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.

scholarly journals Post-Transplant Cyclophosphamide (PTCY) Vs Anti-Thymoglobulin (ATG) As Part of the Gvhd Prophylaxis for Fludarabine/Clofarabine/Busulfan Reduced Intensity Conditioning (RIC) in Allogeneic Stem Cell Transplantation (allo-SCT): Influence on Early Immune Reconstitution

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1955-1955
Author(s):  
Christelle Retiere ◽  
Catherine Willem ◽  
Thierry Guillaume ◽  
Henri Vié ◽  
Laetitia Gautrot-Rolland ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Conditioning Regimen ◽  
Immune Recovery ◽  
Post Transplant ◽  
Gvhd Prophylaxis ◽  
The Impact

Abstract Introduction: The influence of PTCY on early immune reconstitution after allo-SCT has been poorly studied so far, especially in comparison to standard use of ATG as GVHD prophylaxis. Patients and Methods: A prospective study was conducted at the CHU of Nantes with the aim to compare early immune recovery between patients receiving PTCY or ATG as GVHD prophylaxis after a RIC allo-SCT. Secondary objectives were to study the impact of 1 vs 2 days of PTCY (CY1 vs CY2) or ATG (A1 vs A2), and of fludarabine (Flu) vs clofarabine (Clo) as part of the RIC regimen. As such, 3 RIC regimens were considered in both groups: FluCY2 (Baltimore regimen, Luznic, BBMT 2008), FluCY1, CloCY2 (where Clo replaces Flu), on one hand, and FluB2A2, CloB2A2, CloB2A1 (Chevallier, Haematologica, 2014), on the other hand. FluCY2 and FluB2A2 are currently standard of care RIC regimens for patients with haplo-identical and matched donors, respectively. Five patients had to be included in each RIC subgroup, depending on the type of disease and donor (/): FB2A2 (lymphoid/matched); FluCY2 (lymphoid/haplo); CloB2A2 or CloB2A1 (myeloid/matched); CloCY2 (myeloid/haplo), FluCY1 (myeloid or lymphoid/matched). The source of graft was peripheral blood stem cells for all cases. Blood samples were collected before starting the conditioning regimen then 3 times per week from day +0 until day+30, and at days +60 and +90. The following cell subsets were studied: a/b and g/d CD3+, CD8+ and regulatory (Tregs) T cells, B and NK cells and monocytes. The median percentage (%) compared to total lymphocytes was considered for all lymphocytes subsets between days 0-30. Thereafter, median absolute numbers (AN)/mm3 were considered for samples collected at days +30, +60 and +90. The study was approved by the IRB of the CHU of Nantes and all patients provided informed consent. Results: Between August 2014 and May 2015, thirty patients were included, including 15 in both groups and 5 in each RIC subgroups. Median age was 61 years. There were more patients with active disease at transplant (47% vs 7%) and more haplo-identical donors (67% vs 0%) in the PTCY group. All patients engrafted and were alive at day +90. However, 1 PTCY patient with T-ALL relapsed before day+100. Within the first month post-transplant, PTCY group had a significantly higher median % of a/b T cells (69.1 vs 18.9, p<0.0001) and Tregs (3.46 vs 0.45, p<0.0001) while ATG group had higher median % of NK (23 vs 2.57, p<0.0001) and B-cells (0.88 vs 0.43, p=0.0002). Between day+30 and day+90, ATG group had significant higher median counts of a/b