scholarly journals Non-Genetic Determinants of Clonotypic T Cell Expansion Following Allogeneic Stem Cell Transplant

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 646-646
Author(s):  
Albert Yeh ◽  
Motoko Koyama ◽  
Simone A Minnie ◽  
Julie Boiko ◽  
Kathleen S Ensbey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Copy Number ◽  
Research Funding ◽  
Molecular Transport ◽  
Cell Transplant ◽  
Antigen Specificity ◽  
Donor Pool ◽  
Post Transplant ◽  
Low Copy Number

Abstract Background: The immunologic basis of acute GVHD fundamentally involves alloreactive donor T cells that recognize foreign major histocompatibility complex (MHC)-peptide structures derived from both major and minor antigen mismatches with the host. Within this paradigm, the relationship between the donor and recipient genetics represents a closed system that dictates the potential ability of any given T cell receptor (TCR) to expand, raising the question of whether there are predictable aspects of TCR reconstitution at a clonal level. Take a hypothetical example - if genetically identical twins were to receive allogeneic grafts from the same donor and both recipients develop GVHD, would one expect similar TCRs to be clonally expanded? It has been challenging to rigorously explore this phenomenon, however, because of the vast combinatorial diversity of αβ TCRs, the high prevalence of low copy number TCRs, and sampling constraints - all of which render tracking and comparing TCR expansion between the donor and host difficult. Methods: We address these challenges in order to better understand the predictability TCR clonal dynamics through an analysis platform utilizing 1) a series of matched and mismatched murine transplant experiments where genetically identical littermates receive T cells from the same polyclonal donor pool, thus creating multiple transplant replicates simulating the twin transplant system describe above (Fig 1), and 2) probabilistic modeling of individual TCR frequencies to account for partitioning stochasticity (variation in how low copy number TCRs are distributed from donor to recipient). We conduct high-throughput DNA-based TCR amplicon sequencing for both donor and post-transplant recipient samples to generate over 20 million TCRs and model the expansion rates of all identifiable TCRs in each transplant system using a Bayesian approach. Results: While overall V and J gene usage were similar amongst identical recipients (Fig 2), we find that a small fraction of TCR clonotypes appears to have widely disparate clone counts amongst identical recipients receiving the same donor T cell pool. For example, we saw 9,739, 129 and 0 copies of a particular TCR in 3 different recipients in our B6->B6D2F1 system (Fig 3). In order to distinguish whether TCR count discrepancies seen across identical recipients is simply a reflection of donor partitioning stochasticity or true differential expansion (Fig 4), we apply a Bayesian algorithm to identify differential expanders, which represent TCRs that are asymmetrically expanded between recipients of a genetically identical pair (Fig 5). These TCRs can be generated from both memory and naïve T cell compartments. The presence of these differentially expanded clones amongst identical recipients suggests that non-genetic dependent mechanisms may influence which TCRs expand post-transplant. We next show that broad gut decontamination of microbiota with peri-transplant vancomycin, gentamicin, cefoxitin and metronidazole dramatically reduced the fraction of differential expanders (p<0.0001). However, the change in inflammation from microbiome depletion did not appear to drive this difference, as 1) MyD88/TRIF double knockout recipients (deficient TLR signaling) did not show a reduction in differential expanders, and 2) altering conditioning intensity (900cGy to 1300 cGy TBI) also did not change the fraction of differential expanders. Rather, the difference is likely antigenically driven, as differential expanders are enriched in antigen specificity compared to other TCR sequences (p<0.0001) based on published algorithm that identify TCRs with similar amino acid sequence overlap. Conclusions: These results refine our current understanding of clonal T cell selection and expansion after allogeneic BMT and suggests that for a given transplant system, individual TCR selection is not solely dictated by genetic donor and recipient major and/or minor histocompatibility disparities. Rather, microbiota-derived molecules appear to behave as minor antigens to direct systemic clonal TCR selection. These data suggest a novel mechanism by which the microbiome may modulate transplant outcome, challenging current paradigms suggesting the microbiota primarily drive inflammation via their PAMP activities. Figure 1 Figure 1. Disclosures Hill: Applied Molecular Transport: Research Funding; Syndax Pharmaceuticals: Research Funding; Compass Therapeutics: Research Funding; NapaJun Pharma: Consultancy; Generon corporation: Consultancy; iTeos Therapeutics: Consultancy, Research Funding; Neoleukin Therapeutics: Consultancy; Roche: Research Funding.

scholarly journals Increased Early Mortality after Fludarabine and Melphalan Conditioning with Peripheral Blood Grafts in Haploidentical SCT with Post-Transplant Cyclophosphamide

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4496-4496 ◽  
Author(s):  
Luke Eastburg ◽  
David A. Russler-Germain ◽  
Ramzi Abboud ◽  
Peter Westervelt ◽  
John F. DiPersio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Early Mortality ◽  
Cell Transplant ◽  
Advisory Committees ◽  
Post Transplant ◽  
Equity Ownership

