During the last year, the project has focused on chemokine receptors on human helper (CD4+) T cells and on cancer cells. We described the patterns of expression for eight chemokine receptors on blood CD4+ T cells from multiple healthy adults, and we studied in detail the subsets of memory CD4+ T cells that express the receptors CCR5 and CCR2. Our studies suggested a pathway of progressive effector/memory differentiation from the CCR5-CCR2- to CCR5+CCR2- to CCR5+CCR2+ cells. Sensitivity and rapidity of T cell receptor (TCR)-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The CCR5+CCR2+ cells showed the greatest responses to tetanus toxoid (the result of prior vaccination) and herpes simplex virus-1 (the result of prior and persistent (latent) infection). CCR5+CCR2+ cells also expressed the largest repertoire of chemokine receptors. By contrast, the CCR5+CCR2- cells had the greatest percentages of suppressor CD4+ T cells and activated and dividing cells, and were most susceptible to programmed cell death. Data from some mouse models have suggested that CD4+ T cells with effector capabilities, which would be important for initiating immediate protection against infection, are short-lived in vivo. Our results are important because they indicate that, in fact, increasing memory cell differentiation can be uncoupled from susceptibility to cell death and is associated with responsiveness to multiple inflammation-associated chemokines, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells, which are highly enriched in the CCR5+CCR2+ subset and are ideally equipped for migration into tissue in order to mediate rapid recall reactions against infectious agents. Increasing the numbers of such cells in response to immunization might enhance the efficacy of vaccines. In other studies, we analyzed histone modifications at the promoters of the genes encoding CXCR3 and CCR4 in order to understand how expression of these receptors is regulated. CXCR3 and CCR4 are receptors associated with Th1 and Th2 differentiation, respectively, and we analyzed patterns of histone modifications on cord blood cells activated in vitro under Th1 or Th2 polarizing conditions as well as on CD4+ T cells isolated directly from adult blood. Our most notable observation was that the gene for CCR4 could be highly induced in cord blood cells under non-polarizing conditions in vitro without many of the activating changes in histone H3 that were found at CCR4 in Th2 polarized cells and in CXCR3-CCR4+ cells from blood. The low levels of the enabling histone H3 modifications at CCR4 suggested that the gene remained in a relatively inactive configuration in the non-polarized cells, which was supported by our finding that without continued activation, levels of CCR4 expression in the non-polarized cells fell over time. By contrast, levels of CCR4 were maintained on the cells that had been activated initially under Th2 conditions. An important conclusion was that the roles for histone H3 modifications in gene expression can depend on the overall transcriptional environment, producing significant discordance between modifications at promoter histones and expression even for a single gene. Moreover, our data demonstrated that histone modifications contribute to the mechanisms whereby gene expression can be made persistent versus transient. Our studies on chemokine receptors in cancer focused on CXCR4, which has now been reported in more than 23 types of human cancer. The data suggest that CXCR4 is a potential marker on tumors for aggressive behavior and/or poor response to therapy, as well as a possible therapeutic target. Currently, CXCR4 expression in tumors can only be evaluated in biopsy specimens, which provide data limited to the site of the biopsy and to a single time point. AMD3100 is a CXCR4 antagonist that is FDA-approved as plerixafor/MozobilTM for the mobilization of hematopoietic stem cells, and we have evaluated a radiolabeled form of AMD3100 (64CuAMD) in order to detect and quantify CXCR4 expression in cancer using positron emission tomography (PET). For preliminary studies in mice, we used Chinese hamster ovary (CHO) cells and Lewis lung carcinoma (3LL) cells that had been transfected to express CXCR4, along with the parental control cells, to produce subcutaneous and/or lung tumors. CXCR4-expressing but not CXCR4-negative tumors could be visualized by PET in both subcutaneous sites and in the lung at six hours after injection of 64CuAMD. Dosimetry experiments were also done in mice for estimating the allowable dose for administration to humans, which provided a tentative limit of 444 MBq (12 mCi), which might make imaging in patients possible.
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