Mechanisms of Migration and Generation of Allo-Reactive Donor T Cells That Cause Organ Specific Acute GvHD in Allogeneic BMT Recipients.

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.

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Upregulation of Immune Checkpoint Blockade Molecules Post Allogeneic Stem Cell Transplantation Is Necessary but Insufficient to Prevent GvHD

Blood ◽

10.1182/blood.v124.21.1101.1101 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1101-1101

Author(s):

Mohammad Sohrab Hossain ◽

Ghada M Kunter ◽

Vicky Fayez Najjar ◽

David L. Jaye ◽

Edmund K. Waller

Keyword(s):

T Cells ◽

Transcription Factor ◽

Stem Cell ◽

T Cell ◽

Cd8 T Cells ◽

Acute Gvhd ◽

Time Points ◽

Post Transplant ◽

Donor T Cells ◽

Over Time

Abstract 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<0.0005) and B6 PD-L2 KO allo-HSCT recipients (p<0.0005) measured on day 8 after transplant. All PD-L1 KO allo-HSCT recipients died within 10 days post transplant while WT B6 and PD-L2 KO allo-HSCT recipients had 20% mortality until 36 days post transplant. Increased acute GvHD was associated with increased amount of serum inflammatory cytokines and increased numbers of activated PD-1+CD69+CD4+ donor T cells. Interestingly, PD-1 expression on donor CD4+ T cells significantly increased in the spleen of transplant recipients but not in BM, while PD-1 expression was significantly increased on donor CD8+ T cells in both spleen and BM compartments of allo-HSCT recipients compared with the syn-HSCT recipients. CTLA-4 expression on CD4+ and CD8+ donor T cells were significantly increased in spleen in the first two weeks post transplant but decreased at later time points compared with syn-HSCT. Again, CTLA-4 expression on CD4+ donor T cells in the BM remained significantly higher measured on 100+ days post transplant in allo-HSCT recipients compared with the syn-HSCT but similar levels of CTLA-4 expression on CD8+ T cells were measured in BM between these two HSCT recipients. Foxp3 expression on donor T cells and the numbers of CD4+CD25+foxp3+ regulatory T (Tregs) were markedly suppressed in donor T cells on day 4 post HSCT of allo-HSCT recipients compared with the syn-HSCT recipients. Although total numbers of donor T cells in the spleen of allo-HSCT recipients remained low over time, the percentage of PD-L1-expressing donor T cells in spleen were significantly higher (p<0.005) at early time points (day 4) in allo-HSCT recipients compared with the syn-HSCT. While total numbers of host-derived cells in spleen decreased over time in mice that developed GvHD, host-derived PD-L1 expressing CD3+ T cells persisted at higher levels through day 36 post transplant. Additionally, PD-L1 expression was also increased in donor BM-derived T cells and non-T cells populations over time. 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.

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Post-Transplant Cyclophosphamide Treatment Ameliorates Experimental Gvhd While Permitting Lymphopenic Expansion of Non-Host Reactive Donor T Cells.

Blood ◽

10.1182/blood.v116.21.3751.3751 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3751-3751

Author(s):

Monica Jones ◽

Duncan B Ross ◽

Krishna V Komanduri ◽

Robert B. Levy

Keyword(s):

