Donor DC Subsets Polarize Donor T-Cell Immune Responses in Allogeneic BMT.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 573-573
Author(s):  
Jian-Ming Li ◽  
Cynthia Giver ◽  
Doug McMillan ◽  
Wayne Harris ◽  
David L. Jaye ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Hematopoietic Cell ◽  
Post Transplant ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Introduction: Impaired or inappropriate immune reconstitution after allogeneic bone marrow transplantation (BMT) can lead to infection, graft-versus-host disease (GvHD) and leukemia relapse. We have previously reported that BM contains two populations of dendritic cell (DC) subsets, CD11b+ DC and CD11b− DC, and that CD11b depleted donor BM promoted increased donor T-cell chimerism and increased graft-versus-leukemia (GvL) activity in C57BL/6 → B10BR transplants [BBMT, 2004, 10: 540]. To explore the mechanism by which CD11b-depletion improved allo-reactivity, we performed allogeneic hematopoietic cell transplants using defined populations of donor stem cells, DCs, and T-cells in a MHC mis-matched BMT model. Methods: We transplanted FACS purified populations of 50,000 GFP+ CD11b- DC or CD11b+ DC in combination with 5,000 FACS purified Lin- Sca-1+ c-kit+ hematopoietic stem cells (HSC) and 300,000 or 1,000,000 congenic spleen T-cells from C57BL/6 donors into C57BL/6[H-2Kb], B10BR[H-2Kk] and PL/J[H-2Ku] recipients. Proliferation of CFSE stained donor T-cells was measured at 72 hours post-transplant. FACS cytometric bead array and intracellular cytokine staining measured serum and intracellular cytokines in donor T-cells. Results: The initial proliferation and Ki-67 expression of CFSE labeled donor T-cells in allogeneic recipients were much higher than in syngeneic recipients (homeostatic proliferation). Confocal microscopy showed co-localization of donor DC subsets with donor T-cells in the recipient spleens at 3 and 10 days post-transplant. In the allogeneic transplant settings, donor T-cells co-transplanted with CD11b- DC showed increased IFN-γ synthesis at 3 and 10 days post-transplant compared to donor T-cells co-transplanted with HSC plus CD11b+ DC or HSC alone. Increased proliferation of donor T-cells led to increased donor T-cell chimerism at day 10, 30, 60, and day105 post-transplant among recipients of CD11b- DC compared to recipients of HSC alone or HSC plus CD11b+ DC (Figure 1). Transplantation of spleen T-cells and CD11b- DC did not increase GvHD, but was associated with full donor chimerism. In contrast, transplantation of allogeneic CD11b+ DC led to persistence and expansion of residual host T-cells (Figure 2), increased numbers of donor CD4+CD25++Foxp3+ T-cells, and higher serum level of IL-10 supporting early post-transplant expansion of donor T regulatory cells (Treg). Conclusions: Donor CD11b- DC promoted immune reconstitution by polarizing donor T-cells to Th1 immune responses associated with increased IFN-γ synthesis and donor T-cell proliferation, while donor CD11b+ DC suppressed immune reconstitution by inhibiting donor T-cell allogeneic immune responses. These data support a novel paradigm for the regulation of post-transplant immunity and suggest clinical methods to test the hypothesis that manipulation of the DC content of a hematopoietic cell allograft regulates post transplant immunity in the clinical setting. Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells]

The Equilibration of Peripheral Blood and Tissue CD3+ T Cell Engraftment Is Influenced by Graft Versus Host Disease Following Reduced Intensity Conditioning Stem Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2546-2546
Author(s):  
Victoria Harries ◽  
Rachel Dickinson ◽  
Venetia Bigley ◽  
Matthew Collin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Peripheral Blood ◽  
Graft Versus Host ◽  
Post Transplant ◽  
Cell Engraftment ◽  
Donor T Cells ◽  
Patients At Risk ◽  
Reduced Intensity