T cells at days +60 (1316 vs 79.6, p=0.0001) and +90 (795.8 vs 151.6, p=0.03); g/d T cells at day+60 (27.6 vs 1.26, p=0.002); CD8 T cells at day+60 (735 vs 29.6, p=0.008); NK cells at day+30 (203.7 vs 89, p=0.04) and monocytes at days +30 (455.5 vs 221.7, p=0.009) and +60 (832.5 vs 247.2, p=0.004). Compared to the standard FluCY2 regimen, although not significant, FluCY1 was associated with higher median %, between days 0-30 of g/d T cells (2.32 vs 0.8) and higher median AN of g/d T cells at days +30 (9.2 vs 1.02) and +60 (9.22 vs 1.05), of B cells at days +30 (0.4 vs 0.14) and +60 (1.6 vs 0.39) and of NK cells at day+30 (213.9 vs 82.7). Compared to the standard FB2A2 regimen, although not always significant, CloB2A1 was associated with higher median % between days 0-30 of Tregs (0.97 vs 0.25, p=0.002) and higher median AN of g/d T cells at day+30 (6.8 vs 2), B cells at days +30 (2.35 vs 0) and +60 (43.7 vs 0.19), NK cells at day +30 (288 vs 62.1) and Tregs at days +30 (4.3 vs 0.2), +60 (8.8 vs 1.14) and +90 (8.96 vs 3.45). Compared to the standard FB2A2 regimen, although not always significant, CloB2A2 was associated with higher median % between days 0-30 of a/b T cells (28.87 vs 3.78, p=0.01) and B cells (0.76 vs 0.5, p=0.02) and higher median AN of a/b T cells at days +30 (324.3 vs 125.8) and +90 (1594 vs 604.3), of g/d T cells at day +30 (7.35 vs 2), of CD8 + T cells at days +30 (209.7 vs 38.9) and +90 (1211.7 vs 504.6), of B cells at day +30 ( 1.41 vs 0), of NK cells at days +30 (303 vs 62.1), +60 (514.5 vs 225.7) and +90 (647 vs 102.8, p=0.03) and of monocytes at days +30 (688.9 vs 257.4, p=0.03) and +90 (2157.4 vs 611.2). Conclusion: Strong differences exist in term of early immune recovery when using PTCY or ATG as part of the GHVD prophylaxis for RIC allo-SCT. Dose or drug modifications within the standard RIC regimen in both groups may be envisaged to favor some cell population recoveries after allo-SCT in order to increase outcome in patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Impact of Different T Cell Depletion Protocols on Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Amandine Pradier ◽  
Adrien Petitpas ◽  
Anne-Claire Mamez ◽  
Federica Giannotti ◽  
Sarah Morin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Cell Depletion ◽  
Unrelated Donor ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapeutic modality for a variety of hematological malignancies and congenital disorders. One of the major complications of the procedure is graft-versus-host-disease (GVHD) initiated by T cells co-administered with the graft. Removal of donor T cells from the graft is a widely employed and effective strategy to prevent GVHD, although its impact on post-transplant immune reconstitution might significantly affect anti-tumor and anti-infectious responses. Several approaches of T cell depletion (TCD) exist, including in vivo depletion using anti-thymocyte globulin (ATG) and/or post-transplant cyclophosphamide (PTCy) as well as in vitro manipulation of the graft. In this work, we analyzed the impact of different T cell depletion strategies on immune reconstitution after allogeneic HSCT. Methods We retrospectively analysed data from 168 patients transplanted between 2015 and 2019 at Geneva University