Background Antithymocyte globulin (ATG) has been increasingly used to prevent graft-vs-host disease (GVHD), however, its impact on immune reconstitution is relatively unknown. Here we studied (1) immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (2) determined factors influencing the reconstitution, and (3) compared it to non-ATG-conditioned HCT. Methods Immune cell subset counts were determined at 1–24 months posttransplant in 125 HCT recipients who received ATG during conditioning. The subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated). Results (1) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant NKT (iNKT) cells. (2) Faster reconstitution after ATG-conditioned HCT was associated with higher number of cells of the same subset transferred with the graft in case of memory B cells, naïve CD4 T cells, naïve CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in case of naïve CD4 T cells and naïve CD8 T cells; cytomegalovirus recipient seropositivity in case of memory/effector T cells; absence of GVHD in case of naïve B cells; lower ATG serum levels in case of most T cell subsets including iNKT cells, and higher ATG levels in case of NK cells and B cells. (3) Compared to non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells. Conclusions ATG worsens reconstitution of CD4 T cells but improves reconstitution of NK and B cells.

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor. Cancer Res; 70(6); 2435-44. ©2010 AACR.

Reoviruses infect cells that manifest an

MS4A4A is a member of the membrane-spanning, four domain family, subfamily A (MS4A) that includes CD20 (MS4A1), FcRβ (MS4A2) and Htm4 (MS4A3). Like the first three members of this family, transcription of MS4A4A appears to be limited to hematopoietic cells. To evaluate expression of the MS4A4A protein in hematopoietic cell lineages and subsets we generated monoclonal antibodies against extracellular epitopes for use in flow cytometry. In human peripheral blood we found that MS4A4A is expressed at the plasma membrane in monocytes but not in granulocytes or lymphocytes. In vitro differentiation of monocytes demonstrated that MS4A4A is expressed in immature but not activated dendritic cells, and in macrophages generated in the presence of interleukin-4 ('alternatively activated' or M2 macrophages) but not by interferon-γ and lipopolysaccharide ('classically' activated or M1 macrophages). MS4A4A was expressed in the U937 monocytic cell line only after differentiation. In normal bone marrow, MS4A4A was expressed in mature monocytes but was undetected, or detected at only a low level, in myeloid/monocytic precursors, as well as their malignant counterparts in patients with various subtypes of myeloid leukemia. Although MS4A4A was not expressed in healthy B lymphocytes, it was highly expressed in normal plasma cells, CD138+ cells from multiple myeloma patients, and bone marrow B cells from a patient with mantle cell lymphoma. These findings suggest immunotherapeutic potential for MS4A4A antibodies in targeting alternatively activated macrophages such as tumor-associated macrophages, and in the treatment of multiple myeloma and mantle cell lymphoma.

