Donor T-cell Research Articles

Sirolimus Combined with Cyclosporine (CSP) and Mycophenolate Mofetil (MMF) As Graft-Vs-Host Disease (GVHD) Prophylaxis after Nonmyeloablative (NMA) Hematopoietic Cell Transplantation (HCT) Using HLA Class I or Class II Antigen Mismatched Donors: Results from a Phase II Multi-Center Trial

The success of HCT is highly dependent on the availability of compatible donors. Depending on ethnicity, fully HLA-matched donors cannot be identified for 25-84% of patients. In an effort to extend the use of HLA-mismatched donors to patients only eligible for NMA HCT, we previously demonstrated that engraftment and long-term survival can be achieved using CSP and MMF immunosuppression after HLA class I antigen-mismatched HCT (Nakamae et al, 2010). Based on this trial, we designed a phase II multi-center trial to assess the efficacy of GVHD prophylaxis with triple immune suppression with CSP, MMF and sirolimus after HLA class I or class II antigen mismatched NMA HCT (fludarabine 90mg/m2 and 2-3 Gy TBI). Patients ineligible for high-dose conditioning with only HLA class I or class II antigen mismatched donors available were eligible for inclusion. The primary objective was to determine whether the incidence of grades 2-4 acute GVHD can be reduced to less than the historical rate of 70% with the triple-immunosuppression. The evaluation was planned to be carried out separately among HLA class I and class II mismatched patients. Secondary objectives included day 100 non-relapse mortality (NRM) and grades 3-4 acute GVHD. CSP was started on day -3 and continued to day +150 and then tapered off by day +180. Sirolimus was started on day -3 through day +180 and then tapered off by day +365. MMF was given thrice daily from days 0 to +30 then twice daily to day +100 and tapered off by day +150. A total of 76 patients were enrolled and received conditioning with fludarabine and TBI followed by infusion of unmodified G-CSF mobilized peripheral blood stem cell (PBSC) grafts. The median age was 63 (21-76) years, and median follow-up of surviving patients was 47 (4-94) months. 51 (67%) and 25 (33%) of patients had HLA class I or II mismatches, respectively. Transplant demographics are summarized in Table 1. Sustained engraftment was observed in all but 1 (1%) patient with myeloproliferative disease who had autologous recovery after transplant. Median number of days with neutrophils < 500 cells/ml and platelets <20,000 platelets/ml were 14 (0-67) and 2 (0-70), respectively. Full donor T-cell chimerism (>95% donor) was achieved in 48% and 63% at days +28 and 84, respectively. Outcomes are summarized in Table 2.The cumulative incidence of grade 2-4 acute GVHD at day +100 was 34% in all patients (29% and 48% after HLA class I and class II mismatched, respectively; p=0.09) (Figure 1), with only 3% of patients experiencing grade 3 acute GVHD and no patients experienced grade 4 acute GVHD. NRM at day +100, 2 years and 4 years were 4%, 15% and 17%, respectively (HLA class I vs class II: p=0.58) (Figure 2). The 2- and 4-year cumulative incidences of chronic GVHD were 54% and 57% (HLA class I vs. class II: p=0.11). Relapse incidences at 2 and 4 years were 26% and 31% with no differences between the class I and II mismatched patients (p=0.54) (Figure 2). The cumulative incidences at 2 and 4 years of progression-free survival (PFS) were 59% and 52%, and overall survival (OS) were 68% and 62% with no differences between HLA class I and II mismatches for PFS or OS (p=0.64 and 0.90, respectively) (Figure 2). Compared to historical data from our previous trial using MMF and CSP prophylaxis alone in patients with HLA class I mismatches, the addition of sirolimus to GVHD prophylaxis with CSP/MMF improved outcomes both after HLA class I or class II mismatched HCT (Nakamae et al, 2010). In a comparison of HLA class I mismatches only, day +100 grade 2-4 acute GVHD was reduced from 69% in the historical cohort to 29% in the current trial. The relapse incidences were comparable, while 2-year NRM decreased from 47%, in historical controls, to 16%, and translated into improved OS from 26% to 67% at 2 years in the current study. No difference was observed in the incidence of chronic GVHD. Furthermore, in the current trial, similar results were seen using HLA class II mismatched donors, suggesting that the triple drug immunosuppression could be used with either type of mismatch. The current trial demonstrates that triple drug immunosuppression with CSP, MMF, and sirolimus is safe and effective in reducing both acute GVHD and NRM after NMA HCT with either HLA class I or class II antigen mismatched donors. Disclosures Maloney: Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; Celgene,Kite Pharma: Honoraria, Research Funding; BioLine RX, Gilead,Genentech,Novartis: Honoraria; A2 Biotherapeutics: Honoraria, Other: Stock options . OffLabel Disclosure: In the current study cyclosporine, mycophenolate mofetil and sirolimus are used to prevent graft versus host disease after allogeneic hematopoietic cell transplantation. Fludarabine is used together with total body irradiation as at part of the conditioning prior to allogeneic hematopoietic cell transplantation.

