Loss of B1 and marginal zone B cells during ovarian cancer.

Recent advances in immunotherapy have not addressed the challenge presented by ovarian cancer. Although the peritoneum is an "accessible" locus for this disease there has been limited characterization of the immunobiology therein. We investigated the ID8-C57BL/6J ovarian cancer model and found marked depletion of B1 cells from the ascites of the peritoneal cavity. There was also selective loss of the B1 and marginal zone B cell subsets from the spleen. Immunity to antigens that activate these subsets validated their loss rather than relocation. A marked influx of myeloid-derived suppressor cells correlated with B cell subset depletion. These observations are discussed in the context of the housekeeping burden placed on innate B cells during ovarian cancer and to foster consideration of B cell biology in therapeutic strategies to address this challenge.

The somatically generated portion of T cell receptor CDR3α contributes to the MHC allele specificity of the T cell receptor.

Mature T cells bearing αß T cell receptors react with foreign antigens bound to alleles of major histocompatibility complex proteins (MHC) that they were exposed to during their development in the thymus, a phenomenon known as positive selection. The structural basis for positive selection has long been debated. Here, using mice expressing one of two different T cell receptor ß chains and various MHC alleles, we show that positive selection-induced MHC bias of T cell receptors is affected both by the germline encoded elements of the T cell receptor α and ß chain and, surprisingly, dramatically affected by the non germ line encoded portions of CDR3 of the T cell receptor α chain. Thus, in addition to determining specificity for antigen, the non germline encoded elements of T cell receptors may help the proteins cope with the extremely polymorphic nature of major histocompatibility complex products within the species.

Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

The interaction of αß T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

CD28 ligation increases macrophage suppression of T-cell proliferation.

When compared to spleen or lymph node cells, resident peritoneal cavity cells respond poorly to T-cell activation in vitro. The greater proportional representation of macrophages in this cell source has been shown to actively suppress the T-cell response. Peritoneal macrophages exhibit an immature phenotype (MHC class II(lo), B7(lo)) that reduces their efficacy as antigen-presenting cells. Furthermore, these cells readily express inducible nitric oxide synthase (iNOS), an enzyme that promotes T-cell tolerance by catabolism of the limiting amino acid arginine. Here, we investigate the ability of exogenous T-cell costimulation to recover the peritoneal T-cell response. We show that CD28 ligation failed to recover the peritoneal T-cell response and actually suppressed responses that had been recovered by inhibiting iNOS. As indicated by cytokine ELISpot and neutralizing monoclonal antibody (mAb) treatment, this 'cosuppression' response was due to CD28 ligation increasing the number of interferon (IFN)-γ-secreting cells. Our results illustrate that cellular composition and cytokine milieu influence T-cell costimulation biology.Cellular & Molecular Immunology advance online publication, 23 April 2012; doi:10.1038/cmi.2012.13.

Cytokine treatment of macrophage suppression of T cell activation.

High Mphi:T cell ratios suppress the immune response to the retroviral superantigen Mls by IFNgamma-triggered production of the arg- and trp-consuming enzymes iNOS and IDO. Attempts to reverse suppression by treatment with pro-inflammatory cytokines revealed that IL-6 improved the T cell response to Mls and the pro-hematopoietic cyokines IL-3 and GM-CSF increased suppression. GM-CSF treatment increased Mphi expression of CD80, a ligand for the immune suppressive B7H1 and CTLA-4 receptors. These results illustrate potential strategies for reversing the suppression of cell-mediated immunity characteristic of the high Mphi:T cell ratios found in many tumors.

Complementary suppression of T cell activation by peritoneal macrophages and CTLA-4-Ig.

Peritoneal macrophages suppressed T lymphocyte activation by amino acid catabolism. CTLA-4-Ig complemented this form of suppression by blocking the costimulation of T cells. Inhibition of the amino acid catabolizing enzymes or blockade of the IFNgamma signaling essential for macrophage-mediated suppression did not impact CTLA-4-Ig efficacy. Except for phytohemagglutinin, T cell responses to superantigen, mitogen, and anti-CD3 were further reduced when CTLA-4-Ig was combined with peritoneal macrophages. The combination of these complimentary forms of immune regulation potently suppresses adaptive immunity.

Antiinflammatory effect of A3 adenosine receptor agonists in murine autoimmune arthritis models.

OBJECTIVE: CF101, an A3 adenosine receptor (A3AR) agonist, is a small orally bioavailable molecule known to suppress in vitro the production of tumor necrosis factor-alpha (TNF-alpha). We evaluated its therapeutic potential and antiinflammatory effects in 3 murine models of adjuvant induced arthritis (AIA). METHODS: The antiinflammatory effect of CF101 was examined in rat AIA, in mouse collagen induced arthritis, and in tropomyosin induced arthritis. The clinical effect of another A3AR agonist, Cl-IB-MECA, was examined in rat AIA. The effect of low dose (10 or 100 mg/kg/day) A3AR agonists administered orally once daily on arthritis severity was assessed clinically and histologically. The effect of CF101 on the protein expression level of TNF-alpha in the synovial tissue, draining lymph nodes, and spleen cells was determined by Western blot. RESULTS: CF101 and Cl-IB-MECA markedly ameliorated the clinical and histological features of arthritis in the 3 models when administered orally at a low dose of 10 mg/kg body weight in the 3 autoimmune arthritis models. The lower dose of 10 mg/kg of either CF101 or Cl-IB-MECA had better antiinflammatory effect than the higher 100 mg/kg dose. Decreased expression of TNF-alpha was noted in protein extracts of synovia, draining lymph nodes, and spleen tissues. CONCLUSION: The results provide evidence that A3AR agonists exert significant antirheumatic effects in different autoimmune arthritis models by suppression of TNF-alpha production. The beneficial activity of the drugs at the low dose demonstrates that the effect is A3AR mediated.

CF101, an agonist to the A3 adenosine receptor, enhances the chemotherapeutic effect of 5-fluorouracil in a colon carcinoma murine model.

NF-kappaB and the upstream kinase PKB/Akt are highly expressed in chemoresistance tumor cells and may hamper the apoptotic pathway. CF101, a specific agonist to the A3 adenosine receptor (A3AR), inhibits the development of colon carcinoma growth in cell cultures and xenograft murine models. Because CF101 has been shown to downregulate PKB/Akt and NF-kappaB protein expression level, we presumed that its combination with chemotherapy will enhance the antitumor effect of the cytotoxic drug. In this study, we utilized 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and a colon carcinoma xenograft model. It has been shown that a combined treatment of CF101 and 5-fluorouracil (5-FU) enhanced the cytotoxic effect of the latter on HCT-116 human colon carcinoma cell proliferation and tumor growth. Downregulation of PKB/Akt, NF-kappaB, and cyclin D1, and upregulation of caspase-3 protein expression level were observed in cells and tumor lesions on treatment with a combination of CF101 and 5-FU. Moreover, in mice treated with the combined therapy, myelotoxicity was prevented as was evidenced by normal white blood cell and neutrophil counts. These results show that CF101 potentiates the cytotoxic effect of 5-FU, thus preventing drug resistance. The myeloprotective effect of CF101 suggests its development as an add-on treatment to 5-FU.