ALBERT

Description: 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

Description: 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

Description: Abstract 1165 Poster Board I-187 Background Opportunistic CMV infections remain a major cause of morbidity and mortality in allogeneic hematopoetic stem cell transplant (HSCT) recipients, with prolonged periods of viremia associated with cytopenia and life-threatening end-organ infections. Although CMV infection in allogeneic HSCT recipients may be controlled using anti-viral drugs, re-occurrence of viremia when drug therapy is stopped is common, and the emergence of drug resistant viral strains may render the anti-viral agents ineffective. The lack of functional anti-viral immunity that leads to CMV reactivation in immunocompromized HSCT recipients is a complex phenomena and may be due to 1) failure or insufficient donor/host innate immunity to prevent reactivation of CMV from their latent state, 2) failure to effectively process and present CMV antigens by the appropriate APCs to anti-viral T cells due to conditioning regimens used to prevent graft rejection and graft-vs-host disease (GvHD); 3) lack of response of CMV specific memory T cells to control CMV infection. Lack of any or all of these three elements of immune responses are hypothesized to be responsible for clinical CMV reactivation or infections in allogeneic transplant recipients. Determining which of these three elements of effective anti-viral immunity are most critical to preventing CMV infection would help guide novel therapies designed to prevent or treat opportunistic viral infections of high-risk patients. Method Emory is the core immune-monitoring laboratory for multi-center clinical study CTN 0201. As part of this study, we have samples from 69 HLA-A*2+ patients who received myeloablative conditioning and allogeneic HSCT from unrelated donors. HLA-A*2 restricted HCMV peptide pp65 (495-503, NLVPMVATV) specific tetramer+ CD8+ T-cells were determined from the blood samples obtained on 3, 6, and 12 months post transplant. Blood samples from three patients (2 HLA-A*2+ and 1 HLA-B*7+) treated at Emory had simultaneous data on both CMV viral load (measured by RT PCR) and the anti-CMV specific T-cells using the HLA-A*2 and HLA-B*7 [CMV pp65(NLVPMVATV)] tetramer reagents. Results Out of 69 patients, 5% had more than 1% CD8+ T-cells expressing the HLA-A*2 restricted CMV pp65 specific T-cell receptor at 3 months post-transplant, with an increasing proportion of patients showing the presence of at least 1% CMV-specific T-cells at 6 and 12 months post-transplant. Serial simultaneous measurements of CMV viral load and anti-CMV-specific T-cells are shown in Table 1 for 3 pts treated at Emory. In each case, evidence of active CMV replication was accompanied by a rapid expansion of CMV pp65-specific T-cells in the blood. Contraction of the CMV-specific T-cell compartment subsequently occurred, in conjunction with antiviral therapy and successful clearance of the virus. CMV reactivation events were not associated with major changes in the numbers of CD4+ T-cells, monocytes, or granulocytes. Conclusions Successful control of CMV reactivation is associated with a rapid and specific CD8-mediated immune response, which works in conjunction with antiviral therapy to limit viral replication. We would predict that the inability to control CMV reactivation, which is observed in a subset of pts following allogeneic transplantation, may not be associated with the same degree of target-specific immune expansion. This may be due to low absolute T-cell numbers, lack of innate immune responses or suppression of the ability of memory T cells to become effector T cells. Ongoing analyses will correlate measurements of tetramer+ T-cells in the 0201 dataset with laboratory evidence for CMV viremia and clinical infection. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: 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.

