Genome-Wide DNA Methylation Analysis Identifies Aberrant Epigenetic Changes in CD8+ T Cells from Chronic Lymphocytic Leukemia Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3552-3552
Author(s):  
Jiazhu Wu ◽  
Xiaojing Xu ◽  
Lirong Pei ◽  
Eun-Joon Lee ◽  
Austin Shull ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Epigenetic Changes ◽  
Bisulfite Pyrosequencing ◽  
Cpg Sites ◽  
Genome Wide ◽  
Healthy Donors

Abstract Background CD8+ T cells from chronic lymphocytic leukemia (CLL) patients have been demonstrated to exhibit a number of alterations in global gene expression profiles when compared with healthy controls. It has been shown that CD8+ T cells from CLL patients have increased expression of T-cell exhaustion markers like PD-1. CLL-induced functional defects in T cells are thought to directly contribute to the failure of antitumor immunity and are considered a hallmark of this disease. Nevertheless, the molecular regulation of T-cell dysfunction in CLL patients still remains poorly understood. Methods In the present study, CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of patients with CLL (n=10) and healthy donors (n=5), and analyzed by genome-wide DNA methylation profiling using Illumina Infinium 450K methylation array. The differentially methylated genes (KLRG1, CCR6 and TCRA) identified by the 450K array analysis were validated by bisulfite pyrosequencing in additional CLL and healthy control samples. DNA methylation in the first intron, distal upstream, and proximal promoter regions of PD-1 was also examined by pyrosequencing. Luciferase reporter assays were used to determine the effects of DNA methylation on the enhancer activity of a PD-1 upstream sequence. To investigate whether CLL cells can directly alter the methylation of the candidate genes in CD8+ T cells, healthy PBMCs were cultured alone or co-cultured with purified allogeneic CLL cells for 72 hours. In parallel, healthy PBMCs were cultured in CD3mAb-coated plates containing CD28mAb or treated with PMA/ionomycin for 72 hours. Cultured PBMCs were then harvested for flow cytometrc analysis and CD8+ T cells purification. Multicolor flow cytometry was used to characterize T-cell subsets and expression of PD-1, KLRG1 and TCRα/β. Bisulfite pyrosequencing was used to determine the methylation changes of KLRG1, CCR6, TCRA, and PD-1 in CD8+ T cells after co-culture with CLL cells or after T-cell activation. Results The Illumina 450K methylation array analysis identified 312 differentially methylated CpG sites (Student t-test, p<0.05, average methylation difference >0.25) between CD8+ T cells from CLL and healthy controls with 199 hypermethyated and 113 hypomethylated CpG sites that are associated with 206 genes. Interestingly, 4 out of the 7 most significant CpG sites (FDR<0.05) were located in the 3’-end of the TCRA gene. Bisulfite pyrosequencing confirmed the decrease in the methylation levels of CpG sites associated with KLRG1, CCR6 and TCRA in CD8+ T cells from CLL patients as compared to healthy donors. Previous studies have demonstrated the increased expression of exhaustion markers such as PD-1 on the cell surface of CD8+ T cells from CLL patients. We identified a differentially methylation region (DMR) in the distal upstream region of the PD-1 promoter in CD8+ T-cells. This particular DMR shows consistently lower methylation levels in CD8+ T cells from CLL patients as compared to healthy controls. We cloned the DMR sequence into a luciferase reporter vector pGL4.23 with a minimal promoter and demonstrated enhanced luciferase activities from the cloned sequence, suggesting the presence of potential enhancer activity from this region. We observed that co-cultures with allogeneic CLL cells lead to increased expression of TCRα/β and PD-1 in CD8+ T cells from healthy donors. The methylation level of one CpG site from the 3’-end of TCRA was reduced by 50% after co-culture with CLL cells, though no methylation change in the DMR of PD-1 was observed. T-cell activation by CD3/28mAb or PMA/Ionomycin also resulted decrease in the methylation level of the CpG site at the 3-end of TCRA, yet to a lesser extent. Conclusion For the first time, our investigation demonstrates the genome-wide DNA methylation profiles of CD8+ T cells isolated from CLL patients and determined that recurrent epigenetic changes in PD-1, KLRG1, CCR6, and TCRA in CD8+ T cells occur in CLL patients. Our methylation data suggest that the exhaustion phenotype observed in CLL patient CD8+ T cells maybe associated with altered DNA methylation profiles, an event previously seen in antigen-specific CD8+ T cells that undergo chronic viral infection-induced epigenetic changes. Disclosures Awan: Boehringer Ingelheim: Consultancy; Lymphoma Research Foundation: Research Funding. Wang:NIH/NIMHD: Research Funding. Shi:NIH/NCI: Research Funding; Georgia Research Alliance: Research Funding.

