Chimerism Induction Without GVHD Following Low Intensity Conditioning In MHC Disparate Recipients: Combining The Benefit Of Cyclophosphamide Post Transplant With The Tolerogenicity Of Megadose T Cell Depleted HSCT

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3238-3238
Author(s):  
Esther Bachar-Lustig ◽  
Noga Or-Geva ◽  
Yael Zlotnikov Klionsky ◽  
Yair Reisner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Organ Transplantation ◽  
High Dose ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Low Intensity ◽  
Significant Difference ◽  
Donor Type

Abstract Achieving allogeneic T-cell depleted BM (TDBM) engraftment under non-myeloablative conditioning represents a potentially safe and attractive approach for generating tolerance as a platform for organ transplantation and for the treatment of non-malignant diseases. However, overcoming the abundant host anti-donor T-cells (HADTC) surviving such protocols remains a major challenge. The potential role of megadose T cell depleted BM transplants in overcoming this barrier was shown when using 6.0Gy sublethal TBI, but chimerism could not be attained upon further reduction of the conditioning. An alternative approach currently in clinical use is based on the administration of high dose cyclophosphamide at days 3-4 after transplantation. However, attempts have been limited to T cell replete transplants containing at least 2x106 T cells/Kg. The latter are clearly critical for achieving engraftment of mis-matched transplants but they are also associated with a significant risk for GVHD. In the present study, we evaluated the potential benefit of combining these two approaches. Initially, regular (5x106) or high dose (25x106) Balb/c-Nude BM cells (fully depleted of T cells by virtue of the 'nude' phenotype) were transplanted (day 0) into fully allogeneic recipients (C3H/Hen). Conditioning included T cell debulking (TCD) with anti-CD4 and anti-CD8 antibodies (300µg each) delivered on day -6, and exposure to 2.0 Gy total body irradiation (TBI) on day -1. High dose Cyclophosphamide (CY) (100mg/kg) was administered on days +3 and +4. Chimerism analysis on days 35, 95, 180 and 225 revealed that none of the BM recipients that were transplanted with a regular dose of 5x106 T depleted BM in the presence or absence of CY, expressed donor type chimerism. In contrast , 4/7 mice receiving 25x106 cells and CY, exhibited (on day 225) more than 47.7±9. % multi-lineage chimerism. The stability of chimerism over more than 7 months indicated that tolerance was likely achieved. Indeed, this was confirmed by subsequent acceptance of donor type Balb/c skin grafts. Thus, 3/4 chimeric mice accepted Balb/c skin and rejected 3rd party C57BL/6 skin, while all mice that were inoculated with regular dose of BM cells in conjunction with CY (7/7), rejected both donor and 3rd party grafts. Encouraged by these results, we attempted to determine the optimal CY and radiation dose, to improve chimerism induction. Thus, mice receiving increasing CY dose of 125 or 150 mg /kg did not exhibit significant enhancement of chimerism. In addition, as shown in Fig1, while most of the recipients that were irradiated with 2Gy (13/15), 2.5Gy (6/6), 3Gy (4/5), or 3.5Gy, TBI (5/5) did not differ significantly in chimerism level (average= 69.1±24.7% at 300 days post transplant), further reduction of irradiation dose to 1.0Gy TBI, resulted in significantly reduced chimerism (average=25.4±26.2%, p≤0.003 ). Importantly, acceptance of donor type skin graft was further manifested when tested in chimeric mice conditioned with 2GY (9/13), while 3rd party skin was completely rejected (Fig.1). Considering previous suggestions by Ildstad et al. that a subset of CD8+ TCR- BM cells is critical for achieving donor type chimerism, we next determined whether such cells are indeed critical for engraftment of megadose T cell depleted BM transplants. To that end, CD8+ cells were depleted from the Balb/c -'nude' mega dose BM preparation, and chimerism induction was compared to control non-depleted 'nude' BM cells. The results of two independent experiments revealed no significant difference (P≥0.9974) in chimerism levels between mice transplanted in the presence (47.3±31, n=16) or absence (47.3±29, n=15) of CD8+TCR- cells, suggesting that such cells are not essential when using megadose T cell depleted transplants in conjunction with CY. Taken together, our results demonstrate that combination of T cell depleted megadose hematopoietic stem cells with CY administration post transplantation can lead to marked chimerism following low intensity conditioning, in contrast to using each approach alone. Importantly, there is no need to preserve CD8+TCR- cells in the transplant preparation. Such transplants, which are completely free of GVHD risk, could potentially pave the way for a safer tolerance induction modality in organ transplantation and in other non-malignant indications in which the risk associated with GVHD is not justified. Disclosures: Reisner: Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

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<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<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<.001; ATG/pTCD: 127±38, p<.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.

