regulating T cells, we investigated, in depth, the impact of elevated SCFA levels on T cells and tissue inflammation in mice. Orally administered SCFAs induced effector (Th1 and Th17) and regulatory T cells in ureter and kidney tissues, and they induced T cell–mediated ureteritis, leading to kidney hydronephrosis (hereafter called acetate-induced renal disease, or C2RD). Kidney hydronephrosis in C2RD was caused by ureteral obstruction, which was, in turn, induced by SCFA-induced inflammation in the ureteropelvic junction and proximal ureter. Oral administration of all major SCFAs, such as acetate, propionate, and butyrate, induced the disease. We found that C2RD development is dependent on mammalian target of rapamycin activation, T cell–derived inflammatory cytokines such as IFN-γ and IL-17, and gut microbiota. Young or male animals were more susceptible than old or female animals, respectively. However, SCFA receptor (GPR41 or GPR43) deficiency did not affect C2RD development. Thus, SCFAs, when systemically administered at levels higher than physiological levels, cause dysregulated T cell responses and tissue inflammation in the renal system. The results provide insights into the immunological and pathological effects of chronically elevated SCFAs.

The glucocorticoid-induced TNFR family–related protein (GITR, TNFRSF18, CD357) is expressed on effector and regulatory T (Treg) cells. Previous studies demonstrated that GITR triggering by anti-GITR mAb enhanced T and B cell–mediated immune responses. GITR-deficient T cells, however, also proliferate more than normal T cells, and this effect is unexplained. Because the activities of mAbs are controlled by their Fc regions, the true effect of GITR signaling needs to be determined by examining its interaction with authentic ligand. Therefore, we generated a pentamerized form of the GITRL extracellular domain (pGITRL) for ligation to GITR and compared its effect on T cells with that of anti-GITR mAb. The pGITRL was more effective than anti-GITR mAb in enhancing the proliferation of effector and regulatory cells in vitro and in vivo. Nonetheless, the growth of MC38 adenocarcinoma cells in vivo was only suppressed for initial 15 d by pGITRL, whereas it was suppressed indefinitely by anti-GITR mAb. Detailed analysis revealed that pGITRL induced extensive proliferation of Foxp3+CD4+ Treg cells and led to the accumulation of activated Treg cells in tumor tissue and draining lymph nodes. Because GITR signaling could not neutralize the suppressive activity of activated Treg cells, pGITRL seems to lose its adjuvant effect when sufficient activated Treg cells have accumulated in the lymph nodes and tumor tissue. Indeed, the antitumor effects of pGITRL were markedly enhanced by depleting CD4+ cells. These results suggest that GITR signaling has stimulatory effects on effector T cells and inhibitory effects through Treg cells.

The two nuclear hormone receptor ligands progesterone and vitamin D (vit.D) play important roles in regulating T cells. The mechanism that connects these two hormones in regulating T cells has not been established. In this study, we report that progesterone is a novel inducer of vit.D receptor (VDR) in T cells and makes T cells highly sensitive to calcitriol. At the molecular level, the induction by progesterone is mediated by two progesterone receptor-binding elements in the intron region after the first noncoding exon of the human VDR gene. Increased expression of VDR by progesterone allows highly sensitive regulation of T cells by vit.D even when vit.D levels are suboptimal. This novel regulatory pathway allows enhanced induction of regulatory T cells but suppression of Th1 and Th17 cells by the two nuclear hormones. The results have significant ramifications in effective regulation of T cells to prevent adverse immune responses during pregnancy.

Progesterone, a key female sex hormone with pleiotropic functions in maintenance of pregnancy, has profound effects on regulation of immune responses. We report in this work a novel function of progesterone in regulation of naive cord blood (CB) fetal T cell differentiation into key T regulatory cell (Treg) subsets. Progesterone drives allogeneic activation-induced differentiation of CB naive, but not adult peripheral blood, T cells into immune-suppressive Tregs, many of which express FoxP3. Compared with those induced in the absence of progesterone, the FoxP3+ T cells induced in the presence of progesterone highly expressed memory T cell markers. In this regard, the Treg compartment in progesterone-rich CB is enriched with memory-type FoxP3+ T cells. Moreover, CB APCs were more efficient than their peripheral blood counterparts in inducing FoxP3+ T cells. Another related function of progesterone that we discovered was to suppress the differentiation of CB CD4+ T cells into inflammation-associated Th17 cells. Progesterone enhanced activation of STAT5 in response to IL-2, whereas it decreased STAT3 activation in response to IL-6, which is in line with the selective activity of progesterone in generation of Tregs versus Th17 cells. Additionally, progesterone has a suppressive function on the expression of the IL-6 receptor by T cells. The results identified a novel role of progesterone in regulation of fetal T cell differentiation for promotion of immune tolerance.