Weight Loss ◽

T Cells ◽

Lymph Node ◽

T Cell ◽

Cd8 T Cells ◽

Lymph Node Cell ◽

Cell Numbers ◽

Post Transplant ◽

Donor T Cells ◽

Cyclophosphamide Treatment

Abstract Abstract 3751 Following myeloablative as well as reduced intensity conditioning regimens, recipients of allogeneic T cell replete (TCR) HLA matched sibling)or unrelated hematopoietic stem cell transplants (HSCT) experience significant acute as well as chronic graft vs. host disease (GVHD). Recent studies have reported that post-transplant cyclophosphamide (PTC) can function as an effective single agent for prophylaxis of acute and chronic GVHD (Biol. Blood Marrow Transpl. 16:482, 2010; Blood. 116:3224, 2010). To investigate the fate of non-host alloreactive donor cells, which are critical to providing immune reconstitution post-transplant, T cell replete HSCT were performed between MHC-matched allogeneic donor–recipient pairs. C3H.SW (H2b) mice were lethally conditioned 4 hrs prior to receipt of B6-CD45.1 (H2b) T cells and bone marrow. Recipients developed severe GVHD as assessed by weight loss (Fig. 1A) and clinical analyses during the first 4–5 wks post-transplant. Recipients had inverted CD4/CD8 ratios as well as an activated phenotype (CD44hiCD62Llo), severe B and T cell lymphopenia, and virtually undetectable thymic tissue. A single dose of post-transplant cyclophosphamide at day 3 (66mg/kg/body weight) blocked onset of weight loss and clinical signs of GVHD as well as lethality in C3H.SW recipients, consistent with the reduction of alloreactive anti-recipient T cells. In contrast to non-cyclophosphamide treated animals, treated recipients expressed typical CD4/CD8 ratios, significant numbers of spleen and lymph node cells (Fig. 1B) and readily detectable thymi 7–8 weeks post-HSCT. Notably, the majority of T as well as B cells in the periphery of these recipients exhibited a non-activated phenotype and were derived from donor bone marrow. Together with the observation that approximately 50% of T cells exhibited a CD44hiCD62Llo phenotype characteristic of TN, the findings are consistent with de novo derived lymphopoiesis in the thymus and marrow of post-transplant cyclophosphamide treated recipients. To assess the impact of D.3 cyclophosphamide treatment on a non-host reactive T cell population, B6 OT-I CD8 T cells (Vα2/Vβ5 TcR) were added (1-2×106) to the donor inoculum. In non-PTC treated recipients, low numbers of OT-I T cells (below input levels) were detected by 3–4 weeks post-HSCT. In contrast, in PTC-treated recipients, significant numbers of OT-I CD8 T cells were present in the recipient spleen and lymph nodes (Fig. 1C), consistent with the finding of CFSE labeled OT-I cells exhibiting division following cyclophosphamide treatment. Notably, the overall numbers of OT-I CD8 T cells in cyclophosphamide treated recipients was greater than the input levels, indicating that these cells a) survived post-cyclophosphamide treatment and b) underwent lymphopenic expansion. Consistent with these findings, CFSE studies at 72 hrs post-HSCT illustrated OT-I T cells exhibited low proliferation vs. a population of presumed B6 anti-C3H.SW alloreactive T cells. We therefore conclude that non-host reactive donor T cells undergo sufficient repair following cyclophosphamide induced alkylation to enable greater survival in contrast to rapidly dividing anti-host (GVHD) reactive populations. Vaccination studies in cyclophosphamide treated and non-treated HSCT recipients are being performed using heat shock fusion protein-transfected tumor cells to investigate the capacity to elicit anti-tumor (i.e., GVL) responses in the presence and absence of GVHD. Fig. 1: Post-transplant cyclophosphamide treated recipients demonstrate little severe weight loss vs. non-ptc recipients (panel A). Enhanced lymph node cell numbers and non-host reactive donor CD8 OT-I cell levels in ptc recipients 1 month post MHC-matched MiHA-mismatched HSCT (panels B and C). Fig. 1:. Post-transplant cyclophosphamide treated recipients demonstrate little severe weight loss vs. non-ptc recipients (panel A). Enhanced lymph node cell numbers and non-host reactive donor CD8 OT-I cell levels in ptc recipients 1 month post MHC-matched MiHA-mismatched HSCT (panels B and C). Disclosures: No relevant conflicts of interest to declare.

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Flagellin, a TLR5 Agonist, Reduces GvHD in Allogeneic HSCT Recipients While Enhancing Anti-Viral Immunity: A Novel Therapeutic Approach

Blood ◽

10.1182/blood.v118.21.144.144 ◽

2011 ◽

Vol 118 (21) ◽

pp. 144-144

Author(s):

Mohammad S Hossain ◽

David L Jaye ◽

Brian P Pollack ◽

Alton B Farr ◽

John Roback ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Viral Immunity ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Post Transplant ◽

Radiation Chimeras ◽

Donor T Cells

Abstract 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<0.05), and less MCMV in the liver on day 10 post infection (p<0.02) compared with the PBS-treated control recipients. Overall immune reconstitution after flagellin-treatment was robust and associated with larger numbers of CD4+CD25+foxp3+ regulatory T cells in the thymus. 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.

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Post-Transplant Cyclophosphamide Ameliorates Lethal Thymic GVHD By Restoring Regulatory and Effector T Cell Homeostasis in Recipients with Prior PD-1 Blockade Therapy

Blood ◽

10.1182/blood-2018-99-111142 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 479-479

Author(s):

Shuntaro Ikegawa ◽

Yusuke Meguri ◽

Takumi Kondo ◽

Hiroyuki Sugiura ◽

Yasuhisa Sando ◽

...