Rapid T Cell Recovery and Possible Auto-GVHD Following Adoptive Transfer of Ex-Vivo Costimulated Autologous T Cells at Day +2 Post-Transplant.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 769-769 ◽  
Author(s):  
Aaron P. Rapoport ◽  
Stephan A. Grupp ◽  
Edward A. Stadtmauer ◽  
Robert H. Vonderheide ◽  
Bruce L. Levine ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Cell Recovery ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cell Counts ◽  
Engraftment Syndrome ◽  
The Impact

Abstract Retrospective studies suggest that rapid lymphocyte recovery following autologous stem cell transplants (SCT) may be associated with better outcomes. 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 about day 14 post-transplant increased CD4 and CD8 T cell counts at day 42 post-transplant and induced pneumococcal conjugate vaccine-directed T and B-cell responses [Rapoport et al, Nature Medicine, 2005]. In 2 current studies, we are further investigating the impact of ex-vivo costimulated autologous T cells on vaccine responses after SCT. In the first study, we are investigating whether a similar strategy of pre- and post-transplant immunizations along with an early infusion of vaccine-primed ex-T can induce responses to a putative tumor vaccine composed of 4 HLA-A2-restricted peptides derived from survivin and hTERT in pts undergoing SCT for myeloma. In the second (randomized) trial, the impact of early ex-T on immune recovery and vaccine reponses is being tested in pediatric neuroblastoma pts. Compared to the previous study, two methodologic changes were made: The target number of T cells infused was raised 5-fold to 5 x 1010 (109/kg) T cells were infused on day + 2 to take greater advantage of homeostatic expansion mechanisms. Patients were monitored for delayed hematopoietic recovery because of this switch to early ex-T and the fact that survivin and hTERT are also expressed in hematopoietic stem cells. At the time of submission, 16 adult and 30 pediatric patients have been enrolled on these trials of whom 11 and 21, respectively, are evaluable for post-transplant hematopoietic and T-cell recovery. On the myeloma trial, the mean # of T cells infused was 3.95 x 1010 with 96% viability and a CD4/CD8 ratio of 1.8:1. At day 14 post-transplant, the median CD4 count was 1951/mcl (range 651–7668) and the median CD8 count was 4117/mcl (range 1499–39,354). The median # days to achieve an absolute neutrophil count (ANC) > 500 was 12 (range 11–14) and the median # days to achieve a PLT count >20,000/mcl was 13 days (range 0–28). Similarly, in the pediatric cohort, median CD4 and CD8 counts at day 30 were 1500 and 2100/mcl, respectively, compared to 22 and 14 in a group of pts who did not receive d+2 ex-T, with no impact on engraftment. 1 adult and 3 pediatric pts also developed an “engraftment syndrome” characterized by GHVD-like features with or without fever. The adult pt with day 14 CD4 and CD8 counts of 2,724 and 11,571 cells/mcl had clinical and histologic features of (autologous) gut GVHD. 3 pediatric pts developed pruritic rashes clinically and pathologically indistiguishable from GVHD within 14 d of ex-T infusion, with fever seen in 1. In the adult and 1 pediatric pt, steroid treatment led to complete resolution of symptoms. These combined data sets demonstrate that robust CD4 and CD8 T cells counts can be achieved as early as day 14 post-SCT when adults or children receive ex-T at day +2 post-SCT without exogenous IL-2 or other cytokine support. It appears that a subset of patients develop a T cell “engraftment syndrome” similar to autologous GVHD. The mechanisms responsible for this rapid immune cell recovery are currently under investigation.

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.

