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Cytomegalovirus (CMV) infection following allogeneic bone marrow transplant (allo-BMT) is controlled by donor-derived cellular immunity. Vasoactive intestinal peptide (VIP) suppresses Th1 immunity. We hypothesized that blocking VIP-signaling would enhance anti-CMV immunity in murine recipients of allo-BMT. Recipients were transplanted with bone marrow (BM) and T-cells from major histocompatibility complex (MHC)-mismatched VIP-knockout (KO) or wild-type donors, and treated with 7 daily subcutaneous injections of VIPhyb (peptidic VIP-antagonist) or phosphate-buffered saline (PBS). Genetic and pharmacological blockade of VIP-signaling protected allo-BMT recipients from lethal murine CMV (mCMV) infection, improving survival without increasing graft-versus-host disease. Mice treated with VIPhyb or transplanted with VIP-KO allografts had significantly lower viral loads, increased numbers of mCMV-M45-peptide-MHC-tetramer(+) CD8(+) T-cells, with lower PD-1 expression, and enhanced primary and secondary cellular immune responses after mCMV infection than did PBS-treated mice. These results demonstrate that administration of a VIP antagonist after allo-BMT is a promising safely therapeutic approach to enhance antiviral cellular immunity.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2b → CB6F1 and H-2K → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4+CD25+Foxp3+ regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.

Vasoactive intestinal peptide (VIP) induces regulatory dendritic cells (DC) in vitro that inhibit cellular immune responses. We tested the role of physiological levels of VIP on immune responses to murine CMV (mCMV) using VIP-knockout (VIP-KO) mice and radiation chimeras engrafted with syngenic VIP-KO hematopoietic cells. VIP-KO mice had less weight loss and better survival following mCMV infection compared with wild-type (WT) littermates. mCMV-infected VIP-KO mice had lower viral loads, faster clearance of virus, with increased numbers of IFN-γ+ NK and NKT cells, and enhanced cytolytic activity of NK cells. Adaptive antiviral cellular immunity was increased in mCMV-infected VIP-KO mice compared with WT mice, with more Th1/Tc1-polarized T cells, fewer IL-10+ T cells, and more mCMV-M45 epitope peptide MHC class I tetramer+ CD8+ T cells (tetramer+ CD8 T cells). mCMV-immune VIP-KO mice had enhanced ability to clear mCMV peptide-pulsed target cells in vivo. Enhanced antiviral immunity was also seen in WT transplant recipients engrafted with VIP-KO hematopoietic cells, indicating that VIP synthesized by neuronal cells did not suppress immune responses. Following mCMV infection there was a marked upregulation of MHC-II and CD80 costimulatory molecule expression on DC from VIP-KO mice compared with DC from WT mice, whereas programmed death-1 and programmed death ligand-1 expression were upregulated in activated CD8+ T cells and DC, respectively, in WT mice, but not in VIP-KO mice. Because the absence of VIP in immune cells increased innate and adaptive antiviral immunity by altering costimulatory and coinhibitory pathways, selective targeting of VIP signaling represents an attractive therapeutic target to enhance antiviral immunity.

When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform theta (PKCtheta), a key regulator of TCR signaling. In contrast, PKCtheta was required for alloreactivity and GVHD induction. Furthermore, absence of PKCtheta raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCtheta-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCtheta is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.

Inflammatory bowel disease (IBD) severity is positively correlated with cytomegalovirus (CMV) infection. This may reflect CMV triggering and/or exacerbating flares of IBD and/or IBD or immunosuppressive drugs administered to patients with IBD increasing susceptibility to CMV infection. Herein, we performed studies in mice to investigate these possibilities. Mice administered murine CMV (MCMV) developed signs of acute viral infection (malaise and weight loss) and had MCMV loads that were readily detected in numerous organs including the intestine. By 4 weeks, these mice manifested a "latent" infection in which MCMV levels were low but detectable by PCR. Such MCMV infection did not induce acute colitis in either wild-type mice or IL-10(-/-) mice, which are highly prone to developing colitis. However, underlying MCMV infection in an acute or latent state exacerbated the severity of colitis induced by dextran sodium sulfate (DSS). Such potentiation of DSS colitis by latent MCMV infection seemed to occur without viral reactivation. Whereas initial MCMV infection induced acute alterations in serum and intestinal cytokines, such cytokine levels returned to baseline before administration of DSS. However, the initial infection resulted in lasting elevation of antibodies to commensal bacterial antigens, suggesting that MCMV infection may have potentiated colitis via priming of the intestinal immune response to gut microbiota. Thus, underlying CMV infection can alter mucosal immunity, potentially increasing the tendency of CMV-infected hosts to develop colitis.