Single Cell RNA-Seq Reveals Deranged Developmental Hierarchy with Coexisting Oncogenic States and Immune Evasion Programs in ETP T-ALL

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP T-ALL) is a distinct subtype of T-ALL characterized by higher rates of relapse and induction failure. Large-scale genetic sequencing studies have identified frequently mutated oncogenes and gene fusions in ETP T-ALL, while bulk transcriptome analyses have revealed expression features resembling myeloid precursors and myeloid malignancies. However, the contributions of intra-tumoral functional heterogeneity and microenvironment to tumor biology and treatment failure remain unknown. Methods: We performed full-length single-cell RNA-sequencing of 5,077 malignant and normal immune cells from bone marrow or blood from five patients with relapsed/refractory ETP T-ALL (based on immunophenotyping, all with NOTCH1 mutations), before and after targeted therapy against NOTCH1. These patients were enrolled on a phase I trial with the γ-secretase inhibitor (GSI) BMS-906024 (NCT01363817). Expression of selected genes was validated by RT-PCR, flow cytometry and immunohistochemistry. Results: Single cell transcriptome analyses revealed a deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cells implying ineffectual commitment to either lymphoid or myeloid lineage. Most ETP T-ALL cells co-expressed HSC (hematopoietic stem cell), CMP (common myeloid progenitor) and CLP (common lymphoid progenitor) signatures simultaneously (Pearson correlation: CLP-CMP: R= 0.41, p < 2.2e-16; HSC-CLP: R= 0.53; p < 2.2e-16; HSC-CMP: R = 0.39, p <2.2e-16). Only a fraction of cells (less than 15%) demonstrated mutually exclusive CLP or HSC signatures. In contrast, CLP, CMP and HSC signatures were not co-expressed and always negatively correlated in normal bone marrow cells (CLP-CMP: R= -0.11, p < 2.2e-16; HSC-CLP: R= -0.38; p < 2.2e-16; HSC-CMP: R = -0.67, p <2.2e-16). Direct targeting of NOTCH1 as the driving oncogene has shown disappointing results in the clinical setting due to the rapid development of resistance. PI3K activation has been shown as a genetic mechanism of Notch resistance, however it is unclear if transcriptional rewiring can give rise to PI3K dependent cells after Notch inhibition. To address this question, we predicted the activity of signaling pathways in single cells after Notch inhibitor treatment using PROGENy. Most single cells demonstrated loss of Notch signaling. PI3K signaling activity was the most anti-correlated signaling pathway to Notch signaling (Pearson correlation: R= -0.51, p < 2.2e-16). Of note, this population preexisted at a frequency of ~30% in the untreated population, coexisting with cells with high Notch activation. Analysis of the immune microenvironment revealed an oligoclonal T-cell population in ETP T-ALL compared to normal donor T-cells. CD8+ T-cells from ETP patients expressed markers of T-cell exhaustion (PDCD1, TIGIT, LAG3, HAVCR2). Analyses of expression levels of the respective ligands on leukemic blasts and the predicted interaction with their receptors on endogenous CD8+ T-cells demonstrated the highest interaction score between HAVCR2 and its ligand LGALS9. LGALS9 was universally expressed in all leukemic cells, which was confirmed by flow cytometry staining in leukemic blasts and IHC staining in bone marrow of 8 patients with ETP T-ALL and 7 patients with T-ALL. T-ALL supernatant increased expression levels of the exhaustion markers HAVCR2,TIGIT and decreased effector marker GZMB in polyclonal activated normal donor CD8+ T-cells (RT-PCR). This effect was abrogated by neutralizing LGALS9 and could be rescued with recombinant LGALS9. Conclusion: We identified deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cell states and ineffectual commitment to either lymphoid or myeloid lineage in ETP T-ALL. Leukemic blasts demonstrate preexisting heterogeneity of diverse oncogenic states as evidenced by opposing PI3K and Notch activity, suggesting possible novel combination therapies. Notch inhibition abolishes the Notch high state without effecting the PI3K active state. Finally, we demonstrate a possible role for HAVCR2-LGALS9 interactions in causing CD8+ T-cell dysfunction in ETP T-ALL patients, which may provide a novel therapeutic strategy in this disease. Disclosures Silverman: Takeda: Consultancy; Servier: Consultancy, Research Funding. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. DeAngelo:Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria.

Short-term KRP203 and posttransplant cyclophosphamide for graft-versus-host disease prophylaxis.

Posttransplant high-dose cyclophosphamide (PTCY) has been increasingly used as graft-versus-host disease (GVHD) prophylaxis after HLA-haploidentical or matched hematopoietic stem cell transplantation (SCT). However, PTCY alone is insufficient and requires additional immunosuppressants such as calcineurin inhibitors. In the current study, we evaluated effects of a novel GVHD prophylaxis with PTCY in combination with short-term KRP203, a selective agonist of sphingosine-1-phosphate receptor 1 that regulates egress of lymphocytes from the secondary lymphoid organs (SLOs) in mice. Short-term oral administration of KRP203 alone induced apoptosis of donor T cells in the SLOs and ameliorated GVHD. Administration of KRP203 significantly preserved graft-versus-leukemia effects compared to cyclosporin. A combination of KRP203 on days 0 to +4 and PTCY on day +3 synergistically suppressed donor T-cell migration into the intestine and skin, and ameliorated GVHD more potently than PTCY alone. A combination of short-term KRP203 and PTCY is a promising novel calcineurin-free GVHD prophylaxis in HLA-haploidentical SCT.

Identification of the best-suited donor for generating virus-specific T cells.

