Diversity of Intratumoral Regulatory T Cells in Non-Hodgkin Lymphoma

Abstract Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 + T cells, represent a key challenge in the tumor microenvironment by limiting potent antitumor immune responses. While high densities of tumor-infiltrating Tregs are associated with poor prognosis in patients with various types of solid cancers, their prognostic impact in B-cell non-Hodgkin lymphoma (NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotype and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Our in-depth characterization of Tregs in NHL tumors could open new paths for rational drug design, facilitating selective therapeutic manipulation of Tregs to reduce immunosuppression and improve anti-tumor immunity. Methods: Single-cell suspensions from NHL patients (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and healthy donors (tonsils and peripheral blood)) were analyzed by fluorescent flow- and mass cytometry to characterize Tregs, focusing on their expression of co-stimulatory and co-inhibitory checkpoint receptors. The immunosuppressive capacity of Tregs was measured by in vitro co-culture of FACS-sorted subsets of Tregs together with autologous CellTrace Violet-labelled T effector cells as responder cells, using samples from FL and tonsils. Live CD4 + T cells were obtained by FACS sorting from DLBCL (n = 3), FL (n = 3) and healthy donor tonsils (n = 3) and subjected to single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and scTCR-seq by the 10X Genomics platform. The computational framework of CIBERSORTx was used to generate unique signature matrices for the three Treg subsets identified by scRNA-seq, to facilitate validation in separate scRNA-seq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020), and to impute frequencies of the Treg subsets in cohorts with bulk RNA-seq data (Chapuy, Nat Med 2018; Schmitz, NEJM 2018; Pastore, Lancet Oncol 2015). Results: Immunophenotyping by mass cytometry revealed a subset of activated Tregs identified by co-expression of TIGIT, CTLA-4, PD-1, ICOS and OX40, and higher expression of FOXP3, CD25 and CD45RO, that was present in DLBCL and tonsils, but lacking in peripheral blood. This was validated by fluorescent flow cytometry, demonstrating significantly higher frequencies of activated Tregs in NHL tumors compared to PBMCs and tonsils from healthy donors. The phenotypic heterogeneity of intratumoral Tregs reflected different suppressive capacities as activated Tregs more potently suppressed the proliferation of autologous effector CD4 + and CD8 + T cells than naïve Tregs. For global transcriptomic profiling of CD4 + T cells from FL, DLBCL and tonsillar samples, we integrating scRNA-seq and CITE-seq data from 17,774 cells, revealing 13 distinct cellular states including three states of Tregs: naïve, activated and non-conventional LAG3 +FOXP3 - Tregs. Activated Tregs had higher expression of checkpoint receptors (TNFRSF4, TNFRSF18, ICOS), phosphatases (DUSP2, DUSP4), NF-κB pathway (NFKBIA, TNFAIP3, NFKBIZ, REL), chemokine receptors (CXCR4) and transcription factors (JUNB, IRF1, STAT3) as compared to naïve Tregs. We next used a computational approach to develop unique signature matrices for each Treg subset. This approach demonstrated strong concordance between CIBERSORTx estimated cell abundances of the three Treg subsets and the ground truth, and was validated in two external scRNA-seq cohorts. The development of unique signature matrices for Treg subsets facilitated imputation of their frequencies in bulk RNA-seq datasets. These analyses revealed that higher frequency of activated Tregs was enriched in the germinal B cell subtype of DLBCL and was associated with adverse outcome in FL. Conclusion: This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse and include highly immunosuppressive activated Tregs co-expressing several checkpoint receptors, which distinguish them from peripheral blood Tregs. Activated intratumoral Tregs could hamper clinical responses to checkpoint blockade, and identifying and targeting their vulnerabilities has the potential to improve anti-tumor immune responses. Disclosures Holte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Alizadeh: Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding.

Download Full-text

Activated Circulating T Follicular Helper Cells with Increased Function during Chronic Graft Versus Host Disease after Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v126.23.924.924 ◽

2015 ◽

Vol 126 (23) ◽

pp. 924-924

Author(s):

Edouard Forcade ◽

Haesook T. Kim ◽

Corey S Cutler ◽

Kathy S. Wang ◽

Vincent T Ho ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Proliferative Activity ◽

