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Background: AntiCD19 CAR-T cells are effective against chemorefractory B cell lymphoma. Patients (pts) with rapidly progressive disease and urgent need for therapy have very poor prognosis and may not be able to receive CAR-T cells in time. Decreasing the apheresis to infusion time can make CAR-T cells rapidly available. We conducted a dual-center phase I trial using on-site manufacture of CAR-T cells for treatment of relapsed and refractory (r/r) B cell lymphoma. Methods: Adult pts with r/r CD19+ B cell lymphomas who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc, LTG1563) encoding an antiCD19 binding motif, CD8 linker, TNFRS19 transmembrane region, and 4-lBB/CD3z intracellular signaling domains. GMP-compliant manufacture was done using CliniMACS Prodigy in a 12-day culture, subsequently shortened to 8 days. Dose escalation was done using 3+3 design. Lymphodepletion included cyclophosphamide (60mg/kg x 1) and fludarabine (25mg/m2/d x 3). Cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) were graded using the Lee and CARTOX criteria, respectively. CAR-T persistence was measured with qPCR and flow cytometry. Plasma cytokine concentrations were measured using electrochemiluminescence (MesoScale Diagnostics, Inc). Results: Thirty-one pts were enrolled and treated. Baseline patient and disease characteristics are listed in table 1. Twenty-nine (94%) pts were refractory to the prior line of therapy and 21 (68%) had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 45% [range 15-66], median culture expansion was 36-fold [range 3-79]. CAR-T cell doses were 0.5 x 10 6/kg (n = 4), 1 x 10 6/kg (n = 16), and 2 x 10 6/kg (n = 11). Median time from apheresis to lymphodepletion was 7 days (range 2 - 15) and median time from apheresis to CAR-T cell infusion time was 13 days (range 9 - 20). Twenty-eight pts were infused fresh product. Seventeen pts (55%) experienced CRS. Grade 1-2 CRS was observed in 15 pts (48%), grade ≥ 3 was observed in 3 pts (10%). One patient had grade 4 CRS that was later complicated by hemophagocytic syndrome and died on day 21; a second patient had grade 5 CRS in the context of bulky disease and died on day 8. Ten pts (32%) had ICANS and 4 pts had grade 3-4 ICANS. Treatment for CRS / ICANS included tocilizumab (n = 12), siltuximab (n = 4), anakinra (n = 3) and corticosteroids (n = 10). The most common all grade non - hematologic toxicity was fatigue, observed in 19 pts, all grade 1. Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all subjects. Twenty-five (81%) presented disease response and twenty-two pts (71%) achieved complete response (CR). There were no statistically significant differences in the overall and complete response rates between dose levels. After a median follow up of 18 months (range 1 - 32), 5 pts relapsed, and 7 pts have died. Causes of death include progressive disease (n=5), CRS (n=1) and CRS/HLH (n=1). Two-year estimates of PFS and OS for the whole cohort were 67% (95%CI 52-88%) and 75% (95%CI 60-93%)(fig1), respectively. Two-year estimates for patients achieving disease response (CR or PR) were 82% (95%CI 67-99%) and 90% (95%CI 78-100%), respectively. The median duration of response has not been reached (95% CI 74-100). Among pts achieving CR, 94% (95% CI 61-100%) had sustained remission at 12 months. Median time to peak CAR-T expansion, measured by PCR, was 14 days (IQR 14-19), without differences between dose levels, culture duration or fresh vs. cryopreserved infusion. All evaluable subjects had persistent CAR-Ts on PCR measurements done on days 30, 60 and 90. CAR-T cell dose did not have an impact in the time to peak in vivo CAR-T cell expansion or in the rate of CAR-T cell persistence (fig 2). Cytokine measurements have been conducted in 19 pts, with area under the curve (AUC) analyses showing pts with CRS had higher plasma concentrations of multiple cytokines (fig 3). Patients achieving CR had higher plasma concentrations of MIP3B. Conclusions: Second generation antiCD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. On-site manufacture was successful in all pts. This strategy, in combination with fresh product infusion, can make CAR-T cell therapy rapidly available for pts with high-risk r/r B cell lymphoma. Figure 1 Figure 1. Disclosures Caimi: Amgen Therapeutics.: Consultancy; TG Therapeutics: Honoraria; XaTek: Patents & Royalties: Royalties from patents (wife); Kite Pharmaceuticals: Consultancy; Genentech: Research Funding; ADC Theraputics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Verastem: Consultancy. Ghobadi: Wugen: Consultancy; Atara: Consultancy; Amgen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Celgene: Consultancy. Schneider: Lentigen Technology: Current Employment. Boughan: Beigene: Speakers Bureau. Metheny: Incyte: Speakers Bureau; Pharmacosmos: Honoraria. Krueger: Lentigen: Current Employment. Kadan: Lentigen: Current Employment. Orentas: Lentigen: Patents & Royalties. Dropulic: Lentigen: Ended employment in the past 24 months, Patents & Royalties. de Lima: Miltenyi Biotec: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: AntiCD19 CAR-T cells with TNFRSF19 transmembrane domain for treatment of relapsed and refractory B cell lymphomas.