Abstract Abstract 2546 Alemtuzumab-containing reduced intensity transplantation regimens frequently induce a state of partial T cell chimerism in the blood of the recipient. It has been widely shown that partial T cell chimerism is associated with freedom from graft versus host disease (GVHD) and that the occurrence of GVHD is often associated with rapidly rising donor T cell engraftment. The mechanism by which this occurs remains unknown and recipient cells may be killed, out-competed for homeostatic niches or simply diluted out by expanding donor T cells. The skin, a target organ of GVHD, normally contains T cells which enter from the blood in the steady state. Studies in mice have highlighted the gate-keeping function of inflammation in allowing trafficking of host-reactive donor T cells into tissues during conversion from mixed to full donor chimerism in blood. This implies that the equilibration of donor engraftment in the blood and tissue may occur more rapidly in patients at risk for GVHD. To test this hypothesis, we set out to define the relationship between skin and blood donor T cell engraftment in patients with and without GVHD. Methods: We studied a group of 51 patients receiving fludarabine melphalan (FM) conditioning with alemtuzumab 30mg for matched related donors and 60mg for matched unrelated donors. Skin biopsies were obtained at 28 and 100 days post transplant, dermal T cells isolated by migration and chimerism assessed in sex-mismatched transplants by combined immunofluorescence/in situ hybidization for XY chromosomes. Peripheral blood myeloid (CD15+) and T cell (CD3+) chimerism was determined by short tandem repeat amplification at monthly intervals after transplantation. All patients gave consent for clinical follow up and post transplant blood and skin sampling for research purposes, according to protocols approved by the local research ethics committee of Northumberland and North Tyneside. Results: All patients achieved >95% myeloid engraftment by day 100. Median (range) T cell engraftment was variable and significantly higher after MUD transplants: 70% (9-99%) than MRD transplants: 21% (5-85%; Mann Witney p <0.05). The incidence of acute GVHD was also greater after MUD transplantation at 47% (grade I or II) compared with 11% (grade I only) for MRD recipients. Overall a positive correlation was observed between donor T cell engraftment in skin and blood at all time points (r = 0.5792; P 0.0187) and at 100 days (r = 0.6570; P 0.0281). Analysis of the data with respect to GVHD showed a further interesting finding. Patients who developed GVHD had the closest correlation between blood and skin donor engraftment, even when they were in a state of partial T cell chimerism prior to the onset of GVHD. Patients who did not develop GVHD but nonetheless eventually achieved full donor engraftment in the blood tended to show lower levels of donor T cell engraftment in the dermis at day 100. Individual examples of patients who did not develop GVHD are: blood 77%, dermis 37%; blood 77%, dermis 6%; blood 92%, dermis 25%, compared with patients who did develop GVHD: blood 55%, dermis 56%; blood 90%, dermis 75%; blood 100%, dermis 100%. Conclusion: This analysis supports the hypothesis that the equilibration of blood and tissue donor T cells is influenced by GVHD and may offer a means to predict patients at risk of GVHD after withdrawal of immunosuppression or donor lymphocyte infusion. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Innate Lymphoid Cell-Derived IL-22 Regulates Epithelial Recovery From Gvhd

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 339-339
Author(s):  
Alan M Hanash ◽  
Jarrod A Dudakov ◽  
Guoqiang Hua ◽  
Margaret O'Connor ◽  
Lauren F. Young ◽  
...  
Keyword(s):  
T Cells ◽  
Small Intestine ◽  
T Cell ◽  
Large Intestine ◽  
Immune Reconstitution ◽  
Gi Tract ◽  
Post Transplant ◽  
Epithelial Tissues ◽  
Donor T Cells ◽  
Deficient Mice