The use of post-transplant cyclophosphamide (PTCy) in the context of haploidentical stem cell transplant (haplo-SCT) has led to drastically reduced rates of Graft-vs-Host (GvH) disease through selective depletion of highly allo-reactive donor T-cells. Early trials utilized a reduced-intensity Flu/Cy/TBI preparative regimen and bone marrow grafts; however, relapse rates remained relatively high (Luznik et al. BBMT. 2008). This led to the increased use of myeloablative (MA) regimens for haplo-SCT, which have been associated with decreased relapse rates (Bashey et al. J Clin Oncol. 2013). Most studies have used a MA total body irradiation (TBI) based regimen for haplo-SCT. Preparative regimens using fludarabine and melphalan (FluMel), with or without thiotepa, ATG, and/or low dose TBI have also been reported using bone marrow grafts. Reports on the safety and toxicity of FluMel in the haplo-SCT setting with PTCy and peripheral blood stem cell (PBSC) grafts are lacking. In this two-center retrospective analysis, the safety/toxicity of FluMel as conditioning for haplo-SCT was evaluated. We report increased early mortality and toxicity using standard FluMel conditioning and PBSC grafts for patients undergoing haplo-SCT with PTCy. 38 patients at the University of Rochester Medical Center and the Washington University School of Medicine underwent haplo-SCT with FluMel conditioning and PBSC grafts between 2015-2019. Outcomes were measured by retrospective chart review through July 2019. 34 patients (89.5%) received FluMel(140 mg/m2). Two patients received FluMel(100 mg/m2) and two patients received FluMel(140 mg/m2) + ATG. The median age at time of haplo-SCT was 60 years (range 21-73). 20 patients were transplanted for AML, eight for MDS, two for PMF, two for NHL, and five for other malignancies. The median Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) score was 4 (≥3 indicates high risk). 11 patients had a history of prior stem cell transplant, and 16 patients had active disease prior to their haplo-SCT. Seven patients had sex mismatch with their stem cell donor. Median donor age was 42 (range 21-71). 20 patient deaths occurred by July 2019 with a median follow up of 244 days for surviving patients. Nine patients died before day +100 (D100, "early mortality"), with a D100 non-relapse mortality (NRM) rate of 24%. Median overall and relapse free survival (OS and RFS, respectively) were 197 days (95% CI 142-not reached) and 180 days (95% CI 141-not reached), respectively, for the entire cohort. The 1 year OS and NRM were 29% and 50%. The incidence of grades 2-4cytokine release syndrome (CRS) was 66%, and 52% of these patients were treated with tocilizumab. CRS was strongly associated with early mortality, with D100 NRM of 36% in patients with grade 2-4 CRS compared to 0% in those with grade 0-1. The incidence of acute kidney injury (AKI) was 64% in patients with grade 2-4 CRS, and 8% in those without (p < 0.001). 28% of patients with AKI required dialysis. Grade 2-4 CRS was seen in 54% of patients in remission prior to haplo-SCT and in 92% of those with active disease (p = 0.02). Of the 9 patients with early mortality, 89% had AKI, 44% needed dialysis, and 100% had grade 2-4 CRS, compared to 31%, 10%, and 55% in those without early mortality (p = 0.002, p = 0.02, p = 0.01). Early mortality was not significantly associated with age, HCT-CI score, second transplant, disease status at transplant, total dose of melphalan, volume overload/diuretic use, or post-transplant infection. In conclusion, we observed a very high rate of NRM with FluMel conditioning and PBSC grafts for haplo-SCT with PTCy. The pattern of toxicity was strongly associated with grade 2-4 CRS, AKI, and need for dialysis. These complications may be mediated by excessive inflammation in the context of allo-reactive donor T-cell over-activation. Consistent with this, multiple groups have shown that FluMel conditioning in haplo-SCT is safe when using bone marrow or T-cell depleted grafts. Based on our institutional experiences, we would discourage the use of FluMel as conditioning for haplo-SCT with PTCy with T-cell replete PBSC grafts. Alternative regimens or variations on melphalan-based regimens, such as fractionated melphalan dosing or inclusion of TBI may improve outcomes but further study and randomized controlled trials are needed. This study is limited in its retrospective design and sample size. Figure Disclosures DiPersio: WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Karyopharm Therapeutics: Consultancy; Magenta Therapeutics: Equity Ownership; Celgene: Consultancy; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Amphivena Therapeutics: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liesveld:Onconova: Other: Data safety monitoring board; Abbvie: Membership on an entity's Board of Directors or advisory committees.

scholarly journals The Combination of Anti-Tigit and Lenalidomide Promotes Synergistic Myeloma-Specific Immunity after ASCT

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 328-328
Author(s):  
Simone A Minnie ◽  
Nicole S Nemychenkov ◽  
Kathleen S Ensbey ◽  
Christine R Schmidt ◽  
Gregory Driessens ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Molecular Transport ◽  
Immunomodulatory Drugs ◽  
Preclinical Models ◽  
Specific Immunity

Abstract Multiple myeloma is a largely incurable bone marrow (BM) resident plasma cell malignancy that is increasing in incidence. Autologous stem cell transplantation (ASCT) is the current standard consolidation therapy and a subset of patients achieve durable progression free survival that is suggestive of long-term immune control. Utilizing novel preclinical models, we have provided definitive evidence that this is largely mediated by T cell-dependent myeloma-specific immunity. In both patients and preclinical models, myeloma progression is associated with T cell dysfunction and expression of multiple inhibitory receptors suggesting a loss of immunosurveillance. In mice, we have demonstrated potent anti-myeloma efficacy of TIGIT blockade in both ASCT and non-transplant settings. Here we utilized identical TIGIT Abs that do or do not Fc bind to demonstrate that immunological efficacy after ASCT was absolutely dependent on ADCC (median survival was unreached (&gt;110 days) in Fc-binding vs 73 days in Fc-dead and 71 days in control Ig (cIg)-treated mice). Since TIGIT inhibition does not protect against myeloma relapse in all mice, it is apparent that combinational approaches are required to target non-responders. Therefore, we hypothesized that TIGIT blockade could be combined with immunomodulatory drugs (IMiDs) to provide synergistic anti-myeloma activity after ASCT. To that end, we utilized CRBN transgenic mice to investigate the efficacy of TIGIT blockade in combination with lenalidomide, the standard of care IMiD used in maintenance therapy after clinical ASCT. Briefly, B6 Vk*MYC myeloma-bearing (MM-bearing) mice were lethally irradiated and transplanted with B6 bone marrow (BM) and a suboptimal dose of T cells followed by anti-TIGIT or control Ig (100 mg twice weekly) for 5 weeks with lenalidomide (50 mg/kg daily gavage) or control diluent from D+14 for 3 weeks (Figure 1A). The combination of anti-TIGIT and lenalidomide provided synergistic anti-myeloma efficacy evidenced by prolonged median survival (109 days in combination vs &lt; 60 days in monotherapy/control-treated mice, p&lt;0.01; Figure B). Myeloma M bands were also suppressed in the combination treated mice relative to monotherapy or cIg-treated mice (p&lt;0.01; Figure 1). Analysis of BM CD8 T cells 6 weeks after ASCT demonstrated that combination therapy significantly decreased terminal exhaustion (TOX + TIM3 + CD101 + PD-1 + DNAM-1 ─) with an average of only 20% of CD8 T cells with an exhausted phenotype in the combination group compared to greater than 50% exhausted CD8 T cells in monotherapy or cIg-treated mice (p&lt;0.05; Figure 1C-D). The combination also increased the frequency of central memory and tissue-resident memory subsets (CD49b +CD69 +; p&lt;0.05; Figure 1C-D), and increased IFNγ production from activated (PD-1 +; p&lt;0.05; Figure 1E) cells compared to monotherapy or control Ig-treated mice. Importantly, these phenotypic changes were specific to the BM tumor microenvironment as we observed no effect of combination or monotherapy treatment on CD8 or CD4 T cells in peripheral blood. In sum, these data provide a strong rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent the progression of myeloma. Figure 1 Figure 1. Disclosures Driessens: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company. Holmberg: Up-To-Date: Patents & Royalties; Bristol Myers Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding; Millennium-Takeda: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Hill: NeoLeukin Therapeutics: Consultancy; Compass Therapeutics: Research Funding; NapaJen Pharma: Consultancy; Generon Corporation: Consultancy; Roche: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Applied Molecular Transport: Research Funding.