INTRODUCTION: Anti-CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated activity against relapsed/refractory lymphomas. Cytokine release syndrome (CRS) and CAR-T related encephalopathy syndrome (CRES/ICANS) are well-known complications of CAR-T cell therapy. Tocilizumab, a humanized monoclonal antibody targeting the interleukin 6 (IL-6) receptor, is approved for treatment of CRS. Our institutional standard was modified to administer prophylactic tocilizumab before infusion CAR-T cell products. We present the outcomes of subjects treated with locally manufactured antiCD19 CAR-T cells (TNFRSF19 transmembrane domain, CD3Zeta/4-1BB costimulatory signaling) with and without prophylactic tocilizumab. METHODS: Relapsed / refractory (r/r) lymphoma patients (pts) treated with anti-CD19 CAR-T cells at our institution were included. Baseline demographic and clinical characteristics, as well as laboratory results were obtained from our Hematologic Malignancies and Stem Cell Therapy Database. Prior to institution of prophylactic tocilizumab, pts received this agent only if they presented evidence of CRS grade 2 or higher. In May 2019, our institutional practice changed to provide tocilizumab 8mg/kg, 1 hour prior to infusion of CAR-T cell product. CRS was measured according to the ASTCT Consensus Grading, whereas CRES was measured using the CARTOX-10 criteria. Comparisons between groups were done with the Mann-Whitney U test for continuous variables and Fisher's exact test for categorical variables. RESULTS: Twenty-three relapsed / refractory lymphoma pts were treated with antiCD19 CAR-T cells; 15 pts received prophylactic tocilizumab. Median follow up was 312 days (range 64 - 679) days. Baseline characteristics are listed in table 1. Both groups were similar: There were no statistically differences in the rate of bulky, refractory disease, prior ASCT or number or prior lines of therapy. Baseline lymphocyte counts, C - reactive protein (CRP) and were also comparable between groups (Table 2). We did not observe immune adverse reactions to tocilizumab infusion. There were no differences in the incidence of cytopenias or infectious complications between groups. CRS of any grade was observed in 6/8 (75%) of pts without prophylactic tocilizumab vs. 6/15 (40%) in pts treated with prophylactic tocilizumab (p = 0.23), whereas CRS grade >1 was observed in 5 pts (62.5%) without prophylactic tocilizumab and in 3 pts (20%) treated with prophylactic tocilizumab (p = 0.02). There was no significant difference in the incidence of all grade CRES (no prophylaxis, 3/8 [38%] pts; prophylaxis 5/15 [30%] pts, p = 0.2969). There was a statistically significant difference in the peak CRP and peak ferritin without difference in the peak lymphocyte count after CAR-T infusion (Table 2, Figure 1). Patients given prophylactic tocilizumab had higher IL-6 plasma concentrations on day 2 after infusion (Figure 2). Complete response was observed in 4/8 (50%) pts without prophylactic tocilizumab vs. 12/15 (80%) pts with prophylactic tocilizumab (p = 0.18). All pts had detectable Anti-CD19 CAR-T cells on day 30, both groups had peak CAR-T expansion on day 14, with no statistically significant differences in expansion rates between groups. All evaluable subjects have had CAR-T persistence on days 60, 90, 180, and 365. CONCLUSIONS: Use of prophylactic tocilizumab prior to infusion of antiCD19 CAR-T cells is associated with reduced incidence of severe CRS and decreased levels of clinical laboratory markers of inflammation, despite increases in plasma concentration of IL-6. This decreased rate of grade ≥2 CRS is not associated with impaired disease control and did not result in increased rates of neurologic toxicity. Prophylactic tocilizumab does not appear to affect CAR-T cell expansion or persistence. Figure 1 Disclosures Caimi: ADC therapeutics: Other: Advisory Board, Research Funding; Celgene: Speakers Bureau; Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board. Worden:Lentigen, a Miltenyi biotec company: Current Employment. Kadan:Lentigen, a Miltenyi biotec company: Current Employment. Orentas:Lentigen Technology, a Miltenyi Biotec Company: Research Funding. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. de Lima:Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding; Kadmon: Other: Personal Fees, Advisory board; Incyte: Other: Personal Fees, advisory board; BMS: Other: Personal Fees, advisory board. OffLabel Disclosure: Use of tocilizumab as prophylaxis for CRS is not approved, whereas use for treatment is approved and on label.