Hospitals. In our center, several methods for TCD are being used, alone or in combination: 1) In vivo T cell depletion using ATG (ATG-Thymoglobulin 7.5 mg/kg or ATG-Fresenius 25 mg/kg); 2) in vitro partial T cell depletion (pTCD) of the graft obtained through in vitro incubation with alemtuzumab (Campath [Genzyme Corporation, Cambridge, MA]), washed before infusion and administered at day 0, followed on day +1 by an add-back of unmanipulated grafts containing about 100 × 106/kg donor T cells. The procedure is followed by donor lymphocyte infusions at incremental doses starting with 1 × 106 CD3/kg at 3 months to all patients who had received pTCD grafts with RIC in the absence of GVHD; 3) post-transplant cyclophosphamide (PTCy; 50 mg/kg) on days 3 and 4 post-HSCT. Absolute counts of CD3, CD4, CD8, CD19 and NK cells measured by flow cytometry during the first year after allogeneic HSCT were analyzed. Measures obtained from patients with mixed donor chimerism or after therapeutic DLI were excluded from the analysis. Cell numbers during time were compared using mixed-effects linear models depending on the TCD. Multivariable analysis was performed taking into account the impact of clinical factors differing between patients groups (patient's age, donor type and conditioning). Results ATG was administered to 77 (46%) patients, 15 (9%) patients received a pTCD graft and 26 (15%) patients received a combination of both ATG and pTCD graft. 24 (14%) patients were treated with PTCy and 26 (15%) patients received a T replete graft. 60% of patients had a reduced intensity conditioning (RIC). 48 (29%) patients received grafts from a sibling identical donor, 94 (56%) from a matched unrelated donor, 13 (8%) from mismatched unrelated donor and 13 (8%) received haploidentical grafts. TCD protocols had no significant impact on CD3 or CD8 T cell reconstitution during the first year post-HSCT (Figure 1). Conversely, CD4 T cells recovery was affected by the ATG/pTCD combination (coefficient ± SE: -67±28, p=0.019) when compared to the T cell replete group (Figure 1). Analysis of data censored for acute or chronic GVHD requiring treatment or relapse revealed a delay of CD4 T cell reconstitution in the ATG and/or pTCD treated groups on (ATG:-79±27, p=0.004; pTCD:-100±43, p=0.022; ATG/pTCD:-110±33, p&lt;0.001). Interestingly, pTCD alone or in combination with ATG resulted in a better reconstitution of NK cells compared to T replete group (pTCD: 152±45, p&lt;0.001; ATG/pTCD: 94±36, p=0.009; Figure 1). A similar effect of pTCD was also observed for B cells (pTCD: 170±48, p&lt;.001; ATG/pTCD: 127±38, p&lt;.001). The effect of pTCD on NK was confirmed when data were censored for GVHD and relapse (pTCD: 132±60, p=0.028; ATG/pTCD: 106±47, p=0.023) while only ATG/pTCD retained a significant impact on B cells (102±49, p=0.037). The use of PTCy did not affect T, NK or B cell reconstitution when compared to the T cell replete group. Conclusion Our results indicate that all TCD protocols with the only exception of PTCy are associated with a delayed recovery of CD4 T cells whereas pTCD of the graft, alone or in combination with ATG, significantly improves NK and B cell reconstitution. Figure 1 Disclosures No relevant conflicts of interest to declare.

