Abstract: Administration of posttransplant cyclophosphamide (PTCy) has significantly expanded the number of patients undergoing HLA-haploidentical hematopoietic cell transplantation (haplo-HCT). To examine immune reconstitution in these patients, we monitored T- and natural killer (NK)-cell recovery in 60 patients receiving bone marrow or peripheral blood stem cell (PBSC) grafts after haplo-HCT with PTCy and 35 patients receiving HLA-matched donor PBSC grafts with standard graft-versus-host disease (GVHD) prophylaxis. Compared with HLA-matched recipients, early T-cell recovery was delayed in haplo-HCT patients and skewed toward effector memory T cells with markedly reduced naive T cells. We found higher regulatory T (Treg)-cell/conventional T (Tcon)-cell ratios early after HCT and increased PD-1 expression on memory T cells. Within the haplo-HCT, patients who did not develop chronic GVHD (cGVHD) had higher PD-1 expression on central and effector memory CD4+ Treg cells at 1 month after transplant. These findings suggest an immunologic milieu that promotes immune tolerance in haplo-HCT patients. NK cells were decreased early after haplo-HCT with preferential expansion of immature CD56brightCD16− NK cells compared with matched donor transplants. One month after transplant, mass cytometry revealed enrichment of immature NK-cell metaclusters with high NKG2A, low CD57, and low killer-cell immunoglobulin-like receptor expression after haplo-HCT, which partially recovered 3 months post-HCT. At 2 months, immature NK cells from both groups were functionally impaired, but interleukin-15 priming corrected these defects in vitro. Increased immature/mature NK-cell ratios were associated with cytomegalovirus reactivation and increased incidence of cGVHD after haplo-HCT. These homeostatic imbalances in T- and NK-cell reconstitution after haplo-HCT reveal opportunities for early immune-based interventions to optimize clinical outcomes.

Abstract: CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), expressed on antigen presenting cells, epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T-cell activation, proliferation, and trafficking. In this study we examined expression of CD6 by reconstituting T cells in 95 patients after allogeneic cell transplantation and evaluated the effects of itolizumab, an anti- CD6 monoclonal antibody, on T-cell activation. CD6 T cells reconstituted early after transplant with CD4 regulatory T cells (Treg)-expressing lower levels of CD6 compared to conventional CD4 T cells (Tcon) and CD8 T cells. After onset of acute graft-versus-host disease (aGvHD), CD6 expression was further reduced in Treg and CD8 T cells compared to healthy donors, while no difference was observed for Tcon. ALCAM expression was highest in plasmacytoid dendritic cells (pDC), lowest in myeloid dendritic cells (mDC) and intermediate in monocytes and was generally increased after aGvHD onset. Itolizumab inhibited CD4 and CD8 T-cell activation and proliferation in preGvHD samples, but inhibition was less prominent in samples collected after aGvHD onset, especially for CD8 T cells. Functional studies showed that itolizumab did not mediate direct cytolytic activity or antibody-dependent cytotoxicity in vitro. However, itolizumab efficiently abrogated the costimulatory activity of ALCAM on T-cell proliferation, activation and maturation. Our results identify the CD6-ALCAM pathway as a potential target for aGvHD control and a phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGvHD is currently ongoing (clinicaltrials gov. Identifier: NCT03763318).