Abstract Abstract 339 There is little understanding of the maintenance and regeneration of epithelial tissues after allogeneic transplant. Most clinical strategies to limit epithelial damage from graft vs. host disease (GVHD) also limit post-transplant immune function. Damage to the gastrointestinal (GI) tract from GVHD is a major cause of morbidity and mortality, and damage to the thymus from pre-transplant conditioning and GVHD can impair immune reconstitution, predispose patients to infection, and increase the risk of relapse. Therefore, understanding of tissue damage and recovery could lead to strategies selectively protecting epithelial tissues, improving intestinal barrier function, and promoting immune reconstitution without worsening post-transplant immunosuppression. We have recently identified that IL-22 from recipient-derived innate lymphoid cells (ILC) is critical for promoting intestinal recovery from GVHD and for promoting thymic recovery from radiation/pre-transplant conditioning. IL-22 deficient mice demonstrated significantly reduced thymopoiesis after total body irradiation (TBI), and IL-22 deficient murine bone marrow transplant (BMT) recipients demonstrated increased GVHD mortality and intestinal histopathology, deficiency of the antimicrobial molecules Reg3γ and Reg3β, and loss of intestinal stem cells needed for epithelial recovery. The source of thymic and intestinal IL-22 was RORγ+CD3−NKp46−IL-7R+CCR6+ lymphoid-tissue-inducer-like cells. Similar to as had been observed in the thymus, intestinal ILC produced IL-22 in response to IL-23, which was upregulated after TBI (p<.05 small intestine, p<.001 large intestine). IL-22 was also upregulated in response to TBI, but not in p40-deficient mice lacking IL-23 (p<.05 small intestine, p<.01 large intestine). ILC were radioresistant, as lethal TBI led to a three-fold increase in the intestinal ILC:CD4 ratio (p<.05). Furthermore, recipient-derived ILC comprised more than 50% of intestinal lamina propria ILC three months after T cell-depleted BMT, well after donor myeloid reconstitution and after donor reconstitution of the intestinal T cell compartment as well (Figure 1). Although intestinal ILC could survive lethal TBI, they were significantly depleted by both MHC mismatched (B6BALB/c) and MHC matched (LPB6) GVHD. Similarly, GVHD led to depletion of thymic IL-22+ ILC and reduction in thymic IL-22 levels (p<.001). Thymic IL-22 was critical for maintaining thymopoiesis during GVHD, as IL-22 deficient BMT recipients demonstrated significantly greater loss of double positive (DP) thymocytes after MHC-mismatched BMT. We previously identified that IL-21 receptor (IL-21R) signaling contributes to the migration of alloreactive donor T cells to the GI tract and that IL-21R-deficent donor T cells mediate significantly reduced GI GVHD. Given the similar homing molecules involved in the migration of donor T cells to the GI tract and thymus in GVHD, we evaluated the role of IL-21 in thymic GVHD. Donor T cell IL-21R deficiency led to increased thymopoiesis and DP thymocytes (p<.001), but not in IL-22-deficient recipients. ILC evaluation indicated that this IL-22 dependency was because IL-21R-deficiencient donor T cells had a reduced capacity to eliminate thymic ILC during GVHD (Figure 2). Therefore, donor T cell IL-21 signaling was critical for the elimination of recipient thymic ILC during GVHD, and preservation of the ILC compartment allowed for the IL-22 mediated regeneration of thymopoiesis. Finally, we also found that administration of rIL-22 post-BMT could reverse the thymic damage caused by GVHD and elimination of ILC, restoring the numbers of DP thymocytes to a level similar to what was observed after T cell-depleted BMT. In summary, IL-22+ ILC are radioresistant and capable of regulating tissue-specific epithelial recovery after allogeneic BMT. However, recipient ILC are extremely sensitive to GVHD, leading to a loss of the IL-22-mediated recovery response. IL-21 blockade can prevent the elimination of recipient thymic ILC by donor T cells in GVHD, and IL-22 administration can restore the thymopoiesis that is lost in GVHD due to ILC elimination. Maintenance of epithelial function post-BMT is thus an active innate immune response requiring cooperation between both recipient stroma and recipient hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Method of Mobilization: Implication on Cell Subsets in the Graft and Immune Reconstitution Post Autologous Hematopoietic Cell Transplantation (AHCT)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1132-1132
Author(s):  
Melhem M. Solh ◽  
Rathmann Kristin ◽  
Sauvi chang-Fong ◽  
Jeremiah Oyer ◽  
Wesam B. Ahmed ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
P Value ◽  
Autologous Hematopoietic Cell Transplantation ◽  
Graft Composition