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]

Natural Killer Cell Immune Reconstitution Predicts Outcomes for Patients with Chronic Lymphocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3300-3300
Author(s):  
Don Benson ◽  
Leslie Andritsos ◽  
Mehdi Hamadani ◽  
Thomas Lin ◽  
Joseph Flynn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Nk Cell ◽  
Disease Status ◽  
Lymphocytic Leukemia ◽  
T Cell Subsets ◽  
Cell Recovery

Abstract Introduction: Chronic lymphocytic leukemia (CLL), the most common form of leukemia in the Western hemisphere, is associated with severe innate, adaptive and humoral immune dysregulation. CLL remains essentially incurable, with the potential exception of allogeneic stem cell transplantation (ASCT). Natural killer (NK) cells are CD56(+), CD3(−) large granular lymphocytes that comprise a key cellular subset of the innate immune system. Preliminary in vitro data suggest an NK cell versus CLL effect exists, similar to that observed in acute myeloid leukemia (AML) and other blood cancers. Novel immune therapies for CLL (e.g., rituximab, alemtuzumab) likely exert anti-tumor effect, in part, through NK cells, in fact. Although NK cells contribute to the graft-versus-tumor effect following ASCT for other blood cancers, little is known regarding the potential role NK cells may play in the clinical allogeneic transplant setting for CLL. Herein, we provide, to our knowledge, the first report regarding NK cell immune reconstitution following ASCT for CLL. Methods: 27 CLL patients underwent reduced intensity conditioning (RIC) with ASCT. Median age was 52 years (43–69), median number of prior therapies was 3 (2–11). 55% had chemotherapy-refractory disease, and 55% had “high-risk” cytogenetics by FISH (deletion 17p or 11q22-23 abnormality). 14 patients had sibling donors, 15 had volunteerunrelated donors. Conditioning regimens included Fludarabine/TBI/Alemtuzumab (n=8), Fludarabine/Busulfan with (n=9) or without ATG (n=6), and Fludarabine/Cyclophosphamide (n=4). GVHD prophylaxis consisted of tacrolimus/MMF (n=8) or tacrolimus/methotrexate (n=19). Patients underwent bone marrow assessment prior to day +75 following ASCT. Marrow was studied for engraftment, donor chimerism, and disease status as well as lymphoid immune reconstitution by percentage of total lymphocytes and absolute lymphocyte counts by multi-color flow cytometry. Results: NK cell immune reconstitution was predicted by disease status at transplantation. Patients in complete or partial remission at the time of ASCT had more robust NK cell recovery (mean = 45% of total lymphocytes +/− SEM 5%) as compared to patients entering transplant with refractory disease (16% +/− 1, p < 0.01). No differences were observed in CD4(+) or CD8(+) T cells and no lymphocyte subset recovery was associated with CD34(+) or CD3(+) cell dosage. Achieving complete donor chimerism by day +60 was associated with robust NK cell recovery (55% +/− 1 versus 7% +/−1, p = 0.02), recovery of CD4 and CD8 T cells was not associated with chimerism status, however. Patients who went onto exhibit a complete response to ASCT had greater early NK cell reconstitution (31% +/− 3) as compared to those who had no response (8% +/− 1, p = 0.01). No differences in T cell subsets were associated with response. Patients who ultimately achieved complete remission following transplant had a lower CLL:NK cell ratio in marrow (0.35 +/− 0.07) than those who did not (8.1 +/− 1, p = 0.01). However, differences in CLL:CD4(+) and CLL:CD8(+) T cells were not predictive of response. Trends to improvement in progression free survival and overall survival were observed for patients with NK cell reconstitution above the median for the group as compared to those below; no such trends were observed regarding T cell subsets. Greater NK cell reconstitution trended towards ultimate eradication of minimal residual disease following ASCT, but no such trends were observed for T cell subsets. Conclusions: Early NK cell recovery predicts survival following autologous and allogeneic SCT in a number of hematologic malignancies; however, little is known regarding this phenomenon in CLL. To our knowledge, these are the first findings to implicate a potentially important therapeutic role for early NK cell compartment recovery in CLL following ASCT. Further research into restoring and augmenting NK cell function following RIC/ASCT for CLL is warranted.

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.

Influence of Graft Versus-Host Disease Prophylaxis Regimen On T-Cell Repertoire Diversity Following Reduced-Intensity HLA-Matched Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3054-3054 ◽  
Author(s):  
Rachel B. Salit ◽  
Frances T. Hakim ◽  
Michael R. Bishop ◽  
Thea M. Friedman ◽  
Robert Korngold ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Unrelated Donor ◽  
Matched Unrelated Donor ◽  
Cell Repertoire ◽  
Post Transplant ◽  
Gvhd Prophylaxis ◽  
Repertoire Diversity ◽  
Reduced Intensity