We have previously shown that amotosalen-treated splenocytes rescued allorecipients from a lethal dose of mouse CMV (MCMV) administered on day 0 in experimental parent C57BL/6→CB6F1 allogeneic bone marrow transplant. In this study, we investigated the mechanism of antiviral activity of amotosalen-treated donor splenocytes when sublethal MCMV infections were administered 7 days posttransplant. Recipients of 3 × 106 untreated splenocytes were used as control. Following MCMV infection, recipients of untreated splenocytes had 40% early mortality due to acute graft-vs-host disease compared with no deaths among recipients of 10 × 106 treated splenocytes. However, recipients of both types of donor splenocytes effectively cleared MCMV from their liver. Like the untreated CD8+ T cells, amotosalen-treated CD8+ T cells equally retained their in vivo CTL activity against MCMV early peptide-pulsed targets and expressed similar levels of granzyme B within 11 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with both donor spleen- and host-derived anti-MCMV CD8+ T cells in their blood and lymphoid organs, with significantly higher numbers of host-derived CD4−CD8− (double negative) T cells in the spleens of recipients of treated splenocytes compared with the recipients of untreated splenocytes. Additionally, recipients of amotosalen-treated splenocytes had lower levels of serum IFN-γ and TNF-α in response to MCMV infection compared with untreated recipients. Thus, adoptive immunotherapy with treated T cells is a novel therapeutic approach that facilitates hematopoietic engraftment and permits antiviral immunity of both donor and host T cells without graft-vs-host disease.

Chronic graft-versus-host disease (cGvHD) is associated with functional immunodeficiency and an increased risk of opportunistic infections in allogeneic bone marrow transplantation (BMT). We used a parent to F1 model of allogeneic BMT to test the hypothesis that cGvHD leads to impaired antigen-specific antiviral immunity and compared BM transplant recipients with cGvHD to control groups of allogeneic BM transplant recipients without GvHD. Mice with and without cGvHD received a nonlethal dose of murine cytomegalovirus (MCMV) +100 days after transplantation. Recipients with cGvHD had more weight loss and higher viral loads in the spleen and liver. MCMV infection led to greater than 25-fold expansion of donor spleen–derived MCMV peptide–specific tetramer-positive CD8+ T cells in blood of transplant recipients with and without cGvHD, but mice with cGvHD had far fewer antigen-specific T cells in peripheral tissues and secondary lymphoid organs. The immunosuppression associated with cGvHD was confirmed by vaccinating transplant recipients with and without cGvHD using a recombinant Listeria expressing MCMV early protein (Lm-MCMV). Secondary adoptive transfer of lymphocytes from donor mice with or without cGvHD into lymphopenic congenic recipients showed that cGvHD impaired tissue-specific homing of antigen-specific T cells. These results indicate that cGvHD causes an intrinsic immunosuppression and explain, in part, the functional immunodeficiency in allogeneic transplant recipients.

Delayed reconstitution of cellular immunity following T-cell-depleted, CD34-enriched, allogeneic hematopoietic progenitor cell transplantation (HPCT) is the major cause of morbidity and mortality following haploidentical transplantation in adults. This is illustrated in our recent study of 28 high-risk adult patients (median age 31) who were treated with conditioning regimens containing antithymocyte globulin (ATG) before T-cell-depleted, CD34-enriched allogeneic HPCT. Overall mortality was 93% (26/28 patients) with a median survival of 4 months posttransplant. Poor cellular immune reconstitution contributed to death of 21/28 patients, with eight deaths due to opportunistic infections and seven deaths due to relapse. While recovery of normal numbers of circulating NK cells and B-cells occurred within the first 1-2 months posttransplant, recovery of normal numbers of blood T-cells was suppressed for more than 1 year. The mean half-life of active ATG levels in serum was 6 days; rapid clearance suggested that residual ATG did not contribute to the delay of posttransplant T-cell reconstitution. Rapid T-cell reconstitution was seen only in younger patients, indicating that poor thymic function and the absence of T-cells in the graft are the major causes of delayed recovery of cellular immunity. Improved cellular immunity after T-cell-depleted haploidentical HPCT will thus require novel strategies to adoptively transfer antigen specific donor T-cells without inducing lethal graft-versus-host disease (GvHD). This problem has been addressed in a preclinical murine model of MHC-mismatched bone marrow transplantation. Donor T-cells treated ex vivo with fludarabine or a UVA light-activated psoralen compound (amotosalen) have a markedly reduced ability to induce GvHD, yet the treated T-cells confer protection against murine cytomegalovirus and an infused leukemic cell line. Polyclonal donor T-cells reconstituted the blood and lymphoid compartments posttransplant and expanded in vivo. Derivatives of ex-vivo-treated donor T-cells retained the ability to produce cytokines and proliferate in response to antigen challenge. The mechanism of reduced GvHD potential of ex-vivo-treated T-cells appears to be selection of a subset of memory donor T-cells that do not initially home to secondary lymphoid organs and have reduced capacity for producing inflammation in the immediate posttransplant period. Direct selection of the memory subset by high-speed FACS confirmed the improved therapeutic index in the murine model system. Preclinical data indicate the feasibility of treating human T-cells with fludarabine, psoralen, or direct selection based upon the memory phenotype to efficiently produce a population of polyclonal donor T-cells with reduced GvHD activity. A planned clinical phase 1 trial of adoptive therapy utilizing ex vivo psoralen-treated donor T-cells in recipients of T-cell-depleted haploidentical HPCT is presented.

