Using a model of established malignancy, we found that cyclophosphamide (Cy), administered at a dose not requiring hematopoietic stem cell support, is superior to low-dose total body irradiation in augmenting antitumor immunity. We observed that Cy administration resulted in expansion of tumor antigen-specific T cells and transient depletion of CD4+Foxp3+ regulatory T cells (Tregs). The antitumor efficacy of Cy was not improved by administration of anti-CD25 monoclonal antibody given to induce more profound Treg depletion. We found that Cy, through its myelosuppressive action, induced rebound myelopoiesis and perturbed dendritic cell (DC) homeostasis. The resulting DC turnover led to the emergence of tumor-infiltrating DCs that secreted more IL-12 and less IL-10 compared to those from untreated tumor-bearing animals. These newly recruited DCs, originating from proliferating early DC progenitors, were fully capable of priming T cell responses and ineffective in inducing expansion of Tregs. Together, our results show that Cy-mediated antitumor effects extend beyond the well-documented cytotoxicity and lymphodepletion and include resetting the DC homeostasis, thus providing an excellent platform for integration with other immunotherapeutic strategies.

Human immunodeficiency virus (HIV-1) infection causes chronic inflammation. COX-2 derived prostaglandin E2 (PGE2) has been linked to both inflammation and carcinogenesis. We hypothesized that HIV-1 could induce COX-2 in cervical tissue and increase systemic PGE2 levels and that these alterations could play a role in AIDS-related cervical cancer. Levels of cervical COX-2 mRNA and urinary PGE-M, a biomarker of systemic PGE2 levels, were determined in 17 HIV-negative women with a negative cervical HPV test, 18 HIV-infected women with a negative HPV test, and 13 HIV-infected women with cervical HPV and high-grade squamous intraepithelial lesions on cytology. Cervical COX-2 levels were significantly associated with HIV and HPV status (P=0.006 and 0.002, respectively). Median levels of urinary PGE-M were increased in HIV-infected compared to uninfected women (11.2 ng/mg creatinine vs. 6.8 ng/mg creatinine, P=0.02). Among HIV infected women, urinary PGE-M levels were positively correlated with plasma HIV-1 RNA levels (P<0.001). Finally, levels of cervical COX-2 correlated with urinary PGE-M levels (P=0.005). This study demonstrates that HIV-1 infection is associated with increased cervical COX-2 and elevated systemic PGE2 levels. Drugs that inhibit the synthesis of PGE2 may prove useful in reducing the risk of cervical cancer or systemic inflammation in HIV infected women.

Mouse models of minor histocompatibility Ag-mismatched bone marrow transplantation were used to study donor dendritic cell (DC) reconstitution after conditioning, variables influencing the persistence of residual host DCs in different compartments, their phenotype, and their role in governing donor lymphocyte infusion (DLI)-mediated alloresponses. Reconstitution of all splenic DC subsets occurred rapidly after bone marrow transplantation and before T cell reconstitution. However, in contrast to MHC-mismatched chimeras, residual host-derived DCs persisted in the cutaneous lymph nodes (CLNs) of MHC-matched chimeras despite the presence or addition of donor T cells to the graft. The phenotype of these residual host-derived DCs in CLNs was consistent with Langerhans’ cells (LCs). We confirmed their skin origin and found near-complete preservation of host-derived LCs in the skin. Host-derived LCs retained their ability to continuously traffic to the CLNs, expressed homogeneously increased levels of costimulatory molecules, and could capture and carry epicutaneously applied Ags. To determine the role of residual host LCs in governing DLI-mediated alloresponses, we administered DLI alone or after topical application of the TLR7 ligand imiquimod, which is known to enhance the LC emigration from the skin. DLI administration resulted in a decrease in host-derived DCs in the CLNs and increased recruitment of donor-derived DCs to the skin, whereas imiquimod augmented their alloreactivity. These results suggest uniqueness of the MHC-matched setting in relation to the persistence of host-derived DCs in the skin and points to a previously unrecognized role of host-derived LCs in the induction of DLI-mediated graft-vs-host alloresponses.