Abstract: Background Cytokine-release syndrome (CRS) is a life-threatening complication of haploidentical stem cell transplantation (haploSCT) occurring in the first few days after infusion of the stem cell graft prior to administration of post-transplant cyclophosphamide (PTCY). In the last few years, blockade of IL-6 receptor signaling with tocilizumab has emerged as an effective therapy for Grade 3-4 CRS. As IL-6 mediated signaling is a known regulator of the balance between regulatory T cells (Tregs) and other T cell subsets and has effects on NK cell function, questions remain regarding the effect of the blockade of IL-6 signaling on post-transplant immune cell recovery, engraftment, infection, acute graft-versus-host disease (aGVHD), and the graft-versus-tumor effect. We report on our experience with the use of tocilizumab to treat CRS, and we address the hypothesis that tocilizumab has no effect on the reconstitution of the post-haploSCT myeloid or lymphoid immune subsets. Methods A retrospective review of haploSCT patients with malignacies treated at the Dana-Farber Cancer Institute during the period of 2010-2019 was undertaken. Data included treatment with tocilizumab, cumulative incidence of NRM and relapse, day+30 and day+100 post-transplant chimerism, laboratory markers of viral and fungal infection, and immune reconstitution data at 1-month, 2-month, 3-month, and 6 months post-transplant. Kaplan-Meier analysis of overall survival (OS), including competing risk analysis of the cumulative incidence of relapse and non-relapse mortality (NRM) with the Fine-Gray model was preformed with EZR. Flow cytometry identification of relevant lymphoid populations was performed with a customized panel that included Treg (CD4+CD25+), Tcon (CD4+) and NK (CD3-CD56+) cells. Comparison of the lymphoid populations between the Tocilizumab-treated subgroup of haploSCT patients and the remaining patients was done with the Wilcoxon Mann-Whitney test. Results Out of 132 haploSCT patients, 19 received at least one dose of tocilizumab for the treatment of CRS out of whom one patient died from CRS. In tocilizumab-treated patients myeloid engraftment was 100% at day 30 post transplant. Tocilizumab use was not associated with any effect on lymphoid subset reconstitution at any time point post haploSCT (Figure 1A). Specifically, there was no difference in the reconstitution of NK cells (1-month NK: Mann-Whitney U = 120, p = 0.356; 3-month NK: Mann-Whitney U = 130, p = 0.528; 6-month NK: Mann-Whitney U = 110, p = 0.773), Treg (1-month Treg: Mann-Whitney U = 77, p = 0.412; 3-month Treg: Mann-Whitney U = 104, p = 0.203, 6-month Treg: Mann-Whitney U = 80, p = 0.186), or the Treg:Tcon ratio (1-month Treg:Tcon: Mann-Whitney U = 93, p = 0.835; 3-month Treg:Tcon: Mann-Whitney U = 97, p = 0.141; 6-month Treg:Tcon: Mann-Whitney U = 117, p = 0.959). The 1-year OS in the tocilizumab-treated patient subgroup was 62% (95% CI, 36%-80%) while in the rest it was 76% (95%CI, 67%-83%), with no significant difference between the two subgroups (p = 0.13). The 1-year cumulative incidence of relapse in the tocilizumab-treated subgroup was 19% (95% CI, 4%-42%) while in the rest it was 28% (95% CI, 20%-36%), with no difference between the subgroups (p=0.29). The 1-year NRM in the tocilizumab-treated subgroup was 33% (95% CI, 12%-55%) while in the rest it was 10% (95% CI, 5%-16%), and the difference between subgroups was statistically significant (p = 0.02). Fifty-five percent of patients treated with tocilizumab experienced reactivation of one or more of adenovirus, EBV, CMV or a fungal organism, with 50% of reactivations being CMV and 37% being fungal. Among the tocilizumab-treated patients, 42% experienced any aGVHD with only one patient experiencing Grade 3-4 aGVHD. Conclusions Tocilizumab use is not associated with any effect on post-transplant myeloid engraftment or reconstitution of the Treg, Tcon, and NK cell subsets. Although a significant proportion of tocilizumab-treated patients experienced reactivation of CMV or a fungal organism, the majority of these reactivations were not associated with any clinically significant symptoms. Treatment with tocilizumab was not associated with any significant effect on OS or disease relapse, but the tocilizumab-treated group had a higher NRM than the rest of the haploSCT patients. This association is consistent with prior studies correlating severe CRS with a high NRM, and merits further study. Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Biolojic Design Inc: Consultancy; Regeneron: Other: Research Support; BMS: Other: Research Support; Cugene: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Other: Research Support; Equillium: Consultancy; Moderna Therapeutics: Consultancy; Therakos: Membership on an entity's Board of Directors or advisory committees; EMD Serono: Consultancy; Clinigen: Other. Cutler:Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Be The Match/National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Celgene: Other; Juno: Other; Novartis: Consultancy; Alexion: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Gilead: Consultancy. Ritz:TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Rheos Medicines: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy.