Background: Salvage regimens for chemorefractory aggressive lymphoma achieve response rates of approximately 30%. Anti-CD19 CAR-T cells have demonstrated anti-lymphoma activity, but patients (pts) with rapidly progressive disease and urgent need for therapy have worse prognosis and many are not able to receive CAR-T cells in time. Decreasing the time from apheresis to infusion can make CAR-T cells available to pts with rapid progression of their disease. We present the results of a phase I clinical trial using on-site CAR-T manufacture for treatment of relapsed / refractory (r/r) B cell non Hodgkin lymphoma (NHL). Methods: Adult pts with r/r CD19+ B cell lymphomas who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc, LTG1563) encoding an anti-CD19 binding motif, CD8 linker and tumor necrosis receptor superfamily 19 (TNFRS19) transmembrane region, and 4-lBB/CD3z intracellular signaling domains. GMP-compliant manufacture was done using CliniMACS Prodigy, in a 12-day culture. Dose escalation was conducted according to a 3+3 design. Lymphodepletion was done with cyclophosphamide (60mg/kg x 1) and fludarabine (25mg/m2/d x 3). Cytokine release syndrome (CRS) and CAR-T related encephalopathy syndrome (CRES) were graded using the Lee and CARTOX criteria, respectively. Results: As of July 30, 2019 , 12 pts were enrolled and treated. Baseline characteristics are listed in table 1. 10/12 pts were refractory to the prior line of therapy, 5 had bulky disease and 9 had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 48% [range 29-62] with and median culture expansion of 43-fold [range 30-79]. High dimensional flow cytometry showed the infused CD4 and CD8 CAR-T cells express a central memory and transition to memory - like profile, with enrichment for CD27 and high CCR7 expression. In addition, a subset of CD4 and CD8 CAR-T cells expressed effector transcription factors T-BET and GATA3 while CD4 CAR-T clusters express low levels of immune checkpoint blockers PD-1 and BTLA. All enrolled pts received their infusion of anti-CD19 CAR-T cells. CAR-T cell doses were 0.5 x 106/kg (n = 4) and 1 x 106/kg (n = 8). Median apheresis to infusion time was 13 days [range 13-20], 10 products were infused fresh. CAR-T persistence, based on vector sequence, peaked in peripheral blood MNCs between days 14-21. All responding subjects have had CAR-T persistence on follow up PCR measurements (range 1 - 12 months). CAR-T cell dose did not have an impact in the time to peak in vivo CAR-T cell expansion or in the rate of CAR-T cell persistence. Five pts experienced CRS. Grade 1 - 2 CRS was observed in 4 pts; whereas 1 pt died as a consequence of severe CRS in the context of bulky disease. Pharmacologic interventions for CRS included tocilizumab (n = 5), siltuximab (n = 2) and corticosteroids (n = 2). Two subjects presented grade 4 CRES with resolution after corticosteroids, no other grade ≥3 non-hematologic toxicity was observed. The most common all grade non - hematologic toxicity was fatigue, observed in 6 subjects. Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all subjects, with 4 subjects presenting grade 3 neutropenia without fever beyond day 30. Among 11 pts evaluable for response, 8 pts have achieved complete response (CR) and one had partial response (PR). Two pts did not respond. For the intention to treat population (n=12), the CR rate was 67% and overall response rate (ORR) was 75%. Overall response rates were equal between both dose levels (75%), but CR rates were higher in pts treated with 1 x 106 CAR-T cells (75% vs. 50%). Two pts have died, causes of death include progressive disease (n=1) and CRS (n=1). After a median follow up 3 months (range 1 - 12) all responding pts are alive; 1 subject relapsed 6 months after treatment with CD19+ disease and entered CR after anti-CD19 antibody drug immunoconjugate treatment. Conclusions: In this phase 1 study, second generation anti-CD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. The short manufacture times achieved by local CAR-T cell manufacture with the CliniMACS Prodigy enables treatment of a very high risk NHL population that would otherwise not be able to receive CAR-T products due to rapidly progressive disease. Disclosures Caimi: ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Genentech: Research Funding. Schneider:Lentigen Technology, A Miltenyi Biotec Company: Employment. Bakalarz:Genentech: Speakers Bureau. Kruger:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology, A Miltenyi Biotec Company: Employment. Kadan:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Malek:Adaptive: Consultancy; Janssen: Speakers Bureau; Amgen: Speakers Bureau; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Medpacto: Research Funding. Metheny:Takeda: Speakers Bureau; Incyte: Speakers Bureau. Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. OffLabel Disclosure: Clinical Trial of on - site manufactured CAR-T cells. This manufacturing process is under research.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown enormous promise in the treatment of certain B cell malignancies. Access to treatment is still limited due to a variety of issues, including pricing and centralized manufacturing models. Generation of CAR-T cells using an automated platform, followed by rigorous functional phenotyping, may contribute to the development of a robust long-lasting therapy. METHODS: Here, we used the Miltenyi Prodigy (Miltenyi Biotech, Bergisch Gladbach, Germany) to automate the process of manufacturing genetically manipulated T cells in a closed system. The system obviates the need for clean room infrastructure. We tested the feasibility of utilizing the Miltenyi Prodigy to manufacture CAR-T cells using a CD19 scFV vector with the 4-1BB co-stimulatory domain. (Lentigen Technology, Inc, Gaithersburg, MD). The purity, differentiation capacity and effector function of the enriched CAR-T cells was studied using high-dimensional flow cytometry. Finally, the functional potential of these cells was tested in vitro and by treating NOD-SCID-gamma (NSG) mice infused with B cell lymphoma cells (Raji B cell), with the CAR-T cells. RESULTS: Starting with 1 x 108 CD4 and CD8 cells from donor apheresis products, CAR-T cells were expanded for 12 days in culture media containing with TransAct (Miltenyi Biotech), IL7 and IL15. The mean fold-expansion at day 12 was 44 ± 5.6, range 39-50 (n=3). The mean transduction efficiency of CAR-T vector was 20%, range 10-25% (n=3), which is similar to other reported methods. The CD19 CAR-T product was enriched in both the CD4 and CD8 T cells subsets, and showed high-level of cytotoxicity against CD19+ cell lines in vitro and in vivo (Figure 1: Mice treated with the CD19-CAR T demonstrated a marked reduction in disease burden as compared to T cell control as measured by bioluminescence imaging and flow cytometric analysis). The CAR-T product was enriched in cell subsets with both effector (CD27-CCR7-; ~20% of total cells) and central memory phenotypes (CD27+CCR7+; ~30% of total T cells). The effector CD4 and CD8 T cells showed increased expression of major functional T cell differentiation transcription factors (i.e. T-bet and GATA3) critical for the development of anti-tumor responses. Whereas, the central CD4 and CD8 T cells were enriched for the expression of TCF7 (a stemness related member of the WNT signaling known to increase longevity of these cells). The frequencies and phenotypes of these cells were maintained in peripheral blood of NSG mice infused with B cell lymphoma cells (Raji B cells), 1 week after treatment. A significant expansion of CD8+ effector T cells and a dramatic reduction in tumor burden was observed over the next 4 weeks in all major organs. Interestingly, we observed that smaller proportion of central-memory like cells (with higher TCF7 levels) continued to persist 6 weeks post-treatment, potentially contributing to a long-lived recallable response. Based on these data we have initiated a phase 1 clinical trial to test the therapeutic potential of the CAR-T product in patients with advanced/refractory B cell lymphoma. The first clinical grade manufacturing run resulted in a CD19 + cell yield of 1.4 x109. CONCLUSION: Our data highlight that the automated CAR-T generation platform (i.e. Miltenyi Prodigy) is effective at the generating purified functionally competent CAR-T cells. Further, findings from our phenotyping analyses show that the CAR-T product is enriched in both effector and central memory subsets and is effective at tumor clearance in vivo. Thus far, we have treated one patient with CD19 CAR-T manufactured on this platform and 2 more have been enrolled. Though this initial study is based on CD19 CAR-T cells, the approach described here could easily be utilized to genetically engineer T cells with gene constructs that are more relevant for specific cancers and infectious diseases. Disclosures No relevant conflicts of interest to declare.