Abstract Abstract 3054 Background: A clearly superior graft-versus-host disease (GVHD) prophylaxis regimen has not been established for patients undergoing reduced intensity allogeneic hematopoetic stem cell transplantation (HSCT) from matched unrelated donors (URD). Encouraging results have been reported with both the combination of alemtuzumab and cyclosporine (AC) and the regimen of tacrolimus, methotrexate, and sirolimus (TMS) in the URD setting. These two regimens work by biologically distinct mechanisms and may have markedly different effects on immune reconstitution. T-cell receptor (TCR) spectratyping analysis, which provides information on antigen receptor diversity, is a valuable method for monitoring post-transplant immune reconstitution. As part of a randomized pilot study, we prospectively assessed the effects of AC vs. TMS on TCR Vb repertoire diversity in patients undergoing reduced intensity HLA-matched unrelated donor transplantation. Methods: Twenty patients (median age 53 yrs; range 24–70 yrs) with hematologic malignancies received reduced intensity conditioning (fludarabine 30 mg/m2/day and cyclophosphamide 1200 mg/m2/day IV Day -6 to -3) followed by a 10/10 HLA-matched unrelated donor T-cell replete mobilized peripheral blood allograft. Patients were randomized to receive either: AC (n=10): alemtuzumab 20 mg/day IV over 8 hours Days -8 to -4 and cyclosporine starting at Day -1 with a 10% per week taper starting at Day +100 or TMS (n=10): tacrolimus and sirolimus starting at Day -3 with a 33% taper at Day +63 and Day +119 and methotrexate 5 mg/m2 IV, Days +1, +3, +6, and +11. Blood samples were collected from the donor and patient at baseline and the patient at 1, 3, 6 and 12 months post-transplant for TCR spectratyping analysis. All comparisons are based on an exact Wilcoxon rank sum test; p values < 0.01 were significant because of multiple comparisons. Results: Patients on the AC arm had significantly fewer T-cells on Day +14 compared with the TMS arm (median CD3+ = 1 cells/μl vs 356 cells/μl; CD4+ = 0 cells/μl vs 243 cells/μl; CD8+ = 0 cells/μl vs. 59 cells/μl; each p<0.0001); there was less disparity at Day +28 (median CD3+ = 45 cells/μl vs. 398 cells/μl; CD4+ = 36 cells/μl vs. 218 cells/μl; CD8+= 5 cells/μl vs 152 cells/μl; each p 0.002). By Day +100, lymphocyte recovery was not appreciably different between the two arms (median CD3+ = 242 cells/μl vs. 445 cells/μl (p = 0.095): CD4+ = 106 cells/μl vs. 212 cells/μl (p=0.28); CD8+ = 72 cells/μl vs. 135 cells/μl (p = 0.03). NK-cell recovery was slightly less in the AC vs. TMS arm at Day +14 (median NK = 27 cells/μl vs. 70 cells/μl; p = 0.01) and at Day +28 (median NK = 29 cells/μl vs. 150 cells/μl; p=0.02). There was no difference by Day +100 (median NK = 124 cells/μl vs. 88 cells/μl; p=0.31). B-cell reconstitution was negligible in both arms through Day +100. Assessment of CD4+ TCR Vb repertoire diversity by spectratyping demonstrated significantly lower diversity in patients receiving AC at 1 (p = 0.0003), 3 (p = 0.0003) and 6 (p=0.003) months post transplant compared with patients receiving TMS. CD8+ TCR spectratyping similarly revealed significantly reduced diversity in the AC arm at 3 (p = 0.001) and at 6 months (p = 0.003), and a trend toward significance at 12 months (p = 0.07). On each of the 2 arms, 2 of 10 patients developed acute Grade II-IV GVHD. Of the 5 patients on the AC arm who were seropositive for CMV, all 5 reactivated CMV by PCR within the first 60 days and reactivated 2–5 times in the first year. In contrast, only 3 of 5 seropositive patients reactivated CMV on the TMS arm and only one reactivated in the first 60 days. Conclusions: Two factors may have contributed to the loss of repertoire diversity in the AC arm. First, the alemtuzumab regimen may have severely depleted the infused donor T-cells. Second, stimulation by reactivating virus may have induced expansion of CMV-specific memory and effector T-cells, resulting in a skewed and oligoclonal T-cell repertoire. Especially in CD8+ T-cells, CMV has been shown to produce significant oligoclonal expansion (including CD4+: CD8+ ratio inversion). The loss of T-cell numbers and repertoire may in turn have contributed to the prevalence of early CMV reactivation. Thus, despite the similarities in frequency of acute GVHD in this small sample, it appears that these two commonly used GVHD prophylaxis regimens have very different effects on post-transplant immune reconstitution in the first 6 months after allogeneic HSCT. 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.