A major goal of our research is to reduce the graft-vs-host disease (GVHD) activity of allogeneic donor T cells in bone marrow transplantation (BMT), while preserving graft-vs-tumor (GVT) effects. Using ex vivo immunosuppressive strategies and cell-separation techniques to modulate the graft prior to transplantation, we examined the roles of different donor immune cells on GVHD and GVT effects in allogeneic mouse models. Our results demonstrate that donor-memory CD4 T cells facilitate posttransplant immunological reconstitution without causing GVHD, whereas transplantation of equal numbers of donor naïve CD4 T cells leads to fatal GVHD. The initial events of donor T cells interacting with antigen-presenting cells (APCs) in the transplant recipient appear to be critical to the development of GVT, GVHD, or anergy to alloantigens. In the setting of clinical BMT, increased numbers of donor type 2 dendritic cells (DCs) were associated with an increased rate of posttransplant relapse, and decreased rates of chronic GVHD. In a mouse transplant model, manipulation of the DC content of bone marrow grafts was achieved by depletion of CD11b+ cells. Mice transplanted with CD11b- depleted marrow showed enhanced posttransplant expansion of memory T cells with markedly improved GVT activity and limited GVHD compared to recipients of unmanipulated marrow. A model that differentiates GVT from GVHD based on interaction of T-cell subsets with DC subsets is proposed.

Infusion of donor antiviral T cells can provide protective immunity for recipients of hemopoietic progenitor cell transplants, but may cause graft-vs-host disease (GVHD). Current methods of separating antiviral T cells from the alloreactive T cells that produce GVHD are neither routine nor rapid. In a model of lethal murine CMV (MCMV) infection following MHC-mismatched bone marrow transplantation, infusion of MCMV-immune donor lymphocytes pretreated with the DNA cross-linking compound amotosalen prevented MCMV lethality without producing GVHD. Although 95% of mice receiving 30 × 106 pretreated donor lymphocytes survived beyond day +100 without MCMV disease or GVHD, all mice receiving equivalent numbers of untreated lymphocytes rapidly died of GVHD. In vitro, amotosalen blocked T cell proliferation without suppressing MCMV peptide-induced IFN-γ production by MCMV-primed CD8+ T cells. In vivo, pretreated lymphocytes reduced hepatic MCMV load by 4-log10 and promoted full hemopoietic chimerism. Amotosalen-treated, MCMV tetramer-positive memory (CD44high) CD8+ T cells persisted to day +100 following infusion, and expressed IFN-γ when presented with viral peptide. Pretreated T cells were effective at preventing MCMV lethality over a wide range of concentrations. Thus, amotosalen treatment rapidly eliminates the GVHD activity of polyclonal T cells, while preserving long-term antiviral and graft facilitation effects, and may be clinically useful for routine adoptive immunotherapy.

We have previously shown that highly purified cGMP grade recombinant flagellin, CBLB502, a TLR5 agonist, protected allo-HSCT recipients from GvHD without compromising anti-CMV immunity of donor T cells in vivo. A single prophylactic dose (25mg/mouse i.p) of CBLB502 in wild type C57BL/6 (B6) mice also enhanced anti-CMV immunity of NK cells against CMV. CMV is a potential opportunistic infectious agent and most often causes severe life-threatening clinical complications in immunocompromized patients, particularly in cancer patients treated with allogenic bone marrow transplantation (allo-BMT). A mechanism by which CBLB502 protected host against CMV infection has not been well investigated. Transcription factors ROR gammaT, T-bet and EOMES are known to control immune responses of innate and adaptive immunity in vivo. In this study, we have explored the transcription factors ROR gammaT, T-bet and EOMES expressing NK and T cells in CBLB502-treated and MCMV-infected B6 mice. Total numbers of spleen cells increased by 1.7-fold within 48 hours of CBLB502 treatment (day 0 MCMV infection) and by 2.3-fold on day 3 after MCMV infection compared with PBS-treated MCMV-infected mice. T-bet expressing CD8+ T cells (not CD4+ T cells) increased significantly on day 0 of mCMV infection, 48hrs after CBLB502 treatment (CD4, p=0.1; CD8, p90% of CMV-specific CD8+ T cells had persistent expression of T-bet even after 30 days post CMV infection. In contrast, CBLB502 treatment did not have a significant effect on the numbers of ROR gammaT, T-bet and EOMES expressing NK cells compared with PBS-treated mice. These data suggest that 1) CBLB502-induced anti-CMV immunity of T cells is persistently maintained through increased T-bet expression but not by EOMES expression; and 2) CBLB502-induced anti-viral immune regulation is not maintained through transcription factor ROR gammaT. 3) CBLB502-induced increased anti-CMV immunity of NK cells is independent of transcription factors ROR gammaT, T-bet and EOMES. Taken together, these data support the early peri-transplant use of TLR5 agonists as a novel method to enhance antigen-specific anti-viral immunity in recipients of allo-BMT. Disclosures No relevant conflicts of interest to declare.