Administration of virus-specific T cells (VSTs) is a viable antiviral treatment strategy after allogeneic HSCT, even if conventional therapies fail. Third-party donors are often chosen for the generation of the VST product. The eligibility of the donor has to be tested in a rigorous donor screening procedure, since the isolation technology only targets pre-existing VSTs. In a period of 3years, we performed 32 VST treatments for 28 patients. Targeting four different viruses, 284 healthy individuals underwent 417 donor screening procedures. VSTs were counted by flow cytometry detecting interferon-gamma (IFN-γ) producing T cells. Generation of the VSTs was performed from leukapheresis products in a fully automated and closed system using magnetic cell separation. The mean circulating VST frequencies ranged from 0·006% to 0·328%. The average yield of viable VSTs in the product was 1·83·106 cells, while the average VST dose calculated for the patient's body weight was 4·63·104 /kg. The mean purity - percentage of VSTs within the T cells - of all T-cell products was 62·9%. Correlation was identified between the frequency of the VSTs in the peripheral blood of the donor and the VST numbers of the end product; the strongest correlation was seen for CMV. This paper focuses on the T-cell donors, highlighting some key points on the donor selection process. Based on the findings in connection with the CMV therapies, peripheral VST seems to be the best predictor of the VST content of the final product administered to the patient.

Dendritic Cell-Based Immunotherapy in Advanced Sarcoma and Neuroblastoma Pediatric Patients: Anti-cancer Treatment Preceding Monocyte Harvest Impairs the Immunostimulatory and Antigen-Presenting Behavior of DCs and Manufacturing Process Outcome.

Despite efforts to develop novel treatment strategies, refractory and relapsing sarcoma, and high-risk neuroblastoma continue to have poor prognoses and limited overall survival. Monocyte-derived dendritic cell (DC)-based anti-cancer immunotherapy represents a promising treatment modality in these neoplasias. A DC-based anti-cancer vaccine was evaluated for safety in an academic phase-I/II clinical trial for children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors, mainly sarcomas and neuroblastomas. The DC vaccine was loaded with self-tumor antigens obtained from patient tumor tissue. DC vaccine quality was assessed in terms of DC yield, viability, immunophenotype, production of IL-12 and IL-10, and stimulation of allogenic donor T-cells and autologous T-cells in allo-MLR and auto-MLR, respectively. Here, we show that the outcome of the manufacture of DC-based vaccine is highly variable in terms of both DC yield and DC immunostimulatory properties. In 30% of cases, manufacturing resulted in a product that failed to meet medicinal product specifications and therefore was not released for administration to a patient. Focusing on the isolation of monocytes and the pharmacotherapy preceding monocyte harvest, we show that isolation of monocytes by elutriation is not superior to adherence on plastic in terms of DC yield, viability, or immunostimulatory capacity. Trial patients having undergone monocyte-interfering pharmacotherapy prior to monocyte harvest was associated with an impaired DC-based immunotherapy product outcome. Certain combinations of anti-cancer treatment resulted in a similar pattern of inadequate DC parameters, namely, a combination of temozolomide with irinotecan was associated with DCs showing poor maturation and decreased immunostimulatory features, and a combination of pazopanib, topotecan, and MTD-based cyclophosphamide was associated with poor monocyte differentiation and decreased DC immunostimulatory parameters. Searching for a surrogate marker predicting an adverse outcome of DC manufacture in the peripheral blood complete blood count prior to monocyte harvest, we observed an association between an increased number of immature granulocytes in peripheral blood and decreased potency of the DC-based product as quantified by allo-MLR. We conclude that the DC-manufacturing yield and the immunostimulatory quality of anti-cancer DC-based vaccines generated from the monocytes of patients were not influenced by the monocyte isolation modality but were detrimentally affected by the specific combination of anti-cancer agents used prior to monocyte harvest.

91. Differential Impact of Cytomegalovirus (CMV) Donor (D) Serostatus on Rates and Kinetics of CMV Viremia among CMV Seropositive Recipients (R+) of Ex vivo T-cell Depleted (TCD) and Unmodified (CONV) Hematopoietic Cell Transplants (HCT)

BackgroundIn unmodified (CONV) HCT, CMV donor seropositivity (D+) conveys partial protection against CMV disease mediated by the transfer of donor CMV T-cell immunity through the allograft. Ex vivo T-cell depletion by CD34 selection affords a stringent depletion of donor T cells, thus transfer of donor T-cell immunity to CMV would be negligible. We evaluate the impact of CMV D serostatus on rates and kinetics of CMV viremia by Day (D)100 post-HCT in a contemporary cohort of CONV and TCD recipients from a single center.MethodsA retrospective cohort study of R+ adult recipients of first peripheral blood or marrow HCT for hematologic malignancies (excluding multiple myeloma) from June 2010 to December 2017 at MSKCC. Routine CMV monitoring by a quantitative PCR assay occurred weekly from D14 through D100. Patients were treated preemptively. CMV viral burden was assessed as the time-averaged area under the viremia curve over 100 days from HCT (AAUC) calculated as the sum of the area of trapezoids of AUC viral loads divided by the number of weeks of follow-up viremia. The median AAUC for all patients with CMV reactivation (AAUC50) was used to classify patients as CMV controllers (AAUC ≤ AAUC50) or noncontrollers (AAUC >AAUC50).ResultsOf 509 R+, 290 (57%) patients received CONV and 219 (43%) TCD HCT; from 300 (59%) D+ and 209 (41%) D− donors. In CONV, CMV viremia occurred with similar frequency in D+ (65%) and D− (62%), P = 0.6. In contrast, in TCD, CMV viremia occurred more frequently in D+ compared with D− (83% vs. 71%, P = 0.03). Among CONV, D+ was associated with lower CMV burden (median AAUC) compared with D− (0.791 vs. 1.13, respectively, P = 0.0004). In contrast, in TCD, AAUC was similar between D− and D+ (1.19 vs. 1.35; P = 0.86). Among CONV with CMV viremia, D− were more likely to be noncontrollers compared with D+ (56% vs. 31%, respectively, P = 0.001). In contrast, among TCD with CMV viremia the proportion of noncontrollers was similar between D− and D+ (61% vs. 60%, respectively; P = 1).ConclusionDonor CMV serostatus has a differential effect on rates and kinetics of CMV viremia in R+ TCD and CONV HCT recipients. D+ is associated with less CMV viremia and less CMV burden in CONV but not in TCD. Our findings, if confirmed, have implications for donor selection algorithms. Disclosures All Authors: No reported Disclosures.