Hematologic stem cell rescue after highdose cytotoxic therapy is extensively used for the treatment of many hematopoietic and solid cancers. Gene marking studies suggest that occult tumor cells within the autograft may contribute to clinical relapse. To date purging of autografts contaminated with cancer cells has been unsuccessful. The selective oncolytic property of reovirus against myriad malignant histologies in in vitro, in vivo, and ex vivo systems has been previously demonstrated. In the present study we have shown that reovirus can successfully purge cancer cells within autografts. IntroductionAutologous hematopoietic progenitor stem cell (ASC) transplantations following high-dose chemotherapy has gained extensive application as a therapeutic modality in several malignancies. [1][2][3][4][5][6][7] Globally, the number of autologous blood and marrow transplantations now surpasses the number of allotransplantations. [6][7][8] Despite the significant increase in ASC transplantations, controversy still exists as to the contribution of minimal residual disease to the development of relapse after high-dose chemotherapy. Evidence supported by gene marking studies indicates relapse following high-dose ablative therapy followed by ASC transplantation may be due to contaminating cancer cells within the autograft. [9][10][11] It has been estimated that more than 30% of all autografts are contaminated with tumor cells, and this number likely will increase with better detection methodology and increasing use of ASC transplantations in patients with advanced disease.To minimize the number of contaminating tumor cells, a variety of purging techniques to rid the graft of residual tumor cells have been used. The ideal purging technique should preferentially destroy contaminating tumor cells while preserving the number and function of the collected stem cells. Widely cited purging methods of autografts include the use of ex vivo chemotherapy, tumor targeting monoclonal antibodies linked with toxins or selected on immunocolumns, and positive (CD34 ϩ ) selection. 12,13 More recently, photodynamic purging processes, [14][15][16] virus-directed enzyme prodrug therapy, 17 receptor-targeted ligand toxins, 18,19 and attenuated replication-competent virus-based purging techniques 20 have been reported. Yet, to date no method has proved 100% successful in depleting autografts of tumor cells in the clinical setting. Although several studies have suggested that graft manipulation is of clinical benefit, 21-24 until purging techniques lead to a complete eradication of contaminating tumor cells, it will be unclear whether the recurrence of the disease is the result of the contaminating tumor cells or a reflection of a resistant in vivo malignancy or both. In the present study we investigated the use of an unattenuated oncolytic virus, reovirus, as the basis for a novel purging strategy for ASC transplantation.Reovirus is an ubiquitous double-stranded RNA virus that can be isolated from the upper respiratory and gastrointestinal tract...

Purpose: Despite the recent advances made in the treatment of multiple myeloma, the disease still remains incurable. The oncolytic potential of reovirus has previously been shown and is currently in phase III clinical trials for solid tumors. We tested the hypothesis that reovirus can successfully target human multiple myeloma in vitro, ex vivo, and in vivo without affecting human hematopoietic stem cell (HHSC) repopulation/differentiation in a murine model that partially recapitulates human multiple myeloma.Experimental Design: Human myeloma cell lines and ex vivo tumor specimens were exposed to reovirus and oncolysis and mechanisms of cell death were assessed. RPMI 8226GFPþ cells were injected intravenously to non-obese diabetic/severe combined immune deficient (NOD/SCID) mice and treated with live reovirus (LV) or dead virus (DV). Multiple myeloma disease progression was evaluated via whole-body fluorescence and bone marrow infiltration. HHSCs exposed to LV/DV were injected to NOD/SCID mice and repopulation/differentiation was monitored.Results: A total of six of seven myeloma cell lines and five of seven patient tumor specimens exposed to reovirus showed significant in vitro sensitivity. Tumor response of multiple myeloma by LV, but not DV, was confirmed by comparison of total tumor weights (P ¼ 0.05), and bone marrow infiltration (1/6, LV; 5/6, DV). Mice injected with LV-or DV-exposed HHSCs maintained in vivo re-population/lineage differentiation showing a lack of viral effect on the stem cell compartment. Reovirus oncolysis was mediated primarily by activation of the apoptotic pathways.Conclusions: The unique ability of reovirus to selectively kill multiple myeloma while sparing HHSCs places it as a promising systemic multiple myeloma therapeutic for clinical testing.

Graft-versus-host disease (GVHD) is a major transplantation complication. The purpose of this study was to measure immune cell subsets by flow cytometry early after transplantation (before median day of GVHD onset) to identify subsets that may play a role in GVHD pathogenesis. We also measured the subsets later after transplantation to determine which subsets may be influenced by GVHD or its treatment. We studied 219 patients. We found that acute GVHD (aGVHD) was preceded by high counts of CD4 T cells and CD8 T cells. It was followed by low counts of total and naive B cells, total and cytolytic NK cells, and myeloid and plasmacytoid dendritic cells. Chronic GVHD (cGVHD) was preceded by low counts of memory B cells. In conclusion, both CD4 and CD8 T cells appear to play a role in the pathogenesis of aGVHD. Generation of B cells, NK cells, and dendritic cells may be hampered by aGVHD and/or its treatment. Memory B cells may inhibit the development of cGVHD.

The Th1/Th2 paradigm can distinguish CD from UC and may be further associated with response to tumor necrosis factor inhibitor in CD and disease activity in patients with IBD.