scholarly journals Hypomethylation of STAT1 and HLA-DRB1 is associated with type-I interferon-dependent HLA-DRB1 expression in lupus CD8+ T cells

2018 ◽  
Author(s):  
Shaylynn Miller ◽  
Patrick Coit ◽  
Elizabeth Gensterblum-Miller ◽  
Paul Renauer ◽  
Nathan C Kilian ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Type I Interferon ◽  
Interferon Response ◽  
Type I ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Genome Wide

AbstractObjectiveWe examined genome-wide DNA methylation changes in CD8+ T cells from lupus patients and controls, and investigated the functional relevance of some of these changes in lupus.MethodsGenome-wide DNA methylation of lupus and age, sex, and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Measurement of relevant cell subsets was performed via flow cytometry. Gene expression was quantified by qPCR.ResultsLupus CD8+ T cells had 188 hypomethylated CpG sites compared to healthy matched controls. Among the most hypomethylated were sites associated with HLA-DRB1. Genes involved in the type-I interferon response, including STAT1, were also found to be hypomethylated. IFNα upregulated HLA-DRB1 expression on lupus but not control CD8+ T cells. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is induced by IFNα in lupus CD8+ T cells, but not healthy controls. Co-incubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to CD4+ T cell activation, determined by increased expression of CD69, in lupus patients but not in healthy controls. This can be blocked by neutralizing antibodies targeting HLA-DR.ConclusionsLupus CD8+ T cells are epigenetically primed to respond to type-I interferon. We describe an HLA-DRB1+ CD8+ T cell subset that can be induced by IFNα in lupus patients. A possible pathogenic role for CD8+ T cells in lupus that is dependent upon a high type-I interferon environment and epigenetic priming warrants further characterization.

scholarly journals Hypomethylation of STAT1 and HLA-DRB1 is associated with type-I interferon-dependent HLA-DRB1 expression in lupus CD8+ T cells

2019 ◽  
Vol 78 (4) ◽  
pp. 519-528 ◽  
Author(s):  
Shaylynn Miller ◽  
Pei-Suen Tsou ◽  
Patrick Coit ◽  
Elizabeth Gensterblum-Miller ◽  
Paul Renauer ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Type I Interferon ◽  
Interferon Response ◽  
Type I ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Genome Wide

ObjectiveWe examined genome-wide DNA methylation changes in CD8+ T cells from patients with lupus and controls and investigated the functional relevance of some of these changes in lupus.MethodsGenome-wide DNA methylation of lupus and age, sex and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Measurement of relevant cell subsets was performed via flow cytometry. Gene expression was quantified by qPCR. Inhibiting STAT1 and CIITA was performed using fludarabine and CIITA siRNA, respectively.ResultsLupus CD8+ T cells had 188 hypomethylated CpG sites compared with healthy matched controls. Among the most hypomethylated were sites associated with HLA-DRB1. Genes involved in the type-I interferon response, including STAT1, were also found to be hypomethylated. IFNα upregulated HLA-DRB1 expression on lupus but not control CD8+ T cells. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is induced by IFNα in lupus CD8+ T cells, but not healthy controls. CIITA knockdown and STAT1 inhibition experiments revealed that HLA-DRB1 expression in lupus CD8+ T cells is dependent on CIITA and STAT1 signalling. Coincubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to CD4+ T cell activation, determined by increased expression of CD69 and cytokine production, in patients with lupus but not in healthy controls. This can be blocked by neutralising antibodies targeting HLA-DR.ConclusionsLupus CD8+ T cells are epigenetically primed to respond to type-I interferon. We describe an HLA-DRB1+ CD8+ T cell subset that can be induced by IFNα in patients with lupus. A possible pathogenic role for CD8+ T cells in lupus that is dependent on a high type-I interferon environment and epigenetic priming warrants further characterisation.