Similar outcomes of alemtuzumab-based hematopoietic cell transplantation for SAA patients older or younger than 50 years

AbstractSurvival after allogeneic hematopoietic cell transplantation (HSCT) for severe aplastic anemia (SAA) among older patients remains poor and associated with increased risk for graft-versus-host disease (GVHD). In this retrospective study of 65 consecutive patients with acquired SAA who were transplanted using fludarabine, low-dose cyclophosphamide, and alemtuzumab (FCC), outcomes of 27 patients aged at least 50 years were compared with those of 38 patients younger than 50 years. The median age of the older cohort was 61 years (range, 51-71 years); 21 (78%) patients were transplanted from unrelated donors (3 of 21 from HLA 9/10 mismatch donors) and 6 from matched sibling donors. One-year GVHD-free, relapse-free survival (GRFS) was comparable to that of patients younger than 50 years (84% vs 94%, respectively; P = .23). Both groups showed low rates of acute (5% vs 4%) and chronic (18% vs 14%) GVHD, with no cases of severe GVHD among matched donor transplants, and similar 1-year transplant-related mortality (14% vs 5.4%, older vs younger; P = .23). HSCT comorbidity index (HTC-CI) scores were similar between the groups, but overall survival with an HCT-CI of at least 3 was lower compared with a score less than 3 (76% vs 98%; P = .005). Median donor T-cell chimerism among older patients was 64% and 60% at 1 and 3 years, respectively, and was similar to that of younger patients. Increased B regulatory cells potentially contributed to low alloreactivity and mutual donor-recipient tolerance in older patients. Effect of comorbidities rather than age alone may be a more important determinant of suitability for FCC HSCT in older patients.

Donor T-cell–derived GM-CSF drives alloantigen presentation by dendritic cells in the gastrointestinal tract

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF) has recently emerged as an important pathogenic cytokine in acute graft-versus-host disease (GVHD), but the nature of the T-cell lineages secreting the cytokine and the mechanisms of action are less clear. Here we used interleukin 17A-fate reporter systems with transcriptional analysis and assays of alloantigen presentation to interrogate the origins of GM-CSF–secreting T cells and the effects of the cytokine on antigen-presenting cell (APC) function after experimental allogeneic stem cell transplantation (SCT). We demonstrated that although GM-CSF-secreting Th17 and non-Th17 cells expanded in the colon over time after SCT, the Th17 lineage expanded to represent 10% to 20% of the GM-CSF secreting T cells at this site by 4 weeks. Donor T-cell-derived GM-CSF expanded alloantigen-presenting donor dendritic cells (DCs) in the colon and lymph nodes. In the mesenteric lymph nodes, GM-CSF–dependent DCs primed donor T cells and amplified acute GVHD in the colon. We thus describe a feed-forward cascade whereby GM-CSF–secreting donor T cells accumulate and drive alloantigen presentation in the colon to amplify GVHD severity. GM-CSF inhibition may be a tractable clinical intervention to limit donor alloantigen presentation and GVHD in the lower gastrointestinal tract.