Keyword(s):

Overall Survival ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

T Cell Subsets ◽

Post Transplant ◽

Gvhd Prophylaxis ◽

Donor T Cells ◽

The Impact

Abstract Allogeneic HSCT has a curative potential for patients with hematological malignancies. However, graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after HSCT. Regulatory T cells (Tregs) are critical mediator for immune tolerance after HSCT and we recently reported that PD-1 plays an essential role for Treg survival (Asano et al, Blood 2017). Clinical studies suggested that PD-1 blockade prior to HSCT could be a risk of increasing severe GVHD. However, the mechanisms about GVHD induced by PD-1 blockade have largely unclear and there remains a paucity of data on appropriate GVHD prophylaxis for patients who undergo HSCT after PD-1 blockade. To address these issues, we investigated the impact of PD-1 expression on donor T cells on immune reconstitution with murine BMT models. First, lethally irradiated B6D2F1 mice were transplanted with 10 million of C57BL/6-background PD-1+/+ or PD-1-/- spleen cells with 5 million of bone marrow cells from normal C57BL/6, and GVHD scores and overall survival was monitored. Recipients receiving PD-1-/- graft developed severe GVHD resulting in a significant shorter survival than recipients receiving PD-1-/- graft (P<0.0001). We analyzed lymphocytes in spleen and thymus on day3, 7, and 14. We found that CD8 T cells in PD-1-/- group showed markedly higher Ki67 expression and CFSE-dilution until day3. Interestingly, PD-1-/- Tregs increased aggressively at day3 but it could not maintain until day14, while PD-1-/- CD8 T cells and conventional CD4 T cells (CD4 Tcons) continued to increase until day+14, resulting in the significant higher CD8/Treg ratio in PD-1-/- group (P<0.05, vs PD-1+/+ group). PD-1-/- Tregs showed significantly higher expression of Annexin V on day+7 and thymus CD4- and CD8- double-positive (DP) cells were in the extremely low levels in PD-1-/- group on day+14 (P<0.05, vs PD-1+/+ group). Thymic analysis showed that donor PD-1-/- graft-derived CD8 T cells infiltrated thymus in PD-1-/- group, suggesting reconstruction of thymic function was critically disturbed by severe GVHD. These data suggest that loss of PD-1 signaling resulted in unbalanced reconstitution of donor-derived T cell subsets as a consequence of continuous CTL expansion and increased Treg apoptosis. Next, to evaluate the impact of post-transplant cyclophosphamide (PTCy) on the abnormal reconstitution after PD-1 blockade, we administered 50mg/kg of Cy or control vehicle on day3. PTCy efficiently ameliorated GVHD in PD-1-/- group and extended overall survival by safely regulating the proliferation and apoptosis of T cell subsets. Of note, after PTCy, Tregs regained the ability of continuous proliferation in the first 2 weeks, resulting in well-balanced reconstitution of donor-derived T cell subsets. Thymic DP cells on day 14 was markedly increased in PD-1-/- group with PTCy intervention as compared to without PTCy, suggesting PTCy could rescue thymus from PD-1 blockade-related severe GVHD. Finally, to evaluate GVL activity, we performed BMT with co-infusion of P815L tumor cells on day0 and we confirmed that PTCy treatment for PD-1-/- recipients reduced the severity of GVHD with maintaining sufficient GVL effect. In summary, our data suggested three insights about the impact of PD-1 signaling on immune reconstitution. First, PD-1 inhibition influenced graft-derived T cells very differently within T cell subsets. PD-1-/- Tregs increased transiently but it was counterbalanced by accelerated apoptosis, while PD-1-/- CD4+Tcons and CD8 T cells continued the drastic expansion. Second, we found that PD-1-/- donor T cells developed severe GVHD in thymus. Few reports have concentrated on the impact of donor graft PD-1 expression to thymus after BMT and acute GVHD in thymus could lead late central immune disturbance. Third, PTCy successfully ameliorated GVHD induced by PD-1-/- donor T cells preserving GVL effect. Cell proliferation study implied that PD-1-/- graft-derived CD8 T cells might be more susceptible for PTCy because of the high-rate proliferation. In conclusion, PD-1-/- graft cause lethal thymic GVHD and PTCy successfully ameliorated it. The influence of PD-1 inhibition was different within T cell subtypes. PTCy might be appropriate GVHD prophylaxis strategy for patients who had prior usage of PD-1 blockade. Disclosures No relevant conflicts of interest to declare.