Molecular Remission and B Cell Aplasia Induced in a First Cohort of Adults with Relapsed B-ALL Treated with 19–28z CAR-Targeted T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3566-3566
Author(s):  
Marco L Davila ◽  
Isabelle Riviere ◽  
Xiuyan Wang ◽  
Jae H. Park ◽  
Jolanta Stefanski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Induction Chemotherapy ◽  
High Dose ◽  
Molecular Remission ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Initial Cohort

Abstract Abstract 3566 B cell acute lymphoblastic leukemia (B-ALL) in adults has a dismal prognosis. Intensified, combinatorial chemotherapy yields remarkable results in pediatric ALL but less so in adults. As allogeneic hematopoietic stem cell transplantation (HSCT) is curative in only a minority of patients, there remains a dire need for effective therapies in this patient population. Building on our results obtained with genetically targeted T cells in xenogeneic models of ALL and our experience in patients with chronic lymphocytic leukemia (Brentjens, Rivière, Blood, 2011), we have recently initiated a clinical trial (NCT01044069) treating adults with relapsed or refractory B-ALL with autologous T cells expressing the 19–28z chimeric antigen receptor (CAR), a dual CD3zeta/CD28 signaling receptor specific for the B cell antigen CD19. Human peripheral blood T cells retrovirally transduced to express the 19–28z receptor specifically lyse normal and malignant B cells in vitro and eradicate established tumors in vivo in pre-clinical animal models. We report the results from the initial cohort of patients treated on this protocol. The first two patients had relapsed disease that achieved morphologic remission following re-induction chemotherapy. Following adoptive therapy with CAR-modified T cells, one patient achieved a B cell aplasia and molecular remission documented by deep sequencing, which demonstrated the disappearance of IgH rearrangements associated with the malignant clone. The other patient had achieved a molecular remission after re-induction chemotherapy but further developed a complete B cell aplasia following modified T cell infusion. While in molecular remission, both patients successfully underwent allogeneic HSCT and were therefore removed from the study. In contrast to these two patients, the third patient failed to achieve a morphologic remission after re-induction chemotherapy and had >60% blasts in the bone marrow (BM) at the time of T cell infusion. Within 12 hours of completing T cell infusion, the patient developed high-grade fevers, hypotension, and rigors. Serum analyses demonstrated a sharp rise in cytokines (IFNg, TNFa, IL6, IL2, and IL8), reflecting rapid and robust onset of T cell activation. High-dose corticosteroids initiated 5 days after T cell infusion controlled these symptoms. Post-T cell BM aspirate on day 8 demonstrated undetectable blasts. Flow cytometry could not detect blasts or B lineage cells, but readily identified 19–28z CAR+ T cells. Significantly, the patient's B-ALL tumor cells harbored a unique 9p21 cytogenetic deletion that was detected by FISH in 28% of nuclei immediately prior to T cell infusion and was reduced to 5% 8 days after T cell infusion. The next BM aspirate, performed on day 24 after T cell infusion, demonstrated a persistent morphologic remission, a recovering BM, and complete absence of any detectable 9p21 deletions. Furthermore, PCR amplification for IgH rearrangements in the BM 24 days after T cell infusion confirmed a molecular remission and a B cell aplasia. Collectively, the clinical outcomes from this initial cohort of 3 patients demonstrate for the first time the in vivo efficacy of CD19-targeted T cells to induce clinical and molecular remissions as well as B cell aplasia in adults with relapsed or refractory B-ALL. These early results strongly support further investigation of 19–28z targeted T cells to treat leukemias and suggest this is a potential salvage therapy for patients with relapsed/refractory B-ALL who are failing chemotherapy or are ineligible for allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Cardiac Toxicity Of High Dose Cyclophosphamide Post T Cell Replete HLA Haploidentical Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5457-5457
Author(s):  
Laetitia Souchet ◽  
Julien Mayaux ◽  
Damien Roosweil ◽  
Madalina Uzunov ◽  
Alfred Le ◽  
...  
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Cardiac Toxicity ◽  
Early Death ◽  
High Dose ◽  
Control Cohort ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Background T cell replete (TCR) HLA haploidentical hematopoietic stem cell transplantation (SCT) using high dose cyclophosphamide post-transplant (HDCy) to prevent graft versus host disease (GvHD) and graft’s rejection is a promising approach which extends the possibility of grafting patients who lack a conventional donor. Notably, non relapse mortality (NRM) is greatly decreased compared to T-cell depleted haploidentical SCT, giving a low rate of lethal infections or severe GvHD and allowing the possibility to reduce doses of conditioning regiment without impairing the engrafting of the TCR bone marrow. Nevertheless, HDCy post transplant might increase the risk of cardiac toxicity. Methods From March 2012 to July 2013, 11 consecutive adult patients received a TCR haplo-SCT for high risk hematological malignancies: de novo or secondary acute myeloid leukemia (AML) with unfavorable cytogenetics in CR1 (n=5), refractory AML (n=2), chronic myeloid leukemia (n=2), and refractory Hodgkin disease (n=2). Patients with myeloid disease received a myeloablative conditioning (MAC) (Cy 30mg/Kg/d at day -4 and -3 + TBI 12Gy or Busulfan 3.2mg/Kg/d for 4 days + Cy 14.5mg/Kg at day-3 and day-2 + Fludarabine 90mg/m2) or a reduced intensity conditioning (RIC) with Melphalan 140 mg/m2 + Thiotepa 10mg/m2 + Fludarabine 160mg/m2. Two patients with Hodgkin disease and 1 patient who rejected a previous MAC-double cord blood received a RIC with Cy 14.5mg/Kg at day-6 + TBI 2gy + Fludarabine 150mg/m2. Ten patients received HDCy 50 mg/kg/day on days +3 and +4, plus tacrolimus and mycophenolate mophetil for GvHD prophylaxis. In one patient, HDCy was reduced to 50mg/Kg at day+3. All patients but one received a bone marrow graft. Median age was 39.1 (range 22-62). Donors were related for 10, and unrelated for 1 patient with a median of 5.4/10 allele matches (range 5-8). Cardiac evaluation pre-SCT was performed for all patients. Cardiac ultrasound was normal for all (median left ventricular ejection fraction was 62%; range 53-67%). None of the patients had cardiovascular risk factors such as hypertension, diabetes, obesity or history of valvular or ischemic cardiopathy. Median Sorror score was 0.63 (range 0-2). The rate of early deaths (within the 1st month post transplant) related to cardiac toxicity was compared to that of the whole cohort of 875 patients who received an allogeneic HSCT in our center from May 1984 to July 2013 without HDCy post transplantation. Results Among the 11 patients who received HDCy post SCT, 3 patients developed a compressive pericardial effusion at day 4, day 14 and day 41. An extensive microbiological research found no infectious etiology of this cardiac complication and toxicity of HDCy post transplant was the most probable. One patient had received a MAC (TBI 12Gy –Cy) and the 2 other patients had received the RIC Mel-Thio-Flu regimen. Evolution was reversible for 2 patients after the administration of angiotensin-converting enzyme inhibitors and/or pericardial drainage. One patient died at day+16 of a refractory cardiogenic shock. In the control cohort, early death correlated to cardiac toxicity was reported in 6 patients out of 875 (0.69%), as compared to 1/11 (9%) in the haploidentical cohort. In the control cohort, median age of patients who died from cardiac toxicity was 40.1 (range 30-51). All but 1 received a MAC including HDCy pre-transplantation. No HDCy was administered after SCT. The death occurred at a median time of 14.6 days post-transplant. No early death correlated to cardiac toxicity was reported after 1 month post SCT. Conclusion Although limited by the small number of patients, the rate of cardiac toxicity seems to be increased after HDCy post TCR haplo-SCT. This toxicity is probably multi-factorial and HLA mismatches syndrom may contribute to the severity of symptoms. Nevertheless, such severe and early cardiac complication is an unusal observation in patients without any particular cardiovascular risk factor or other co-morbidities at the time of transplant. Caution should be taken during the days following the administration of HDCy in collaboration with intensive care specialists. Disclosures: Leblond: Roche : Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Efficacy of anti-CD19 chimeric antigen receptor modified T(CAR-T) cell therapy in Chinese patients with relapsed/refractory acute lymphocytic leukemia in a multicenter trial.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 7028-7028 ◽  
Author(s):  
Lei Xiao ◽  
He Huang ◽  
Xiaojun Huang ◽  
Xiaoyan Ke ◽  
Yu Hu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Disease Free Survival ◽  
Lymphocytic Leukemia ◽  
Chinese Patients ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T Cell ◽  
Significant Difference ◽  
Car T