Agonistic anti–4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8+ T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8+ T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB–triggered suppression of autoimmune responses using IFN-γ- or IFN-γR–deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3+CD4+ regulatory T (Treg) cells, particularly among CD4+ T cells. This was not due to a direct effect of 4-1BB signaling on CD4+ T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8+ T cells, but also did so in IFN-γ–deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.

Stimulation of 4-1BB (CD137) was shown to produce strong anticancer effects in vivo. In contrast, 4-1BB–deficient (4-1BB−/−) B6 mice are remarkably resistant to tumor growth. We set out to determine the mechanisms involved in these seemingly contradictory observations. We found that the therapeutic effects of 4-1BB triggering were mainly dependent on CD8+ T cells and partially on NK cells, whereas CD8+ T and NK cells were equally needed to suppress tumor growth in 4-1BB−/− mice. Cellular analysis showed that the frequency and number of NK cells in the spleen and bone marrow were decreased by 4-1BB triggering but were increased in the absence of 4-1BB signaling in tumor-challenged mice. The 4-1BB–mediated downregulation of NK cell development was primarily dependent on IFN-γ, which was produced by peripheral CD8+ T and NK cells. The suppression of NK cell development by 4-1BB–mediated IFN-γ production occurred in the bone marrow. As 4-1BB signaling increased in the periphery, more CD8+ T cells but fewer NK cells contributed to the antitumor immunity. As 4-1BB signaling decreased, more NK cells participated in the antitumor immunity. We conclude that 4-1BB signaling results in a shift of the dominant type of immune cell in antitumor immunity from the innate NK cell to the adaptive CD8+ T cell and that the level of IFN-γ is critical for this 4-1BB–mediated shift.

Th17 cells are major effector T cells in the intestine, but the regulation of their tissue tropism within the gut is poorly understood. We investigated the roles of vitamin A and retinoic acid in generation of inflammatory Th17 cells with distinct tissue tropisms within the intestine. We found that Th17 cells with distinct tissue tropisms and pathogenic activities are generated depending on the available concentration of retinoic acid (RA). In contrast to the widespread perception that RA would suppress the generation of Th17 cells, we provide evidence that RA is actually required for generation of Th17 cells with specific tissue tropisms within the gut. Th17 cells induced at suboptimal serum concentrations of RA migrated and induced moderate inflammation mainly in the large intestine, whereas the Th17 cells induced with optimal levels of exogenous RA (∼10 nM) migrated to the small intestine and induced more severe inflammation. The Th17 cells, induced in the presence or absence of RA, differentially expressed the trafficking receptors CCR9 and α4β7. CCR9 is required for Th17 cell migration to the small intestine, whereas α4β7 is required for the migration of Th17 cells throughout the whole intestine. Our results identified RA as a major signal that regulates the generation of gut Th17 cells with distinct capacities in migration and inflammatory activities. The results indicate also that specific gut tropism of Th17 cells is determined by the combination of trafficking receptors regulated by the RA signal.

We have demonstrated in this study the existence of a PDCA-expressing functional B cell population (PDCA+ B lymphocytes), which differentiates from activated conventional B (PDCA−IgM+) lymphocytes. Stimulation with anti-μ, LPS, CpG oligodeoxynucleotide, HSV-1, or CTLA-4 Ig activates the PDCA+ B lymphocytes, leading to cell division and induction of type I IFNs and IDO. Notably, the PDCA+ B lymphocytes are capable of Ag-specific Ab production and Ig class switching, which is corroborated by transfer experiments in B- and PDCA+ B lymphocyte-deficient μMT mice. Importantly, in lupus-prone MRL-Faslpr mice, PDCA+ B lymphocytes remain the principal source of autoantibodies. The PDCA+ B lymphocytes have phenotypes with plasmacytoid dendritic cells, but are a distinct cell population in that they develop from C-kit+B220+ pro-B precursors. Thus, our data suggest that not all PDCA+ cells are dendritic cell-derived plasmacytoid dendritic cells and that a significant majority is the PDCA+ B lymphocyte population having distinct phenotype and function.