scholarly journals Mogamulizumab within 55 Days of Allogeneic Transplant: A Report of 3 Cases

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5297-5297
Author(s):  
Mary Jo Lechowicz ◽  
Dolores Caballero ◽  
Mollie Leoni ◽  
Marilyn Tabachri ◽  
Fiona Herr ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lymphoma ◽  
The United States ◽  
T Cell Lymphoma ◽  
Cell Transplant ◽  
Open Label ◽  
Pharmaceutical Development ◽  
Post Transplant ◽  
T Cell Malignancies

Introduction: Mogamulizumab is a humanized monoclonal antibody against CCR4 (CC chemokine receptor 4), which is frequently expressed on certain T-cell malignancies such as adult T-cell leukemia-lymphoma (ATL), peripheral T-cell lymphoma (PTCL), and cutaneous T-cell lymphoma (CTCL), and in addition, on normal immune T-cells such as regulatory T-cells (Tregs). Mogamulizumab is approved as monotherapy in Japan for relapsed or refractory CCR4-positive ATL, PTCL, and CTCL, and in the United States for adult patients (pts) with relapsed or refractory mycosis fungoides (MF) and Sézary syndrome (SS) - two subtypes of CTCL - after ≥1 prior systemic therapy. Based on retrospective analysis of ATL pts in Japan, evidence suggests that a shorter interval (<50 days) between the last dose of mogamulizumab and allogeneic hematopoietic stem cell transplant (allo-HSCT) might increase the risk of graft versus host disease (GVHD)-related complications and negatively impact clinical outcomes compared to a longer interval (Fuji S et al, J Clin Oncol 2016). Outcomes of allo-HSCT within 50 days of mogamulizumab treatment outside of Japan have not been reported. A retrospective review of Western clinical trials of mogamulizumab in T-cell malignancies identified 3 non-ATL pts who went on to receive allo-HSCT within approximately 50 days of their last dose of mogamulizumab. Here, we report safety and outcome information for these cases. Methods: A review of clinical trial pts in the West identified two pts from the open-label, phase 3, randomized controlled MAVORIC trial (ClinicalTrials.gov number: NCT01728805), which compared treatment with mogamulizumab to vorinostat in patients with relapsed or refractory MF/SS; crossover was allowed from the vorinostat to the mogamulizumab treatment arm upon progressive disease or intolerable toxicity. A third pt was identified from an open-label, single-arm phase 2 trial for previously treated PTCL (NCT01611142) in patients with relapsed or refractory CCR4-positive PTCL. In both studies, pts received mogamulizumab 1.0 mg/kg intravenously (IV) on a weekly basis for the first 28-day cycle, then on days 1 and 15 of subsequent cycles. Results: Pts were 49, 61, and 22 years of age with MF, MF/SS, and PTCL, respectively (Table). All pts had received at least 1 prior systemic treatment regimen for their disease. The last dose of mogamulizumab was administered 54, 17, and 45 days prior to allo-HSCT, respectively. Post-transplant, all three pts experienced at least one occurrence of GVHD, both steroid refractory and responsive, with grades ranging from 1 to 4 in the skin, abdomen, colon, and eye. The pts with MF and SS were alive at 1399 days and 1187 days post-transplant, respectively. The pt with PTCL died on day 330 post-transplant. Conclusions: This is the first published case study report to describe three pts with non-ATL T-cell malignancies who received mogamulizumab approximately 50 days or less prior to allo-HSCT and who experienced varying types and severity of GVHD. Two pts with MF/SS-type CTCL, who received mogamulizumab within 55 days of transplant, survived ≥1187 days. Further research is needed to understand the efficacy, safety, and appropriate timing of mogamulizumab prior to allo-HSCT in pts with T-cell malignancies. Disclosures Lechowicz: Kyowa Kirin Inc: Consultancy; Spectrum: Consultancy. Leoni:Kyowa Kirin Pharmaceutical Development, Inc.: Employment. Tabachri:Kyowa Kirin Pharmaceutical Development, Inc.: Employment. Herr:Kyowa Kirin, Inc.: Employment. Lamar:Kyowa: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy.

Autologous Whole Tumor Cell Vaccination Early After Allogeneic Stem Cell Transplantation Elicits Anti-Tumor T Cell Responses in Patients with Advanced Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1892-1892
Author(s):  
Ute E. Burkhardt ◽  
Ursula Hainz ◽  
Kristen E. Stevenson ◽  
Di Wu ◽  
Vincent T. Ho ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
T Cell Responses ◽  
Lymphocytic Leukemia ◽  
Autologous Tumor ◽  
Post Transplant ◽  
Cell Responses

