Adenosine is a purine nucleoside that acts as a regulatory molecule by binding to specific G-protein-coupled A1, A 2A , A 2B , and A3 cell surface receptors. We have recently demonstrated that adenosine inhibits tumour cell growth and concomitantly stimulates bone marrow cell proliferation via activation of the A3 adenosine receptor (A3AR). In the present study, we show that a synthetic agonist to the A3AR, CF101, at the low nanomolar concentration range, inhibits HCT-116 human colon carcinoma cell growth. This effect was reversed by the selective A3AR antagonist MRS1523, demonstrating the specificity of the response. CF101 (given orally) was efficacious in inhibiting the development of primary tumours in xenograft and syngeneic models in which mice were inoculated subcutaneously with human HCT-116 or murine CT-26 colon carcinoma cells, respectively. Moreover, CF101 suppressed (50%, Po0.01) colon cancer liver metastases in syngeneic mice inoculated to the spleen with CT-26 cells. The mechanism of action entailed upregulation of interleukin-12 production in the CF101-treated groups and potentiation of NK cell activity. In the HCT-116 xenograft model in which a combined therapy of CF101 and 5-fluorouracyl (5-FU) was examined, an additive antitumour effect was demonstrated. Moreover, CF101 prevented the 5-FU-induced myelotoxicity, resulting in normal values of white blood cell and neutrophil counts. We conclude that the A3AR agonist CF101, a small orally bioavailable molecule, exerts systemic anticancer, antimetastatic, and myeloprotective effects in colon carcinoma-bearing mice, and may serve as an adjuvant treatment to enhance the chemotherapeutic index and prevent myelotoxicity.
SummaryT-lymphocyte activation triggered by anti-CD3, endogenous or exogenous superantigen, and mitogens was suppressed in a cell-dose-dependent fashion by peritoneal cavity (PerC) leucocytes. Study of lymphocyte-deficient mice and the use of multiparameter fluorescence-activated cell sorter analyses revealed that macrophages were responsible for this form of immune regulation. Interferon-c was essential to trigger suppression, which, by enzyme inhibition studies, was shown to be the result of tryptophan and arginine catabolism. These results illustrate that macrophages, which are classically defined by their innate effector function as antigen-presenting cells, have the potential to temper adaptive immunity.
The T cell composition of the peritoneal cavity (PerC) in naïve BALB/c, C57BL/6, DBA/2J, and B-1 B cell-defective BALB.xid mice was investigated. The BALB.xid PerC T cell pool had a high CD4:CD8 T cell ratio relative to the other strains whose ratios were similar to those found in their lymph node and spleen. All mice had significant representation of T cells with an activated (CD25 + , GITR hi , CD44 hi , CD45RB lo , CD62L lo ) phenotype and low numbers of Foxp3 + T reg cells in their PerC. Despite a phenotype indicative of activation, peritoneal T cell responses to CD3 ligation were very low for C57BL/6 and BALB.xid, but not BALB/c, mice. Enzyme inhibition and cytokine neutralization studies revealed active suppression of the T cell response mediated by the macrophages that represent a significant portion of PerC leucocytes. Driven by IFNγ to express iNOS, macrophages suppressed T cell activation in vitro by arginine catabolism. Although BALB/ c T cells were also in a macrophage-dense environment their limited IFNγ production failed to trigger suppression. This difference between BALB/c and BALB.xid PerC T cells suggests a role for xid in shaping macrophage-mediated immune regulation.
Mature T cells bearing αβ 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 β 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 β 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.
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.
The interaction of αβ 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.T he genes for immunoglobulins (Igs), αβ T-cell receptors (TCRs), and antigen-presenting MHC proteins appeared at least 450 million years ago in the cartilaginous fish and are present in all modern vertebrates (1-3). The more primitive hagfish and lampreys lack these genes and have an adaptive immune system comprised of unrelated proteins (4). The main ligands for αβ TCRs are short peptides derived from self and foreign proteins, captured in a specialized groove of MHC class I (MHCI) and class II (MHCII) molecules and presented to T cells (5, 6). Functional Igs and TCRs are created by very similar recombination mechanisms involving fusion of V, J, and sometimes D gene segments with additional variations at the junctions to create an enormous potential repertoire of Igs and TCRs, suggesting a common, unknown evolutionary origin for these loci.These observations have raised several unanswered questions. For example,why did a separate TCR-rearranging gene system develop for lymphocytes recognizing peptide-MHC ligands? How did the extraordinarily polymorphic MHC genes stay functionally connected to TCR genes throughout 450 million years of evolution? One long-standing hypothesis has been that certain features of TCRs and MHC molecules are evolutionarily conserved to promote their interaction (7-10). Like Igs, the antigen-recognition portions of TCRs are partially encoded in the complementary determining region (CDR) CDR1 and CDR2 loops of germ-line TCR Vα (TRAV) and Vβ (TRBV) genes and are partially generated by somatic recombination processes that form the CDR3 loops. This initial repertoire is culled dramatically during T-cell development in the thymus. First, only those T cells whose TCRs have at least some minimal affinity for the selfpeptide-MHC molecules expressed in the thymus are positively selected for fu...
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 IIlo, B7lo) 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 mAb treatment, this “co-suppression” response was due to CD28 ligation increasing the number of IFNγ-secreting cells. Our results illustrate that cellular composition and cytokine milieu influence T cell costimulation biology.
High Mφ:T cell ratios suppress the immune response to the retroviral superantigen Mls by IFNγ-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 Mφ 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 Mφ:T cell ratios found in many tumors.
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