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Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4 + and CD8 + T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8 + T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4 + T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

Acute gastrointestinal (GI) graft-versus-host disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem cell transplantation (alloSCT). The condition is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN-γ, IL-17A, or GM-CSF and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between Th cell states during priming in mesenteric lymph nodes (mLNs) and effector function in the GI tract remain undefined at genome scale. We applied scRNA-Seq and computational modeling to a mouse model of donor DC-mediated GVHD exacerbation, creating an atlas of putative CD4+ T cell differentiation pathways in vivo. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLNs. Importantly, we inferred an unexpected second trajectory, categorized by little proliferation or cytokine expression, reduced glycolysis, and high tcf7 expression. TCF1hi cells upregulated α4β7 before gut migration and failed to express cytokines. These cells exhibited recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced T cell factor 1 (TCF1). Thus, scRNA-Seq suggested divergence of alloreactive CD4+ T cells into quiescent and effector states during gut GVHD exacerbation by donor DC, reflecting putative heterogeneous priming in vivo. These findings, which are potentially the first at a single-cell level during GVHD over time, may assist in examination of T cell differentiation in patients undergoing alloSCT.

Cytomegalovirus latently infects myeloid cells; however, the acute effects of the virus on this cell subset are poorly characterised. We demonstrate that systemic cytomegalovirus infection induced rapid activation of monocytes in the bone marrow, characterised by upregulation of CD69, CD11c, Ly6C and M-CSF receptor. Activated bone marrow monocytes were more sensitive to M-CSF and less sensitive to granulocyte-monocyte colony stimulating factor in vitro, resulting in the generation of more macrophages and fewer dendritic cells, respectively. Monocyte activation was also observed in the periphery and resulted in significant accumulation of monocytes in the spleen. MyD88 expression was required within the haematopoietic compartment to initiate monocyte activation and recruitment. However, monocytes lacking MyD88 were activated and recruited in the presence of MyD88-sufficient cells in mixed bone marrow chimeras, indicating that once initiated, the process was MyD88 independent. Interestingly, we found that monocyte activation occurred in the absence of the common inflammatory cytokines, namely type I interferons (IFNs), IL-6, TNF-α and IL-1 as well as the NLRP3 inflammasome adaptor protein, ASC. We also excluded a role for the chemokine-like protein MCK-2 (m131/129) expressed by murine CMV. Taken together, these results challenge the notion that a single inflammatory cytokine mediates activation and recruitment of monocytes in response to infection., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of the mononuclear phagocyte system requires macrophage colony-stimulating factor (CSF-1) signaling through the CSF-1 receptor (CSF1R, CD115). We examined the effect of an antibody against CSF1R on macrophage homeostasis and function using the MacGreen transgenic mouse (csf1r-enhanced green fluorescent protein) as a reporter. The administration of a novel CSF1R blocking antibody selectively reduced the CD115(+)Gr-1(neg) monocyte precursor of resident tissue macrophages. CD115(+)Gr-1(+) inflammatory monocytes were correspondingly increased, supporting the view that monocytes are a developmental series. Within tissue, the antibody almost completely depleted resident macrophage populations in the peritoneum, gastrointestinal tract, liver, kidney, and skin, but not in the lung or female reproductive organs. CSF1R blockade reduced the numbers of tumor-associated macrophages in syngeneic tumor models, suggesting that these cells are resident type macrophages. Conversely, it had no effect on inflammatory monocyte recruitment in models, including lipopolysaccharide-induced lung inflammation, wound healing, peritonitis, and severe acute graft-versus-host disease. Depletion of resident tissue macrophages from bone marrow transplantation recipients actually resulted in accelerated pathology and exaggerated donor T-cell activation. The data indicate that CSF1R signaling is required only for the maturation and replacement of resident-type monocytes and tissue macrophages, and is not required for monocyte production or inflammatory function.