scholarly journals Mass Cytometry Reveals Heterogeneity in the Exhaustion Profile of T-Cells in Chronic Lymphocytic Leukemia and the CD4:CD8 Ratio May be a Reliable Predictor of CD8+ T Cell Compartment "Fitness"

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 353-353 ◽  
Author(s):  
Muharrem Muftuoglu ◽  
Li Li ◽  
Han Chen ◽  
Duncan Mak ◽  
Elif Gokdemir ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Healthy Controls ◽  
Inhibitory Receptors ◽  
Cell Exhaustion ◽  
Healthy Donors ◽  
Checkpoint Receptors ◽  
Inhibitory Molecules

Abstract T cell exhaustion is characterized by coordinated expression of a series of negative checkpoint receptors such as programmed death-1 (PD-1), 2B4, CD160 and TIGIT, resulting in T cell dysfunction and immune evasion. Under physiological states, these inhibitory molecules maintain self-tolerance and prevent autoimmunity by applying a break on cytotoxic T cells. In cancer, T-cells exhibit features of T-cell exhaustion including increased expression of PD-1, 2B4 and CD160, coupled with reduced T cell proliferation, altered synapse formation and impaired cytotoxicity. Although the role of the PD-1/PD-L1 axis in mediating T cell defects in chronic lymphocytic leukemia (CLL) is well-studied, the contribution of other checkpoint molecules such as 2B4, CD160 and TIGIT in mediating tumor-induced immune dysfunction remains to be determined. Checkpoint inhibitors have provided a paradigm-shifting approach to cancer treatment. We hypothesized that the expression levels of checkpoint receptors on T-cells, as well as the "fitness" of the T cell compartment may provide a prognostic stratification system to predict response to checkpoint inhibitors in CLL. To determine if the number of inhibitory receptors per cell and their expression level may identify patient-to-patient differences that may not be easily deciphered using conventional research tools, we performed a detailed single-cell analysis of the T-cell repertoire, using 40-parameter mass cytometry (CyTOF) in 12 untreated CLL and 12 healthy controls. Consistent with previous reports, we found that expression of 2B4 (43.7% vs 30.8%), PD1 (28.8% vs 21%) and CD160 (17% vs. 9.7%) was significantly higher on CLL CD8+ T cells compared to healthy controls. In addition, CD8+T cells in CLL expressed higher levels of TIGIT (48.2% vs 25.2%), CD57 (43.9% vs 17.9%) and KLRG1 (49.5% vs. 29.7%). We clearly distinguished 2 patterns of exhaustion marker distribution in CLL. In one group of patients, the expression of checkpoint receptors was similar to that seen in healthy controls, whereas in the second group, CD8+ T-cells expressed higher levels of PD1, 2B4, TIGIT, CD160 as well as markers of terminal differentiation such as CD57 and KLRG1. Compared to healthy donors, CLL was characterized by an inversion in the CD4:CD8 ratio. Interestingly, CD8+ T cells in patients with a low CD4:CD8 ratio (defined as <2.5) expressed significantly higher levels of 2B4 (56.6% vs 31.25%), TIGIT (62.9% vs 37%), CD160 (22.8% vs 12.6%), CD57 (57% vs 28.7), PD-1 (34.6% vs 24.5%) and KLRG1 (62.3% vs 36.3%). In contrast, the expression levels of PD-1, 2B4 and CD160 in CLL patients with a CD4:CD8 ratio of ≥2.5 were similar to that seen in healthy controls, suggesting that the CD4:CD8 ratio may be a valuable marker of T cell exhaustion in CLL. Next, we compared the number of checkpoint molecules expressed per CD8+ T-cells in CLL patients versus healthy donors. Whereas a similar proportion of CD8+ T-cells in CLL (mean 19.56%, range 18.34-31.73%) and healthy donors (mean 22.13%, 14.17-41.19%) expressed one inhibitory receptor, a significantly higher proportion of CLL patients expressed 2 and more inhibitory receptors (mean 28.4, range 10.52-48.78%) compared to healthy controls (mean 15.38%, range 9.67-21.94%). PD-1 was mostly co-expressed with TIGIT, although TIGIT+PD-1+CD4+ and CD8+ T-cells were higher in CLL compared to healthy controls (12.9% vs 7.1%). Interestingly the predominant population of PD-1+CD8+ T cells in CLL was also positive for 2B4 and TIGIT, whereas expression of TIGIT was more diverse and was seen in association with PD-1, 2B4, KLRG1 or CD57. Taken together, our findings indicate a remarkable heterogeneity in the expression patterns of inhibitory molecules on CD8+ and CD4+ T-cells in CLL. While CLL patients with a normal CD4:CD8 ratio expressed comparable levels of inhibitory molecules to that seen in healthy controls, a low CD4:CD8 ratio was indicative of higher expression of checkpoint molecules. On a per cell basis, CLL CD8+ T cells expressed more inhibitory receptors compared to healthy controls, suggesting that certain patients may benefit from combinational use of checkpoint molecules. A more detailed data and analysis, including transcription and functional profile of exhausted CLL T cells, will be presented in the meeting. Disclosures Wierda: Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding. Jain:Incyte: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Abbvie: Research Funding; Infinity: Research Funding; BMS: Research Funding; Genentech: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Seattle Genetics: Research Funding; Celgene: Research Funding; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria.

