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.

BackgroundHigh concentrations of plasmatic IgE have been related to distinct systemic inflammatory conditions that frequently predispose individuals to hypersensitivity reactions. Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce. MC are an important component in tumor microenvironment where they seem to secrete a number of immunomodulatory and angiogenic mediators, such as the Vascular Endothelial Growth Factor (VEGF) by not well-described mechanisms. In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth.MethodsFor in vitro studies, murine bone marrow-derived mast cells (BMMCs) were used. Pharmacological inhibitors and phosphorylation of key elements controlling VEGF secretion and protein translation were used to characterize the mechanism of VEGF production triggered by IgE.In vivo, the effect of a single i.v. administration of monomeric IgE on B16 melanoma tumor weight, intratumoral blood vessel formation and tumor-associated MC was assessed in four groups of mice: MC-proficient (WT), MC-deficient (Wsh), Wsh reconstituted with MC derived from WT mice (Wsh Rec WT) and Wsh reconstituted with MC derived from Fyn −/− mice (Wsh Rec Fyn −/−).ResultsMonomeric IgE induced VEGF secretion through a Fyn kinase-dependent mechanism and modulated de novo protein synthesis modifying the activity of the translational regulator 4E-BP1 in BMMCs. In vivo, monomeric IgE increased melanoma tumor growth, peritumoral MC and blood vessel numbers in WT but not in Wsh mice. The positive effects of IgE on melanoma tumor growth were reproduced after reconstitution of Wsh mice with WT but not with Fyn −/− BMMCs.ConclusionOur data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.

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Murine peritoneal cavity (PerC) T cells from C57BL/6J mice respond poorly to CD3 ligation due to the high concentration of macrophages (mf) found in this anatomic location. Mfs suppressed T cell activation by their catabolism of ARG by iNOS. To further investigate PerC mf-T cell interaction, we characterized the immunobiology of these cells from C57BL/6J, BALB/c, BALB.xid, and DBA/2J mice. C57BL/6J mice had the greatest number of PerC T cells and the lowest CD4:CD8 ratio. Comprehensive FACS analysis revealed an increased frequency of activated (CD44hi, CD62lo, CD45Rblo, GITRhi) T cells in the PerC relative to conventional lymphoid (lymph node, spleen) tissue. Although there were significant numbers of CD25+, GITR+ T cells in the PerC, Foxp3+ Treg cells were not enriched relative to LN or SP cells. The degree of suppression of T cell activation varied among the strains as well as the mechanism involved. Immunosuppressive prostaglandin production was a modest factor in all strains. Suppression best correlated with the number of IFNg-secreting cells: PerC cells from the “Th1 strain” C57BL/6J and the “Th2 strain” BALB/c were the most and least suppressed, respectively. These results reveal strain-specific differences in PerC mf suppression of T cells. Supported by NIH AREA R15-CA136901-01.

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Although cells essential for immunity exist within tumors they often fail to prevent cancer progression. T cell activation is often suppressed in tumors. Our model mimics this aspect of tumors. As seen when tumor macrophage (Mf) numbers are increased, we find high Mf:T cell ratios foster suppression. Mf catabolism of ARG and TRP contributes to reduced T cell activation as IDO and iNOS inhibitors permit T cell activation. Using FACS we assessed expression of costimulatory ligand‐receptor combinations to determine if their aberrant expression correlated with suppression. The MFI of Mf Class II expression was reduced in suppressed cultures. In contrast, CD80 and CD86 MFI increased. The greatest change was a 20X increase in B7‐H1 expression. Marked up‐regulation was evident by 24 hrs for C57BL/6 but not BALB/c mice. This reflects the Th1/Th2 dichotomy of these strains with IFNg driving more rapid B7‐H1 expression on C57BL/6 Mfs. Mfs lacking the IFNgR failed to suppress and expressed less B7‐H1. Mf‐like B‐1 B cells constitutively expressed B7‐H1. As the Mf:T cell ratio was reduced, T cell activation returned, Class II MHC and CD80 expression increased, and CD86 and B7‐H1 expression decreased. These data reveal targets to curb the suppression evident in tumors with aberrant Mf:T cell ratios. Supported by NIH AREA R15‐AI060356‐01.