Invariant natural killer cells are immunomodulatory T cells with a proven effect in Graft versus Host disease. Recent data have shown that the infused iNKT cell dose is associated with decreased risk of aGVHD in matched sibling graft recipients. This study was conducted to assess the effect of the infused iNKT cell dose and early INKT recover at day 30 and 60 post HCT on relapse and GVHD. We also assessed the effect of INKT cells among patients receiving thymoglobulin as part of their HCT protocol.Methods: 48 adult allogeneic HCT recipients were enrolled on a single arm prospective trial between 2012 and 2014. All donors were mobilized with G-CSF per institutional and NMDP guidelines. Conditioning regimen consisted of Flu/Bu4 in the myeloablative setting and flu/Bu2 in RIC setting. Thymoglobulin at 1.5mg/kg/day x3 days was used for unrelated donor recipients. A small portion (2.5mL) of the donor peripheral blood stem cell product was collected prior to transplant and recipient peripheral blood (~10 mL) was collected on days +30, and +60 post HCT. Flow cytometric analysis was performed on the samples with an antibody cocktail that contained the following pre-conjugated monoclonal antibodies: CD56-PE (Miltenyi Biotech, Auburn, CA), CD3-APC, CD16-FITC, (Beckman Coulter, Brea, CA), CD19-PE-CY7 (BD Biosciences, San Jose, CA). For iNKT analysis and for CD4/CD8 analysis, 2 x 106 total cells were stained with CD3-APC, TCR Vα24-PE, TCR Vβ11-FITC (Beckman Coulter), CD4-APC-H7 and CD8-PE-CY7 (BD Biosciences). Data were acquired using BD FACSCanto II and analyzed with the FACSDiva software (BD Biosciences) to quantify CD3+ T cells, CD3+ CD56+ NK-like T cells, CD56+ CD16+ and CD56+ CD16- NK cells as well as CD19+ B cells. INKTs were quantified as CD3+, TCR Vα+, TCR Vβ+ lymphocytes. A total of 30,000 lymphocytes were collected for NK, T, and Bcell analysis. For iNKT, an end point of 200 iNKT or 500,000 total events was set. The graft and early cell subsets were assessed for their impact on acute GVHD and relapse.Results: 48 patients with a median age of 55 years received Flu/Bu4 (n= 29) and Flu/Bu2 (n=19). Donor source was a matched sibling (n=13, 27%) or a matched unrelated donor (n=35, 73%). The collected and infused graft cell doses are shown in table 1. Median time to Neutrophil and platelet engraftment was 11.5 and 18.8 days respectively. The propability of one year survival was 76% with a 1 year cumulative incidence of relapse of 34%. The pre-transplant parameters that predicted a more robust iNKT cell recovery by multivariate analysis were young recipient age, young donor age, dose of CD34 infused and RIC regimen (p<0.02 for all). A multivariate analysis to assess predictors of relapse showed that RIC (p=0.002), older donor age (p=0.03), and a smaller dose of infused INKT (p=0.012) were all significantly correlated with higher relapse rates. Day 30 and day 60 absolute iNKT and iNKT/T cell ratios were not predictors of relapse. None of the 22 patients with iNKT/T ratio of >10-3developed CMV reactivation.Conclusion: The infused iNKT cell dose in the peripheral graft inversely affects relapse rates post allogeneic HCT. This effect is independent of thymoglobulin use or donor source. Trials aiming at expanding the INT cell population in the infused grafts may help decrease risk of relapse post HCT.Table 1:Cellular Content of the Infused GraftCell SubsetMean ± sdMedian (min-max)CD3/kg infused250 ± 111251 (23-428)CD 34 infused6.2 ± 2.16.0 (1.9-13.5)NK cell x106/kg infused9.5 ± 11.06.6 (0-67)T cell x106/kg infused192 ± 102187 (0-516)B cell x106/kg infused39.8 ± 3335 (0-151)NKT x106/kg infused9.5 ± 11.06.6 (0 -67)iNKT x106/kg infused0.43 ± 0.70.23 (0-4.6)%CD34 product0.99 ± 0.50.89 (0.15-2.7)%NK product2.4 ± 1.51.9 (0.19-6.5)%T cell product27.0 ± 11.027 (1.21-51.8)%NKT product1.3 ± 1.21.0 (0.5- 5.9)%iNKT product0.26 ± 0.70.12 (0 -4.5)%B cell product5.4 ± 3.84.5 (0 -19.7)%WBC product236 ± 100209 (115-508)% Lymph product32.4 ± 10.332 (13-51.8)NK cells, T cell, NKT and B cells are percentage of total nucleated cellsiNKT cells are percentage of CD3+ cells DisclosuresNo relevant conflicts of interest to declare.