Background The human microbiome has been associated with allogeneic hematopoietic cell transplantation (HCT) outcomes, namely infections, graft-versus-host disease and relapse. There are no studies describing the longitudinal changes in the oral or gastrointestinal microbiome in the setting of autologous HCT. We conducted a prospective study to describe the changes in microbial diversity in patients undergoing HCT for multiple myeloma (MM), and whether these correlate with HCT outcomes and/or toxicities. Methods Samples were collected from 15 MM patients on admission (baseline, T-2), during marrow aplasia (T+7) and after engraftment (T+30) (Table 1- summarizes baseline characteristics). We evaluated the bacterial and fungal microbiome of 15 patients using Ion-Torrent PGM workflow. The amplicons generated from the 16s rRNA and the ITS genes were sequenced for bacterial and fungal identification, respectively. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance) and taxonomically classified via Qiime bioinformatics pipeline. Diversity was calculated using Shannon diversity index and richness using the R package 'vegan'. Longitudinal analysis was performed using all pairwise Multiple Comparison of Mean Ranks as implemented PMCMR plus R package, employing Kruskal & Wallis test followed by Bonferroni-Dunn post-hoc adjustment. Results Diversity and richness of the oral mycobiome decreased at T+7 compared to pre-transplant levels with further decrease noted at T+30, without reaching significance. Fecal mycobiome diversity and richness decreased from baseline to T+7 meeting statistical significance for diversity (T-2 vs T+7, p=0.05) and richness trended towards significance (p=0.06) with a further decrease noted at T+30. The temporal changes in bacterial diversity and richness in both oral and fecal samples did not reach statistical significance. (Figure1- Box and whisker plots of diversity and richness of the bacteriome and mycobiome at the genus levels from oral rinse and fecal samples) In fecal samples, bacterial diversity noted at T+7 during count nadir was associated with the severity of diarrhea experienced after myeloablation, with lower diversity correlating with more severe diarrhea (p= 0.03). Anaerobic targeting antibiotic exposure on or before T+7 affected both the genus diversity and richness at T+7 (p=0.015 and p=0.014, respectively). The bacterial genus richness at baseline (p=0.03) as well as the diversity and richness noted at T+7 (p=0.01) was associated with the development of fever on or after T+7. For the oral mycobiome, exposure to anaerobic depleting antibiotics correlated with genus richness in T+30 samples (p=0.04). There was a trend towards significance between the diversity of fecal samples at baseline and the development of nausea post transplant, such that higher diversity was associated with lower incidence/severity of nausea (p=0.06). Conclusion and Future Directions While acknowledging the limitation inherent in the small sample size of this pilot study, our results highlight several aspects of the longitudinal changes in the microbiome during HCT. Oral and lower gastrointestinal microbial diversity and richness is altered during HCT with trends significantly different between the oral and fecal bacteriome and mycobiome. This change is likely multifactorial owing to the conditioning regimen, antimicrobial exposure and immune dysregulation. Our data suggest a possible correlation between exposure to anaerobic organism depleting antimicrobials and these changes in microbial diversity and richness at the genus level. Baseline microbial diversity and richness as well as changes coinciding with marrow aplasia could correlate with the incidence and severity of transplant related toxicities. Amifostine was used as a cytoprotectant before high dose melphalan for our patients. The effect of this organic thiophosphate on the microbiota is unclear and it would be of interest to compare matched patient samples to explore the effect of this cytoprotectant on the microbiota. Further studies conducted on a larger scale and incorporating metabolomics and proteomics will help elucidate the interactions between the host and the microbiome and their effect on short term and long term transplantation outcomes as well as toxicities. Disclosures Lazarus: Pluristem Ltd.: Consultancy. Caimi:Celgene: Speakers Bureau; Kite Pharma: Other: Advisory Board Participation; Kite Pharma: Other: Advisory Board Participation; Genentech: Other: Advisory Board PArticipation, Research Funding. Malek:Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau.

Background The human microbiome has been associated with allogeneic hematopoietic cell transplantation (HCT) outcomes. To date, there are no studies describing the longitudinal changes in the oral or gastrointestinal microbiome in the setting of autologous HCT. We conducted a prospective study to determine the longitudinal microbial profile in patients undergoing HCT for multiple myeloma (MM), and whether changes in microbial abundance correlate with HCT outcomes and/or toxicities. Methods Samples were collected from 15 MM patients on admission (baseline, T-2), during marrow aplasia (T+7) and after engraftment (T+30) (Table 1- summarizes baseline characteristics). We evaluated the bacterial and fungal microbiome of 15 patients using Ion-Torrent PGM workflow. The amplicons generated from the 16s rRNA and the ITS genes were sequenced for bacterial and fungal identification, respectively. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance) and taxonomically classified via Qiime bioinformatics pipeline. Statistical analysis was performed using the statistical programming language R. Multivariate distance based association between communities and outcome was performed using the Adonis function as implemented in the R package vegan using Bray-curtis dissimilarities distances with and without presence/absence standardization (BrayPA). Non-parametric Spearman correlation and wilcoxon rank-sum test were used for association with continuous outcome and binary outcome respectively. Results Relative abundance was determined at the phylum and genus levels across each time point, in the oral and fecal microbiome, both bacterial and fungal. At the bacterial phylum level, the oral bacterial community composition significantly changed at T+30 compared to baseline (Bray-curtis, p=0.046) and T+7 (Bray-curtis, p=0.025). When examining the changes in the mycobiome composition, test for dissimilarity showed a significant difference in the fecal fungal genus between baseline and T+30 (BrayPA, p=0.025). No other statistically significant differences were noted. Next, we correlated the microbial community (Table 2) and individual composition with outcome and transplant related toxicity, the later will be reported here. In fecal samples, the bacterial phylum Bacteriodetes present at T+7 was associated with the development and severity of diarrhea, such that patients with higher abundance of Bacteroidetes experienced lower gastrointestinal toxicity (pAdj=0.03). Additionally, the 2 genera Blautia and Ruminococcus, both belonging to the Firmicutes phylum and Clostridium Class, when detected at T+7 were associated with a higher development and severity of vomiting after exposure to high dose melphan (pAdj=0.05 for both respectively). In oral samples, the presence of the genus Glomerella at T+7 was negatively associated with rates of neutrophil engraftment (pAdj=0.03). Conclusion and Future Directions While acknowledging the limitation inherent in the small sample size, our results show that oral and fecal microbiota undergo dynamic changes in their diversity (Abstract ID 120038), composition and relative abundance during the course of transplantation. This change is likely multifactorial owing to the conditioning regimen, antimicrobial exposure and immune dysregulation. Our data suggest the bacterial and fungal microbiota present specifically at count nadir could be important players in this population of patients. Interestingly, the relative abundance of particularly the anaerobic bacterial phyla, Bacteroidetes and Firciumtes (Blautia and Ruminococcus) during marrow aplasia, correlated with transplant related toxicities in our cohort. This could further contribute to our knowledge of the role anaerobic organisms play in HCT outcomes, in accordance to what has been described in the allogeneic HCT literature. Amifostine was used as a cytoprotectant before high dose melphalan for our patients. The effect of this organic thiophosphate on the microbiota is unclear and warrants future investigations. Further studies conducted on a larger scale and incorporating metabolomics and proteomics will help elucidate the interactions between the host and the microbiome and their effect on short term and long term transplantation outcomes as well as toxicities. Disclosures Lazarus: Pluristem Ltd.: Consultancy. Malek:Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

In chronic HIV infection, most untreated patients lose naive CD4+ and CD8+ T cells, whereas a minority preserve them despite persistent high viremia. Although antiretroviral therapy (ART)–mediated viral suppression generally results in a rise of naive and total CD4+ T cells, certain patients experience very little or no T-cell reconstitution. High peripheral T-cell activation has been linked to poor clinical outcomes, interfering with previous evaluations of thymic function in disease progression and therapy-mediated T-cell recovery. To circumvent this, we used the sj/βTREC ratio, a robust index of thymopoiesis that is independent of peripheral T-cell proliferation, to evaluate the thymic contribution to the preservation and restoration of naive CD4+ T cells. We show that the loss of naive and total CD4+ T cells is the result of or is exacerbated by a sustained thymic defect, whereas efficient thymopoiesis supports naive and total CD4+ T-cell maintenance in slow progressor patients. In ART-treated patients, CD4+ T-cell recovery was associated with the normalization of thymopoiesis, whereas the thymic defect persisted in aviremic patients who failed to recover CD4+ T-cell counts. Overall, we demonstrate that efficient thymopoiesis is key in the natural maintenance and in therapy-mediated recovery of naive and total CD4+ T cells.

Allogeneic hematopoietic stem cell transplantation (AHSCT) leads to a prolonged state of immunodeficiency characterized by low peripheral naive T-cell counts. To identify the mechanisms leading to this defect we quantitatively and qualitatively analyzed thymic function through quantification of T-cell receptor excision circle (TREC) frequencies (both the signal-joint TREC [sjTREC] and 6 different DbetaJbeta TRECs, by-products of T-cell receptor [TCR] alpha and beta gene rearrangement, respectively), in conjunction with immunophenotype and spectratype analyses in a cohort of patients sampled from 1 to 10 years following AHSCT. In this cohort, reduced thymic function was associated only with ongoing clinical chronic graft-versus-host disease (cGVHD). Nonetheless, the diversity of thymic production remained unchanged irrespective of the patient's cGVHD status. Interestingly, increased homeostatic proliferation was found in the naive T-cell compartment of cGVHD- patients who underwent transplantation. However, reduced expression of both the interleukin-7 receptor alpha (IL-7Ralpha) (CD127) chain and the antiapoptotic protein Bcl-2 was observed. Taken together, these data indicate that the inability to reconstitute the naive T-cell compartment for several years after AHSCT, in the absence of cGVHD, is a consequence of impaired naive T-cell survival rather than thymic dysfunction.