Research Funding ◽

Advisory Committees ◽

Follicular Helper ◽

Immune Pathology

Abstract T follicular helper cells (TFH) interact with B cells in the germinal center to support B cell proliferation, differentiation and immunoglobulin class switch. Previous studies have demonstrated that donor B cells contribute to the immune pathology of chronic GVHD (cGVHD) and studies in murine models of cGVHD have suggested that TFH play a central role in this process. Although TFH are located primarily within germinal centers of secondary lymphoid organs, TFH can also be found in the peripheral blood and are identified as a subset of memory CD4 T cells expressing CXCR5. Circulating TFH (cTFH) share functional and phenotypic characteristics of TFH including the ability to interact with B cells and provide helper signals. To examine the potential role of cTFH in the pathogenesis of cGVHD we monitored the reconstitution of cTFH (CD4+CD45RA-CXCR5+ T cells) in fresh blood samples obtained at different times after allogeneic HSCT. Recovery of cTFH mirrored the recovery of CD4 T cells and absolute numbers of cTFH gradually increased over time, reaching a plateau at 24 months post-HSCT in the range of normal values. Within the CD4 T cell population, the frequency of cTFH was low initially, but reached normal levels 6-9 months post-HSCT. Detailed phenotypic and functional analysis of cTFH was undertaken in a cohort of 66 adult patients (>9 months post-HSCT): 16 had no cGVHD, 12 had resolved cGVHD, and 38 had active cGVHD (mild = 15, moderate = 14, severe = 9). Results were compared to 22 healthy donors (HD). Overall, the frequency of cTFH was significantly reduced in HSCT patients compared to HD (median: 9.87 vs. 13.65 % of CD4+ T-cells, respectively, p =0.0003). cTFH frequency was lower in active cGVHD compared to no cGVHD patients (median: 9.44 vs. 11.65 % of CD4+ T-cells, p = 0.029); while cTFH in patients with resolved cGVHD was similar to patients without cGVHD (Figure 1). CXCL13 chemokine facilitates T-B interaction, thus promoting the GC reaction. CXCL13 levels were increased in active cGVHD compared to no cGVHD (137.7 pg/mL vs. 33.74 pg/mL, p < 10-4) suggesting that trafficking of cTFH to lymphoid organs was promoted in cGVHD. cTFH activation results in increased surface expression of ICOS and PD1 and increased proliferation. ICOShi PD1hi cTFH were increased in active cGVHD compared to no cGVHD (2.035 % vs 1.065 %, p = 0.016) in association with high proliferative activity (Ki67) (3.38 % vs. 2.31 % respectively, p = 0.0086). Functional cTFH subsets are also characterized by expression of CXCR3 and CCR6 as follows: Th1 (CXCR3+CCR6-), Th2 (CXCR3-CCR6-), Th17 (CXCR3-CCR6+). Within these subsets, Th2 and Th17 cTFH provide greater levels of B cell help measured by in vitro co-cultureassays (plasmablast generation and IgG production). Although not statistically significant, Th1 cTFH frequency was decreased during active cGVHD (33.95 vs 37.6 for no cGVHD) and Th2 and Th17 cTFH frequencies were increased in the moderate-severe cGVHD group (31.1 and 30.5, respectively) compared to no cGVHD (27.05 and 24.45, respectively). Within active cGVHD patients, the frequencies of Th1 and Th17 were associated with clinical grade of cGVHD (Th1: 40, 34, 19, p=0.008; Th17: 21, 26, 37, p=0.02; for mild, moderate, severe, respectively). We used the ratio (Th2+Th17)/Th1 cTFH to normalize B cell help capacity, and found a higher ratio in severe cGVHD compared to no cGVHD (3.59 vs 1.36, p = 0.0006). To measure cTFH functional activity, cTFH and naive B cells were purified from patient samples by cell sorting and co-cultured for 6 days. cTFH from patients with active cGVHD induced greater plasmablast generation (CD27hi CD38hi B cells) compared to cTFH from non-active cGVHD (Fig. 2). Increased functional activity of cTFH was confirmed when patient cTFH were purified and cocultured with naive B cells from a normal allogeneic donor (Fig. 2). In patients with cGVHD, there was also a significant correlation between the proliferative activity of cTFH and the generation of CD27hi CD38hi B cells (r = 0.48, p = 0.0029). These results indicate that active cGVHD is characterized by phenotypic and functional abnormalities of cTFH. In cGVHD, cTFH show high levels of activation, proliferation and switch toward Th2 and Th17 phenotype. These changes enhance cTFH function, promoting GC reactions and B cell differentiation. These data support the involvement of cTFH in the immune pathology of human cGVHD, suggesting the investigation of novel therapies targeting the GC reaction. Disclosures Armand: Merck: Consultancy, Research Funding; Infinity Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Research Funding. Antin:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Heterogeneity of Regulatory T Cells in B-Cell Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2020-142380 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Ivana Spasevska ◽