7028 Background: r/r B-ALL was reported as the most-threatening disease because of the low disease free survival even treatment with allogeneic hematopoietic stem cell transplantation. For overcoming conventional therapies limitation, autologous CD19CAR-T was performed in our clinical trials to induce remission in patients with r/r disease. 30 patients (from 7 clinical centers, in China) as volunteers with r/r B-ALL were treated by autologous CD19 CAR-T. Methods: 5 juveniles and 25 adults with r/r ALL received autologous CD19 targeted CAR-T, the doses between 1.03 × 106 CAR-T cells/kg and 10.09 × 106CAR-T cells/kg. These 30 cases(from 7 clinical centers, in China) were treated with CAR-T cells from May. 8 2015 to January. 4 2017 (Table1). Patients were monitored for a response. Highly standardized CAR T cell preparation protocol and manageable CRS in most were kept for no significant difference in 7 clinical centers. Results: After treated with CAR-T, a total of 30 cases (5 juveniles and 25 adults coming from 7 clinical centers, in China) with r/r B-ALL were all detected the CAR-T cells proliferated in the blood and bone marrow. The results showed that complete remission (CR) is 26/30(86.67% ) between day7-14 after CD19 CART cell infusion, and 25/30(83.33%) cases arrived at MRD negative. There is about 1/3 of the total cases receiving a repeat infusions following initial ones since these patients have no safety concerns. Additionaly, the severe Cytokine release syndrome (CRS) was 8/30(26.67%) of cases and 24/30(80%) of cases was seen CRS. The anti-IL6R agent tocilizumab and Methylprednisolone were effective confrontation Severe CRS. Conclusions: This is the first multicentre report to our knowledge of successful treatment of r/r ALL with anti-CD19 CAR T cells in China. Even r/r B-ALL with high-burden leukemia patients also was effective and associated with a high remission rate after infused autologous CD19 CAR-T).(NCT 02813837).