Abstract: Background Relapse of acute myeloid leukemia (AML) after allogeneic stem cell transplant has a poor prognosis with limited treatment options. Cytokine-induced memory like natural killer (CIML NK) cells are a novel therapy with enhanced cytotoxicity independent of KIR ligand interactions able to induce remission of relapsed/refractory AML (Romee et al, Science TM 2016). We are evaluating the safety and potential efficacy of donor-derived CIML NK cells in patients with relapsed myeloid malignancies after haploidentical donor transplant (haploSCT) in a phase 1/1b study (clinicaltrials.gov: NCT040247761). Here we report on manufacturing, safety, and in vivo correlative biology of the adoptively transferred CIML NK cells in the first 3 enrolled patients. Methods The primary endpoint is to identify the maximum-tolerated dose (MTD) of CIML NK cells in patients with MDS, MPN, or AML who relapsed after haplo-SCT. NK cells were enriched from donor-derived non-mobilized leukapheresis product via two-step CD3 depletion followed by selection of CD56+ cells using the CliniMACS reagent system (Miltenyi Biotec). The enriched NK cell product was cultured for 12-16 hours in X-VIVO 15 media containing rhIL-12, rhIL-15, and rhIL-18 to generate CIML NK cells (Figure 1A). Patients in the current cohort were lymphodepleted with fludarabine 25mg/m2 daily for 3-5 days and cyclophosphamide 60mg/kg daily for 2 days followed by CIML NK cells at a dose of 5-10 x 106 cells/kg and IL-2 106 IU/m2 QOD for 7 doses. Dose-limiting toxicities were evaluated for 6 weeks following NK cell infusion. Response to therapy was assessed at day+28 following CIML NK cell infusion. Results Patient #001 has FLT3-ITD AML that relapsed 5 months after a reduced intensity (RIC) haploSCT. She received 7.4x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. Her day+28 bone marrow had no leukemia blasts although FLT3-ITD mutation remained detectable. Two months post-CIML NK the leukocyte and granulocyte chimerism were 88% and 89%, respectively. Patient #002 has AML with multiple pathogenic variants, including a potentially pathogenic variant in TP53. His disease relapsed 15 months post haplo-SCT with repeat marrow showing all the original mutations, including the TP53 variant (VAF 50.5%). He received 9.5x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. His day+28 marrow had trilineage hematopoiesis without any mutations, including no TP53 mutation (Figure 1B). Two months post-CIML NK the leukocyte and granulocyte chimerism were both 99%. Patient #003 has MDS whose disease relapsed 8 months post RIC haploSCT with persistence of all her diagnostic pathogenic mutations. She received approximately 9.2x106 NK cells/kg and has only just completed IL-2 106 IU/m2 QOD for 7 doses. Among the 3 patients, the main toxicity was prolonged cytopenia requiring stem cell boost in one case. Donor NK cells demonstrated a dramatic shift from a predominantly CD56dimCD16hi (88% of NK cells) to a CD56dimCD16lo phenotype (49% of NK cells) as CIML NK cells. Infused CIML NK cells expanded massively, with approximately 50-fold, 10-fold, and 15-fold maximum in vivo expansion in the first three patients, respectively (Figure 1C). CIML NK cells were the major population in the day+28 marrows in both patients #001 and #002, with CD56+CD7+ cells constituting 89% of the cellularity in the former and 48% in the latter (Figure 1C). CIML NK cells persisted for 3 and 6 months post-infusion in the first two patients. The NK cells were predominantly mature, with most expressing CD16 and low levels of the inhibitory receptor NKG2A. PD-1 expression was much lower on the expanded NK cells vs the pre-infusion donor-derived NK cells. There was minimal concurrent expansion of CD4+CD25+ T-regulatory cells (Figure 1D). Conclusion We show with the first 3 patients in this trial that CIML NK cells can be generated and infused safely, can expand massively in the peripheral blood and bone marrow within the first 30 days post-infusion, and can persist for several months. In addition, CIML NK cell infusion can reduce the burden of pathogenic variant alleles to below the limit of detection, including the burden of high-risk mutations such as in TP53. Though our results are preliminary, the massive in vivo expansion and long-term persistence of adoptively transferred CIML NK cells underscores the unique biology of these cells that makes them an attractive option for cellular therapy protocols. Disclosures Nikiforow: Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Equillium: Research Funding. Cutler:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; Clinigen: Other; Miltenyi: Other: Research Support; BMS: Other: Research Support; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Amgen: Consultancy; Moderna Therapeutics: Consultancy. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; alexion: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Ritz:TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Falcon Therapeutics: Consultancy; Infinity Pharmaceuticals: Consultancy.