Host antigen-presenting cells (APCs) are known to be critical for the induction of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but the relative contribution of specific APC subsets remains unclear. We have studied the role of host B cells in GVHD by using B-cell-deficient microMT mice as BMT recipients in a model of CD4-dependent GVHD to major histocompatibility complex antigens. We demonstrate that acute GVHD is initially augmented in microMT recipients relative to wild-type recipients (mortality: 85% vs 44%, P < .01), and this is the result of an increase in donor T-cell proliferation, expansion, and inflammatory cytokine production early after BMT. Recipient B cells were depleted 28-fold at the time of BMT by total body irradiation (TBI) administered 24 hours earlier, and we demonstrate that TBI rapidly induces sustained interleukin-10 (IL-10) generation from B cells but not dendritic cells (DCs) or other cellular populations within the spleen. Finally, recipient mice in which B cells are unable to produce IL-10 due to homologous gene deletion develop more severe acute GVHD than recipient mice in which B cells are wild type. Thus, the induction of IL-10 in host B cells during conditioning attenuates experimental acute GVHD.

4.1B is a member of the protein 4.1 superfamily of proteins that link transmembrane proteins to the actin cytoskeleton. The 4.1B gene localizes to chromosome 18p11.3, which undergoes loss of heterozygosity in mammary tumors. Here, we examine the expression of 4.1B in murine mammary epithelium and find that 4.1B is dramatically upregulated in mammary epithelial cells during pregnancy when there is extensive cell proliferation. In contrast, 4.1B is not expressed in virgin, lactating, or involuting mammary epithelium. To examine the consequence of 4.1B loss on mammary epithelial cell proliferation, we analysed mammary glands in 4.1B-null mice. 4.1B loss results in a significant increase in mammary epithelial cell proliferation during pregnancy, but has no effect on mammary epithelial cell proliferation, in virgin or involuting mice. Furthermore, we show that 4.1B inhibits the proliferation of mammary epithelial cell lines by inducing a G1 cell cycle arrest, characterized by decreased cyclin A expression and reduced Rb phosphorylation, and accompanied by reduced erbB2 phosphorylation. This cell cycle arrest does not involve alterations in the activities of MAPK, JNK, or Akt. Collectively, our findings demonstrate that 4.1B regulates mammary epithelial cell proliferation during pregnancy and suggest that its loss may influence mammary carcinoma pathogenesis in multiparous women.

Donor treatment with granulocyte-colony-stimulating factor (G-CSF) attenuates the ability of donor T cells to induce acute graft-versus-host disease (aGVHD) but increases the severity of chronic GVHD (cGVHD). We investigated the role of the regulatory cytokine transforming growth factor beta (TGF-beta) in this paradox in well-established murine models of aGVHD and cGVHD wherein recipients undergo transplantation with splenocytes from donors treated with G-CSF. Neutralization of TGF-beta after stem-cell transplantation (SCT) significantly increased the severity of aGVHD, and the concurrent prevention of interleukin-10 (IL-10) production further exaggerated this effect. Early after SCT, donor T cells were the predominant source of TGF-beta and were able to attenuate aGVHD in a TGF-beta-dependent fashion. Although the neutralization of TGF-beta augmented the proliferation and expansion of donor T cells after SCT, it paradoxically impaired cellular cytotoxicity to host antigens and associated graft-versus-leukemia (GVL) effects. In cGVHD, neutralization of TGF-beta from day 14 after SCT attenuated histologic abnormalities, and CD11b+ mononuclear cells infiltrating sclerodermatous skin produced 50-fold more TGF-beta than corresponding T cells. Thus, though the production of TGF-beta by donor T cells early after transplantation attenuates aGVHD and is required for optimal GVL, the production of TGF-beta late after SCT is preferentially from mononuclear cells and mediates cGVHD. These data have important implications for the timing of therapeutic TGF-beta neutralization to prevent cGVHD after allogeneic SCT.