Comparison of T Cell (CD3+) Chimerism after Myeloablative (MY) and Nonmyeloablative (NM) Allogeneic Hematopoietic Stem Cell Transplantation (AHSCT).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2960-2960
Author(s):  
Ronald Sobecks ◽  
Karl Theil ◽  
Lisa Rybicki ◽  
Jennifer Bates ◽  
Matt Kalaycio ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Hla Class I ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Immune Effector ◽  
Post Transplant ◽  
Significant Difference

Abstract Assessment of chimerism after AHSCT has been important to monitor donor hematopoietic engraftment and to assess for residual disease or relapse. Achievement of T cell (CD3+) complete donor chimerism (CDC), particularly after NM AHSCT, has been considered important to achieve a graft-vs.-malignancy effect. Formal comparisons of the onset and frequency of T cell CDC in pts treated with MY vs. NM conditioning regimens have not been well described. The current analysis compared rates of achieving T cell CDC for 116 pts transplanted from 1/10/00–5/19/06 who were categorized into the following 4 groups based upon type of transplant conditioning: 1) 200 cGy total body irradiation (TBI) + fludarabine 30 mg/m2/d × 3 days (NM200; n=47); 2) 400 cGy TBI + fludarabine (NM400; n=23); 3) MY AHSCT with busulfan/cyclophosphamide without TBI (MY-noTBI; n=31); 4) MY AHSCT with TBI (MY-TBI; n=15). All NM AHSCT pts received peripheral blood stem cells and all MY AHSCT patients received bone marrow as their stem cell source. The total nucleated cell (TNC) and CD34+ cell doses were higher in the NM AHSCT patients. 36 (31%) pts had matched unrelated donors, all with at least an 8/8 match (HLA-A, -B, -Cw, -DR) by HLA class I and II DNA-based typing [10 (21%) NM200, 7 (30%) NM400, 9 (29%) MY-noTBI, 10 (67%) MY-TBI; p=0.011]. Graft-vs-host disease prophylaxis consisted of mycophenolate mofetil and cyclosporine or tacrolimus for most pts. Short tandem repeat analysis for T cell (CD3+) chimerism was performed on peripheral blood post transplant and CDC was defined as achievement of >95% DNA of donor origin isolated from CD3+ T cells. Post-transplant T cell chimerism was found in both the MY and NM AHSCT groups. The number of pts who achieved CDC and the median time to its occurrence for each group was as follows: NM200 - 34 (72%) at 4 mos; NM400 - 18 (78%) at 2.7 mos; MY-noTBI - 20 (65%) at 3.3 mos; and MY-TBI - 13 (87%) at 1.3 mos. The Kaplan-Meier curves for achievement of CDC are shown above (p=0.23). The group of pts who received MY-TBI developed T cell CDC more rapidly than the NM200 pts (p=0.05). No significant differences were observed between the NM400, MY-noTBI and MY-TBI groups with regards to achieving CDC. NM conditioning with either 200 cGy or 400 cGy TBI did not show a significant difference in rate of achieving T cell CDC as compared to MY conditioning with busulfan/cyclophosphamide without TBI. This may be related in part to the higher TNC and CD34+ cell doses in the NM AHSCT pts. However, the increased TBI doses utilized for MY conditioning may more effectively suppress anti-donor immune effector cells from the recipient, which resulted in the increased CDC compared to the NM200 group. In conclusion, post-transplant monitoring for T cell CDC is important in both MY and NM AHSCT to allow for immune manipulation to maintain a state of donor-host tolerance in order to prevent graft rejection. Figure Figure