Abstract Abstract 1892 Patients with advanced hematological malignancies remain at high risk for eventual disease progression following reduced intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We hypothesized that vaccination with whole leukemia cells during the critical period of immune reconstitution early after transplant may enhance antitumor immunity and facilitate expansion of leukemia-reactive T cell responses. We tested this hypothesis in a prospective clinical trial, in which patients with advanced chronic lymphocytic leukemia (CLL) received up to 6 vaccine doses initiated between day 30–45 following RIC allo-HSCT. Each vaccine consisted of 1×107 irradiated autologous tumor cells admixed with 1×107 irradiated K562 bystander cells secreting GM-CSF (GM-K562). All patients received tacrolimus and mini-methotrexate as graft-versus-disease (GvHD) prophylaxis. Tacrolimus was maintained at therapeutic levels during the vaccination period without taper. Twenty-two patients were enrolled, all with advanced disease (median number of prior therapies 3; range 2–11). Many of the leukemias expressed markers associated with aggressive disease (e.g. unmutated IgVH - 68%) and displayed high-risk cytogenetic abnormalities (sole del(11q) - 41%; sole del(17p) - 23%; del(11q and 17p) - 18%). Greater than 50% (n=13) of patients had persistent marrow involvement (≥10%) at time of allo-HSCT. Eighteen of 22 subjects were vaccinated after allo-HSCT and received a median of 6 (range 1–6) vaccines. The remaining 4 patients were precluded from vaccination due to development of acute GvHD before day 45. Vaccines were generally well tolerated, but mild, transient injection site erythema was common. Only one grade 4 event (neutropenia) with a possible attribution to treatment occurred. We observed a similar incidence of grade II-IV aGvHD at 1 year in the 18 vaccinated patients (39%; 95% CI: 17–61%) and 42 control CLL patients that underwent RIC allo-HSCT at our institution from 2004–2009 (31%; 95%CI: 18–46%). At a median follow-up of 2.9 (range 1–4) years, the estimated 2-year rates of progression-free survival and overall survival of vaccinated study participants were 80% (95% CI: 54–92%) and 84% (95% CI: 58–95%). With these promising clinical results, we next focused on gaining insight into the mechanism that generated the observed clinical graft-versus-leukemia (GvL) responses. To delineate the specific contribution of vaccination to the overall GvL effect, we performed T cell assays to detect CLL-specific reactivity in serial pre- and post-HSCT samples obtained from vaccinated patients (n=9) who received median of 6 vaccines (range 3–6). In comparison, we examined T cell responses in study subjects (n=4) that developed aGvHD at a median of 44.5 days (range 26–56) after HSCT; and control CLL patients (n=4; no vaccine, no GvHD in the early post-transplant period) that were not enrolled in the study. Although early post-transplant vaccination had no impact on recovering absolute T cell numbers, reactivity of CD8+ T cells from the vaccinated patients was consistently directed against autologous tumor cells but not alloantigen bearing-recipient cells (PHA T cell blasts and fibroblasts) in IFNγ ELISpot assays. A peak response against autologous tumor cells was reached at day 60 after allo-HSCT (average 221 SFC/5×105 cells vs. 29 and 33 average SFC/5×105cells for PHA blasts and fibroblasts, respectively). CD8+ T cell clones were isolated from 4 vaccinated study subjects by limiting dilution and 17% (range 13–33%) reacted solely against CLL-associated antigens. In contrast, broad CD8+ T cell reactivity indicating an alloantigen response was observed in GvHD patients, while no increase in T cell reactivity against tumor-associated or alloantigens was seen in control patients. Tumor-reactive CD8+ T cells isolated from vaccinated patients secreted a broad profile of effector cytokines (GM-CSF, TNFα and IP10). Moreover, the amount of cytokines secreted by these CLL-specific CD8+ T cells steadily increased following early post-transplant vaccination, but not after allo-HSCT alone or in relation to GvHD. Our studies reveal that vaccination with autologous whole CLL/GM-K562 cells between days 30–100 after allo-HSCT is associated with induction of immunity against recipient CLL cells, and suggest that this is an effective strategy for promoting GvL following RIC allo-HSCT. Disclosures: Brown: Genzyme, Celgene: Research Funding; Calistoga, Celgene, Genentech, Pharmacyclics, Novartis, Avila: Consultancy. Cutler:Pfizer, inc: Research Funding; Astellas, Inc: Consultancy, Research Funding.

PD-1 Is Elevated on the Peripheral Blood T Cell Subsets of Patients with Relapsed/Refractory Hodgkin Lymphoma Compared to Normal Volunteers

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4860-4860 ◽  
Author(s):  
Catherine S. Diefenbach ◽  
Rachel Sabado ◽  
Sean Clark-Garvey ◽  
Crystal Cruz ◽  
Isabelita Vengco ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Cell Transplant ◽  
Normal Volunteers ◽  
Control Subjects ◽  
Potential Biomarker ◽  
Healthy Control ◽  
Programmed Death 1

Abstract Abstract 4860 Background: Programmed death-1(PD-1) and programmed death-1 ligand (PD-L) signaling are involved in the functional impairment and “exhaustion” of T cells in conditions such as chronic viral infection and in tumor immune evasion. The interaction of PD-1 with its ligand PD-L suppresses T cell function. Up-regulation of PD-L has been demonstrated both in Hodgkin lymphoma (HL) cell lines, and in primary Hodgkin Reed Sternberg (HRS) cells. PD-1 is markedly elevated in the tumor infiltrating lymphocytes of HL patients, leading to the hypothesis that T cell exhaustion and deficient anti-tumor immunity induced by the activation of the PD-1-PD-L signaling pathway may play a key role in creating a permissive milieu for HL. In this setting, elevated PD-1 expression in HL patient T cells may be a biomarker of disease activity. We investigated this hypothesis by examining PD-1 expression in the peripheral T cells of patients with relapsed/ refractory HL. Methods: Patients with relapsed or refractory HL who enrolled in a clinical trial of an HDAC inhibitor in combination with Niacinamide between December of 2010 and July of 2011 were eligible to participate. PD-1 levels were assessed pre-treatment, and at selected timepoints during therapy. The levels of PD-1 expression for 5 relapsed/refractory HL patients and 4 healthy control subjects were evaluated by flow cytometry. An aliquot of cells (1 × 106/mL) was washed and stained with: CD3 APC-H7, CD8 PerCP-Cy5.5, CD4 FITC and PD-1-APC, for 20 minutes at 4°C. Dead cells were excluded using the Live/Dead Fixable Staining Kit (Invitrogen), and stained cells were acquired using the LSR II flow cytometer (BD). Compensation (parallel controls using cells singly stained for each color) and data analysis were performed using FlowJo flow cytometry analysis software (TreeStar). The percentage and/or mean fluorescence intensity (MFI) of PD-1+ cells within the live CD3+CD4+ and CD3+CD8+ populations was compared to isotype controls to establish baseline values and to normal control subjects. Results: The median age of the 5 HL patients was 32 (range 25–73). The median age of the healthy control subjects was 43 (range 39–49). Three of the HL subjects were male; all 4 of the normal controls were female. All 5 of the HL patients were heavily pretreated with an average number of prior regimens of 7.8 (range 5–13). Three of the 5 patients underwent previous autologous stem cell transplant, and 2 had a prior allogeneic stem cell transplant. There was a clear shift of PD1+ cells within the CD4+ T cell population in the HL patients compared to normal controls. PD-1 was significantly elevated in the peripheral blood CD4+ cells of HL patients compared to normal volunteers (mean MFI 1034 vs 123, p < 0.025). PD-1 was also elevated on the CD8+ T cells of the HL patients compared to normal volunteers (MFI 808 vs 221), but did not reach significance. There was no correlation between the level of PD-1 elevation on CD4+ or CD8+ cells, and prior autologous or allogeneic transplantation. Conclusion: PD-1 expression in peripheral blood CD4+ T cells may be a potential biomarker of systemic immune dysregulation in heavily pre-treated HL patients. PD-1 warrants further exploration in relapsed/refractory HL both as a potential biomarker, and as a target for directed immunotherapy. Additional studies by our group, investigating the role PD-1 as a biomarker in relapsed/refractory HL are ongoing. Disclosures: O'Connor: Spectrum: Research Funding; Novartis: Research Funding; Merck: Research Funding; Celgene: Consultancy, Research Funding.