2313 Graft-versus-Host-Disease Following Liver Transplant

INTRODUCTION: Graft versus host disease (GVHD) is a rare, life-threatening complication of liver transplantation that may affect the skin, GI tract and bone marrow. Donor T-lymphocytes remaining in the transplanted graft primarily target the recipient causing organ injury. Presentation may be non-specific and mimic other post-transplant complications, leading to delay in diagnosis and a mortality rate of greater than 80%. There is no consensus regarding optimal management of GVHD in the setting of liver transplantation. CASE DESCRIPTION/METHODS: A 65-year-old male with a history of NASH cirrhosis underwent deceased-donor liver transplant. Induction therapy was given with basiliximab and solumedrol then immunosuppression was maintained with tacrolimus, mycophenolate mofetil (MMF) and prednisone. Three weeks post-transplant, diarrhea developed and MMF was dose-reduced. Given persistence of diarrhea, fever, neutropenia and bilateral lower extremity petechial rash, additional work-up was performed. Imaging revealed colonic inflammation and subsequent flexible sigmoidoscopy demonstrated diffuse inflammation in the sigmoid. Biopsies of the colonic mucosa and lower extremity skin rash were consistent with GVHD. Peripheral blood confirmed the presence of donor chimerism. The patient was started on empiric antibiotics for febrile neutropenia, received granulocyte stimulating factor for leukopenia, intravenous steroids, and oral budesonide. MMF was initially discontinued but later resumed at increased dose; tacrolimus was continued. The patient's condition continued to deteriorate with diffuse mucosal GI bleeding. He received antithymocyte globulin and etanercept but his illness progressed secondary to Enterococcus bacteremia leading to septic shock. The patient died 8-weeks post-transplant in the setting of multiorgan failure. DISCUSSION: While rare, GVHD after liver transplantation should be considered in patients with new onset, unexplained symptoms. By virtue of its rarity after liver transplantation, data are sparse and management decisions are challenging. This case illustrates the need for increased awareness and standardized treatment guidelines. Management decisions are currently based on small case series often with discordant strategies including increasing or decreasing immunosuppression exposure. Global registries and multicenter collaboration will be essential to increase knowledge and improve outcomes.

SP-05 - Unrelated donorship in Russia: donors’ register (Kirov hematological institute)

The importance of chimerism status in the very early period after hematopoietic stem cell transplantation is unclear. We determined PBMC and T-cell donor chimerism 50 days after transplantation and related this to disease relapse and overall survival.144 sequential patients underwent transplantation of which 90 had AML/MDS and 54 had lymphoma. ‘Full donor chimerism’ was defined as ≥99% donor cells and three patient groups were defined: 40% with full donor chimerism (FC) in both PBMC and T-cells; 25% with mixed chimerism (MC) within both compartments and 35% with ‘split’ chimerism (SC) characterised by full donor chimerism within PBMC and mixed chimerism within T-cells.In patients with myeloid disease a pattern of mixed chimerism (MC) was associated with a one year relapse rate of 45% and a five year overall survival of 40% compared to values of 8% and 75%, and 17% and 60%, for those with SC or FC respectively. The pattern of chimerism had no impact on clinical outcome for lymphoma.The pattern of lineage-specific chimerism at 50 days after transplantation is highly predictive of clinical outcome for patients with myeloid malignancy and may help to guide subsequent clinical management.

Activated protein C ameliorates chronic graft-versus-host disease by PAR1-dependent biased cell signaling on T cells

AbstractSoluble thrombomodulin plasma concentrations are elevated in steroid-resistant graft-versus-host disease (GVHD), implying endothelial hypofunctioning for thrombomodulin-dependent generation of activated protein C’s (APC) anticoagulant, anti-inflammatory, and antiapoptotic functions. Recombinant thrombomodulin or APC administration decreases acute GVHD, manifested by intense inflammation and tissue destruction. Here, we administered recombinant murine wild-type (WT) APC to mice with established chronic GVHD (cGVHD), a less-inflammatory autoimmune-like disease. WT APC normalized bronchiolitis obliterans–induced pulmonary dysfunction. Signaling-selective APC variants (3A-APC [APC with lysine 191-193 replaced with 3 alanines] or 5A-APC [APC with lysine 191-193 replaced with 3 alanines and arginine 229/230 replaced with 2 alanines]) with normal cytoprotective properties, but greatly reduced anticoagulant activity, provided similar results. Mechanistically, WT APC and signaling-selective variants reduced T follicular helper cells, germinal center formation, immunoglobulin, and collagen deposition. WT APC can potentially cleave protease-activated receptor 1 (PAR1) at Arg41 or Arg46, the latter causing anti-inflammatory signaling. cGVHD was reduced in recipients of T cells from WT PAR1 or mutated Gln41-PAR1 donors but not from mutated Gln46-PAR1 donors. These data implicate donor T-cell APC-induced noncanonical cleavage at Arg46-PAR1, which is known to confer cytoprotective and anti-inflammatory activities. Together, these data indicate that APC anticoagulant activity is dispensable, whereas anti-inflammatory signaling and cytoprotective cell signaling by APC are essential. Because a phase 2 ischemic stroke clinical trial did not raise any safety issues for 3A-APC treatment, our studies provide a foundational platform for testing in clinical cGVHD therapy.

Donor Lymphocyte Infusions After Allogeneic Transplantation: A Single Center Experience

Allogeneic hematopoietic cell transplantation (AHCT) represents the only curative therapy for many haematological malignancies. The graft-versus-leukemia effect (GVL), driven by donor T-cells plays a major role in its curative potential. This effect is sometimes very evident when AML and MDS relapses post AHCT are treated with donor lymphocyte infusions (DLI).

Diverse and highly cross-reactive T-cell responses in ragweed allergic patients independent of geographical region.