Abstract Method of Mobilization: Implication on Cell Subsets in The Graft and Immune Reconstitution post Autologous Hematopoietic Cell Transplantation (AHCT) The optimal mobilization method for either myeloma or lymphoma patients undergoing AHCT is still debatable and strategies for graft collection vary between different institutions. Plerixafor, a CXCR4 antagonist is used for peripheral blood stem cell mobilization in multiple myeloma and non-Hodgkins lymphoma patients requiring AHCT. The effect of plerixafor on graft composition has scarce data that are based mostly on cryopreserved samples. Moreover; the effect of plerixafor on immune reconstitution and hematologic recovery post AHCT has not been well evaluated. The goal of our study was to compare graft composition, hematologic and immune reconstitution recovery among patients mobilized with plerixafor plus G-CSF to those mobilized with G-CSF alone. Methods: 49 patients eligible for AHCT were enrolled on a single arm prospective trial at a single transplant center. All patients were mobilized with G-CSF 10µg/kg/day for 4 consecutive days. A peripheral blood CD34 level of <20/µl on day 4 was used as a cutoff to use plerixafor 0.24mg/kg in addition to G-CSf on 9pm of the fourth day. Peripheral blood collection was started on day 5 and was continued till the target dose is achieved or a minimum CD 34+ cell dose of >2x106 cells/Kg was obtained after 3 collection days. Samples from the freshly collected graft and patients' peripheral blood on days +30 and +60 were analyzed by flow cytometry (BD FACSCanto II) . A single platform assay was used (Beckman-Coulter Stem kit) via a ISHAGE protocol. The antibody cocktail contained the following pre-conjugated monoclonal antibodies: CD56-PE (Miltenyi Biotech, Auburn, CA), CD3-APC, CD16-FITC, (Beckman Coulter, Brea, CA), CD19-PE-CY7 (BD Biosciences, San Jose, CA). Data were acquired using BD FACSCanto II (BD Biosciences) and analyzed with the FACSDiva software (BD Biosciences) to quantify CD3+ T cells, CD3+ CD56+ NK-like T cells, CD56+ CD16+ and CD56+ CD16- NK cells as well as CD19+ B cells. Results: 49 patients with a median age of 58 years (range 21-75) were mobilized with either G-CSF alone (N=16) or plerixafor +G-CSF (G+P)(N=33).The median number of collection days was 1.42 and 1.81 (p=0.2) and the median collected CD34+ dose was 8.28x106/kg and 5.24x106 /kg (p=022) in the G+P and G-CSF alone groups respectively. Both groups had similar times to neutrophil and platelet engraftment. The graft analysis showed a white blood count of 309x109/l and 262x109/l (p=0.38), median percentage of CD34+ cells of 0.75% and 0.73% (p=0.81), percentage of CD3+ T cells of 25.6% and 22% (p=0.6) in the G+P and G-CSF alone groups resepectively. Both groups had similar proportions of CD3+, CD4+,CD8+, NK, NKT and iNKT cells in the mobilized grafts. Peripheral blood samples at day +30 and day +60 were analyzed for T cell markers and hematologic recovery (table 1). There was no significant difference between absolute lymphocyte counts, NK cell counts, T cells and absolute neutrophil count. Conclusion: Plerixafor when combined with G-CSF helps in achieving mobilization goals in patients predicted to be poor mobilizers based on peripheral CD34 levels. The addition of plerixafor doesn't not seem to affect T cell composition of the graft and yields similar hematologic and immune recovery when compared to mobilization with G-CSF alone. Table 1: Immune Reconstitution at Day 30 and Day 60 post Autologous Transplantation Treatment Group G-CSF (N=16) Plerixafor + G-CSF (N=33) P-value G-CSF (N=16) Plerixafor + G-CSF (N=33) P-value Day 30 Day 60 WBC 5.08 5.41 0.873 4.94 5.38 0.654 HGB 10.86 11.19 0.353 11.22 11.17 0.757 HCT 32.35 33.66 0.321 33.36 33.53 0.565 PLT 119.88 161.42 0.068 166.94 173.73 0.949 Abs Lymph 1.09 1.44 0.296 1.41 1.50 0.974 % NK 26.14 30.38 0.277 11.53 20.09 0.095 Abs NK 0.31 0.35 0.186 0.17 0.21 0.470 % T cell 67 60 0.183 76.15 67.39 0.340 Abs T cell 0.72 0.96 0.717 1.35 .82 0.095 NKT%* 5.28 3.33 8.25 3.38 B cell % 2.38 1.52 0.922 2.63 5.58 0.424 Abs. Neut count 2.99 2.64 0.488 2.85 3.01 0.848 Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 771-771 ◽  
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