scholarly journals Phase 2 Study of Combination of Cytarabine, Idarubicin, and Nivolumab for Initial Therapy of Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 815-815
Author(s):  
Farhad Ravandi ◽  
Naval Daver ◽  
Guillermo Garcia-Manero ◽  
Christopher B Benton ◽  
Philip A Thompson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
T Cell Subsets ◽  
Newly Diagnosed ◽  
Effector Cells ◽  
T Effector Cells ◽  
Grade 3

Abstract Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p&lt;0.05) and a trend towards higher percentage of CD4 T effector cells co-expressing PD1 and LAG3 compared to responders (Fig 1B). Co-expression of TIM3 or LAG3 on PD1+ T cells have been shown to be associated with an exhausted immune phenotype in AML (Zhou et al., Blood 2011). Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Sabine Schmied ◽  
Anne Richter ◽  
Mario Assenmacher ◽  
Juergen Schmitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Target Cells ◽  
Clinical Use ◽  
Healthy Donors ◽  
Effector Cytokines

Background The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization of CD4+ and CD8+WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge. In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use. Methods For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers. For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells. Results Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells. To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. Stainings using multiple WT1-MHCI-tetramers furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed. Conclusions Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use. Disclosures: Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.

T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3781-3781
Author(s):  
Eolia Brissot ◽  
Sawa Ito ◽  
Kit Lu ◽  
Carly Cantilena ◽  
B. Douglas Smith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Research Funding ◽  
Immune Escape ◽  
Nk Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Healthy Donors

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an “immune editing” effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p<0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

New Insights into the Mechanism of Action (MoA) of First-in-Class IgG-Based Bcma T-Cell Bispecific Antibody (TCB) for the Treatment of Multiple Myeloma (MM)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2096-2096 ◽  
Author(s):  
Laura Moreno ◽  
Aintzane Zabaleta ◽  
Diego Alignani ◽  
Marta Lasa ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Activation ◽  
Rational Combination ◽  
Dose Dependent ◽  
Tumor Cell Lysis