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Donor-Derived T Cells Can Be Rendered Hyporesponsive to Alloantigen without Loss of Pathogen or Tumor Immune Responses.

Blood ◽

10.1182/blood.v110.11.771.771 ◽

2007 ◽

Vol 110 (11) ◽

pp. 771-771 ◽

Cited By ~ 2

Author(s):

Jeff Davies ◽

Dongin Yuk ◽

Lee Nadler ◽

Eva Guinan

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Acute Gvhd ◽

Alloreactive T Cells ◽

Cell Pool ◽

Donor T Cells ◽

Leukemia Antigen

Abstract The prevention of severe acute Graft-versus-Host Disease (GvHD) without impairment of immune reconstitution is the major challenge in HLA-mismatched hematopoietic stem cell transplantation (HSCT). One alternative to experimental strategies to selectively destroy or remove alloreactive T cells from the donor T cell pool is to induce hyporesponsiveness (anergy) in alloreactive T cells within the donor T cell pool and thus preserve the vast majority of T cell repertoire. We previously reported early clinical data of HLA-mismatched HSCT after alloanergization of donor bone marrow via ex vivo allostimulation in the presence of co-stimulatory blockade (CSB) with Cytotoxic T Lymphocyte Antigen-4 Immunoglobulin (CTLA4-Ig). Analysis of a larger cohort of such patients revealed a low rate of severe acute GvHD and very few clinically significant viral infections, with over 30% of patients (pts) surviving long-term without disease relapse. This suggested that CSB might indeed be controlling alloreactivity with preservation of pathogen-specific immunity and a graft-versus-leukemia (GvL) effect. We therefore sought to directly determine the effect of alloanergization of human donor T cells on alloreactivity, pathogen- and leukemia-antigen-specific immunity. After alloanergization via blockade of CD28-mediated co-stimulation with clinical-grade humanized anti-B7.1 and anti B7.2 antibodies, HLA-mismatched alloproliferative responses were reduced by 2 logs, a more efficient reduction in alloreactivity than previously reported with the use of CTLA4 Ig. Using CFSE-based labeling of human responder T cells we have demonstrated directly for the first time that alloanergization efficiently abrogates stimulator-specific alloproliferation in both CD4 and CD8 donor T cells, whereas third party responses are retained (Figure 1). Importantly, the strategy does not diminish the capacity of donor CD4 and CD8 T cells to mount a range of functional immune responses, including proliferation, cytokine production and cytotoxic responses, in response to stimulation with several human herpes viruses. We have also demonstrated that frequencies of WT1-specific IFN-g+ CD4 and CD8 T cells are not diminished after the process of alloanergization, showing that a T cell mediated GvL effect may be retained. Importantly we demonstrated retention of pathogen and leukemia antigen-specific responses to both MHC Class I- and II-restricted antigens and in both HLA-A2+ and non-HLA-A2+ responders. These data confirm that the technique of alloanergization can be used to provide non-alloreactive donor T cells without loss of beneficial CD4 and CD8 donor immunity. The optimal dose of HLA-mismatched alloanergized donor T cells that will improve immune reconstitution whilst controlling acute GvHD after HLA-mismatched HSCT remains to be defined. To answer this question, we have embarked on a dose-escalating clinical study of delayed alloanergized donor T cell infusion to improve immune reconstitution after haploidentical HSCT. Figure Figure

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Abrogation of Donor T Cell IL-21 Signaling Leads to Tissue-Specific Modulation of Immunity and Separation of Gvhd From GVL

Blood ◽

10.1182/blood.v116.21.729.729 ◽

2010 ◽

Vol 116 (21) ◽

pp. 729-729

Author(s):

Alan M. Hanash ◽

Lucy W. Kappel ◽

Nury L. Yim ◽

Rebecca A. Nejat ◽

Gabrielle L. Goldberg ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lymph Nodes ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Inflammatory Cytokine ◽