scholarly journals Analysis of transgene-specific immune responses that limit the in vivo persistence of adoptively transferred HSV-TK–modified donor T cells after allogeneic hematopoietic cell transplantation

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2294-2302 ◽  
Author(s):  
Carolina Berger ◽  
Mary E. Flowers ◽  
Edus H. Warren ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
T Cell Responses ◽  
Hematopoietic Cell ◽  
Allogeneic Hematopoietic Cell Transplantation ◽  
Cell Responses ◽  
Donor T Cells

AbstractThe introduction of an inducible suicide gene such as the herpes simplex virus thymidine kinase (HSV-TK) might allow exploitation of the antitumor activity of donor T cells after allogeneic hematopoietic cell transplantation (HCT) without graft versus host disease. However, HSV-TK is foreign, and immune responses to gene-modified T cells could lead to their premature elimination. We show that after the infusion of HSV-TK–modified donor T cells to HCT recipients, CD8+ and CD4+ T-cell responses to HSV-TK are rapidly induced and coincide with the disappearance of transferred cells. Cytokine flow cytometry using an overlapping panel of HSV-TK peptides allowed rapid detection and quantitation of HSV-TK–specific T cells in the blood and identified multiple immunogenic epitopes. Repeated infusion of modified T cells boosted the induced HSV-TK–specific T cells, which persisted as memory cells. These studies demonstrate the need for nonimmunogenic suicide genes and identify a strategy for detection of CD4+ and CD8+ T-cell responses to transgene products that should be generally applicable to monitoring patients on gene therapy trials. The potency of gene-modified T cells to elicit robust and durable immune responses imply this approach might be used for vaccination to elicit T-cell responses to viral or tumor antigens.