Ragweed frequently causes seasonal allergies in North America and Europe. In the United States, several related ragweed species with diverse geographical distribution cause allergic symptoms. Cross-reactivity towards related ragweed species of IgE and treatment-induced IgG4 has been demonstrated previously. However, less is known about the underlying T-cell cross-reactivity. The allergen content of ragweed extracts was determined by mass spectrometry and related to T-cell epitopes of Amb a allergens (group 1, 3, 4, 5, 8 and 11) in 20 American ragweed allergic patients determined by FluoroSpot and proliferation assays. T-cell responses to 50 frequently recognized Amb a-derived T-cell epitopes and homologous peptides from western ragweed (Amb p), giant ragweed (Amb t) and mugwort (Art v) were investigated in an additional 11 American and 14 Slovakian ragweed allergic donors. Ragweed extracts contained all known allergens and isoallergens thereof. Donor T-cell responses were diverse and directed against all Amb a 1 isoallergens and to most minor allergens investigated. Similar response patterns were seen in American and Slovakia donors. Several epitopes were cross-reactive between isoallergens and ragweed species, some even including mugwort. T-cell cross-reactivity generally correlated with allergen sequence homology. T-cell epitopes of multiple allergens/isoallergens are involved in the diverse T-cell responses in ragweed allergic individuals. T-cell lines were highly cross-reactive to epitopes of related ragweed species without any apparent geographical response bias. These data support that different ragweed species can be considered an allergen homology group with Amb a as the representative species regarding diagnosis as well as allergy immunotherapy.

P2A.07: Mucosal T-cell kinetic in the early phases of rat model of allogenic small bowel transplantationMucosal T-cell kinetic in the early phases of rat model of allogenic small bowel transplantation

Introduction: Intestinal transplantation (IT) faces many challenges, among them, the necessity to early detect and treat rejection processes. Improving our understanding on the dynamics of allogenic response will be useful to bring candidate targets to these aims. The objective of our study was to determine the kinetics of mucosal T-cells population in the early phase of small bowel rejection using a Heterotopic IT rat model. We made emphasis in phenotypical, activation and exhaustion markers for the early detection of the rejection phenomena. Methods: Allogeneic heterotopic small bowel IT (n=5) was performed following standard procedure. Brown Norway animals were used as donors and Lewis as recipients. Rats did not receive any immune suppressor treatment. Rejection was monitored by clinical scoring and hematoxylin-eosin staining of intestinal grafts (Gf). T-cells from the peripheral blood (PB) and small bowel mucosa were assessed by flow cytometry. CD3, CD4, CD8, CD25, CD45, CD45RC, PD-1, FoxP3 and anti-Lewis MHC I expression was analyzed. Gf samples were taken at day 0, 3 and 7 post-operative (POD). Results: No clinical signs of rejection or significant histological changes were observed at day 3. At this time, in PB 7.9 % ± 0,7 of T cells were from the donor, meanwhile in the Gf 42,7% ± 4,6 of T-cells were recipient cells. We observed a significant increase of CD3+CD4+CD25+, CD3+CD4+CD45RC+ and CD3+CD8+CD45RC+ % in comparison with basal levels (PB:p<0,05;Gf:p<0,01). CD3+CD4+CD25+FoxP3+ cells were also increased in the Gf. At day 7 animals present significant weight loss and histological features compatible with mild to moderate cellular rejection. Differently from day 3 POD, in PB we could not detect donor T-cells. In the Gf 70% ± 7 of T-cells were from the recipient. CD3+CD4+CD45RC+ and CD3+CD8+CD45RC+ % were significantly higher than at day 3 POD (PB p<0,001;Gf:p<0,01). We detected increased levels of CD3+CD4+PD-1+ and CD3+CD8+PD-1+ cells in the Gf in comparison to basal levels(p<0,01). No differences in CD25 expression was observed between day 3 and 7. Conclusion: T-cell replacement in the Gf mucosa is a very fast process and CD25 expression in both PB and the Gf is an early event that precede the histological changes and should be analyzed as a potential biomarker. Considering that T-cells expressing CD45RC have been described as key players in the Gf rejection processes, our results also suggest they could be a relevant target to treat/prevent ACR.

Very early lineage-specific chimerism after reduced intensity stem cell transplantation is highly predictive of clinical outcome for patients with myeloid disease

BackgroundThe importance of chimerism status in the very early period after hematopoietic stem cell transplantation is unclear. We determined PBMC and T-cell donor chimerism 50 days after transplantation and related this to disease relapse and overall survival. Methods144 sequential patients underwent transplantation of which 90 had AML/MDS and 54 had lymphoma. ‘Full donor chimerism’ was defined as ≥99% donor cells and three patient groups were defined: 40% with full donor chimerism (FC) in both PBMC and T-cells; 25% with mixed chimerism (MC) within both compartments and 35% with ‘split’ chimerism (SC) characterised by full donor chimerism within PBMC and mixed chimerism within T-cells. ResultsIn patients with myeloid disease a pattern of mixed chimerism (MC) was associated with a one year relapse rate of 45% and a five year overall survival of 40% compared to values of 8% and 75%, and 17% and 60%, for those with SC or FC respectively. The pattern of chimerism had no impact on clinical outcome for lymphoma. ConclusionThe pattern of lineage-specific chimerism at 50 days after transplantation is highly predictive of clinical outcome for patients with myeloid malignancy and may help to guide subsequent clinical management.

Myeloid differentiation factor 88 signaling in donor T cells accelerates graft-versus-host disease.