scholarly journals Exploring LAG-3 Expression in Multiple Myeloma Patients Following Autologous Stem Cell Transplant

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3434-3434 ◽  
Author(s):  
Lucas Fabienne ◽  
Michael Pennell ◽  
Don M. Benson ◽  
Yvonne Efebera ◽  
Maria Chaudhry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mrna Expression ◽  
Stem Cell Transplant ◽  
Autologous Stem Cell Transplant ◽  
Cell Transplant ◽  
Post Transplant ◽  
Cell Dysfunction ◽  
T Cell Dysfunction ◽  
Mrna Markers

Abstract Background: Multiple Myeloma (MM) is associated with T-cell dysfunction. MM-related T-cell deficiencies are reported as both quantitative and qualitative defects with variable functional abnormalities. The clinical significance of these T-cell abnormalities in MM is not fully characterized. Furthermore, the impact of Autologous Stem Cell Transplant (ASCT) on T-cell subsets and immune checkpoint molecules, including PD-L1, PD-1, BLIMP-1, CTLA-4/CD28, TIM-3, and LAG-3, is being explored. Here we examined these features along with other mRNA markers of cellular senescence, immunosenescence, and exhaustion in MM patients pre- and post-ASCT. These studies aimed to improve understanding of the immunologic changes in MM and relationship to disease progression. Evaluating T-cell immune reconstitution post-transplant is invaluable for future therapies to identify targets or stimulate T-cell response to mitigate relapse. Methods: 100 MM patients were prospectively enrolled in a longitudinal study prior to ASCT for analysis of clinical and biologic factors related to clinical outcomes and event free survival (EFS) post-transplant. Paired peripheral blood T-cells (PBTL) were analyzed (n=31) before ASCT and 90 days post-ASCT. PBTL mRNA targets were anayzed using a custom Nanostring platform (OSU_Senescence) evaluating markers of cellular senescence, immunosenescence, and exhaustion. T-cell populations and subsets were further characterized by flow cytometry pre- and post-ASCT in 20 representative trial patients and 10 age- and sex-matched controls. Serum samples were analyzed for soluble ligands using Luminix MAGPIX multiplex analysis. Results: Increased PBTL LAG-3 mRNA expression at 90 days post-ASCT was significantly associated with EFS, HR 5.44 (95%CI 1.92-15.46, p=0.001, adjusted p* controlling for false discovery rate=0.038). When adjusted for age, a similar relationship of increased PBTL LAG-3 mRNA expression and EFS was found, HR 5.66 (95%CI 1.83-17.47, p=0.003, p*=0.056). No relationship was observed between other mRNA markers of immunosenesence or exhaustion and EFS. Flow cytometric analysis of post-transplant PBTLs revealed an inverted CD4:CD8 ratio, reduced CD28 expression on CD8+ T-cells, and increased levels of CD4+ T-regulatory cells. LAG-3 expression was significantly increased in CD4+ T-cells post-ASCT, predominately in the CD4 naïve and central memory subsets. Soluble LAG-3 (sLAG3) serum concentrations were similar pre- and post-ASCT, and in comparison to controls. Conclusions: We found increased expression of Lymphocyte-Activation Gene, LAG-3 (CD223), on CD4+T-cells post-ASCT. PBTL LAG-3 mRNA expression 90 days post-ASCT was associated with EFS and may serve as an early indicator of adverse outcomes. LAG-3 is expressed on activated T-cells and modulates T-cell expansion and function. Future studies targeting the LAG-3 pathway are warranted to restore T-cell dysfunction and augment immunity in MM. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Dendritic Cell Tumor Fusion Vaccination in Conjunction with Autologous Transplantation for Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 783-783
Author(s):  
Jacalyn Rosenblatt ◽  
Irit Avivi ◽  
Baldev Vasir ◽  
Tami Katz ◽  
Lynne Uhl ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Board ◽  
Advisory Committees ◽  
Cd8 Cells ◽  
Post Transplant