Sarah Josefsson ◽

Yngvild Nuvin Blaker ◽

Chloe B. Steen ◽

Ankush Sharma ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Single Cell ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Research Funding ◽

Median Frequency ◽

Non Hodgkin Lymphoma ◽

Healthy Donors ◽

Checkpoint Receptors

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 T cells, are enriched in B-cell non-Hodgkin lymphoma (NHL) and constitute a barrier to potent antitumor immune responses. Despite extensive studies, the significance of tumor-infiltrating Tregs on disease outcome is unclear and while Tregs may express co-inhibitory and co-stimulatory receptors, the role of intratumoral Tregs in the context of immune checkpoint therapy remains elusive. Emerging evidence suggests heterogeneity among Tregs and their suppressive capacities in cancer, emphasizing the need for additional markers to identify highly suppressive Tregs. Therefore, an in-depth characterization of Treg heterogeneity in NHL could provide important insight into the disease pathogenesis and have implications for rational drug design. Methods: Expression of checkpoint receptors in Tregs was characterized by fluorescence flow cytometry and mass cytometry analysis of single-cell suspensions from diffuse large B-cell lymphoma (DLBCL; n = 16), follicular lymphoma (FL; n = 8), mantle cell lymphoma (MCL; n = 10), marginal zone lymphoma (MZL; n = 2), chronic lymphocytic lymphoma (CLL; n = 7), as well as tonsils (n = 8) and peripheral blood (n = 4) from healthy donors. Functional characterization of intratumoral Tregs was performed by a proliferation assay using FACS-sorted Tregs as suppressor cells and autologous CellTrace Violet-labelled T effector cells as responder cells. Single-cell RNA sequencing (scRNA-seq) was performed on FACS-sorted CD4 T cells from 3 DLBCL, 3 FL and 3 healthy donor tonsils using the 10X Genomics single cell 5' based library construction and VDJ libraries for TCR-sequencing. Additionally, for simultaneous profiling of phenotypic features with the mRNA expression in single cells, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) was applied. The Treg compartment was characterized by clustering into distinct transcriptional Treg states and differential expression of marker genes. Results: TIGIT and CTLA-4 were identified as common markers of intratumoral Tregs, in addition to FOXP3 and CD25. Unsupervised computational analysis revealed two distinct Treg subsets, based on contrasting expression of PD-1, OX40, CD226 and ICOS (Figure 1A). One subset displayed a checkpoint receptorlow phenotype that corresponded to peripheral blood Tregs. The second subset had a checkpoint receptorhigh phenotype with elevated levels of PD-1, OX40, ICOS, TIGIT, CTLA-4 and increased levels of activation markers CD28, CD69 and CD95/Fas. The frequency of checkpoint receptorhigh Tregs was significantly increased in NHL tumors, compared to PBMCs and tonsils from healthy donors. FL tumors had the highest frequency of Tregs with receptorhigh phenotype among the NHL entities (median frequency of 86%, range 71-92%) and DLBCL had the highest donor-to-donor variation (median frequency of 77%, range 35-98%) (Figure 1B). This phenotypic heterogeneity of the Treg compartment reflected different suppressive capacities of the two subsets. Checkpoint receptorhigh Tregs were more potent mediators of immunosuppression in terms of suppressing the proliferation of autologous effector CD4 and CD8 T cells (Figure 1C). Furthermore, transcriptomic analysis of CD4 T cells by scRNA-seq and CITEseq revealed distinct transcriptomic signatures of the checkpoint receptorhigh and -receptorlow subsets. In addition, a third subset of Tregs, characterized by increased expression of LAG3 and immunosuppression-associated genes (CTLA-4, IL10, CD38, KLRB1) but lack of FOXP3, was identified (Figure 1D-E). Analysis of scTCR-sequences to compare TCR repertoires and to identify developmental trajectories will further add to our knowledge of intratumoral Tregs. Conclusions: These results reveal heterogeneity within the Treg compartment in NHL based on expression of checkpoint receptors, transcriptional profiles and suppressive capacities. As intratumoral Treg phenotypes differ from peripheral blood Tregs, this presents new therapeutic opportunities. Specific targeting of intratumoral Tregs would lead to stronger antitumor effects while limiting immune-related adverse events. A deeper understanding of Treg heterogeneity within the tumor microenvironment could therefore open new paths for rational design of immune checkpoint therapy. Disclosures Kolstad: Merck: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