The competence of cellular immunity depends on a diverse T-cell receptor (TCR) repertoire arising from thymic output. Normal thymopoiesis arises from marrow-derived CD3(-)CD4(-)CD8(-) triple-negative T-cell progenitors (TN), which develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive, DP) transiently, leading to de novo T-cell production. Interleukin-7 (IL7) is a singularly important common γ-chain IL involved in normal thymic development. Our previous work has demonstrated that γc cytokines fused with granulocyte-macrophage colony stimulating factor (GMCSF) at the N-terminus acquire unheralded biological properties. Therefore, to enhance thymopoiesis, we developed a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 leads to cortical thymic hyperplasia including the specific expansion of CD44(int)CD25(-) double-negative 1 (DN1) thymic progenitors. During murine cytomegalovirus (mCMV) infection of aged animals, GIFT7-mediated neo-thymopoiesis led to increased absolute numbers of viral-specific CD8(+) T cell. Our work demonstrated that thymic precursors can be therapeutically repopulated and its reconstitution leads to meaningful central and peripheral T-cell neogenesis, correcting immune dysfunction arising from age-associated thymic atrophy.

Donor T-lymphocytes are effective adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), but life threatening complications related to GVHD limits its clinical application. Recent advancement in the field of immunotherapy has directed our interest to enhancing the anti-tumor response of donor T cells by modulating expression of checkpoint blockade molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and foxp3, the transcription factor associated with regulatory T cells. The two ligands of PD-1, PD-L1 or PD-L2 are highly expressed in the presence of inflammatory signal induced by infection or cancer and PD-1/PD-L1 interaction negatively regulates T-cell antigen receptor (TCR) signaling and dampen T cell cytotoxic activity. Herein, we studied the role of PD-1, CTLA-4 and transcription factor foxp3 expressing donor CD4+ and CD8+ T cells in the development of GVHD. Methods: We have used two established allo-HSCT murine GvHD models. Lethally irradiated wild type (WT) B6, PD-L1 knock out (KO) B6 and PD-L2 KO B6 mice were transplanted with 2 x 106 splenic T cells and 2 x 106 T cell depleted bone marrow (TCD BM) cells from H-2Kdonors. Lethally irradiated CB6F1 recipients were similarly transplanted with splenocytes and TCD BM cells from B6 donors. Acute GvHD scores were determined by combining scores obtained from histological tissue sections and weight-loss, posture, activity, fur texture and skin integrity following standard published procedures. The activation status of donor T-cells and BM and host-derived non-T cells in GvHD target organs was analyzed by flow cytometry. Data from allo-HSCT recipients were compared with the respective data obtained from B6 à B6 syngenic HSCT (syn-HSCT) recipients. Serum cytokines were determined by Luminex assay. Results: PD-L1 KO B6 allo-HSCT recipients had significantly increased acute GvHD scores compared with WT B6 allo-HSCT recipients (p Collectively, these data indicate that severe GvHD occurs in allo-HSCT recipients in spite of increased numbers of PD-1, CTLA-4 and PD-L1 expressing donor and host cells. The occurrence of severe GvHD in these allo-HSCT models systems was associated with markedly reduced levels of CTLA-4 and foxp3 transcription factor expressing Tregs indicating that these pathways may be more relevant to controlling GvHD than PD-1:PD-L1 expression. Disclosures No relevant conflicts of interest to declare.

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1-mediated cellular immunity. We previously reported that VIP-knockout (VIP-KO) mice have enhanced cellular immune responses and increased survival following murine cytomegalovirus (mCMV) infection in C57BL/6 mice. In this study, we tested whether treatment with a VIP receptor antagonistic peptide protects C57BL/6 and BALB/c mice from mCMV-infection. One week of daily subcutaneous injections of VIPhyb was non-toxic and did not alter frequencies of immune cell subsets in non-infected mice. VIPhyb administration to mCMV-infected C57BL/6 and BALB/c mice markedly enhanced survival, viral clearance, and reduced liver and lung pathology compared with saline-treated controls. The numbers of effector/memory CD8+ T-cells and mature NK cells were increased in VIPhyb-treated mice compared with PBS-treated groups. Pharmacological blockade of VIP-receptor binding or genetic blockade of VIP-signaling prevented the up-regulation of PD-L1 and PD-1 expression on DC and activated CD8+ T-cells, respectively, in mCMV-infected mice, and enhanced CD80, CD86, and MHC-II expression on conventional and plasmacytoid DC. VIPhyb-treatment increased type-I IFN synthesis, numbers of IFN-γ- and TNF-α-expressing NK cells and T-cells, and the numbers of mCMV-M45 epitope-peptide-MHC-I tetramer CD8+ T-cells following mCMV infection. VIP-treatment lowered the percentage of Treg cells in spleens compared with PBS-treated WT mice following mCMV infection, while significantly decreasing levels of serum VEGF induced by mCMV-infection. The mice in all treated groups exhibited similar levels of anti-mCMV antibody titers. Short-term administration of a VIP-receptor antagonist represents a novel approach to enhance innate and adaptive cellular immunity in a murine model of CMV infection.