Myeloid differentiation factor 88 (MyD88) signaling has a crucial role in activation of both innate and adoptive immunity. MyD88 transduces signals via Toll-like receptor and interleukin-1 receptor superfamily to the NFκB pathway and inflammasome by forming a molecular complex with interleukin-1 receptor-associated kinase 4. The MyD88/interleukin-1 receptor-associated kinase 4 pathway plays an important role, not only in innate immunity, but also T-cell immunity; however, its role in donor T cells on the pathophysiology of graft-versus-host disease (GvHD) remains to be elucidated. We addressed this issue by using MyD88-deficient T cells in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-SCT). While MyD88-deficient and wild-type T cells proliferated equivalently after transplantation, MyD88-deficient T cells demonstrated impaired survival and differentiation toward Th1, Tc1, and Th17, and induced less severe GvHD compared to wild-type T cells. Administration of interleukin-1 receptor-associated kinase 4 inhibitor PF-06650833 significantly ameliorated GvHD after allo-SCT. These results thus demonstrate that donor T-cell MyD88/interleukin-1 receptor-associated kinase 4 pathway is a novel therapeutic target against GvHD after allo-SCT.

Graft plasmacytoid dendritic cells improve thymic function in allogeneic hematopoietic cell transplantation

Abstract Allogeneic hematopoietic stem cell transplantation(allo-HSCT) is a well established cure for a variety of hematologic diseases. However, graft-versus-host disease(GVHD) remain the primary obstacles to successful transplant. GVHD results from donor T-cell recognition of a genetically disparate recipient. Thymus has been recognized as a recipient tissue that can be severely damaged by GVHD. Plasmacytoid dendritic cells(pDCs) is a distinct subset of DC, which have both immunogenic and tolerogenic effect in allo-HSCT, licensed by the microenvironment to respond to the milieu in which they migrate. Impaired thymic DC has been the major causation of dysfunctional thymic negative selection, thus generating allo-reactive T cells and GVHD. To further demonstrate pDCs from the graft are migrating to thymus to protecting host from GVHD. We transplanted B10.BR mouse with pDCs from MHC mismatched GFP-labeled C57/B6 mouse, along with hematopoietic stem cells and T-cells from normal C57/B6 mouse. The results from the confocal imaging suggest that pDCs are migrating to thymus 14 days after allo-HSCT. CCR9 expression is important for pDCs migrating to thymus. We transplated mice with either wild type pDCs or CCR9 knock out pDCs. Survival rate is significantly lower in group transplanted with CCR9-knockout pDCs suggesting thymus is a critical place for pDCs to induce tolerogenic response. We also tranplanted wild tipe pDCs with thymectomized host and normal host. Thymectomy group have better survival rate compared to non-thymectomized group, further confirming the association between pDCs and thymus. Improved thymic function post transplant with pDCs from the graft may have a potential to decrease GVHD.

Cytopeutics® umbilical cord mesenchymal stromal cells (Cyto-MSC) for patients with grade II–IV acute graft-versus-host disease: a phase I/II clinical study – protocol overview

Background & Aim Background Graft-versus-host disease (GVHD) is a devastating complication following allogeneic hematopoietic stem cell transplantation (HSCT) mediated by stimulation of antigen presenting cells (APCs) which leads to donor T-lymphocytes activation and target tissue destruction, particularly affecting the skin, gastrointestinal tract, and liver in acute setting. In recent years, researchers have discovered that the application of mesenchymal stromal cells (MSCs) as salvage treatment among steroid refractory GVHD patients improves outcomes without long-term risk association. On the other hand, the use of MSCs concurrently with steroids as front-line treatment for acute GVHD has yet to be researched on. We hypothesize that this approach, as the MSCs will be administered at earlier stage of the disease, will increase survival rate and reduce mortality among aGVHD patients. Objective In this study, we aim to determine the efficacy and safety of allogeneic infusion of Cytopeutics® umbilical cord-derived mesenchymal stromal cells (Cyto-MSC) in combination of standard corticosteroid therapy as front-line approach for treatment of grade II-IV acute GVHD patients. Methods, Results & Conclusion Study Design This is a phase I/II clinical study involving patients who underwent an allogeneic HSCT for malignant or non malignant haematological disorders and developed grade II-IV acute GVHD. A total of 40 eligible patients will be recruited in this study. For Phase I open labelled study, 5 eligible patients will be recruited to receive Cyto-MSC (5 × 106 MSC per kg bodyweight) and standard treatment. Meanwhile, for Phase II double blinded placebo controlled study, another 35 eligible patients will be recruited and randomized into 2 study groups where 15 patients will be assigned into Group A to receive Cyto-MSC (5 × 106 MSC per kg bodyweight) and standard treatment, meanwhile another 20 patients will be assigned into Group B to receive Placebo and standard treatment. Cyto-MSC or Placebo will be administered at Day 1 and Day 4. Another infusion of Cyto-MSC or Placebo will be given at Day 7 if the patient shows no or partial response based on GVHD grading criteria. All patients will be assessed up until 6 months follow-up which include medical history, clinical and physical evaluations, pathology investigations, biomarkers and immune cell subsets analysis, as well as quality of life questionnaires.