The cytoadherence-linked asexual gene 9 (clag 9) of Plasmodium falciparum has been implicated in the cytoadherence of infected erythrocytes. To determine the immunogenicity of the clag 9 gene product (CLAG 9 protein) in humans, we measured antibody responses to 11 synthetic CLAG 9 peptides in a group of 177 asymptomatic children and adults subject to intense malaria exposure in Madang, Papua New Guinea. The CLAG 9 peptides were immunogenic in adults and children. Antibody responses to peptides 4 and 10 were high across all age groups and detectable in a majority of children less than five years of age. While CLAG 9 peptides are immunogenic in humans, longitudinal studies will be required to determine the longevity of antibody responses to CLAG 9 and their role in protection from disease.

The erbB2 receptor tyrosine kinase and the CD44 transmembrane glycoprotein interact with one another in numerous cell types. This interaction helps to maintain erbB2 activity that contributes to tumor progression. We investigated whether CD44 and erbB2 similarly interact in endometrial carcinomas in vitro and in situ. In contrast to other carcinomas, CD44 did not colocalize with erbB2 in any of the 51 cases of endometrial cancer analyzed. CD44 also did not coimmunoprecipitate or colocalize with erbB2 in two endometrial carcinoma cell lines. We propose that the lack of CD44-erbB2 interactions may reduce the contribution of erbB2 to endometrial carcinoma progression.

Objective: The CD44 transmembrane glycoprotein family has been implicated in the growth and metastasis of numerous human cancers. CD44 may function in some cells through interactions with type I receptor tyrosine kinases, including erbB2. Here, we tested whether CD44 interacts with erbB2 and another type I receptor, the epidermal growth factor receptor (EGFR), in human cervical carcinoma tissues and cell lines and whether these interactions influence erbB2 signaling., Methods: CD44, EGFR, and erbB2 colocalization were examined in 36 pT1b-pT2b cervical cancer cases and in the CaSki and SiHa cervical carcinoma cell lines by immunohistochemistry and laser scanning confocal microscopy. The role of CD44-EGFR-erbB2 interactions in erbB2 signaling was examined by immunoprecipitation and using antisense CD44 oligonucleotides., Results: CD44, erbB2, and EGFR coexpression and colocalization were observed in 42% (15/36) of cervical carcinoma cases and in both cervical carcinoma cell lines. Colocalization occurred to an equivalent extent in all tumor grades examined. CD44 coimmunoprecipitated with erbB2 and EGFR in cervical carcinoma cell lysates, indicating that these proteins interact with each other. Reduction of CD44 expression inhibited constitutive erbB2 activity. High CD44 expression was linked to EGFR activity using dominant negative EGFR, suggesting that type I receptors may autoregulate their activity in these cells., Conclusions: Our data indicate that CD44 can mediate type I receptor function in cervical carcinoma cells that overexpress both CD44 and either erbB2 or EGFR and suggest a novel mechanism by which these proteins may contribute to cervical carcinoma tumor growth and metastasis., (Copyright 2001 Academic Press.)

Prolactin (PRL) shares several characteristics with growth factors and cytokines, many of which are known to bind to heparan sulfate proteoglycans. In this study we examined the heparin-binding properties of selected members of the PRL/GH family, using heparin affinity columns followed by gel electrophoresis/Western blotting. Purified human PRL and its cleaved 16K fragment, but not human GH or placental lactogen, were retained on the heparin column and were displaced by 0.5 M NaCl. Native PRL in human pituitary extracts and amniotic fluid showed a similar binding affinity to heparin as the purified hormone. None of the other hormones tested, e.g., rat, ovine and bovine PRL, glycosylated ovine PRL or rat GH, bound to heparin. Two consensus heparin-binding sequences are present in human PRL but not in the other hormones included in this study. We postulate that the heparin-binding capability of PRL affects its biological activity as a growth factor and the angiostatic actions of its 16K fragment.