Wild Type ◽

Mesenteric Lymph Nodes ◽

Mesenteric Lymph ◽

Donor T Cells

Abstract Abstract 729 Allogeneic hematopoietic transplantation is frequently the only curative therapy available to patients with hematopoietic malignancies, however transplant success continues to be limited by complications including graft vs. host disease (GVHD) and disease relapse. Separation of GVHD from graft vs. leukemia/lymphoma (GVL) responses continues to be a major goal of experimental and clinical transplantation, and better understanding of T cell immunobiology may lead to novel strategies to accomplish this goal. Interleukin 21 (IL-21) is a pro-inflammatory cytokine produced by Th17 helper T cells, and abrogation of IL-21 signaling has recently been demonstrated to reduce GVHD while retaining GVL. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL are incompletely understood. In order to characterize the effect of IL-21 on GVH and GVL T cell responses, we compared wild type and IL-21 receptor knockout (IL-21R KO) donor T cells in a C57BL/6 into BALB/c murine MHC-mismatched bone marrow transplant (BMT) model. Lethally irradiated BMT recipients of IL-21R KO T cells demonstrated decreased GVHD-related morbidity (p<.05) and mortality (p<.01), and decreased histopathologic evidence of GVHD within the small intestine (p<.05). While this reduction in IL-21R KO T cell-mediated GVHD was associated with increased donor regulatory T cells two to three weeks post-BMT (p<.001), IL-21 signaling in both donor CD4 and donor CD8 T cells was found to contribute to GVHD mortality (p<.01 for CD4, p<.05 for CD8). Analysis of IL-21R expression by wild type T cells demonstrated receptor upregulation upon polyclonal activation in vitro and upon alloactivation in vivo (p<.01). However, this IL-21R upregulation was not required for in vivo alloactivation, as IL-21R KO and wild type donor T cells demonstrated equivalently greater proliferation in allogeneic vs. syngeneic recipients (p<.001), equivalent upregulation of CD25 (p<.001), and equivalent downregulation of CD62L (p<.01 for CD8 T cells). Despite this equivalent alloactivation, IL-21R KO T cells demonstrated decreased infiltration within the small intestine (p<.05), decreased infiltration in mesenteric lymph nodes (p<.05 for CD8 T cells, p<.001 for CD4 T cells), and decreased inflammatory cytokine-producing CD4 T cells within mesenteric lymph nodes (p<.01 for IFN-g, p<.001 for TNF-a, Figure 1A). Consistent with this, transplanted IL-21R KO donor T cells demonstrated decreased expression of a4b7 integrin (LPAM, p<.05), a molecule known to be involved in homing of GVHD-mediating donor T cells to the gut. However, in contrast to the reduced inflammatory cytokine-producing CD4 T cells observed in mesenteric lymph nodes, IL-21R KO helper T cell cytokine production was maintained in spleen (Figure 1B) and peripheral lymph nodes, and IL-21R KO T cells were able to protect recipient mice from lethality due to A20 lymphoma (p<.001). In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T cell function and GVL capacity are retained. Targeting IL-21 for therapeutic intervention is an exciting strategy to separate GVHD from GVL, and this novel approach should be considered for clinical investigation to improve transplant outcomes and prevent malignant relapse. Disclosures: No relevant conflicts of interest to declare.

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Peroxisome Proliferator Activated Receptor-Delta Controls Fat Oxidation in Alloreactive T Cells

Blood ◽

10.1182/blood-2018-99-116727 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 476-476

Author(s):

Gail Waltz ◽

Arati Rajeevan ◽

Andrea Dobbs ◽

Elisabeth Denby ◽

Craig Byersdorfer

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Fat Oxidation ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Post Transplant ◽