Description: Abstract 1002 Background: Vasoactive intestinal peptide (VIP) is a neuropeptide hormone and type 2 cytokine that inhibits Th1 immunity and induces the generation of regulatory T-cells. We have recently reported that non-transplanted mice “knocked-out” for VIP and syngeneic transplant recipients of VIP-knockout (KO) BM had dramatically improved survival, viral clearance, and increased numbers of specific antiviral CD8+ T-cells following murine cytomegalovirus (mCMV) infection (JI 2011. 187:1057–65). In this study, we used a small molecule VIP antagonist as well as VIP-KO mice to further investigate effects and mechanisms of VIP-signaling on antiviral immune responses in wild type (WT) non-transplanted mice and following allogeneic BMT. Methods: B10BR (CD45.2, H-2Kk) and CB6/J F1 (CD45.2, H-2Kb/d) mice were transplanted with 3 × 103 FACS-sorted hematopoietic stem cells (HSC), 5 × 104 dendritic cells (DC), and 0.3, 1, or 3 × 106 splenic T-cells either from VIP-KO (CD45.2, H-2Kb) or WT donors after myeloablative conditioning (11Gy). WT mice and BMT recipients transplanted with WT grafts were treated with daily subcutaneous injection of VIP antagonist (10 μg/100μL per mouse) or PBS for 7 days (from one day prior to infection to 6 days post-infection). BALB/C mice, B6 VIP-KO and WT littermates, as well as CB6/J F1 BMT recipients, were infected with graded doses (LD10, LD50 and LD90) of mCMV by intraperitoneal injection. Survival, viral load, antigen specific T-cells, and clinical scores of graft versus host disease (GvHD) were assessed at distinct time-points post-BMT or after mCMV infection. The expression of co-stimulatory or co-inhibitory markers (CD25, CD62L, CD69, PD-1, FoxP3, PD-L1, CD80, CD86, and MHC-II) and intracellular expression of cytokines (IL-10, IFN-γ, TNF-α, and IL-12) on T-cells and DC from the mice were measured by flow cytometry. Results: Improved survival was seen in mCMV-infected allogeneic B6→CB6/J F1 transplant recipients of VIP-KO grafts (100%) compared with recipients of WT grafts treated with PBS (40%). Allogeneic recipients of VIP-KO grafts and allogeneic recipients of WT grafts treated with VIP antagonist had increased viral clearance and enhanced in vivo killing of viral-peptide-pulsed targets compared with PBS-treated recipients of WT grafts. No difference in the incidence or severity of acute GvHD was seen in allogeneic BMT recipients of graded doses of VIP-KO versus WT splenic T-cells (0.3, 1, and 3 × 106) in murine MHC mis-matched BMT models. Allogeneic transplant recipients of VIP-KO grafts and WT grafts treated with VIP antagonist, infected with low dose mCMV, had lower levels of PD-L1 and PD-1 expression on DC and T-cells, respectively, and higher levels of CD80, CD86 and MHC-II expression on conventional DC (cDC) and plasmacytoid DC (pDC) compared with recipients of WT allografts treated with PBS. Recipients of VIP-KO grafts and recipients treated with VIP antagonist had higher-levels of IL-12+ cDC, activated CD25+/CD69+ CD4 and CD8 T-cells, and more mCMV-M45-peptide MHC-I tetramer+ CD8+ T-cells compared with recipients of WT grafts treated with PBS. Absence of VIP-signaling led to enhanced intracellular expression of IFN-γ and less IL-10 expression in T-cells from mCMV-infected recipients of VIP-KO B6→CB6/J F1 allogeneic transplants, and mCMV-infected, VIP antagonist-treated recipients of WT allogeneic transplants. In the absence of mCMV infection, the numbers of regulatory T cells (Treg) were similar among VIP-KO mice, WT mice treated with VIP antagonist, and PBS-treated WT controls. In contrast, mCMV-infected VIP-KO mice had significantly fewer Treg compared with mCMV- infected WT mice, non-infected WT mice and non-infected VIP-KO mice. Conclusion: Genetic or pharmacological blockade of VIP-signaling enhanced both innate and adaptive antiviral immune responses in allogeneic BMT recipients without significantly elevating GvHD. Selective targeting of VIP-signaling represents a novel therapeutic approach to enhance antiviral immunity in the setting of immunodeficiency and allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3561 Poster Board III-498 Background Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Immunosuppressive drugs limit clinical GvHD but increase relapse and susceptibility to opportunistic infections and also result in drug related toxicities. To develop an alternative approach to control GvHD, we tested the immunomodulatory immune properties of flagellin, a bacterial protein that agonistically binds with TLR5 and protects mice from radiation-induced gut injury, in murine allo-BMT models. Methods We used established BA.B10 (H-2K) → C57BL/6 (H-2b) MHC mismatched experimental models of allogeneic HSCT in which GvHD is a major complication. 50 μg LPS-free purified flagellin in PBS or PBS alone were administered intraperitoneally in two doses: 3 hours before fractionated irradiation (5.5Gy X 2 fractions) and 1 day post-transplant. Allografts were performed 1 day after irradiation and contained 5 million (M) T-cell depleted bone marrow (BM) cells and 5 M plastic non-adherent splenocytes from naïve BA.B10 donors. The primary end-points was survival; HSCT recipients were monitored twice a day for mortality and GvHD signs and recipients having more than 25% weight loss were sacrificed. Blood, spleen, thymus and BM were collected from surviving mice on day 132 post transplant, live cells counted, and immune phenotypes were analyzed by FACS. The numbers and phenotype of immune cells in organs from flagellin-treated HSCT recipients were compared to the similar immune cells per organs analyzed from a normal B6 mouse having similar age of HSCT recipients. Results Flagellin treated recipients had 15% weight-loss and 33% transplant-related death by 132 days post transplant versus severe acute GvHD and 100% early post-transplant mortality among control HSCT recipients that received PBS. Flagellin-treated recipients had 100% donor chimerism with limited clinical signs of GvHD. While total cell numbers per spleen (8.2 ± 5.4M) and thymus (7.1 ± 4.9M) were very low in flagellin-treated recipients compared to normal B6 mice (〉100M/organ), the cell numbers isolated from blood (8.9 ± 2.6 M/ml) and BM (104.5 ± 37.4 M) were similar to non-transplanted B6 mice (11.4M/ml blood and 108 M/BM, respectively). BM of flagellin-treated HSCT recipients contained similar numbers of CD4+ T cells (4.6 ± 2.7 M) and CD8+ T cells (2.5 ± 1.4 M) as normal B6 mice (4.03M and 1.3M, respectively). Numbers of naïve and memory CD4+ T-cells in the BM were similar between flagellin-treated and control mice: CD4+CD62L+(0.7 ± 0.2 versus 0.5M); CD4+CD62L- (3.9 ± 2.5 versus 3.5 M); CD4+ CD44hi (2.8 ± 1.4 versus B6 3.6M); and CD44lo (1.7 ± 1.3 M versus 0.44M). In contrast, flagellin-treated HSCT recipients had more naïve CD8+ T-cells but similar memory CD8+ T-cells in their BM compared with control mice: CD8+CD62L+(2.6 ± 1.4 versus 1.0M); CD8+CD62L- (1.7 ± 1.2M versus 0.3 M); CD8+CD44hi(0.8 ± 1.1 versus 0.7M); and CD8+CD44lo (0.7 ± 0.3M versus 0.6 M). The numbers of total CD3+ T cells, NK cells, and lin-CD11b-Sca-1+Ckit+ Stem cells in the BM were also similar comparing flagellin-treated recipients with non-transplanted B6 control mice. The number of CD3-B220+ B cells in the BM were lower in flagellin-treated recipients compared to B6 mouse (18.1 ± 3.2M versus 43.1M) as were the numbers of T-cells and B-cells per mL blood of flagellin-treated mice were found lower compared with the blood of normal B6 mouse: 0.8 ± 0.2M T-cells/mL versus 2.1M/mL; 5.5 ± 2.5M B cells/mL versus 9.1M/mL. Although the cellularity of the thymus in flagellin-treated animals was very low compared to normal B6 mice, a usual percentage (62.5 ± 10.5%) of thymocytes were of CD4/CD8 double positive, indicating functional thymopoiesis in these recipients. Conclusion Flagellin protected allogeneic HSCT recipients from irradiation-induced BM damage and prevented lethal GvHD in a major MHC mis-matched model of GvHD. Flagellin and other TLR5 agonists may be novel therapeutic approaches to prevent or reduce GvHD in allogeneic HSCT recipients. Disclosures: No relevant conflicts of interest to declare.