Abstract Abstract 783 Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to explore the role of immunotherapeutic strategies in eradicating malignancy. Patients achieve tumor cytoreduction following ASCT, however ultimately experience disease relapse from a persistent reservoir of chemotherapy resistant disease. Cancer vaccines that educate host immunity to target myeloma cells can be used to eradicate residual disease following ASCT. Our group has developed a cancer vaccine whereby dendritic cells (DCs) are fused with autologous tumor cells. DC/MM fusion cells present a broad array of tumor antigens in the context of DC derived costimulatory molecules. We are conducting a clinical trial in which patients with MM undergo ASCT followed by post-transplant vaccination with 3 doses of DC/MM fusions (cohort 1). A second cohort of patients receive an additional vaccination prior to stem cell collection in order to induce the expansion of tumor specific lymphocytes that are collected in the stem cell product (cohort 2). The infusion of educated lymphocytes provides a platform for subsequent post-transplant vaccination. To date, 26 patients have been enrolled in cohort 1 and 9 patient have been enrolled in cohort 2. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 70%) and maturation markers (mean CD83 55%). MM cells were isolated from bone marrow and were identified by their expression of CD38 or CD138. DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean yield of the DC and myeloma preparations was 1.72 × 108 and 6.6 × 107 cells, respectively. Mean fusion efficiency was 38% and the mean cell dose generated was 3.6 × 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 87%, 87%, and 78%, respectively. As a measure of their potency as antigen presenting cells, fusion cells potently stimulated allogeneic T cell proliferation in vitro. Mean stimulation indexes were 13, 60, and 32 for T cells stimulated by myeloma cells, DCs, and fusion cells at an APC: T cell ratio of 1:10. Adverse events judged to be potentially vaccine related were mild, and included injection site reactions, pruritis, myalgias, fever, chills, and tachycardia. ASCT was associated with suppression of measures of cellular immunity. Circulating CD4 cells were depressed in the post-transplant period and CD4:CD8 ratios remained inverted for greater than 10 months. Similarly, 65% of patients had a positive DTH response to candida antigen prior to transplant while only 21% demonstrated a positive response in the early post-transplant period. T cell response to PHA mitogen was transiently depressed post-transplant with mean stimulation indexes of 79, 10, 26, 36, and 63 prior to transplant, 1, 2, 3, and 6 months post-transplant, respectively. Consistent with these findings, in vitro T cell responses to tetanus toxoid were blunted in the post-transplant period. In contrast, a significant increase in circulating tumor reactive lymphocytes was noted, as determined by T cell expression of IFN by CD4 and CD8 cells following ex vivo coculture with autologous myeloma cell lysate (Mean percentage of tumor reactive CD8 cells was 1 and 7.7 pre and post-transplant, respectively; mean percentage of CD4 cells was 0.9 and 3.2). A further amplification of tumor reactive lymphocytes was seen with vaccination in a subset of patients (mean percentage of CD4 and CD8 tumor reactive T cells was 6.4 and 13.4, respectively). In the post-transplant period, regulatory T cells fell to minimal levels. To date, 23 patients have completed follow up and were evaluable for clinical response. 3 patients achieved CR at 1 month following ASCT. Of note, an additional 7 patients obtained a CR following completion of vaccinations, suggesting a role for post-transplant immunotherapy in mediating elimination of disease. In summary, fusion cell vaccination in conjunction with ASCT was well tolerated, stimulated anti-tumor immunity and was associated with the induction of post-transplant complete response. Disclosures: Richardson: Millenium (Research Funding and Advisory Board), Celgene, Keryx, BMS, Merck, Johnson and Johnson (All Advisory Board): Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Millenium (Research Funding and Advisory Board: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Keryx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees.

Recombinant Human Interleukin-7 (CYT107) Enhances CD4 and CD8 T Cell Recovery Following T-Cell Depleted Allogeneic Hematopoietic Stem Cell Transplant In Patients with Myeloid Malignancies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 674-674
Author(s):  
Miguel-Angel Perales ◽  
Jenna D. Goldberg ◽  
Leuren Lechner ◽  
Jianda Yuan ◽  
Esperanza Papadopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Cell Transplant ◽  
Immune Recovery ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Interleukin 7 ◽  
T Cell Reconstitution

Abstract Abstract 674 Immune recovery is an important determinant in multiple outcomes following allogeneic hematopoietic stem cell transplant (allo-HSCT). Delays in B and T cell reconstitution are associated with an increased risk of infection, relapse and secondary malignancy. Strategies to enhance post-transplant T-cell reconstitution could therefore improve morbidity and mortality after allo-HSCT. The cytokine Interleukin-7 (IL-7) is a unique therapeutic candidate to promote immune reconstitution because it has a central role in T cell development and survival. Murine models of allo-HSCT have demonstrated that IL-7 can enhance thymopoiesis as well as promote peripheral T cell survival and expansion. Initial clinical trials performed with recombinant human IL-7 (rhIL-7) have demonstrated a dose-dependent expansion of CD4+ and CD8+ T cells with an acceptable toxicity profile in patients with solid tumors or HIV infection. Hence we are conducting a phase I trial of post-transplant administration of rhIL-7 (CYT107, Cytheris Inc) in recipients of a T cell depleted (TCD) allo-HSCT to determine the safety, toxicity and biological activity on T cell reconstitution. To date, 9 patients (AML=7, MDS=2), with a median age of 59.3 years (range 27–67 years) have been treated with escalating doses of rhIL-7 (3 at 10 mcg/kg, 6 at 20 mcg/kg) administered subcutaneously weekly for 3 weeks following TCD allo-HSCT from an HLA compatible donor. Accrual is ongoing in the final cohort (30 mcg/kg). Recombinant hIL-7 was started at a median of 96 days post allo-HSCT (range 61–244 days). Most patients experienced transient minor injection site reactions. One patient (20 mcg/kg) developed a biopsy proven hypersensitivity drug rash a week after the first injection and was removed from the study (evaluable for toxicity but not immune recovery endpoints). No other significant injection-related toxicities have occurred, and no patients have developed GVHD. No anti-IL-7 antibodies or neutralizing antibodies have developed following rhIL-7 injection. Two of 9 patients with high-risk AML have relapsed (4 and 9 months post rhIL-7), an incidence consistent with published data in patients undergoing allo-HSCT for AML in CR, irrespective of T-cell depletion. Eight patients remain alive with a median follow-up of 14.5 months post rhIL-7 administration. At baseline, the median T cell counts were 91/mm3 (range 5 – 219 /mm3), 43/mm3 (range 9 – 299 /mm3) and 0 (range 0 – 17 /mm3) for CD4+, CD8+ and CD45RA+ T cells, respectively. Preliminary assessment of the immunological effects of rhIL-7 in 8 evaluable patients has demonstrated an increase in CD4+ T cells exhibiting a naïve or central memory phenotype (69% median increase over baseline at day 21 – range 8% to 35-fold increase), and CD8+ T cells exhibiting a naïve or effector memory phenotype (94% median increase over baseline at day 28 – range 0 to 11-fold increase). There was no observed effect on the frequency of CD4+CD25+FoxP3+ T cells or CD19+ B cells. TCR excision circles (TREC) analysis performed on CD4+ and CD8+ subsets in the first 6 patients, using absolute quantification real-time PCR, demonstrated increases in TRECs in 5/6 patients indicating enhanced T cell production. Finally, all 3 CMV-seropositive patients developed CD8+ T cell CMV-specific responses detected by intracellular IFNγ production to overlapping CMV-pp65 pentadecapeptides peptide pools after administration of rhIL-7. In one patient, we also analyzed CMV-specific T-cell frequency using HLA-A*0201 restricted MHC-tetramers. The highest CMV-specific response levels were noted in this patient with a history of CMV viremia and low-level CMV-specific CD8+ T cells prior to rhIL-7 (5.3-fold increase to the A0201-restricted immunodominant NLV peptide by tetramer assay after rhIL-7). Our pre-clinical data and early clinical results suggest that administration of rhIL-7 in recipients of a TCD allo-HSCT has minimal toxicity and can enhance post-transplant immune recovery without causing GVHD. Disclosures: Perales: Cytheris: Research Funding. Croughs:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morre:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van den Brink:Cytheris: Research Funding.