Alloreactive T Cells ◽

Donor T Cells

Abstract Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and multiple non-malignant hematologic disorders. However, the routine use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that GVHD-causing T cells increase fat oxidation compared to both syngeneic and naive T cells. To explore this adaptation mechanistically, we studied the role of the transcription factor Peroxisome Proliferator Activated Receptor delta (PPAR-δ) in alloreactive donor T cells during the initiation of GVHD. By day 7 post-transplant, alloreactive T cells up-regulated PPAR-δ >5-fold compared to pre-transplant naive T cells (p<0.0001, Figure 1A). Furthermore, PPAR-δ was necessary for maximally severe GVHD, as major-MHC mismatched B6xDBA2 F1 mice receiving donor T cells deficient in exon 4 of PPAR-δ (PPAR-δ KO) survived longer than mice receiving wildtype (WT) T cells (p<0.007, Figure 1B). We next investigated the mechanism underlying this observed decrease in GVHD severity. As a transcription factor, PPAR-δ controls expression of multiple genes involved in fat transport and oxidation. To determine its role in alloreactive cells, RNA was collected from CD4 and CD8 T cells on day 7 post-transplant and levels of 8 known PPAR-δ targets quantitated by RT-PCR. These 8 targets were selected from a longer list of genes known to be up-regulated in alloreactive cells. Transcript levels of both carnitine palmitoyl transferase-1a (CPT-1a) and CD36 decreased in PPAR-δ KO CD8 T cells (Figure 2A), with decreases in CD36 protein levels confirmed by immunoblot (Figure 2B). Interestingly, changes in CPT-1a and CD36 did not occur in PPAR-δ KO CD4 T cells. To assess the functional consequence of these changes, day 7 WT versus PPAR-δ KO CD8 T cells were plated with 3H-palmitate and fat oxidation measured ex vivo. Consistent with a decrease in expression of genes involved in fat transport and mitochondrial fat import, fat oxidation decreased by >75% in PPAR-δ KO CD8 cells (Figure 2C). However, despite these decreases, the number of PPAR-δ KO CD8 T cells recovered on day 7 post-transplant was equivalent to WT T cells (Figure 3A, left panel). In contrast, PPAR-δ KO CD4 T cell numbers decreased by 30% on day 7, despite equivalent levels of CD36 and CPT1a (Figure 3A, right panel). Finally, we addressed whether pharmacologic inhibition of PPAR-δ might also effectively mitigate GVHD. Administration of the PPAR-δ inhibitor GSK3787 on days 3-6 post-transplant substantially decreased the number of donor T cell recovered on day 7 (Figure 3B), with PPAR-δ impairment corroborated by a decrease in CPT1a gene transcription. However, instead of improving recipient health, GSK3787 treatment instead worsened weight loss and increased rates of post-transplant morbidity and mortality. From these data, we conclude that PPAR-δ is necessary in alloreactive T cells to cause maximally severe GVHD and that mechanistically, an absence of PPAR-δ impairs fat oxidation in CD8 T cells without impacting CD8 T cell numbers. In contrast, PPAR-δ deficiency decreases the number of CD4 T cells post-transplant, but does so without impacting CPT1a or CD36 levels, highlighting clear differences in metabolic reprogramming between CD4 and CD8 alloreactive cells. Finally, our data suggest that systemic inhibition of PPAR-δ post-transplant is not feasible given a sharp increase in toxicity. Future work will elucidate the mechanism of PPAR-δ in CD4 T cells, define the additional metabolic adaptations of CD8 cells which lack PPAR-δ, and determine if similar changes occur in human T cells. Together, these studies will test whether cellular inhibition of PPAR-δ represents a clinically-relevant, future therapy for GVHD. Disclosures No relevant conflicts of interest to declare.

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Blockade of VIP-Signaling Enhanced Anti-Leukemic Activity in Murine Allogeneic BMT without Significantly Increased GvHD.

Blood ◽

10.1182/blood.v120.21.3001.3001 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3001-3001

Author(s):

Jian-Ming Li ◽

Hyun Don Yun ◽

Edmund K. Waller

Keyword(s):