Abstract Novel agents have improved outcomes in MM, but prognosis after patients relapse remains poor and new drugs with novel MoA are needed. The breakthrough of immuno-oncology has rendered new therapeutic options, and most recently we reported on EM801, a novel BCMA-TCB that showed remarkably efficacy when used as single agent in primary bone marrow (BM) samples from MM patients (Seckinger, Blood 2015;126: abstr 117). Because of its novelty, further knowledge about the MoA of BCMA-TCB is of utmost importance to improve its efficacy by designing rational treatment combinations. In order to optimize the in vitro efficacy of the BCMA-TCB, we started by investigating in primary BM samples from 6 MM patients whether longer treatment periods with BCMA-TCB2 (a BCMA-TCB candidate sharing similar "2+1" structure of EM801 but displaying higher affinity to BCMA) would increase MM cell death. Upon treating samples with BCMA-TCB2 for 48h vs 96h, we noted a 2-fold increment in MM tumor cell lysis at 1nM and 10nM concentrations (Panel A). In parallel, the phenotypic profiles of CD4 and CD8 T cells showed that BCMA-TCB2 induced robust activation (ie. dose-dependent increment in CD69, CD25, HLADR after exposure to 100pM, 1nM and 10nM of BCMA-TCB2), but also led to the natural emergence of the checkpoint inhibitor PD-1 in the surface of activated CD4 and CD8 T cells (Panel B). We then investigated if there was a correlation between the percentage of PD-1 positive CD4 and CD8 T cells and the efficacy of BCMA-TCB2; interestingly, those patients with lower frequencies of PD-1 positive CD4 and CD8 T cells prior to treatment showed the highest rates of MM tumor cell lysis after 48h and 96h of BCMA-TCB2 at 10nM of (r=0.6, P=0.04; Panel C). By contrast, upon measuring the concentration of soluble BCMA and APRIL in the supernatants of primary BM samples from 16 MM patients treated with BCMA-TCB, we found no significant differences between responding (n=11) and non-responding (n=5) patients. Similar results were observed upon comparing the density of BCMA in the surface of MM tumor cells from responding vs non-responding patients (1256 vs 1522 SABC units; P=87). Since the efficacy of BCMA-TCB2 was found to be intrinsically related to the phenotype and activation status of T cells, we then investigated whether we could further harness immune cells by combining BCMA-TCB2 with three drugs representing different types of immunotherapy: lenalidomide (IMIDs), anti-PD1 (checkpoint inhibitors) and daratumumab (mAb). H929 MM cells were co-cultured with human leukocytes (n=5) and challenged to suboptimal concentrations of BCMA-TCB2 (10pM) alone, or in combination with standard doses of lenalidomide (1µM), anti-PD1 (10µg/ml) and daratumumab (10µg/ml) (Panel D). Interestingly, we observed that combining BCMA-TCB2 with lenalidomide or daratumumab significantly increased their anti-MM efficacy by 4-fold and 2.5-fold, respectively. Because lenalidomide and daratumumab share in common that they rely, at least in part, on activated NK cells to eradicate MM cells, we hypothesized whether such robust T cell activation induced by BCMA-TCB2 was leading to co-stimulation of NK cells. First, we demonstrated by analyzing the transcriptomes of T cells prior and after treatment exposure (n=3), that BCMA-TCB2 modulated the transcriptomes of CD4 and CD8 T cells (159 and 141 deregulated genes, respectively), consistent with enhanced activation and T-cell mediated inflammatory response (eg. TNFRS18, STAT1, CCL4). Furthermore, we observed a dose-dependent and significant increment of the CD69 (2-fold), CD25 (2.5-fold) and HLADR (4-fold) activation markers in the surface of NK cells from primary BM samples of 11 MM patients treated with BCMA-TCB2 (Panel E), suggesting a functional crosstalk between activated T cells and NK cells. In conclusion, we showed that the promising pre-clinical activity of the first-in-class IgG-based BCMA-TCB can be further enhanced by longer treatment periods followed by robust T cell activation. The observation that the efficacy of BCMA-TCB is intrinsically related to the activation status of T cells suggests its rational combination with IMIDs as demonstrated here. Most interestingly, potential crosstalk between activated T and NK cells could lead to enhanced function of the later immune subset, and provide a rational combination between BCMA-TCB and anti-CD38 antibodies to eradicate MM cells through highly activated T and NK cells. Figure Figure. Disclosures Strein: EngMab: Employment. Vu:EngMab: Employment. Paiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

scholarly journals CD4+ T Cell Fate in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4494-4494
Author(s):  
Rachel Elizabeth Cooke ◽  
Jessica Chung ◽  
Sarah Gabriel ◽  
Hang Quach ◽  
Simon J. Harrison ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
Advisory Committees ◽  
Mitochondrial Mass ◽  
Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Somatic STAT3 Mutations in CD8+ T Cells of HTLV-2 Positive Blood Donors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3133-3133
Author(s):  
Daehong Kim ◽  
Mikko Myllymäki ◽  
Matti Kankainen ◽  
Timo Jarvinen ◽  
Giljun Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Blood Donors ◽  
Somatic Mutations ◽  
Amplicon Sequencing ◽  
Viral Antigens ◽  
Stat3 Mutations ◽  
Lgl Leukemia