Extramedullary myelopoiesis occurs in peripheral organs such as spleen and produces many types of myeloid cells with diverse functions in response to inflammation and infection. It is increased during immune responses and chronic inflammation and is a significant factor in regulating inflammatory diseases and immunity. Increased myeloid cells are found in FoxP3-deficient mice but the mechanism has been unclear. We investigated the mechanism by which FoxP3+ regulatory T cells regulate the extramedullary myelopoiesis. We found that Ab or genetic depletion of FoxP3+ regulatory T cells greatly increased the number of the myeloid progenitors in spleen during immune responses. Consistently, the splenic myelopoiesis was effectively suppressed by increased numbers of natural or induced FoxP3+ regulatory T cells. We demonstrated that myelopoiesis is positively regulated by splenic CD4+ T cells that produce myelopoietic cytokines (GM-CSF and IL-3), and these effector CD4+ T cells are induced from naive CD4+ T cells in response to antigenic stimulation. FoxP3+ regulatory T cells were able to effectively suppress the differentiation of naive T cells into myelopoietic cytokine-producing T cells. This suppression was found to be dependent on cell contact but independent of TGFβ. Unlike splenic myelopoiesis, marrow myelopoiesis is not significantly affected by FoxP3+ regulatory T cells. We conclude that FoxP3+ T cells can negatively regulate splenic extramedullary myelopoiesis by suppressing the naive T cell differentiation into myelopoietic cytokine-producing CD4+ T cells. Our results provide new insights into regulation of extramedullary myelopoiesis.

It is a question of interest whether Th17 cells express trafficking receptors unique to this Th cell lineage and migrate specifically to certain tissue sites. We found several Th17 cell subsets at different developing stages in a human secondary lymphoid organ (tonsils) and adult, but not in neonatal, blood. These Th17 cell subsets include a novel in vivo-stimulated tonsil IL17+ T cell subset detected without any artificial stimulation in vitro. We investigated in depth the trafficking receptor phenotype of the Th17 cell subsets in tonsils and adult blood. The developing Th17 cells in tonsils highly expressed both Th1- (CCR2, CXCR3, CCR5, and CXCR6) and Th2-associated (CCR4) trafficking receptors. Moreover, Th17 cells share major non-lymphoid tissue trafficking receptors, such as CCR4, CCR5, CCR6, CXCR3, and CXCR6, with FOXP3+ T regulatory cells. In addition, many Th17 cells express homeostatic chemokine receptors (CD62L, CCR6, CCR7, CXCR4, and CXCR5) implicated in T cell migration to and within lymphoid tissues. Expression of CCR6 and CCR4 by some Th17 cells is not a feature unique to Th17 cells but shared with FOXP3+ T cells. Interestingly, the IL17+IFN-γ+ Th17 cells have the features of both IL17−IFN-γ+ Th1 and IL17+IFN-γ− Th17 cells in expression of trafficking receptors. Taken together, our results revealed that Th17 cells are highly heterogeneous, in terms of trafficking receptors, and programmed to share major trafficking receptors with other T cell lineages. These findings have important implications in their distribution in the human body in relation to other regulatory T cell subsets.

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Rα (CD127) and the common γ-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage. In this study, we report that loss of CD127 defines terminally differentiated B cell-helping effector T cells in human tonsils. Although naive CD4+ T cells uniformly express CD127, the memory/effector (non-FOXP3+) CD4+ T cells are divided into CD127+ and CD127− cells. The CD127− T cells are exclusively localized within the germinal centers where B cells become plasma and memory B cells, whereas CD127+ T cells are found in T cell areas and the area surrounding B cell follicles. Consistently, the CD127− T cells highly express the B cell zone homing receptor CXCR5 with concomitant loss of CCR7. Compared with CD127+ memory T cells, CD127− T cells have considerably shorter telomeres, do not proliferate in response to IL-7, and are prone to cell death. The CD127− T cells produce a large amount of the B cell follicle-forming chemokine CXCL13 upon stimulation with B cells and Ags. Most importantly, they are highly efficient in helping B cells produce Igs of all isotypes in a manner dependent on CD40L and ICOS and inducing activation-induced cytidine deaminase and Ig class switch recombination. The selective loss of CD127 on the B cell-helping effector T cells would have implications in regulation and termination of Ig responses.