scholarly journals Modifying a Xenogeneic Gvhd Model to Allow for Use of CRISPR-Treated Primary Human T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4788-4788
Author(s):  
Archana Ramgopal ◽  
Darlene A. Monlish ◽  
Manda Ramsey ◽  
Craig Byersdorfer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Human T Cells ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia, immunodeficiencies, and bone marrow failure syndromes. Therapeutic use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues. We have previously shown that GVHD-causing T cells increase activation of AMP-activated protein kinase (AMPK), a cellular energy sensor, and that T cell-specific ablation of AMPK in murine models decreases GVHD severity. To study human T cell biology, we modified a previous xenogeneic model. Current models transplant whole peripheral blood mononuclear cells (PBMCs) from healthy human donors into lightly irradiated immunodeficient NOD-scid IL2Rgamma null (NSG) mice. However, in our hands, CRISPR-treatment of primary human T cells requires up to 10 days of culturing to obtain sufficient cells for transplantation. Therefore, prior to assessing GVHD severity using AMPK-deficient human T cells, we optimized the xenogeneic GVHD model to allow time for the manipulation and subsequent injection of CRISPR-engineered cells. Aim: To develop a xenogeneic model compatible with CRISPR/Cas9 manipulated T cells to determine whether changes in human T cells decrease GVHD severity similar to what is seen using murine T cells. Results: We first demonstrated that expanded T cells alone cause minimal xenogeneic GVHD, but that disease could be significantly facilitated with addition of non-T cell antigen presenting cells (APCs). Transplanting T cells plus non-T cell APCs increased numbers of human CD45+CD3+ T cells recovered on day 25 post-transplant (Figure 1A-B) and elevated levels of human interferon (IFN)-γ (Figure 1C). Liver sections from recipients of T cells + APCs subjectively had more perivascular infiltrates than mice receiving T cells alone (data not shown). Additionally, as seen in murine T cells, xenogeneic human T cells increased fatty acid oxidation, additional evidence that our model recapitulates the murine findings (Figure 1D). We next wished to fix the number of co-administered APCs and optimize the number of activated T cells to reliably reproduce xenogeneic GVHD without inducing overt toxicity. To accomplish this goal, we performed xenogeneic transplants with serially decreasing numbers of expanded human T cells (starting with 6×10 6/recipient) and administered 1×10 6 APCs in all cohorts. We also trialed inclusion of recently thawed non-T cell APCs in place of freshly derived cells. Reassuringly, the number of human CD45+CD3+ cells recovered on day 25 post-transplant remained proportional to the number of cells injected (Figure 2A-B), as did levels of human IFN-γ (detected by serum ELISA (Figure 2C). These data indicate that robust xenogeneic GVHD can be induced with as few as 2×10 6 expanded T cells and 1×10 6 autologous APCs, with a concomitant increase in GVHD-associated proinflammatory cytokines. Importantly, these data also demonstrate that 1×10 6 APCs are sufficient to cause reproducibly severe disease and may be recovered from a cryopreserved source. Finally, we wished to extend the assessment of GVHD severity following administration of varying doses of donor T cells. Serum from recipient mice on day 25 post-transplant was analyzed for the production of murine-derived cytokines via a LEGENDplex assay. Of 13 cytokines tested, both murine MCP-1 and TNF-alpha were proportional to the number of T cells injected (Figure 3A-B), with the MCP-1 result confirmed by ELISA (data not shown). Thus, both MCP-1 and TNF-α, cytokines commonly implicated in acute GVHD pathogenesis, provide additional host-derived soluble factors that can be utilized to quantitate the severity of GVHD in our modified xenogeneic model. Expression of these proteins will serve as valuable biomarkers in the assessment of xenogeneic GVHD using CRISPR-treated cells. Conclusions: We have successfully adapted a xenogeneic model of GVHD using in vitro expanded T cells and cryopreserved APCs, thereby allowing for expanded testing of genetically manipulated human T cells in an in vivo model. Future studies will compare the necessity of genes in human donor T cells using CRISPR-mediated gene editing and compare CRISPR techniques with novel pharmacological inhibition using this modified xenogeneic approach. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Host and Donor Immune Responses Contribute to Anti-Viral Effects of Amotosalen-Treated Donor Lymphocytes Following Early Post-Transplant CMV Infection.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3217-3217
Author(s):  
Mohammad S. Hossain ◽  
John D. Roback ◽  
Ned Waller
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Opportunistic Infections ◽  
Lethal Dose ◽  
Mcmv Infection ◽  
Post Transplant ◽  
Allogeneic Bmt ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ

Abstract 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.

Schedule Dependent Effects of Adoptively Transferred Autologous T Cells After AutoSCT for Myeloma: Accelerated Immune Recovery Is Associated with Improved Event-Free Survival.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 784-784
Author(s):  
Aaron P. Rapoport ◽  
Nicole A. Aqui ◽  
Edward A. Stadtmauer ◽  
Dan T. Vogl ◽  
Hong-Bin Fang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Conjugate Vaccine ◽  
Adoptive Transfer ◽  
Pneumococcal Conjugate Vaccine ◽  
Immune Reconstitution ◽  
Cd8 T Cell ◽  
Post Transplant ◽  
Log Ratio