Abstract A171: A fully human tissue-based ex vivo cell migration analysis model to study T-cell infiltration and distribution in colorectal cancer liver metastases

Abstract T-cell exclusion by tumors is one of the major obstacles for cancer immunotherapies. In colorectal cancer liver metastasis (CRC-LM) patients, effector T-cells are mainly found in the invasive margin and not in the tumor epithelium. Such T-cell exclusion might hinder an effective antitumor immune response and could account for therapy failures with immune checkpoint inhibitors, which is observed in the majority of CRC-LM patients. As T-cell infiltration and distribution are difficult to detect in patients, little is known about detailed immunotherapy effects on tumor-infiltrating lymphocytes (TIL). Therefore, we established a fully human tissue-based ex vivo cell migration analysis model to monitor T-cell infiltration and positioning in the authentic tumor microenvironment of CRC-LM patients. In brief, we isolated T-cells from fresh resected CRC-LM patient tissue samples, labeled the cells with a fluorescent dye and returned the labeled T-cells to the tissues, which were cultured for a certain time period. Besides autologous T-cells, several tissue samples were treated with non-autologous labeled T-cells isolated from a healthy donor. After tissue processing, immunostaining and cell quantification, we observed that remigration of the labeled autologous and donor T-cells into the tissues was significantly associated with numbers of endogenous TIL. The labeled autologous and donor T-cells showed a similar distribution pattern to endogenous TIL with highest densities in the invasive margin of patient tissues. No relevant changes in tumor cell numbers or apoptosis protein concentrations could be observed by both treatments. Furthermore, quantification of the chemokines CXCL9, CXCL10 and CCL5 showed significant associations with labeled autologous and donor T-cell infiltration. Interestingly, treatment with a PD1 inhibitor did not support infiltration of exogenous or endogenous T-cells into the tumor epithelium. Our findings highlight that infiltrating T-cells in CRC-LM are always positioned into the invasive margin independently of the T-cell´s origin. Especially microenvironmental factors such as CXCL9, CXCL10 and CCL5 seem to be involved in this process, preventing T-cell contact with the tumor epithelium, which could also not be abrogated by immune checkpoint inhibition. The similar infiltration and distribution pattern of T-cells in the ex vivo and in vivo settings highlights the functionality and reliability of the human tissue-based cell migration analysis model, emphasizing its use for studying therapy effects on TIL in CRC-LM patient tissues. Citation Format: Anna Berthel, Meggy Suarez-Carmona, Jakob N. Kather, Rodrigo Rojas-Moraleda, Pornpimol Chaorentong, Nektarios A. Valous, Fee Klupp, Martin Schneider, Alexis Ulrich, Markus Buechler, Inka Zoernig, Dirk Jaeger, Niels Halama. A fully human tissue-based ex vivo cell migration analysis model to study T-cell infiltration and distribution in colorectal cancer liver metastases [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A171.

Abstract A040: Hijacking CAR19 T-cells for use in targeting diverse hematopoietic and solid tumors

Abstract Introduction: CAR T-cells targeting CD19 (CAR19s) can eradicate B cell leukemias and lymphomas. The effectiveness of CAR19s is linked to their robust expansion properties but also their long-term persistence. Persistence is maintained by normal CD19+ B cells: a non-tumor dependent, self-renewing source of antigen. In this manner, CAR19s are quite unique. We have re-engineered CAR19s to secrete a wide variety of retargeting fusion proteins (FPs) by encoding expression cassettes downstream of the CAR sequence in lentiviral vectors. By hijacking CAR19s, we utilize their inherent persistence properties. By designing multispecific FP, we directly counter the clinically critical issues of tumor heterogeneity and antigen loss. Experimental Procedures: A lentiviral vector with an MCSV promoter was used to express the CAR19 construct and FPs. FPs and multispecific-FPs were designed to encode the extracellular domain of the CD19 protein, followed by one or two scFv sequences, separated from the CAR sequence by a P2A cleavage site, a design termed IMPACTtm (Integrated Modular Proteins for Adoptive Cell Therapy). The FPs therefore consist of the CD19 extracellular domain linked to one or more scFvs. The FPs redirect CAR19 T-cell cytotoxic activity to any tumor antigen of interest by coating that antigen with CD19 via the scFv. Further, multiple antigens can be coated with CD19 by encoding multiple scFv in the FP. Hijacked CAR19s therefore serve as a platform for targeting diverse antigens. Results: Here we describe one example in detail, focusing on Her2+ solid tumors. The CD19/anti-Her2 FP was highly potent in cytotoxicity assays targeting Her2+/CD19- solid tumor cell lines. The concentration of fusion protein required to reduce tumor cell number by 50% was 10 pM (0.7 ng/ml). Primary donor T-cells transduced with the CAR19 - CD19/anti-Her2 FP lentiviral vector secreted &amp;gt; 20 ng/ml of FP in cell culture. Cytotoxic activity of this redirected CAR19 against Her2+ SKOV3 tumor cells was demonstrated in vitro and in vivo. CD19-mediated persistence was demonstrated in serial restimulation assays. A bispecific FP containing CD19 linked to anti-Her2 and anti-EGFR scFv had specific activity against both antigens with a potency of 0.75 pM. For each antigen, the potent cytotoxicity was specifically mediated by the secreted fusion protein. Additional program examples of multispecific targeting for diverse hematologic and solid tumor types will be shown. Conclusions: The IMPACT platform addresses critical issues in cell therapy including CAR persistence, antigen escape and antigen heterogeneity, and provides important solutions for treating both hematologic and solid tumors. The potency of redirected cytotoxicity supports clinical development of CAR19/IMPACT programs, four of which are now ready for IND enabling studies. Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Christine Ambrose, Roy Lobb. Hijacking CAR19 T-cells for use in targeting diverse hematopoietic and solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A040.