Adoptive T-Cell Therapy with 3rd Party CMV-pp65-Specific CTLs for CMV Viremia and Disease Arising after Allogeneic Hematopoietic Stem Cell Transplant

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 747-747
Author(s):  
Susan Prockop ◽  
Ekaterina Doubrovina ◽  
Irene Rodriguez-Sanchez ◽  
Aisha N. Hasan ◽  
Juliet Barker ◽  
...  
Keyword(s):  
Overall Survival ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Hematopoietic Stem Cell Transplant ◽  
Response Rate ◽  
Third Party ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Cmv Disease

Abstract Adoptive immunotherapy with transplant donor-derived virus-specific T-cells is effective in the treatment of CMV viremia and disease complicating allogeneic hematopoietic stem cell transplant (HCT), but is not available if the donor is seronegative or unavailable to provide lymphocytes. In addition, CMV-specific T-cell lines (CMV-CTLs) from non-identical donors may be restricted by HLA alleles not shared by the patient, rendering them ineffective. This limitation has become more problematic with increased use of haploidentical HCT donors. We treated 50 transplant recipients with third party donor-derived CMVpp65-specific T-cells between 10/14/11 and 11/28/16, evaluable for response assessment as of 6/20/17. Patients had received an unmodified (n=11) or T-cell depleted HCT (n=33) or a cord blood (n=6), transplant. Fifteen were treated for overt CMV disease involving CNS (N=6) GI (N=10) and Lung (N=2) and 35 for CMV viremia persisting despite &gt;2 weeks of induction therapy with 1-3 antiviral agents. Treatment with CMVpp65-CTLs was initiated at a median of 151 (29-4940) days post transplant and 128 (7-564) days after CMV reactivation. One patient was treated for CMV colitis developing more than 10 years after transplant due to immune suppression for chronic graft versus host disease. Patients had received a median of 3 (1-6) prior antiviral treatments. Third party CMVpp65-CTLs were selected from a bank of 186 lines generated under GMP conditions from normal HCT donors who specifically consented to use of their T cells in patients other than their designated transplant recipient. Selection was made on the basis of HLA restriction by at least one HLA allele shared by the patient and HCT donor, and matching for &gt; 2/10 recipient alleles. If such a line was not available, a patient could be treated with a line matched at only one HLA allele as long as the restriction was through that matched allele. Patients received 3 weekly infusions of approximately 1x106 CMVpp65-CTL/kg/infusion. Patients were sequentially evaluated for clinical and radiographic changes, quantifications of CMV DNA by PCR and IFN+ CMVpp65-specific T-cells in the blood. Responses were assessed 28-42 days after the first of each cycle of CMVpp65-CTLs. Response in patients with CMV disease was considered complete (CR) if all sites were cleared of virus by biopsy and blood sampling and partial (PR) if symptoms resolved and viremia met criteria of PR. In patients treated for persistent viremia, responses were complete if CMV DNA was cleared in repeated testing, and partial if the level of CMV fell based on the testing method by &gt;50% (N=2) or by 2log10 (N=12). Of the 50 patients 18 had a complete and 14 a partial response for an overall response rate of 64%. Response rates in patients with disease (5CR+4PR/15) were similar to those of patients with persistent viremia (13CR+10PR/35). In patients treated for viremia alone, survival at 6 months was 65.7% and in those with disease 60.0% (a). More extensively pretreated patients who received CMVpp65 CTLs &gt; 100 days post CMV initial detection fared as well as those treated earlier (62.1% vs. 66.7% OS) (b). Patients who responded to CMVpp65-CTL therapy (CR or PR) had an improved survival with 6 month overall survival of 81.3% (b) and 12 month overall survival of 62.1% (c); only 1 of these 32 patients died of CMV. In contrast 7 of 18 non-responding patients died of CMV; overall survival in this cohort was 33.3% at 6 months. By 12 months, 8 non-responding patients had died of CMV and overall survival had decreased to 22.2%. Toxicities associated with CMVpp65-CTL infusions in this cohort are limited with 5 patients experiencing adverse events of &gt; grade 3 severity deemed possibly related to CMVpp65-CTL therapy. Two of these patients died, one due to sepsis and one due to progression of CMV. This study demonstrates a high response rate among patients with otherwise refractory CMV viremia and disease. The bank of CMVpp65-CTLs can provide an immediate source of HLA partially-matched appropriately restricted T cells for adoptive immunotherapy to treat persistent CMV viremia and CMV disease, including disease isolated to the CNS. The availability of 3rd party CMVpp65-CTLs enables treatment early in the course of disease and may thereby improve response rates while minimizing toxicity from anti-viral therapy. Figure 1 Figure 1. Disclosures Doubrovina: Atara: Consultancy, Research Funding. Hasan: Atara Biotherapeutics: Consultancy, Other: During time of this study, Research Funding; Merck: Employment. Kernan: Gentium: Other: Received grants from Gentium during the conduct of the study and research was supported by The National Cancer Institute of the National Institutes of Health under award number P30 CA 008748, Research Funding. Koehne: Atara: Consultancy, Patents & Royalties. O'Reilly: Atara: Patents & Royalties, Research Funding.