Abstract: CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation and trafficking and is central to inflammation. Early studies by Soiffer et al. demonstrated that ex vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized anti-CD6 monoclonal antibody, has been shown to modulate T cell activation and proliferation. The aim of this study was to characterize: (1) expression of CD6 and ALCAM, and (2) activity of itolizumab on T cell responses in peripheral blood from HCT patients pre- and post-aGvHD. We analyzed immune reconstitution in 31 adult patients who underwent HLA matched donor HCT for hematological malignancies. Patients received peripheral blood stem cell grafts and GVHD prophylaxis with tacrolimus and methotrexate. Twelve of 31 patients developed aGVHD at a median of 58 days, range 27-208, after HCT and systemic treatment was started in 83% of these cases. aGVHD grade severity was 25%, 58.3% and 16.7% of grade I, II and IV, respectively. Patient samples were collected at 1, 2 and 3 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of itolizumab was tested using peripheral blood mononuclear cells (PBMC) obtained from HD and patients before (preGVHD) and after (postGVHD) aGvHD onset (within 30 days). PBMC were stimulated with antiCD3/CD2/CD28 coated beads in the presence of itolizumab or isotype control (cetuximab) for 72 hours. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation was measured by expression of CD25 and CD45RO, respectively. For statistical analysis, non-parametric unpaired (Mann-Whitney) or paired (Wilcoxon matched-pairs signed rank) test were used. CD6+ T cells reconstituted early after transplant, accounting for 95% of positive CD3 T cells, range 57-100 at 1 month. Similar to HD PBMC, in the first 3 months after HCT, CD4 Tcon had the highest CD6 expression, while CD4 Treg had a lower CD6 expression compared to both CD4 Tcon and CD8 T cells (Fig 1A and 1B). To characterize the expression of CD6 on different T cell subsets, we used a t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and visualized the data using a viSNE map (Fig 1C). Within the Tcon compartment, there were no differences in expression of CD6 between HD and patients at all 3 time points. Within CD4 Treg and CD8 T cells, CD6 expression was reduced in naïve CD8 T cells and CM Treg after transplant compared to HD. In HD, ALCAM expression was detected in 35% of CD14+ monocytes, 23% of CD19+ B cells, 20% of myeloid (CD11c+ CD123-) DCs and 97% of plasmacytoid (CD11c-CD123+) DCs. After HCT, expression of ALCAM in DC compartments was similar to HD. In functional studies, itolizumab inhibited CD4 and CD8 T cell proliferation in preGVHD samples, similar to HD controls. This effect was less prominent in samples collected from patients who had developed GVHD and were already receiving immunosuppressive medications, potentially confounding the ability to assess the effect of itolizumab in this assay (Fig 2A). Similar results were observed for CD25 (Fig 2B) and CD45RO (Fig 2C) expression pre- and post-aGVHD. Finally, itolizumab did not increase rates of cell death in samples from HCT patients as assessed by Annexin V expression, suggesting that itolizumab-mediated T cell inhibition was not due to increased T cell apoptosis. There was a slight increase in Annexin V expression in HD vs isotype control (21%, range 10-43 vs 15%, range 11-31, p= 0.0273). In conclusion, we demonstrate for the first time that CD6+ T cells reconstitute rapidly in peripheral blood after HCT and that CD6 expression is highest in Tcon while lowest in Treg (Tcon 〉 CD8 〉 Treg). Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMC from aGvHD patients, thus representing a potential therapeutic for treating aGvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Cugene: Consultancy; Equillium: Consultancy. Cutler:Pharmacyclics: Consultancy; Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; BMS: Consultancy; Jazz: Consultancy; Incyte: Consultancy; Fate Therapeutics: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board. Ampudia:Equillium: Employment. Ng:Equillium: Employment, Equity Ownership. Connelly:Equillium: Employment, Equity Ownership. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy.