Intrathymic clonal deletion is thought to be the major mechanism responsible for tolerance to nonsequestered antigens such as the ones expressed by bone marrow-derived cells. In the case of sequestered antigens that potentially do not come in contact with T cells in the thymus, it is thought that autoreactive T cells are present in periphery but are tightly regulated to prevent autoimmune disease. Indeed, autoreactive T cells to sequestered antigens can be isolated in healthy individuals. However, the presence of autoreactive T cells to nonsequestered circulating antigens had not been observed. In this report, we present evidence for the presence, in the periphery of all healthy individuals tested (n = 25), of autoreactive T cells to GpIIb- IIIa, a membrane antigen present on bone marrow-derived cells that is expressed on circulating platelets and on the cell surface of the epithelial cells of the thymic stroma early in intrauterine life. Using an in vitro T-cell proliferation assay, we have demonstrated that activation of these specific GpIIb-IIIa autoreactive alpha beta TCR+ CD4+ CD8- T cells requires internalization and processing of the GpIIb- IIIa by antigen-presenting cells and its presentation by HLA-DR class II molecules in the presence of exogenous interleukin 2 (IL-2). This indicates that some autoreactive T cells directed against membrane antigens present on bone marrow-derived cells and also expressed in the thymus are not necessarily eliminated by intrathymic deletion.

Immediate-early host-virus interactions that occur during the first weeks after HIV infection have a major impact on disease progression. The mechanisms underlying the failure of HIV-specific CD8 T-cell response to persist and control viral replication early in infection are yet to be characterized. In this study, we performed a thorough phenotypic, gene expression and functional analysis to compare HIV-specific CD8 T cells in acutely and chronically infected subjects. We showed that HIV-specific CD8 T cells in primary infection can be distinguished by their metabolic state, rate of proliferation, and susceptibility to apoptosis. HIV-specific CD8 T cells in acute/early HIV infection secreted less IFN-γ but were more cytotoxic than their counterparts in chronic infection. Importantly, we showed that the levels of IL-7R expression and the capacity of HIV-specific CD8 T cells to secrete IL-2 on antigenic restimulation during primary infection were inversely correlated with the viral set-point. Altogether, these data suggest an altered metabolic state of HIV-specific CD8 T cells in primary infection resulting from hyperproliferation and stress induced signals, demonstrate the discordant function of HIV-specific CD8 T cells during early/acute infection, and highlight the importance of T-cell maintenance for viral control.

We demonstrated the enhancement of CD8-specific responses following the administration of an immune-based therapy consisting of dendritic cells (DC) electroporated with autologous amplified HIV-1 RNA and CD40 ligand (CD40 L) RNA manufactured by the Arcelis™ process in HIV patients receiving antiretroviral therapy (ART). We conducted a sub study on circulating B cell populations to further assess changes induced by this autologous DC therapy as CD40L is a major B cell co-stimulatory factor. To this end, we assessed B cell subset changes in relation to the proliferative capacity of CD4+ and CD8+ T cells response to DC targets containing the 4 HIV-1 antigens (Gag, Vpr, Rev, Nef). The co-expression of CD19, CD38, IgD, CD10, CD23, CD27, CD5, and CD138 were analyzed by multi-parametric flow cytometry to assess circulating B cell subsets such as naïve resting B-cells (Bm1), activated naïve B cells (Bm2), GC founder cells (Bm2’), centroblasts and centrocytes (Bm3 and Bm4), early memory B cells (eBm5), memory B cells (Bm5), IgD memory cells, plasma cells, and B-1 cells. Changes in B cells subsets were analyzed before and after the four intradermal injections of this immunotherapeutic product containing 1.2 × 107 DC. Ten ART treated subjects with undetectable viral load (< 50 copies/ml), median CD4+ count of 440 cells/μl (range: 316–1102), and with a CD4+ nadir > 200 cells/μl were studied. Throughout the study, no significant changes in CD4+ cell count, CD4/CD8 ratio, and no viral blips were noticed. The percentage of total B cells, Bm1, Bm2, Bm2′, eBm5, IgD memory, plasma cells, and B-1 cell subsets did not significantly change. However, a decrease in the percentage of Bm3 and Bm4 cells was found (0.36 [0.06–0.86] versus 0.11 [0.04–0.36]; P=0.05). Conversely, an important increase in the Bm5 cell subset was evidenced (10.4 [1.6–24.2] versus 18.1 [5.1–27.5]; P=0.005) suggesting a proliferation of B memory cells induced by DC immunization. In addition, the multifunctional and polyvalent CD8+ T cell proliferative responses to the 4 HIV genes used in this immunotherapy were noticed in 8 out of 9 subjects available for analysis and characterized by an effector memory phenotype. No CD4+ T cell immune responses were detected, consistent with the endogenous HLA class I loading of the antigens. Collectively, these results indicate that this immunotherapy induces an increase in the B memory cell population in the absence of inducing any clinically apparent autoimmunity along with strong HIV specific multifunctional CD8+ T cell specific immune responses.