Efficient Selection of WT1-Specific Donor Lymphocytes by Streptamer Technology.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3311-3311
Author(s):  
Xinchao Wang ◽  
Anita Schmitt ◽  
Xun Xu ◽  
Bao-An Chen ◽  
Mathias Freund ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Effector T Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Cytotoxicity Assays ◽  
Significant Difference ◽  
Selection Of ◽  
Leukemia Antigen

Abstract Abstract 3311 Poster Board III-199 BACKGROUND After allogeneic hematopoietic stem cell transplantation, donor lymphocyte infusions (DLIs) have the potential to generate a desirable graft-versus-leukemia (GVL) effect, but bear the risk of eliciting a noxious graft-versus-host disease (GVHD). To minimize the risk of a GVHD and to improve the GVL effect, a positive selection of leukemia (antigen)-specific T cells would be highly desirable. In this study we focused on the leukemia-antigen Wilms Tumor gene 1 (WT1). In patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), a good correlation of WT1 expression and the number of leukemia blasts could be demonstrated. Several groups described immunogenic T cell epitopes derived from the WT1 protein. MATERIAL AND METHODS Here, we used the technology of streptamers available on a GMP level to detect the frequency of HLA-A2 restricted CD8+ T cells in the naïve peripheral blood (PB) from both healthy donors (HDs) and AML patients. Such WT1-specific CD8+ T cells were further characterized for the expression of CD27, CD28, CD45RA, CCR7 and CD107a. In the next step, WT1-specific cells were positive selected by magnetic cell separation (MACS™) columns after labeling with streptamers or tetramers and thereafter immunophenotyped. Moreover, mixed lymphocyte peptide cultures (MLPCs) were performed to enrich WT1-specific T cells derived from the PB of HDs. At last, WT1 specific T cells were evaluated in CFSE-based proliferation and cytotoxicity assays. Streptamer selected WT1-specific CD8+ T cells were compared with tetramer selected WT1-specific CD8+ T cells. RESULTS 21 of 40 HDs showed naïve WT1 specific T cell frequencies of 0.5 to 2.0% of all CD8+ T cells. In two of ten AML patients, also 0.4 to 3.6% of WT1-specific T cells could be detected. Through streptamer based MACS™, a purity of more than 90% of all CD8+ T cells could be achieved for WT1-specific CD8+ T cells. These cells revealed to be CD8+WT1 _Streptamer+CD28+ CD45RA+CD69-CD107a+CCR7-CD137- effector T cells in flow cytometry. Proliferation assays proved that the streptamer technology does not alter the proliferative potential of the WT1-specific CD8+ T cells. In cytotoxicity assays, WT1-specific CD8+ T cells after streptamer selection were able to lyse HLA-A2+WT1+ cells at an effector/target ratio of 20:1. No significant difference between streptamer and tetramer selection could be found for the phenotype, as well as for the proliferative and cytotoxic potential of the cells. After a maximum of three rounds of MLPC, only a frequency of 2-5% could be achieved, thus demonstrating the power of the streptamer technology. CONCLUSION In summary, the streptamer technology allows to select a highly purified fraction of WT1-specific effector T cells with proliferative and cytotoxic properties. In analogy to DLIs specific for viral antigens, production of leukemia specific DLIs is feasible on a GMP level. Further leukemia antigens are currently evaluated by our group. Disclosures No relevant conflicts of interest to declare.

Rapid Immune Reconstitution Following Unmanipulated Haploidentical BMT with Post-Transplant High Dose Cyclophosphamide

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3050-3050 ◽  
Author(s):  
Alida Dominietto ◽  
Anna Maria Raiola ◽  
Barbara Bruno ◽  
Maria Teresa van Lint ◽  
Francesco Frassoni ◽  
...  
Keyword(s):  
Immune Reconstitution ◽  
Conflicts Of Interest ◽  
Conditioning Regimen ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Cd4 Counts ◽  
Post Transplant ◽  
Gvhd Prophylaxis ◽  
Donor Type ◽  
Unrelated Cord Blood