Abstract: CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen-presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway modulates T cell activation, proliferation and trafficking playing a central role in inflammation. Early studies by Soiffer and colleagues demonstrated that ex-vivo depletion of CD6+ donor T cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized IgG1 anti-CD6 monoclonal antibody, has been shown to block CD6 signaling, leading to a reduction in T cell activation and proliferation. The aim of this study was to characterize expression of CD6 early after HCT and to examine the effects of Itolizumab on T cell responses in the setting of aGVHD. We analyzed immune reconstitution in 95 adult patients who underwent allogeneic HCT for hematological malignancies. Patients received myeloablative (50.5%) or reduced intensity (49.5%) conditioning. Almost all patients received peripheral blood stem cell grafts from HLA-matched related (MRD; 27.4%), matched unrelated (MUD; 66.3%) or mismatched unrelated donors (MMUD; 6.3%). Tacrolimus and methotrexate were used for GVHD prophylaxis. Forty-two of 95 patients developed grade I-IV aGVHD at a median of 50 days (range 20-294) after HCT and systemic immunosuppression was started in 81% of these cases. aGVHD grade severity was 38.1%, 40.5%, 7.1% and 14.3% of grade I, II, III and IV, respectively. Patient samples were collected at 1, 2, 3 and 6 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of Itolizumab was tested in vitro using peripheral blood mononuclear cells (PBMCs) obtained from HD and patients before (preGVHD) and after aGvHD onset (postGVHD) after 72 hour stimulation with antiCD3/CD2/CD28 coated beads. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation were monitored by expression of CD25 and CD45RO, respectively. To test if Itolizumab activity was dependent on the presence of the CD6 cognate ligand, ALCAM, HD CD3+ T cells were stimulated with anti-CD3 antibody with or without ALCAM-Fc for 96 hours. CD6+ T cells reconstituted early after transplant, accounting for 77.8% (range, 69.9-93.7) of CD4Treg, 98.1% (range, 96.5-99.5) of CD4Tcon, 84% (range, 77.1-92.1) of CD8 T cells at 1 month. This pattern remained unchanged at 2, 3 and 6 months and in HD. CD6 expression (MFI) was the highest in Tcon and lowest in Treg in patients as well as in HD. Compared to HD, CD6 MFI in CD4 Treg was significantly lower in patients at 1,2,3 and 6 months after HCT (p & lt;0.002 at 1,2,3 months and 0.01 at 6 months) (Fig 1A and 1B). CD6 MFI was significantly lower in post-GVHD Tcon (P=0.0004) and Treg (P=0.04) compared to patients without GVHD (Fig 1C). Prior to GVHD onset, Itolizumab inhibited CD4 and CD8 T cell proliferation in a similar fashion to HD control. This effect was less prominent in T cells collected after GVHD onset, when patients were already receiving immunosuppressive medications, especially for the CD8 T cells (Fig 1D). Similar results were observed for CD25 and CD45RO expression (Fig 1D). Addition of ALCAM-Fc to anti-CD3 antibody enhanced T cell proliferation, activation and maturation. Itolizumab efficiently inhibited T cell proliferation, activation and maturation in the presence of ALCAM-Fc and anti-CD3 antibody, while no effect was observed in the presence of anti-CD3 antibody alone (Fig 2A). Finally, Itolizumab alone did not induce significant CDC, ADC or ADCC in vitro (Fig 2B). In conclusion, we demonstrate that CD6 positive T cells reconstitute rapidly after HCT with higher levels of expression in Tcon compared to Treg and CD8 T cells. Percentages of CD6+ T cells do not change during aGVHD, but levels of CD6 expression decrease in Tcon and Treg after aGVHD. Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMCs from patients with aGvHD. Functional inhibition of the CD6-ALCAM pathway without T cell depletion may be a novel therapeutic strategy for treating aGvHD. A phase I/II study using Itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; BMS: Other: Research Support; Miltenyi: Other: Research Support; Clinigen: Other; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; Moderna Therapeutics: Consultancy; Amgen: Consultancy. Cutler:Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Cugene: Consultancy; alexion: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Consultancy; Mana Therapeutics: Consultancy; Rheos Therapeutics: Consultancy; Novartis: Consultancy; VOR Biopharma: Consultancy. Ampudia:Equillium: Current Employment, Current equity holder in publicly-traded company. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ritz:Rheos Medicines: Consultancy; Amgen: Research Funding; Avrobio: Consultancy; Talaris Therapeutics: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Equillium: Research Funding; Falcon Therapeutics: Consultancy; Kite Pharma: Research Funding; TScan Therapeutics: Consultancy.