We have recently shown that high levels of interleukin-7 (IL-7) predict poor virological response in HIV-1-infected patients. To understand the underlying mechanisms associated with increase in IL-7 levels, we assess the expression of IL-7 receptor and activation markers on different CD8 subsets in there well defined groups of patients including successfully treated (ST; n=8), untreated (UT; n=6), failing HAART due to drug resistance (FT; n=12) and healthy subjects (n=5). The expression of IL-7/IL-2 receptors (CD127/CD25) and activation markers (CD38/HLA-DR) on CD8 naïve cells (CD45RA+CCR7+CD27+), central memory (TCM; CD45RA−CCR7+CD27+), pre-terminal effector memory (TPEM; CD45RA−CCR7−CD27−) and terminal effector memory cells (TTEM; CD45RA+CCR7−CD27−) was measured in fresh blood samples using 8-color flow cytometry. Mann Whitney U test and Spearman correlation were used. Although there was a trend toward a decrease in naïve, TCM, TPEM and TTEM cells from ST to UT and then to FT, this did not reach significance (P=0.09). FT and UT subjects showed higher number of CD8 activated cells than ST or controls (22%, 20%, 3% and 4% respectively; P=0.07). Similarly, the percentages of TCM activated cells were significantly higher in FT and UT than ST or controls (28%, 27%, 7% and 0.07%; P=0.02), whereas naïve and TTEM activated cells were similar in all groups. Interestingly, the percentage of TCM cells expressing CD127+CD25+ was lower in FT compared to UT or ST (0.7%, 2.1 and 7.4%; P=0.02). A strong positive correlation was observed between viral load and CD8+ or TCM activated cells (r=0.46, P=0.02; r=0.65, P=0.004 respectively) but not with naïve CD8+ cells. These data suggest that a substantial proportion of TCM but not naïve, TPEM and TTEM cells expressing immune activation markers with lower IL-7 receptor expression is present in subjects with persistent antigen stimulation, thus providing an explanation for increasing circulating IL-7 levels.

Virus-specific CD4 T-cells are believed to play a critical role in determining the persistence of memory and effector CD8 T-cell responses during viral infections. However, the reasons for which human immunodeficiency virus (HIV)-specific CD4 cells fail to generate effective CD8 cell responses remain incompletely understood. In this study, we analyzed the HIV-specific CD4 cells in 10 aviremic (viral load < 50 copies/ml) and 8 viremic (mean viral load 45,295 copies/ml) patients treated during primary HIV infection and followed for up to 6 years. At the time of apheresis, median CD4 T cell counts for aviremic and viremic patients were 671 cells/μl and 485 cells/μl respectively. Using Gag and Nef overlapping HIV peptides, the highly sensitive CFSE-based proliferation assay and intracellular staining techniques, we observed that proliferative Gag and Nef peptide-specific CD4 cell responses were 30-fold higher in aviremic patients compared to viremic. Several subsets of HIV-specific memory CD4 cells endowed with different proliferative and functional capacities were identified. We observed two subsets of HIV-specific memory CD4 cells in aviremic patients, CD45RA− CCR7+ central memory cells (Tcm) producing exclusively IL-2 and CD45RA− CCR7− effector memory cells (Tem) that produced both IL-2 and IFN-γ. In contrast, in viremic patients, Tem were found to unexpectedly produce IFN-γ exclusively. Longitudinal data showed that only cells, which were capable of producing IL-2, persisted as long-term memory cells. In viremic patients, HIV-specific CD4 cells that produce INF-γ were found only during periods of elevated viral loads. To test if the presence of IL-2 could restore the proliferation of these cells, we stimulated CD4 cells from viremic patients with a pool of peptides, which gave strong IFN-γ responses in these patients in the presence of exogenous IL-2. The addition of IL-2 during the in vitro peptide stimulation dramatically increased the fraction of proliferating cells. This experiment strongly suggests that the impaired proliferation of CD4 cells from viremic patients is not caused by a virus-mediated destruction of proliferating cells but a lack of producing IL-2. Altogether, these results indicate that long-term memory HIV-specific CD4 cell produce mainly IL-2, while those producing IFN-γ are short-lived. These findings favor a model in which Tcm are continuously produced in limited numbers but under continuous viral stimulation are rapidly induced to differentiate into IFN-γ only-producing cells that lack the capacity for self-renewal.These findings also indicate that treatment strategies aimed to increase the long-term memory CD4 cells are needed for future HIV vaccine development.