Abstract Abstract 3050 Background. Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for the majority of hematological malignancies. Early and successful immunologic reconstitution after HSCT reduces morbidity and mortality due to infection complications and improves survival. Aim of the study. We analyzed immune recovery after HSCT in 444 patients according to donor source. Patients and Methods. From January 2005 to June 2011 176 patients were grafted from HLA identical siblings (MSD), 125 from alternative donors (1 antigen mismatched family or unrelated donors) (ALT), 103 from unrelated cord blood grafted intra bone (CBIB) and 40 from haplo-identical mismatched family donors (HAPLO). All patients received unmanipulated bone marrow: 283 after a myeloablative (MA) conditioning regimen (CY-TBI or BU-CY) and 161 after a fludarabine based reduced intensity regimen (RIC). Graft versus host disease (GvHD) prophylaxis was cyclosporin methotrexate (CyA+MTX) for all patients except for CBIB (CyA and mycophenolate, MMF) and for HAPLO transplants which consisted of CyA+MMF and post-transplant high dose cyclophosphamide (HDCY) according to the Baltimore protocol (Lutznik et al BBMT 2008). Anti-thymocyte globulin (ATG) was used only for ALT transplants. Results. We compared immune reconstitution in MA and RIC transplants according to donor type at different time points post BMT. CD3+ absolute median counts/μl in MA conditioning on day+30, +90, +180 were respectively in MSD 477, 565, 700; in ALT donors were 146, 404, 470; in CBIB were 30, 57, 196; for HAPLO transplants they were 195, 182, 499. CD3+ absolute median counts/μl in RIC conditioning on day+30, +90, +180 were respectively in MSD 301, 660, 700; in ALT donors were 506, 186, 721; in CBIB 234, 399, 522; in HAPLO were 178, 276, 1300. CD4+ absolute median counts/μl in MA conditioning on day+30, +90, +180 were respectively in MSD 166, 170, 198; in ALT donors were 36, 86, 111; in CBIB 7, 36, 106; for HAPLO transplants they were 45, 127, 211. CD4+ absolute median counts/μl in RIC conditioning on day+30, +90, +180 were respectively in MSD 89, 189, 274; in ALT donors were 131, 210, 220; in CBIB 52, 110, 130; for HAPLO transplants they were 41, 205, 385. CD8+ absolute median counts/μl in MA conditioning on day+30, +90, +180 were respectively in MSD 280, 389, 500; in ALT donors were 102, 278, 413; in CBIB 42, 16, 51; in HAPLO transplants were 73, 424, 408. CD8+ absolute median counts/μl in RIC conditioning on day+30, +90, +180 were respectively in MSD 196, 432, 300; in ALT donors were 366, 65, 494; in CBIB 71, 167, 199; for HAPLO transplants they were 137, 129, 900. CD3, CD8, and CD4 counts in HAPLO transplants were not statistically different from MSD with the only exception of day +30, both for MA and RIC conditioning. Platelet median counts/μl on day+30, +90, +180 in MA conditioning were in MSD 142, 129, 180, in ALT 75, 101, 147, in CBIB were 19, 77, 128 and for HAPLO transplants were 67, 126, 128; in RIC conditioning platelets counts were in MSD 137, 156, 168, in ALT 33, 134, 142, in HAPLO were 77, 95, 188. Acute GvHD II-IV developed in 29% (MSD) 38% (ALT) 16% (CBIB) and 12% (HAPLO) (p=0.004) in MA conditioning and 40% (MSD) 18% (ALT) 25% (CBIB) and 10% (HAPLO) (p=0.07). Overall Cumulative Incidence of Non-Relapse Mortality (CI-NRM) was respectively 18% (MSD), 35% (ALT), 34% (CBIB), 22% (HAPLO) (p=0.02) in MA conditioning (p=0.02) and was 30% (MSD), 33% (ALT), 45% (CBIB), 0% (HAPLO) (p=0.02) in RIC conditioning (p=0.02). Day+100 CI-NRM was respectively 10% (MSD), 21% (ALT), 19% (CBIB), 12% (HAPLO) in MA conditioning (p=0.01) and 11% (MSD), 19% (ALT), 26% (CBIB), 0% (HAPLO) in RIC conditioning (p=0.02). Death due to infections were respectively 6% (MSD), 26% (ALT), 30% (IBCB), 17% (HAPLO) in MA conditioning and for RIC were 15 (MSD), 36% (ALT), 32% (IBCB), 0% (HAPLO). Conclusions. HAPLO transplant with HDCY post transplant as proposed by the Baltimore group, is associated with (1) rapid immunologic (CD3, CD4, CD8) recovery (2) low infectious death rate, (3) low overall and Day+100 CI-NRM, (4) rapid hematologic recovery. These results are comparable with those achieved with MSD and warrant further studies with HDCY post transplant as a GvHD prophylaxis. Figure: absolute CD4+ counts/μl on day+30, +90, +180, according to donor type in MA conditioning regimen. Disclosures: No relevant conflicts of interest to declare.