Forkhead box P3 (FoxP3)-positive T cells are a specialized T cell subset for immune regulation and tolerance. We investigated the trafficking receptor switches of FoxP3+ T cells in thymus and secondary lymphoid tissues and the functional consequences of these switches in migration. We found that FoxP3+ T cells undergo two discrete developmental switches in trafficking receptors to migrate from primary to secondary and then to nonlymphoid tissues in a manner similar to conventional CD4+ T cells as well as unique to the FoxP3+ cell lineage. In the thymus, precursors of FoxP3+ cells undergo the first trafficking receptor switch (CCR8/CCR9→CXCR4→CCR7), generating mostly homogeneous CD62L+CCR7+CXCR4lowFoxP3+ T cells. CXCR4 expression is regained in FoxP3+ thymic emigrants in the periphery. Consistent with this switch, recent FoxP3+ thymic emigrants migrate exclusively to secondary lymphoid tissues but poorly to nonlymphoid tissues. The FoxP3+ thymic emigrants undergo the second switch in trafficking receptors for migration to nonlymphoid tissues upon Ag priming. This second switch involves down-regulation of CCR7 and CXCR4 but up-regulation of a number of memory/effector type homing receptors, resulting in generation of heterogeneous FoxP3+ T cell subsets expressing various combinations of trafficking receptors including CCR2, CCR4, CCR6, CCR8, and CCR9. A notable difference between the FoxP3+ and FoxP3− T cell populations is that FoxP3+ T cells undergo the second homing receptor switch at a highly accelerated rate compared with FoxP3− T cells, generating FoxP3+ T cells with unconventionally efficient migratory capacity to major nonlymphoid tissues.

Forkhead Box P3+ (FOXP3+) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3+ T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3+ T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3+ T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3+ T cells are divided into CD45RA+ (naive type) and CD45RO+ (memory type) FOXP3+ T cells in neonatal blood, adult blood, and tonsils. CD45RA+FOXP3+ T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO+FOXP3+ T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA+FOXP3+ T cells and is completed after their differentiation to CD45RO+ cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3+ cells at a specific stage (CD69+CD45RO+) in tonsils. Our in vitro differentiation study demonstrated that CD45RA+FOXP3+ T cells indeed undergo chemokine receptor switch from CD45RA+ (secondary lymphoid tissue homing) to CD45RO+ type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3+ T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.

T cells undergo chemokine receptor switches during activation and differentiation in secondary lymphoid tissues. Here we present evidence that dendritic cells can induce changes in T cell expression of chemokine receptors in two continuous steps. In the first switch over a 4–5 day period, dendritic cells up-regulate T cell expression of CXCR3 and CXCR5. Additional stimulation leads to the second switch: down-regulation of lymphoid tissue homing related CCR7 and CXCR5, and up-regulation of Th1/2 effector tissue-targeting chemoattractant receptors such as CCR4, CCR5, CXCR6, and CRTH2. We show that IL-4 and IL-12 can determine the fate of the secondary chemokine receptor switch. IL-4 enhances the generation of CCR4+ and CRTH2+ T cells, and suppresses the generation of CXCR3+ T cells and CCR7− T cells, while IL-12 suppresses the level of CCR4 in responding T cells. Furthermore, IL-4 has positive effects on generation of CXCR5+ and CCR7+ T cells during the second switch. Our study suggests that the sequential switches in chemokine receptor expression occur during naive T cell interaction with dendritic cells. The first switch of T cell chemokine receptor expression is consistent with the fact that activated T cells migrate within lymphoid tissues for interaction with B and dendritic cells, while the second switch predicts the trafficking behavior of effector T cells away from lymphoid tissues to effector tissue sites.

CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4+ thymocytes and on a major fraction of blood nonintestinal (α4β7−) memory CD4 lymphocytes, including almost all skin memory CD4+ cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4+ memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4+ CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4+ memory CD4 lymphocytes (including both CLA+ and CLA− fractions) readily express the Th1 cytokine IFN-γ after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.