scholarly journals Frequency of Marrow Infiltrating PD-1 Expressing CD4 Effectors Following Autologous Stem Cell Transplant (ASCT) for Multiple Myeloma Is Prognostic for Clinical Outcome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4368-4368
Author(s):  
Nouf Alrasheed ◽  
Lydia Lee ◽  
Huw Richards ◽  
William Wilson ◽  
Oliver C Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Outcomes ◽  
Cd8 T Cells ◽  
High Frequency ◽  
Research Funding ◽  
Autologous Stem Cell Transplant ◽  
T Cell Subsets ◽  
Cell Transplant ◽  
Ki 67

Introduction: High dose therapy and autologous stem cell transplant (ASCT) remain standard first line therapy for young fit patients with multiple myeloma (MM). Beyond cytoreduction, ASCT may also alter the bone marrow (BM) immune environment to augment anti-tumour immune responses. A more precise understanding of the immune microenvironment post ASCT and its relationship with clinical outcomes may identify biomarkers to refine prognostication and provide opportunity for therapeutic intervention. Aim: We aimed to define T cell subsets and expression of co-activating and co-inhibitory proteins in the BM early post ASCT, and explore associations with clinical outcomes. Methods: BM samples were obtained at 100 days (D100) post ASCT from 61 MM patients, and 15 healthy donors (HD). BM mononuclear cells (MNCs) were stained for surface (CD3, CD4, CD8, PD-1, LAG-3, and ICOS) and intracellular markers (GzmB, Ki-67, CTLA-4, and FoxP3). Data were acquired on a BD LSR Fortessa and analysed using FlowJo. Progression free survival (PFS) was defined from ASCT as per IMWG criteria, and patient groups were compared using Mann-Whitney U test. Adverse risk genetics was defined as t(4;14), t(14;16) or del(17p). Results: Median age of patients at ASCT was 58yrs (36-71), 70.5% were male. Median follow up was 17 months (3-54). At diagnosis, 36.1% were ISS stage I, 8.2% had extramedullary disease, and 14.7% had adverse risk genetics. As induction therapy, 96.7% received a proteasome inhibitor, 45.9% an immunomodulatory drug, 54.1% both, and 21.3% received post ASCT maintenance/consolidation. Median PFS was 24 months. At D100, 24.6% had achieved CR, 47.5% VGPR, 21.3% PR, 4.9% SD and none had PD. Improved PFS was found in patients with deeper response (CR/VGPR vs. rest, p=0.003), and with ISS stage I vs II/III (p=0.013). There was a trend for improved PFS with standard risk genetics (p=0.088 cf high risk). MM BM contained lower frequencies of CD3 and CD4 T cells compared to HD (CD3, 28% of live MNCs, vs 41.2%, p=0.012; CD4, 4.1% vs 14.0%, p<0.0001), while CD8 frequency was similar. The frequency of regulatory T cells (CD4+FoxP3+, Tregs) was higher in MM patients compared to HD (0.23% of live MNCs vs 0.07%, p=0.02; 4.2% of CD4+ vs 1.2%, p<0.0001). Thus CD4 effector (CD4+FoxP3-, CD4eff):Treg ratio was lower in MM patients (16.8 vs 140.2, p<0.00001). There was no difference in CD8:Treg ratio. Immune checkpoint proteins were highly expressed on BM T cell subsets in post ASCT compared to HD. CD4eff in MM patients expressed higher frequency of PD-1, LAG-3, ICOS, CTLA-4, GzmB, and Ki-67 (p<0.0001, p=0.0016, p=0.0018, p=0.003, p<0.0001, and p<0.0001, respectively). CD8 T cells in MM patients expressed higher frequency of LAG-3, GzmB and Ki-67 (p=0.0003, p<0.0001, and p<0.0001 respectively). We observed higher frequencies of ICOS and CTLA-4 on Tregs in MM patients (vs HD p=0.0025 for both) indicating Treg activation and enhanced suppressive function. There were considerable correlations between the frequency of LAG-3 on different T cell subsets; (CD4eff LAG-3+ vs CD8 LAG-3+ r=0.82, p<0.0001; CD4eff LAG-3+ vs Tregs LAG-3+, r=0.56, p<0.0001; Tregs LAG-3 vs CD8 LAG-3, r=0.5, P<0.0001). Clinical outcomes were not influenced by T cell subsets (CD3, CD4eff, CD8, and Treg) but there was an inverse association with co-inhibitory receptor expression. High frequency (>median) of LAG-3 on CD4eff and CD8, and Tregs was associated with shorter PFS (median 18mo vs 53, p=0.0028; 20mo vs 53, p=0.0057; 20mo vs 53, p=0.046 respectively). Further, patients with high frequency of PD-1 on CD4eff and Ki-67 on CD8 T cells had shorter PFS (median 18mo vs 36, p=0.042 and 18mo vs unreached, p=0.019 respectively). A multivariate Cox regression model was built to include ISS stage, depth of response at D100, and the immune phenotypes identified to correlate with PFS. In this model, high frequency of PD-1 on CD4eff retained independent prognostic value, along with ISS stage and CD8 Ki67+ T cells. Conclusion: We present data indicating that high frequency of PD-1+ CD4eff and Ki67+ CD8 T cells post ASCT associate with shorter PFS in MM patients. Pending confirmation in larger cohorts, these data reveal potential immune biomarkers for poor outcomes, and prompt deeper phenotypic analysis and mechanistic studies to uncover the immune drivers of sustained remissions post ASCT. Disclosures Lee: Autolus Therapeutics: Equity Ownership, Research Funding. Yong:Takeda: Research Funding, Speakers Bureau; Autolus: Consultancy; Amgen: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Speakers Bureau.

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