Developing Novel Approaches To Comprehensively Assess T Cell Repertoire Dynamics In The Early Post-Transplant Period

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4618-4618
Author(s):  
Ute E. Burkhardt ◽  
Joseph Kaplinsky ◽  
Cindy Desmarais ◽  
Kristen E. Stevenson ◽  
Edwin P. Alyea ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Healthy Controls ◽  
Transplant Recipients ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
T Cell Reconstitution ◽  
Tcr Repertoire ◽  
Control Samples

Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a highly effective treatment modality for many hematologic malignancies, a major treatment-associated toxicity is the induction of a prolonged state of T cell immunodeficiency in the transplant recipient, which in turn contributes to critical clinical outcomes such as infectious complications, and the risk of relapse. Targeted deep sequencing of the T cell receptor beta-chain (TCRβ) has emerged as a promising technology for enabling the qualitative and quantitative monitoring of T cell recovery following transplant with unprecedented resolution. Major challenges remain, however, in the establishment of informative analysis tools for characterization of global TCRβ repertoire dynamics. In the current work, we developed and applied a novel analysis approach as a mean to gain detailed biological insight into T cell reconstitution following allo-HSCT. To this end, we isolated naïve and memory CD4+ and CD8+ T cells from peripheral blood mononuclear cells of 14 patients with advanced chronic lymphocytic leukemia who underwent allo-HSCT following reduced-intensity doses of fludarabine and busulfan. From these T cell subpopulations, genomic DNA was extracted at post-transplant day 30 (d30) and later time points informative for thymic-independent (4 month post-transplant; d120) and thymic-dependent (1 year post-transplant; d365) T cell immune recovery. Subsequently, a template library for sequencing on an Illumina GA2 system was generated through PCR amplification of the TCRβ CDR3 region using an established panel of 45 Vβ- and 13 Jβ-specific primers. We obtained a median of 394,872 (range 0-26,426,784) productive reads across our 168 samples. As a comparison group, we further studied repertoire data from naïve and memory CD4+ and CD8+ T cells collected from 9 healthy adult volunteers. To characterize how transplant perturbs the TCR repertoire, we first compared VDJ usage between the transplanted patients and the healthy controls. For each of the post-transplant and control samples, we tallied the number of clones from all sequenced compartments (CD4+ and CD8+, naïve and memory) that used each of the several thousand possible VDJ combinations. We performed pairwise comparisons of the resulting VDJ distributions for all 253 sample pairs at days 30, 120 and 365 by calculating the R2 and, separately, X2 statistics. Permutation analysis demonstrated that control samples were more similar to each other than either post-transplant day 30, 120 or 365 samples (P=2.5-5.0x10-5, 2.5-5.0x10-5 and ≤2.5x10-5 by X2; 2.5-5.0x10-5, 5.5-5.7x10-4 and 1.0-1.2x10-4 by R2, respectively). Of note, whereas control samples demonstrated a similar VDJ usage, such similarity was not observed among post-transplant samples at day 30, 120 or 365 (P=0.65, 0.53, and 0.60 by X2; P=0.014, 0.38, and 0.43 by R2, respectively). These results demonstrate that VDJ usage in transplant recipients remains more heterogeneous than in healthy controls throughout the entire first year of reconstitution. To understand whether this heterogeneity reflects equilibrium or dynamic changes of the TCR repertoire, we visualized the time course of reconstitution using principal component analysis of VDJ usage. We observed marked dynamism, in which most transplant recipients both experienced a greater degree of change than was represented by the controls, and explored regions of VDJ usage very different from that of controls. Preliminarily, we observed that several transplant recipients became more similar to controls over time, while others did not. Our results demonstrate that post-transplant T cell reconstitution follows both personal and highly dynamic trajectories across a range of clinical courses, and suggest that TCR sequencing in larger sample sizes is a promising avenue for future study. Ongoing analyses focus on investigating the correlates of this dynamism among the 14 transplant recipients through subgroup analysis based on their clinical course and sequence-level analysis. The results obtained through these novel computational and systems methods will be integrated with other experimental measures of immune reconstitution including immunophenotyping and TCR excision circle (TREC) analysis. Disclosures: No relevant conflicts of interest to declare.

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