Abstract Abstract 784 BACKGROUND: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 & CD8 levels at day +42 and augmented vaccine-specific immune responses in pts with myeloma (MM)[Nat Med 2005; 11: 1230-7]. DESIGN: We conducted a phase I/II 2-arm clinical trial in which 54 pts with advanced MM received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Pts also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine (PCV) only (ARM B; n = 26) or the PCV plus an HLA-A2 restricted multipeptide tumor antigen vaccine (3 hTERT peptides + 1 survivin peptide + 1 CMV peptide) for HLA-A2+ patients (ARM A; n = 28). After adoptive transfer of 4.26 × 10e10 costimulated T cells (range 1.59-5.0), the median CD3, CD4 and CD8 counts were 4198, 1545 and 2858 cells per microliter at day +14 post-transplant and 16% of pts developed significant autologous GVHD [Clin Cancer Res 2009;15: 4499 – 507]. Here we first report clinical outcomes from this trial and describe several novel observations about immune reconstitution after day +2 T-cell transfers. CLINICAL RESULTS: For the 54 total transplant pts, the median age was 55 (range 37-68), 28 (52%) were male and 21 (39%) were AA while 32 (59%) were Caucasian and 1 (2%) was Asian. 66% had IgG and 28% had IgA paraproteins while 6% excreted light chains only; 41% had abnormal cytogenetics. With a median followup of 1 year (0.25 - 2.5 years) 50 pts (93%) are alive and 31 (57%) remain event-free (EFS). The projected median EFS is 15 mos with no difference between ARMS A and B. Based on the Cox regression analysis, EFS did not correlate significantly to gender, race nor absolute lymphocyte counts (ALC). However, age correlated inversely to EFS with older pts exhibiting a lower EFS (coefficient = -0.0667, p-value = 0.0398). MM responses at day +60 and day +100 did not correlate to EFS, but the response at day +180 did correlate to EFS (p <0.0001). Notably, higher CD3+ T-cell levels at day +14 and higher CD8+ T-cell levels at day +14 and day +60 were significantly associated with improved EFS (p = 0.0104, 0.00117, 0.0111, respectively). IMMUNOLOGICAL RESULTS: Preliminary results indicated that 6 of 15 pts (40%) in ARM A had positive tetramer responses to hTERT and/or survivin peptides at one or more timepoints after immunization, defined as tetramer staining by flow cytometry > 0.1% (and > 3-fold increase). Based on analysis of the first 36 patients, the mean sum of the antibody responses to 4 of the 7 serotypes of the pneumococcal conjugate vaccine (PCV; serotypes 6B, 14, 19F, 23F) were 83.55 mcg/ml at day +100 and 74.13 mcg/ml at day +180, highly protective levels, compared to 5.17 mcg/ml at enrollment. We also observed several novel features of immune system recovery after early T-cell transfers. Compared to an historical cohort of 103 MM pts who were autografted without ex-T, post-transplant quantitative IgG, IgA and IgM levels at day +180 (and days +60/+100 for IgG and IgA) were significantly higher in the pts who received day +2 T-cell transfers (IgG: 1525 mg/dl vs 1137 mg/dl, p= 0.014; IgA: 264 mg/dl vs 76 mg/dl, p = 0.0029; IgM: 58 mg/dl vs 39 mg/dl, p = 0.029). Furthermore, when IgG recovery was examined for the IgA MM pts only, the IgG level at day +180 was significantly higher in the ex-T trial (mean IgG = 810 mg/dl vs 611 mg/dl, p = 0.0478). Conversely, when IgA recovery was examined for the IgG MM pts only, the IgA level was significantly higher in the ex-T trial at days +60 (mean IgA = 129.82 mg/dl vs 71.70 mg/dl, p = 0.0036), day +100 (mean IgA = 114.43 mg/dl vs 59.44 mg/dl, p = 0.0075) and day +180 (mean IgA = 98.38 mg/dl vs 48.74 mg/dl, p = 0.0037). Remarkably, a pattern of CD8 T-cell “reprogramming” was also observed after day +2 T-cell transfers such that the % of CD8+/CD45RA+ T-cells at day +60 post-transplant was 75% vs 36% in a cohort of MM transplant pts who did not receive ex-T (p< 0.0001). In addition, the log ratio of Teffector/Tregulatory cells was significantly higher among the patients who received early T-cell transfers (2.57 vs. 1.93, p = 0.0066). CONCLUSIONS: Early adoptive transfers of anti-CD3/anti-CD28 costimulated autologous T-cells significantly enhanced post-transplant humoral and cellular immune reconstitution, a pattern which may be associated with better myeloma control and protection from infection. These data may have important implications for efforts to induce clinically significant immune responses to cancer vaccines in the transplant setting. Disclosures: Vonderheide: University of Pennsylvania and Dana Farber Cancer Institute: Patents & Royalties. June:University of Pennsylvania: Patents & Royalties.

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