Abstract 3289 Functional T cell immunity depends on the diversity of T cell receptor (TCR) repertoire. Numerous cytolytic assaults can perturb effector cell fitness by inhibiting thymic de novo T cell production or predisposing peripheral CD8+ T cells to exhaustion. In fact, age-associated thymic atrophy and PD-1 upregulation remain the critical components of immune dysfunction. Normal thymopoiesis arises from marrow-derived CD3−CD4−CD8− triple-negative T cell progenitors (TN) which seed the thymic cortex. Thereafter, TN develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive-DP) transiently, leading to de novo T cell production. Interleukin-7 (IL7) is a singularly important common γ-chain interleukin involved in this process. In order to pharmacologically enhance thymopoiesis, we report the development of a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 compared to IL7 in young immune competent mice leads to a transient increase in the number of DN1 (5.48±1.01 v.s. 2.74±1.07 × 106, p=0.046) by day 7. Total thymic cellularity significantly increases in GIFT7-treated group by day 14 (237±51.3 v.s. 123±25.6 × 106, p=0.026). GIFT7-mediated hypertrophic effect is significantly more pronounced in aged thymi: 111±21.8, 67.8±13.9, 60.3±4.6 × 106 for 7-dose of (5ug kg−1) GIFT7, IL7, or PBS treatment respectively. We observed a significant increase in DN1 and DN4 subsets with the greatest fold difference in the CD44int DN1 subset. Histologically, GIFT7 administration leads to significant cortical thymic hyperplasia (cortex: medulla ratio 2.67:1). Functionally, GIFT7 enhances viral-specific CTL responsiveness as modeled by murine cytomegalovirus (MCMV) peptide MHC+ tetramer enumeration (10.4±3.8 v.s. 2.9±1.4 × 106 for GIFT7 and untreated respectively, p In the periphery, GIFT7 selectively expand a CD8+ subset with a Central Memory (CM) phenotype defined as CD8+CD44+CD62L+CCR7+KLRG−CD27+PD1−. This unique expansive effect of GIFT7 is also demonstrated on peripheral blood mononuclear cells (PBMC) derived from non-human primates (NHP), in that GIFT7 stimulation of pre-activated NHP-PBMC leads to a two-fold increase in total cell number after 3 days and >80% of Ki67+ expression in both CD4+ and CD8+ compartments. Interestingly, GIFT7-mediated proliferation in CD8+ T cells is accompanied by a 40% decrease in the mean fluorescent intensity (MFI) of PD1 expression. Our work identifies a CD44intCD25− subset of DN thymocytes most responsive to IL7R-mediated STAT5 hypersignalling and their function as T cell progenitors in GIFT7-driven thymic reconstitution. This points to the translational potential of GIFT7 or GIFT7-primed T lineage cells as treatment of immune malfunction. Disclosures: No relevant conflicts of interest to declare.

Abstract 255 Background: Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods: To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results: Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures: No relevant conflicts of interest to declare.

Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients. Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures: No relevant conflicts of interest to declare.

Abstract 2458 Poster Board II-435 Introduction: When used as therapy for hematopoietic malignances, allogeneic hematopoietic stem cell transplantation (HCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host-disease (GVHD) is a potentially lethal complication of allogeneic BMT. Thus, inhibition of GVHD, while preserving GVL and protective responses against infectious agents, can enhance the therapeutic potential of BMT. GVHD is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues. Protein kinase C theta isoform (PKCθ) is crucial in TCR signaling and regulates the nature of lymphocyte-specific effector responses. The aim of this study was to investigate the significance of PKCθ in donor T-cell-mediated GVHD, anti-leukemia, and antiviral infection. Methods: Our results were validated using clinically relevant murine bone marrow transplantation (BMT) models. GVHD was measured by recipient survival and clinical signs such as body weight loss. Leukemia/lymphoma was induced by intravenous injection of a luciferase expressing A20 clone (a B lymphoma cell line derived from BALB/c mice). Tumor invasion was quantitated using the Xenogen IVIS-200 bioluminescence imaging system. Murine cytomegalovirus (MCMV) infection was induced by intraperitoneally injecting MCMV in the recipients after GVHD was established. Results: Using 3 distinct mouse models of allogeneic BMT, we found that T cells lacking PKCθ are unable to undergo robust expansion and cause damage to recipient hematopoietic or epithelial tissues, demonstrating that an essential requirement for PKCθ in alloreactivity and GVHD development. In contrast, PKCθ-/- T cells retain the ability to respond to virus infection post-BMT. Mechanistically, absence of PKCθ raises the threshold for T cell activation that selectively impacts alloresponses, but T cell responses triggered by infection or following administration of antigen plus microbial adjuvant are relatively well preserved in the absence of PKCθ Furthermore, we evaluated the role of PKCθ in GVHD and GVL responses, and found that PKCθ-/- T cells have at least a 10-fold reduction in the ability to induce GVHD but only a ∼2.5-fold reduction in GVL compared to WT T cells. Thus, absence of PKCθ impacts GVHD responses more significantly than GVL responses. Conclusion: These findings validate PKCθ as a potentially unique therapeutic target that is required for GVHD induction but not for GVL or protective responses to infectious agents in allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