T Cells ◽

T Cell ◽

Small Molecule ◽

Cd8 T Cells ◽

Low Dose ◽

Transplant Recipients ◽

Tumor Bearing ◽

Allogeneic Bmt ◽

Donor T Cells ◽

Clinical Scoring

Abstract Abstract 3001 Background and Objective: Vasoactive intestinal peptide (VIP) has potent immune-suppressive activity and can generate tolerogenic dendritic cells (DC) in vitro that block graft versus host disease (GvHD) in mouse model of BMT. We have previously published that absence of VIP signaling dramatically decreases PD-1 expression on activated CD8 T-cells and increases cellular antiviral immunity (JI 2011, 187:1057). To determine whether blockade of VIP-signaling enhances the graft-versus-leukemia (GvL) activity of donor T-cells in an allogeneic BMT model, we treated tumor bearing B6B̂10BR allogeneic transplant recipients with a short course of daily s.c. injections of a small molecule VIP antagonist - VIPhyb or used VIP-knockout (VIP-KO) mice as BM donors. Methods: Recipient mice were inoculated with luciferase+ murine acute T-cell lymphoma cells (Luc+ LBRM) by i.v. injection one day after lethal total body irradiation, then transplanted with the combination of 5 × 106 T cell-depleted BM (TCD-BM) plus splenocytes from either VIP-KO mice or wild-type (WT) littermates two days after irradiation. One group transplanted with WT BM and splenocytes received daily injections of 10 μg VIPhyb for one week; another group received saline injections. Survival, GvHD clinical sores (body weight, activity, posture, fur texture and skin), and bioluminescence imaging (BLI) were collected daily, twice a week, and weekly, respectively. Results: Transplantation of low dose (0.5 × 106) splenocytes from VIP-KO donors or low dose WT splenocytes in conjunction with VIPhyb-treatment dramatically improved tumor-free survival in the B6B̂10BR allogeneic BMT model compared with PBS-treated recipients of WT grafts (Figure 1). The best overall survival (70%) and lowest number of mice with detectable tumor (10%) were seen in the VIPhyb-treated group. VIPhyb-treated mice did not have increased GvHD as assessed by clinical scoring. Recipients of VIP-KO grafts had 40% survival with no detectable tumors by BLI, and were without significant GvHD by clinical scoring. In contrast, the recipients transplanted with TCD-BM alone (without added splenocytes) and recipients that received 0.5 × 106 splenocytes and TCD-BM from WT donors and treated with PBS had increased tumor growth detected by BLI following BMT, and all of the mice died by 2 months post-BMT. Moreover, in non-tumor bearing mice transplanted with an intermediate dose (1 × 106) of splenocytes, survival was not different among recipients engrafted with VIP-KO BM and T-cells (84 ± 6 %), WT BM and T-cells treated with VIPhyb (89 ± 8 %) and WT BM and T-cells treated with PBS (94 ± 5 %). A similar enhancement of the GvL effect and a corresponding survival advantage for VIP-signaling blockade was seen in tumor-bearing transplant recipients of TCD-BM plus 1 × 106 splenocytes, with significantly better survival among recipients of VIP-KO donor cells (50%), recipients of WT cells treated with VIPhyb (60%) compared with recipients of WT cells treated with PBS (20%; p=0.04). Furthermore, in non-tumor bearing mice that received a higher dose (3 × 106) of splenocytes, recipients of VIP-KO BM and recipients of WT BM treated with VIPhyb had no significant increase in GvHD compared with recipients of WT BM treated with PBS (66 ± 9 %, 71 ± 8 % and 71 ± 8 % survival at 80 days, respectively). The mechanism by which administration of a VIP antagonist enhanced anti-tumor immunity includes the effect of blocking VIP-signaling induction of cAMP, leading to fewer Treg and fewer tolerogenic DC. Of note, blocking VIP-signaling led to significant decreases in expression of PD-1 and PD-L1 on CD8+ T-cells and DCs, respectively. Conclusion: Treatment with a small molecule antagonist of VIP-signaling, VIPhyb, dramatically increased anti-leukemic activity of donor T-cells without significantly increased GvHD. Disclosures: No relevant conflicts of interest to declare.

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Impaired Donor T Cell Egress From Recipient Lymphoid Tissue Profoundly Diminishes Acute GvHD,

Blood ◽

10.1182/blood.v118.21.4022.4022 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4022-4022

Author(s):