Abstract Introduction: T-cell large granular lymphocyte (T-LGL) leukemia is a rare lymphoproliferative disorder with recurrent somatic STAT3 mutations. It has been suggested that viral antigens act as the initial stimuli resulting in clonal expansion of CD8+ cells in the disease. However, less is known whether chronic exposure to viral antigens is associated with acquisition of somatic mutations in CD8+ T cells among individuals without clinically detectable lymphoproliferations. Human T-cell leukemia virus type 2 (HTLV-2) antibody positivity has been detected in patients with T-LGL leukemia. Here, we examined whether CD8+ T cells from HTLV-2 positive healthy blood donors harbor somatic mutations in STAT3 or other immune-associated genes, potentially identifying individuals at risk of subsequent lymphoproliferative diseases. Methods: We analyzed HTLV-2 infected (n=30) and uninfected (n=35) healthy blood donor samples obtained from University of California San Francisco and Vitalant Research Institute, which were enrolled in the United States-based HTLV Outcomes Study (HOST) cohort. All cases had serologic evaluation for HTLV-2 infection at the time of sampling. We examined somatic mutations of STAT3 in CD4+ and CD8+ T-cell populations using ultra-deep targeted amplicon sequencing. In addition, we applied a custom sequencing panel covering the coding regions of 2,533 immune-related genes to characterize a larger spectrum of somatic mutations in CD8+ T cells. Results: Somatic STAT3 mutations were detected in CD8+ but not in CD4+ T cells of four (13.3%, 4/30) HTLV-2 positive healthy blood donors (p.Y640F, p.N647I, p.D661Y, and p.Y657_K658insY with variant allele frequencies of 11.9%, 0.5%, 4.9%, and 1.2%, respectively) using amplicon sequencing. The detected STAT3 mutations have been previously described and reported in T-LGL leukemia. Total white blood cell and lymphocyte counts were similar between STAT3 mutated and non-mutated cases. No STAT3 mutations were discovered in HTLV-2 negative donors with amplicon sequencing. Of the 28 HTLV-2 positive cases, 19 had at least one somatic variant in CD8+ T cells based on the immunogene panel sequencing (n=28). 8 cases had variants in genes previously identified in T-LGLL (STAT3, KMT2D, TYRO3, DIDO1, BCL11B, CACNB2, KRAS, LRBA and FANCA), and 5 cases had variants in genes involved in JAK-STAT signaling (NFKBIA, PIK3R5, MAPK14, EP300, MPL, IFNAR1, IL6ST and IL20RA). Three recurrently mutated genes were detected: VWF, SMAD7 and MXRA5. The four HTLV-2 positive blood donors harboring STAT3 mutations had more somatic mutations (median=6) than HTLV-2 positive donors without STAT3 mutations (median=1, p=0.06). Conclusion: We report the presence of somatic gain-of-function STAT3 mutations in CD8+ T cells of 13% of HTLV-2 positive healthy blood donors. We identified additional somatic mutations in genes associated with JAK-STAT signaling, immune regulation and lymphoproliferation in CD8+ T cells of HTLV-2 positive cases. While STAT3 activation, with or without mutations, is considered as a hallmark of T-LGLL, our results reveal the presence of STAT3 mutations in CD8+ T cells of healthy blood donors harboring HTLV-2 without clinical history of lymphoproliferative disease. Additional research is warranted to elucidate whether HTLV-2 carriers harboring STAT3 and other mutations are at increased risk of subsequent T-LGL leukemia or other lymphoproliferative diseases. Disclosures Mustjoki: Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding; Janpix: Research Funding.

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.

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