Background: Allo-reactive donor T cells are primarily responsible for GvHD in allogeneic BMT. A number of studies have shown that increased allo-reactivity is found among the CD62L+ subset of donor T-cells, but the mechanisms for organ specific allo-reactivity are poorly defined. Our hypothesis is that rapid proliferation and migration of CD62L+ naive donor CD4+ and CD8+ T cells to specific organs leads to acute GvHD. Methods: We used a parent (C57BL/6) to (C57BL/6 × BALB/c) CB6F1 allogeneic BMT model with a combination of T cell depleted BM (TCD BM) and splenocytes. 30 × 106 congeneic donor splenocytes labeled with CFSE were transplanted with 5 × 106 TCD congeneic BM into lethally irradiated (11Gy) CB6F1 mice. Recipients were sacrificed within 3.5 days of transplant and FACS was used to measure proliferation of CFSE-labeled donor T-cells isolated from blood, spleen, liver, lungs, thymus, BM, lymph nodes, and peritoneal exudates cells (PEC). Syngeneic C57BL/6 recipients served as controls. At least 5 mice per group were used in each experiment. Results: There was increased homing of CFSE-labeled donor T-cells to most organs in allogeneic compared to syngeneic BMT recipients. CD45.1+ donor cells were 4-fold higher in spleen, p=0.01; 9-fold higher in liver, p=0.002; 14-fold higher in PEC, p=0.017; 136-fold higher in lung, p=0.0006; 126-fold higher in BM, P=0.002, 1482-fold higher in thymus p=0.002 compared to syngeneic recipients. Allogeneic and syngeneic recipients had equivalent numbers of donor CFSE-labeled lymphocytes in PBMC and lymph nodes. The tissue specific homing of CD4+ and CD8+ donor T-cells was also found significantly higher in most organs except the PBMC and LNs. Donor splenocytes were 80% CD62L+ before transplant, but the frequency of CD62L+ donor T-cells had declined to 15–16% in BM, 4–10% in liver, 17–30% in spleen and 10 to 25% in the thymus within 3.5 days post-transplant. In syngeneic recipients, 80% of donor T-cells remained CD62L+ within 3.5 days post-transplant. Most donor T-cells that divided rapidly lost expression of CD62L, while non-replicating donor CD4+ and CD8+ T cells remained predominately CD62L+. The expression of CD44 on donor T-cells were the opposite, with CD44+ cells undergoing less, and CD44− cells dividing more in allogeneic transplant recipients. In syngeneic BMT, donor CD4+ and CD8+ T-cells underwent minimal proliferation within the first 3.5 days post-transplant. Intracellular cytokine staining showed that high levels of IFN-g and TNF-a synthesis was seen among CD62L+ CD4+ and CD8+ T cells that had yet to divide (and had un-diluted CFSE staining). Conclusion: Migration of allogeneic donor T cells to tissues and local proliferation occurs rapidly after allogeneic BMT compared to recipients of syngeneic transplants. The dissociation of CD62L expression from lymph node homing suggests lack of the CD62L-receptor expression in lymph node HEV following irradiation, or a dominant effect of other chemokine receptors in directing donor T-cell preferentially to other organs. The marked and preferential homing of donor T-cells to the recipient thymus and bone marrow may play a role in achieving donor hematopoietic and T-cell chimerism in recipients of allogeneic BMT. Tissue specific homing of naive CD62L+ donor T-cells, with a high proliferative capacity, is likely responsible for the initiation of acute GvHD at these sites.

Background: GvHD and opportunistic infections are the major causes of morbidity and mortality in cancer patients treated with allogeneic BMT. In allogeneic BMT patients, donor derived T-cells help eradicate residual cancer and fight against opportunistic infections but they also cause the major deleterious effects, including GvHD which is the result of host allo-antigens recognition by the donor T-cells. Moreover, donor T-cells also play a critical role in promoting stem cell engraftment, encouraging rapid recovery of cellular immunity, and decreasing the probability of disease relapse. Thus, to establish a therapeutically useful adoptive immunotherapy using donor T-cells, separation of the beneficial anti-opportunistic infection and anti-tumor effects of donor T-cells from the deleterious GvHD effect are highly desirable. We previously showed that amotosalen-treated splenocytes rescued recipients from a lethal dose of MCMV administered on day 0 in experimental parent to F1 allogeneic bone marrow transplant (BMT). To model early post-transplant CMV reaction, in this study, we investigated the anti-viral immune responses and GvHD activity of treated donor T-cells after infecting allogeneic BMT recipients with a lethal dose of MCMV on 7 days post transplant. Methods: Using a parent to F1 mouse BMT model, splenocytes (3×106 untreated or 10×106 amotosalen-treated) harvested from the MCMV immunized C57BL/6 donors were transplanted along with 5×106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 (C57BL/6 × Balb/C) recipients. Recipient mice were infected i.p. with a sublethal dose (5×104 pfu per mouse) of MCMV 7 days after transplant. Flow cytometry was used to quantitate T cell chimerism (in recipient spleen and thymus) and MCMV-peptide specific tetramer+ CD8+ T-cells. Serum IFN-γ and TNF-α were determined by ELISA. Liver viral load was determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: MCMV infection in recipients of amotosalen-treated splenocytes did not cause any mortality whereas recipients of untreated splenocytes had 40% early mortality due to acute GvHD. Like the recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes effectively cleared MCMV from their liver within 10 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with donor spleen- and host-derived MCMV peptide specific tetramer+ CD8+ T cells that proliferated following day 7 post MCMV infection. Significantly higher numbers of host derived CD4−CD8− (DN) TCRαβT-cells appeared in the spleen with peak on day 3 post MCMV infection among recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes. Lower levels of serum IFN-γ and TNF-α and preservation of thymic function were also noted in the recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes following MCMV infection. Conclusion: Adoptive immunotherapy with amotosalen-treated T cells is an ideal therapeutic approach that facilitates early hematopoietic engraftment, anti-viral donor immune reconstitution and preserves early post-transplant host immunity leading to protection from lethal viral infection without causing aGvHD.