CK beta-11 chemoattracts T cells, B cells, dendritic cells, macrophage progenitors, and NK cells and facilitates dendritic cell and T cell interactions in secondary lymphoid tissues. We hypothesized that expression of CK beta-11 in tumor cells may generate antitumor immunity through these interactions. After transduction with the retroviral vector L(CK beta 11)SN, the murine breast cancer cell line C3L5 (C3L5-CK beta 11) showed expression of retroviral mRNA by Northern analysis and production of functional CK beta-11 by chemotaxis of human NK cells to C3L5-CK beta 11 supernatant. Only 10% of mice injected with C3L5-CK beta 11 developed tumors, compared with 100% of mice injected with a transduced control C3L5 line (C3L5-G1N). Importantly, the in vitro growth characteristics of the CK beta-11-transduced cell line were unaffected, suggesting the difference in growth in vivo was a result of chemokine production. Vaccination with C3L5-CK beta 11 partially protected animals from parental C3L5 challenge. Immunodepletion with anti-asialo-GM1 or anti-CD4 during C3L5-CK beta 11 vaccination significantly reduced CK beta-11 antitumor activity compared with control and anti-CD8-treated groups. Splenocytes from NK-depleted animals transferred the acquired immunity generated with C3L5-CK beta 11 vaccination, while splenocytes from the CD4-depleted animals did not. These results indicate, for the first time, that expression of CK beta-11 in a breast cancer cell line mediates rejection of the transduced tumor through a mechanism involving NK and CD4+ cells. Furthermore, CK beta-11-transduced tumor cells generate long-term antitumor immunity that requires CD4+ cells. These studies demonstrate the potential role of CK beta-11 as an adjuvant in stimulating antitumor responses.

Chemoattractants are potential factors influencing cell migration. Stromal cell-derived factor-1, a CXC chemokine, is the only chemokine reported to have chemotactic activity for hemopoietic progenitor cells (HPC). We report in this work another chemokine of the CC subfamily, which is chemotactic for HPC. Macrophage-inflammatory protein (MIP)-3β/EBI1-ligand chemokine/CKβ-11 attracted bone marrow and cord blood CD34+ cells. In contrast to stromal cell-derived factor-1, which attracts multiple types of HPC, MIP-3β attracted mainly CFU granulocyte macrophage, but not other HPC such as burst-forming unit erythrocyte or CFU granulocyte, erythrocyte, macrophage, and megakaryocyte. Chemoattracted CD34+ cells formed CFU granulocyte macrophage-like colonies, which were morphologically determined as large macrophages. These progenitors were selectively responsive to stimulation by macrophage CSF, demonstrating that MIP-3β attracts macrophage progenitors. Expression of CCR7, the receptor for MIP-3β, was detected at a mRNA level in the attracted CD34+ cells as well as input CD34+HPC. Expression of MIP-3β mRNA was not constitutive, but was inducible in bone marrow stromal cells by inflammatory agents such as bacterial LPS, IFN-γ, and TNF-α. Taken together, our findings suggest that MIP-3β is expressed in the bone marrow environment after induction with certain inflammatory cytokines and LPS, and may play a role in trafficking of macrophage progenitors in and out of the bone marrow in inflammatory conditions.

We examined the functional properties of CKβ-11/MIP-3β/ELC, a recently reported CC chemokine that specifically binds to a chemokine receptor, EBI1/BLR2/CCR7. CKβ-11/MIP-3β/ELC is distantly related to other CC and CXC chemokines in primary amino acid sequence structure. Recombinant human CKβ-11/MIP-3β/ELC expressed from a mammalian cell system showed potent chemotactic activity for T cells and B cells but not for granulocytes and monocytes. An optimal concentration of CKβ-11/MIP-3β/ELC attracted most input T cells within 3 h, a chemotactic activity comparable with that of stromal cell derived factor 1 (SDF-1), a highly efficacious CXC chemokine. CKβ-11/MIP-3β/ELC equally attracted naive CD45RA+ and memory type CD45RO+ T cells. CKβ-11/MIP-3β/ELC also strongly attracted both CD4+ and CD8+ T cells, but the attraction for CD4+ T cells was greater. CKβ-11/MIP-3β/ELC was also a more efficacious chemoattractant for B cells than MIP-1α, a known B cell chemoattractant. CKβ-11/MIP-3β/ELC induced actin polymerization in lymphocytes, and chemotaxis was completely blocked by pertussis toxin showing its receptor, most likely EBI1/BLR2/CCR7, is coupled to a Gαi protein. CKβ-11/MIP-3β/ELC induced calcium mobilization in lymphocytes, which could be desensitized by SDF-1, suggesting possible cross-regulation in their signaling. Human CKβ-11/MIP-3β/ELC attracted murine splenocytes suggesting functional conservation of CKβ-11/MIP-3β/ELC between human and mouse. The efficacy of chemoattraction by CKβ-11/MIP-3β/ELC and tissue expression of its mRNA suggest that CKβ-11/MIP-3β/ELC may be important in trafficking of T cells in thymus, and T cell and B cell migration to secondary lymphoid organs.