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

Blood ◽  
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.

scholarly journals Ecto-5′-Nucleotidase (CD73) Regulates the Survival of CD8+ T Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Mariana V. Rosemblatt ◽  
Brian Parra-Tello ◽  
Pedro Briceño ◽  
Elizabeth Rivas-Yáñez ◽  
Suat Tucer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
Cd8 T Cell ◽  
Antigenic Stimulation ◽  
T Cell Subsets ◽  
Limited Information ◽  
Α Chain ◽  
The Impact

Ecto-5′-nucleotidase (CD73) is an enzyme present on the surface of tumor cells whose primary described function is the production of extracellular adenosine. Due to the immunosuppressive properties of adenosine, CD73 is being investigated as a target for new antitumor therapies. We and others have described that CD73 is present at the surface of different CD8+ T cell subsets. Nonetheless, there is limited information as to whether CD73 affects CD8+ T cell proliferation and survival. In this study, we assessed the impact of CD73 deficiency on CD8+ T cells by analyzing their proliferation and survival in antigenic and homeostatic conditions. Results obtained from adoptive transfer experiments demonstrate a paradoxical role of CD73. On one side, it favors the expression of interleukin-7 receptor α chain on CD8+ T cells and their homeostatic survival; on the other side, it reduces the survival of activated CD8+ T cells under antigenic stimulation. Also, upon in vitro antigenic stimulation, CD73 decreases the expression of interleukin-2 receptor α chain and the anti-apoptotic molecule Bcl-2, findings that may explain the reduced CD8+ T cell survival observed in this condition. These results indicate that CD73 has a dual effect on CD8+ T cells depending on whether they are subject to an antigenic or homeostatic stimulus, and thus, special attention should be given to these aspects when considering CD73 blockade in the design of novel antitumor therapies.

Rapid Helper T-Cell Recovery at 3 Months Correlates to Successful Transplant Outcomes after Allogeneic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5201-5201
Author(s):  
Joon Ho Moon ◽  
Jin Ho Baek ◽  
Dong Hwan Kim ◽  
Sang Kyun Sohn ◽  
Jong Gwang Kim ◽  
...  
Keyword(s):  
Overall Survival ◽  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Opportunistic Infections ◽  
Helper T Cells ◽  
Cell Recovery ◽  
Helper T Cell ◽  
Post Transplant ◽  
Cell Counts

Abstract Background: The current study attempted to evaluate the role of a simple quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes, including overall survival (OS), non-relapse mortality (NRM), and opportunistic infections, after allogeneic stem cell transplantation (SCT). Methods: A total of 69 patients receiving an allogeneic SCT were included. The disease entities were as follows: AML 42, ALL 5, CML 15, NHL 5, and high-risk MDS 2. The peripheral lymphocyte subset counts, such as CD3+ T-cells, CD3+4+ helper T-cells, CD3+8+ cytotoxic T-cells, CD19+ B-cells, and CD56+ natural killer (NK) cells, were measured 3, 6, and 12 months post-transplant. Results: The CD19+ B-cell reconstitution was slow, while a rapid CD56+ NK cell recovery was noted. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (p&lt;0.0001), NRM (p=0.0007), and opportunistic infections (p=0.0108) when stratifying patients with cut-off value of 200×106/L CD4+ helper T-cells. A rapid CD4+ helper T-cell recovery was also independently associated with a higher CD4+ helper T-cell transplant dose (p=0.006) and donor type (p&lt;0.001) in a regression analysis. An early CD4+ helper T-cell recovery at 3 months was associated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate survival analysis, a combination of a higher CD34+ cell dose and rapid recovery of CD4+ helper T-cells at 3 months was found to a have favorable prognosis in terms of OS (p=0.001, hazard ratio [HR] 3.653) and NRM (p=0.005, HR 4.836), yet not relapse. Conclusion: A rapid recovery of the CD4+ helper T-cell count above 200×106/L at 3 months seemed to correlate with a faster immune reconstitution and predict a successful transplant outcome. Figure. The overall survival according to the helper T-cell counts at 3 months (A) and the difference of total T-(B) and helper T-cell (C) immune reconstitution within 1-year post-transplant according to helper T-cell counts at 3 months Figure. The overall survival according to the helper T-cell counts at 3 months (A) and the difference of total T-(B) and helper T-cell (C) immune reconstitution within 1-year post-transplant according to helper T-cell counts at 3 months

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