Background: In allogeneic BMT patients, the presence of allo-reactive donor CD4+ T cells in the graft were reported to be the primary cause of GvHD. Moreover, donor T-cells are required to promote the stem cell engraftment and to decrease the disease relapse. A number of studies also reported that a subset of CD4+CD25+ T cells usually generated de novo from the thymus that expressed FoxP3 regulate the T cells allo-reactivity in vivo. Thus, to establish a therapeutically useful adoptive T-cells immunotherapy, we depleted the CD4+ T cells from the graft and transplanted along with T cell depleted (TCD) BM cells in clinically relevant parent to F1 experimental allogeneic BMT model. Our hypothesis is that CD4-depleted graft will not cause GvHD, preserve the thymic function, homeostatically produce donor BM-derived CD4+ T cells along with FoxP3+CD4+CD25+ regulatory T cells with beneficial anti-opportunistic infection and anti-tumor effects. Methods: We used a parent (C57BL/6) to (C57BL/6 X BALB/c)CB6F1 allogeneic BMT model with a combination of TCD BM and splenocytes as the hematopoietic graft. CD4+ or CD8+ cells were selectively depleted from the splenocytes of C57BL/6 donor mice using MACS column. 1×106 CD4-depleted splenocytes or a mixture of 2×106 CD8-depleted and 1×106 CD4-depleted splenocytes and/or grafts containing 10×106 unfractionated splenocytes along with 5×106 TCD BM cells harvested from the congeneic C57BL/6 donor mice, were adoptively transferred to lethally irradiated (11Gy) CB6F1 mice. GvHD was monitored twice weekly by weight loss and other clinical signs. After 50 days post transplant recipients mice were bled or sacrificed and lymphocytes isolated from blood and different organs were analyzed by multicolor FACS. Results: Within 50 days of transplant the recipients of CD4-depleted splenocytes had 100% survival without GvHD whereas recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes suffered from severe GvHD (%weight loss below 20%) with 50% survival. Surprisingly, very significantly expansion of total CD4+ T cells (37% ± 7% of lymphocytes, CD4:CD8 ratio 6:1) occurred in the blood of recipients of CD4-depleted splenocytes. In contrast the recipients of mixture of CD4- and CD8-depleted splenocytes DLI or whole splenocytes had only few CD4+ T cells (~2% ± 2% of lymphocytes, CD4:CD8 ratio 1:2). Over 90% of the CD4+ T cells in the blood of recipients of CD4-depleted splenocytes were from the donor BM and included significantly higher number of CD25+CD4+ T cells compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes. Similarly, significantly increased numbers of FoxP3+CD25+CD4+ regularity T cells were also found in the spleen and thymus of recipients of CD4-depleted splenocytes compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes (p Conclusion: Adoptive immunotherapy with the CD4-depleted hematopoietic graft results better immune reconstitution, caused extensive homeostatic expansion of donor stem cell-derived CD4+ T cells including significantly increased levels of FoxP3+CD25+ CD4+ regulatory T cells derived from de novo thymopoiesis without GvHD. The presence of donor FoxP3+CD25+ CD4+ regulatory T cells in the hematopoietic graft are not necessary for post-transplant expansion of donor stem-cell-derived regulatory T-cells via thymopoiesis.