HIV-1 infection is associated with a progressive and functional decline in the CD4+ lymphoid Th1 subset. Here, we propose that the HIV nef gene product may function as a specific regulator of Th1 cytokine production. By use of a T cell-specific inducible expression system, we show that upon T cell activation, induced nef expression down-regulated both IL-2 and IFN-gamma production in a dose-dependent manner, whereas IL-4, IL-9, IL-13, IL-8, and TNF-alpha production remained unaffected. In addition to this, independent transfected clones expressing various nef genes, including nef sequences amplified directly from an HIV-1 primary clinical isolate, displayed a similar pattern of cytokine expression. The specific Th1 impairment induced by nef, therefore, seems to be an important and conserved feature of HIV-1 infection and may represent a significant function of this viral gene in AIDS pathogenesis.

Short chain acids (SCFAs) are highly produced by gut commensal bacteria and play important roles in regulating immune responses. SCFAs can regulate epithelial cells an dimmune cells, and some of the functions are mediated through GPR41 and GPR43. Epithelial cells highly express both GPR41 and GPR43, but the functions of these receptors in regulation of epithelial innate immunity remain unclear. To investigate this, we utilized mice deficient in expression of GPR41 or GPR43. When the gut barrier function was breached with 50% ethanol, GPR41-/- and GPR43-/- mice were defective in expression of inflammatory cytokines (IL-6, IL1b and TNFa) and chemokines (CXCL1 and CXCL2) in response to invading bacteria. When the mice were infected with C. rodentium, the KO mice were delayed in mounting immune responses and clearing the pathogen. On the other hand, wild type mice with increased levels of SCFAs were more efficient in expression of the inflammatory factors. The results of our bone marrow transfer and in vitro culture experiments indicate that epithelial GPR41 and GPR43 play critical roles in mediating the SCFA signal. SCFAs induced a series of signaling events from the surface receptors to ERKs and to ATF2 activation to express the inflammatory factors. These processes are required to recruit and activate immune cells to clear pathogenic bacteria. Our results revealed a novel positive role of short chain fatty acids and their receptors in regulating intestinal immunity.

T helper cells that produce IL-17 (Th17 cells) play important roles in regulation of immunity to bacteria and fungi and can cause organ-specific inflammation. The intestine is a tissue where Th17 cells are highly enriched, but migration and trafficking receptors of Th17 cells to the intestine have been unclear. It has been established that CCR6 is the signature chemokine receptor for Th17 cells but the role of this trafficking receptor in Th17 cell trafficking to the intestine has not been determined. We report here that Th17 cells preferentially migrate to the intestine and associated-lymphoid tissues, and CCR6 is the homing receptor important for Th17 cell migration to certain tissue microenvironments of the intestine where its ligand CCL20 is highly expressed. We determined the function of CCR6 of Th17 cells by comparing the migration and effector function of Th17 cells derived from wild type and CCR6-deficient mice. CCR6-deficient Th17 cells were defective in migration to Peyer's patches and small intestine but their migration to the large intestine was normal. Surprisingly, administration of CCR6-deficient Th17 cells into severe combined immunodeficiency (SCID) mice led to excessive intestinal inflammation. Following the administration of Th17 cells, we found that Th17 cells fail to propagate in the gut in the absence of CCR6 and transdifferentiate into inflammatory Th1 cells. In addition, CCR6 deficiency led to aberrantly wide-spread effector T cells in the inflamed intestine of the SCID mice. Finally, we found that the expression of CCR6 by Th17 cells is induced by TGF-β1 but suppressed by IL-2 in vitro and in vivo. Taken together, we conclude that CCR6 is a key trafficking receptor that regulates the migration and effector function of Th17 cells in the intestine.