LeShara M Fulton ◽

James Coghill ◽

Michelle L. West ◽

Niko Foger ◽

James Bear ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lymph Nodes ◽

Lymphoid Tissue ◽

Acute Gvhd ◽

Actin Binding ◽

Cell Transplant ◽

Post Transplantation ◽

Target Organs ◽

Donor T Cells

Abstract Abstract 4022 The migration of donor T cells into and out of host lymphoid tissue is critical for the induction of acute Graft-versus-Host Disease (GvHD). A number of groups including ours have found that the lack of lymphoid tissue or impaired migration into that tissue prevented acute GvHD. However, it is not clear if preventing egress of effector T cells from lymphoid tissue can inhibit GvHD. Coronin 1A (Coro 1A) is an actin binding protein involved in cell migration and motility, and has previously been shown to contribute to the ability of T cells to migrate within lymph nodes. To elucidate a role for Coro 1A in GvHD pathogenesis we used mice deficient in Coro 1A (Coro 1A−/−). Methods : A haplotype-matched Hematopoietic Stem Cell Transplant (HSCT) model was used. C57BL/6 (H-2b, “B6”) mice were used as donors while C57BL/6 x DBA2 F1 (H-2bxd, “B6D2”) functioned as recipients. Na•ve conventional T cells (Tns) were isolated from the spleens of wild type B6 (WT), enhanced green fluorescent protein expressing B6 (WT eGFP), Coro 1A−/−B6, and Coro 1A−/−eGFP mice. B6D2 mice were lethally irradiated to 900 rads on day −1 and injected with 4 × 106 Tns from WT, WT eGFP, Coro 1A−/−, or Coro 1A−/−eGFP mice supplemented with 3 × 106 WT B6 T cell depleted bone marrow cells on day 0. Recipients were followed for survival and assessed for GVHD twice weekly using a validated scoring system. In those mice given WT eGFP or Coro 1A−/− eGFP Tns, recipient organs were imaged by stereofluorescence microscopy on days 3 and 14 post transplantation. Results : Mice that received Coro 1A−/− Tns displayed significantly attenuated GvHD with greater than 90% surviving to transplant day 60. In contrast, those animals receiving WT Tns demonstrated a 100% mortality rate by the end of the study period (Fig 1A, p<0.05 by Fisher's exact test). Stereomicroscopy performed 3 days after transplantation demonstrated a lower eGFP signal within the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) of those mice given Coro 1A−/−eGFP T cells versus those receiving WT eGFP cells. By 14 days post transplantation, however, the converse was observed, with more cells found within the MLNs and PPs of recipient mice receiving Coro 1A−/−eGFP cells (Fig 1B). Total T cells numbers in the liver and colon, however, were decreased at this time point in Coro 1A−/−eGFP recipients, indicating an impaired ability for these cells to exit host lymphatic tissue and traffic to GvHD target organs. Conclusion : These data indicate that impaired egress from lymphoid tissue, by blocking the function of Coro 1A, leads to a profound decrease in GvHD pathology and donor T cells in GvHD target organs. These data suggest that blocking the migration of donor T cells out of lymphoid tissue is a new viable approach to prevent acute GvHD. Disclosures: No relevant conflicts of interest to declare.

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Intravenous Arginine Administration Benefits CD4+ T-Cell Homeostasis and Attenuates Liver Inflammation in Mice with Polymicrobial Sepsis

Nutrients ◽

10.3390/nu12041047 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1047

Author(s):

Chiu-Li Yeh ◽

Sharon Angela Tanuseputero ◽

Jin-Ming Wu ◽

Yi-Ru Tseng ◽

Po-Jen Yang ◽

...

Keyword(s):

T Cells ◽

Single Dose ◽

Lymph Node ◽

T Cell ◽

Lymph Nodes ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

Liver Inflammation ◽

Aortic Lymph Node ◽

Inos Inhibitor

This study investigated the effects of a single dose of arginine (Arg) administration at the beginning of sepsis on CD4+ T-cell regulation and liver inflammation in C57BL/6J mice. Mice were divided into normal control (NC), sham (SH), sepsis saline (SS), and sepsis Arg (SA) groups. An inducible nitric oxide (NO) synthase (iNOS) inhibitor was administered to additional sepsis groups to evaluate the role of NO during sepsis. Sepsis was induced using cecal ligation and puncture (CLP). The SS and SA groups received saline or Arg (300 mg/kg body weight) via tail vein 1 h after CLP. Mice were euthanized at 12 and 24 h post-CLP. Blood, para-aortic lymph nodes, and liver tissues were collected for further measurement. The findings showed that sepsis resulted in decreases in blood and para-aortic lymph node CD4+ T-cell percentages, whereas percentages of interleukin (IL)-4- and IL-17-expressing CD4+ T cells were upregulated. Compared to the SS group, Arg administration resulted in maintained circulating and para-aortic lymph node CD4+ T cells, an increased Th1/Th2 ratio, and a reduced Th17/Treg ratio post-CLP. In addition, levels of plasma liver injury markers and expression of inflammatory genes in liver decreased. These results suggest that a single dose of Arg administered after CLP increased Arg availability, sustained CD4+ T-cell populations, elicited more-balanced Th1/Th2/Th17/Treg polarization in the circulation and the para-aortic lymph nodes, and attenuated liver inflammation in sepsis. The favorable effects of Arg were abrogated when an iNOS inhibitor was administered, which indicated that NO may be participated in regulating the homeostasis of Th/Treg cells and subsequent liver inflammation during sepsis.

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