A major problem in allogeneic BMT is post transplant immunodeficiency leading to opportunistic infection and relapse. Previously we showed that amotosalen-treated allogeneic donor T cells given at the time of BMT and experimental murine cytomegalovirus (MCMV) infection could prevent lethal MCMV disease without producing GvHD. In this study we have focused on a more clinically applicable model where prophylactic amotosalen-treated allogeneic donor splenocytes are given at the time of BMT, followed by MCMV infection 100 days later. We observed that amotosalen-treated donor T-cells significantly expanded and responded well in presence of viral infection without inducing any GvHD, protected recipients against viral disease, and were associated with significantly improved hematopoietic engraftment and immune reconstitution. Methods: Using a parent to F1 mouse BMT model, splenocytes (3x106 untreated or 10x106 amotosalen-treated) from MCMV immunized C57BL/6 donors were transplanted along with 5x106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C). Recipient mice were infected i.p. with a sublethal dose (5x104 pfu per mouse) of MCMV 100 days or more after transplant. Clinical chronic GvHD was monitored by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. Flow cytometry was used to quantitate T cell chimerism (in recipient PBMC, spleen, liver and thymus) and MCMV-peptide specific CD8+ T-cells (tetramer+ and IFN-γ producing). Serum IFN-γ and TNF-α were determined by ELISA. Liver and spleen viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: Recipients of untreated control donor splenocytes suffered from chronic GvHD within 100 days of transplant, while those that received amotosalen-treated splenocytes experienced no GvHD. In response to MCMV infection at 100 days post transplant, residual amotosalen-treated donor T-cells rapidly expanded over 25-fold within 10 days, but did not cause lethality or detectable GvHD. Expanded amotosalen-treated T-cells showed activated anti-viral responses and developed a memory phenotype at late phases of viral infection. PBMC, spleen and liver showed elevated levels of MCMV specific tetramer+, IFN-γ+, and TNF-α+ CD8+ T-cells that were associated with accelerated viral clearance within day 3 after viral infection. While expansion and generation of amotosalen-treated donor T-cells mostly occurred in the liver, the generation of donor bone marrow-derived new T-cells occurred through both the thymus and the liver. In contrast, recipients of untreated donor splenocytes had reduced thymic function, resulting in severely impaired immune reconstitution and decreased anti-viral immunity. Conclusion: Prophylactically administered amotosalen-treated allogeneic donor T cells 1) were almost completely devoid of GvHD activity, 2) promoted hematopoietic engraftment and improved immune reconstitution, and 3) persisted long-term (>100 days) and successfully protected recipients from sublethal MCMV infection. Thus, infusion of amotosalen-treated donor T-cells at the time of transplantation is a clinically-attractive approach to adoptive anti-viral immunotherapy without chronic GvHD following hematopoietic progenitor cell transplantation.

CMV infection is reported to increase the incidence and severity of chronic GvHD and clinical data have shown that preemptive antiviral therapy decreased the risk of extensive chronic GvHD. Using mouse model of Allogeneic BMT, we investigated the mechanism for the association of MCMV infection and GvHD. We hypothesize that MCMV infection leads to generalized immune activation and increases the number of donor derived allo-reactive T cells and GvHD activity. Methods: A parenteral to F1 mouse BMT model was used to study anti-CMV immunity and GvHD. Low dose splenocytes (3x106) from MCMV immunized C57BL/6 donors (H-2b, Thy1.2+, CD45.1+) were transplanted with 5x106 T cell depleted bone marrow (TCD BM) from congeneic mice (H-2b, Thy1.1+, CD45.2+) into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C, H-2b/d, Thy1.2+, CD45.2+). Previous work has established this as a dose that protects against CMV without immediate lethality from GvHD. Non-GvHD control mice received a dose of Amotosalen treated splenocytes (10x106) and 5x106 TCD BM that protects against CMV without GvHD. Recipient mice were infected i.p. with a supralethal dose (2.5x104 pfu) of MCMV 7 days post transplant. Clinical GvHD was monitored twice weekly by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. T cell chimerism in recipient spleen and thymus, and MCMV peptide specific tetramer+CD8+ T cells were determined by flow cytometry. Liver and lung viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: During the acute phase of MCMV infection (day 3 to 14 post infection), recipient mice that received 3x106 untreated donor splenocytes developed GvHD characterized by weight loss and higher mortality than the non-GvHD control mice. Although both GvHD+ and control mice effectively cleared the virus from their liver, delayed viral clearance from the lung was found in non-GvHD recipients. Viral clearance was associated with expansion of donor spleen-derived MCMV peptide specific tetramer+ CD8+ T cells. The kinetics of donor T-cell expansion varied significantly between the two treatment groups, with GvHD+ recipients showed rapid early expansion of donor derived T-cells followed by the development of GvHD with subsequent lymphopenia. Recipients of Amotosalen-treated splenocytes had more gradual expansion of total and 400-fold expansion of antigen specific T-cells with sustained lymphoid reconstitution. GvHD+ recipients of untreated splenocytes had complete chimerism comprised of >80% of CD8+ donor T cells whereas non-GvHD controls had significant expansion of host derived T cells following MCMV infection and lacked allo-reactive of donor- spleen-derived T cells. Thymic function was inhibited among animals that developed GvHD and preserved among control mice throughout the infectious phase. Very delayed CMV infection (on day 60 after transplant) in mice with established chronic GvHD exacerbated GvHD and was associated with delayed lung viral clearance. Conclusion: After CMV infection there is extensive expansion of allo-reactive T cells in GvHD+ mice with associated damage to the microenvironment in the spleen and thymus. Amelioration of the immuno-suppressive effect of CMV infection (in clinical transplantation) will likely require more effective prophylaxis and treatment of GvHD in allotransplant recipients.