Murine models of bone marrow transplantation were used to study the mechanisms governing the activation of donor lymphocyte infusions (DLIs) manifesting as lymphohematopoietic graft-versushost (LH-GVH) and graft-versus-leukemia (GVL) reactivities. We demonstrate here that established mixed chimerism influences the potency of DLI-mediated alloreactivity only in the MHC-mismatched but not MHC-matched setting. In the MHCmatched setting, high levels (> 40%) of residual host chimerism correlated negatively with DLI-mediated alloreactivity irrespective of the timing of their administration, the donor's previous sensitization to host antigens, or the level of residual host APCs. In vivo administration of Tolllike receptor (TLR) ligands was required to maximize DLI-mediated LH-GVH and GVL reactivities in chimeras with low levels (< 15%) of residual host chimerism. In contrast, coadministration of DLI with antigen-presenting cell (

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The role of host antigen presenting cells (APCs), particularly DCs in governing DLI-mediated alloresponses post MHC-matched alloBMT is unclear. To evaluate whether the amount of residual host-derived APCs determines the alloresponse of DLI we constructed MHC-matched B10.D2 (H2d)→BALB/c (H2d) chimeras with varying degrees of residual host APCs. After 8 weeks of rest, chimeras received DLI in the form of 2 x 107 splenocytes from B10.D2 (Thy1.1+) mice that differ from BM donors by the expression of Thy 1.1+ marker on the lymphocytes. The differential expression of the Thy 1.1+ marker on DLI-derived T cells allowed us to precisely monitor their alloresponses in vivo. We found that in the MHC-matched model of alloBMT, contrary to the MHC-mismatched model, the amount of residual host APCs does not influence DLI-mediated T-cell alloresponses measured as the alloantigen-driven expansion of DLI-derived T cells and changes in donor chimerism. However, the early administration of DLI, 4 weeks after the conditioning resulted in statistically significant expansion of DLI-derived CD8+ and CD4+ T cells (p&lt;0.01). Since these data suggest the importance of timing of DLI administration on the DLI-mediated alloresponses in MHC-matched setting, we next evaluated whether the augmented alloresponses may be reflective of kinetics of donor DC reconstitution, changes in DC populations or their qualitative characteristics at different time points post-transplant. To test these variables we used C3H.SW (H-2Kb, CD45.2+)→B6Ly5.2 (H-2Kb, CD45.1+) MHC-matched model that enabled us to dissociate the origin of DCs at different time points post-transplant based on the differential expression of CD45 marker on all host and donor hematopoietic tissues. We found that host to donor DC turnover is rapid, results in near full conversion to donor DC chimerism in the spleen (&gt;99 %) and mesenteric lymph nodes (LN) (&gt;99%), but not in the subcutaneous LNs ( 90%). Comprehensive phenotypic analysis showed that donor-derived DC populations in the spleen and LNs do not differ in the chimeras early or late after conditioning, however, residual host-derived DCs express significantly higher levels of costimulatory molecules, CD80, CD40 and especially CD86 at early time points after conditioning (3 weeks vs 8 weeks). These finding suggest that residual host DC activation status may be the critical factor influencing the potency of DLI-mediated responses. To further investigate the role of DC activation status, we co-administered DLI with ex vivo-matured DCs or systemically activated them with agonistic anti-CD40 antibody or CpG immunostimulatory oligonucleotides that stimulate Toll-like receptor (TLR)-9. While both approaches augmented the in vivo proliferation of DLI-derived T cells, only CpGs augmented effector function of DLI-derived T cells, particularly the production of interferon-γ (p&lt;0.005) and conversion to full donor chimerism. Additionally, in the post-transplant C1498 leukemia relapse model survival of C3H.SW→C57BL/6 chimeras that received DLI and CpG immunostimulatory molecules was superior to the chimeras that received DLI only or DLI + ex vivo-matured host DCs (10/10 vs 2/10 vs 2/10; p&lt;0.01). These data indicate that DLI-mediated alloresponses in the setting of MHC-matched alloBMT are critically influenced by the activation status of residual host DCs and can be further manipulated by in vivo administering TLR-9 ligands but not by administering ex vivo-matured host DCs.