Download Full-text

Site to Site Comparison of Follicular Lymphoma Biopsies By Single Cell RNA Sequencing

Blood ◽

10.1182/blood-2019-129445 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 297-297 ◽

Cited By ~ 4

Author(s):

Sarah Haebe ◽

Tanaya Shree ◽

Anuja Sathe ◽

Grady Day ◽

HoJoon Lee ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Single Cell ◽

Board Of Directors ◽

Rna Sequencing ◽

Peripheral Blood ◽

Advisory Committees ◽

Blood Samples ◽

Single Cell Rna Sequencing

Follicular lymphoma (FL) originates from a single B cell that has rearranged one copy of its BCL2 gene on chromosome 18 to the Ig locus on chromosome 14 and in addition has acquired a mutation in a histone modifying gene such as CREBBP or KMTD2. By the time the disease is diagnosed the progeny of this original cell harbors additional mutations and is usually found at multiple lymphoid sites throughout the body. At each of these sites the malignant cells are accompanied by a rich network of follicular dendritic cells, T cells and other immune cells. This tumor microenvironment (TME) is clearly an important feature of the biology of FL and can impact the clinical behavior of the disease (Dave et al., NEJM, 2004). It remains unknown whether tumor clonal heterogeneity and the composition of the TME differ between various lymphoma sites within the same patient. Single cell RNA sequencing facilitates a detailed and unbiased view of both the tumor clone and the complex TME. To profile the TME and explore FL tumor evolution, we obtained fine needle aspirates (FNAs) at 2 different sites in the body and peripheral blood specimens all on the same day and subjected these samples to single cell RNA sequencing and immune repertoire analysis. These biopsies were taken prior to therapy from patients entering immunotherapy clinical trials (NCT02927964, NCT03410901). Single cell RNA sequencing of FNA and blood samples was performed using the 10X Genomics platform to an average targeted depth of 50,000 reads/cell. We have prepared sequencing libraries from 15 tumor FNA and peripheral blood samples from 5 patients thus far. Typically, 3,000-10,000 cells have been sequenced per sample, with excellent sequencing quality metrics. By applying Uniform Manifold Approximation and Projection (UMAP), a dimensionality reduction algorithm, we found the TME of these FL patients to be richly populated by many phenotypically discrete non-malignant cells, including many subpopulations of T cells, B-cells, myeloid cells, NK cells and dendritic cells. Evaluating the combined dataset containing all tumor samples for all 5 patients, we found that malignant B cells from different patients clearly clustered apart from each other, a feature not dependent on immunoglobulin clonality or HLA type. Each patient's tumor population contained 3-5 distinct subpopulations, presumably a result of multiclonal tumor evolution. Nonetheless, we were able to define several malignant B-cell sub-phenotypes common to all patients. Intriguingly, compared to malignant B cells, infiltrating non-malignant B cells showed higher MHC I expression, activation markers, and an enrichment in interferon-induced genes. Of note, we could also detect circulating tumor cells in peripheral blood samples of several patients, and these exhibited a distinct gene expression profile compared to their counterparts within lymph nodes. Analysis of the diverse T cell subpopulations within tumors revealed distinct functional states. For example, in regulatory and T follicular helper cells, we identified activated clusters (CD27, BATF, TNFRSF4) and putative resting clusters (SELL, KLF2, IL7R), while effector T cells resided in separate cytotoxic (GZMA, GZMB, GNLY) and exhausted (TIGIT, CXCL13, LAG3) clusters. Tumor B cell gene expression and composition of the TME from site to site within the same patient were similar in some cases and remarkably divergent in others. For example, we detected a significant upregulation of interferon signaling pathways in the tumor B cells and an enrichment of effector T cells in only one of the two sites within one patient. Analysis of B cell and T cell antigen receptor sequences to evaluate tumor subclonality and TCR clonotype diversity are ongoing. To the best of our knowledge, this is the first study to compare different sites of FL in the same patients at the single cell level. Our analyses characterize inter- and intra-patient heterogeneity in malignant and immune cell subsets and provide a baseline for eventual comparison of alterations occurring over time as these patients receive experimental immunotherapy interventions. Disclosures Levy: XCella: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Five Prime: Membership on an entity's Board of Directors or advisory committees; Corvus: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Nohla: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Reprogramming Aberrant B Cell-Subsets to Improve Immune Function in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.3986.3986 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3986-3986

Author(s):

Rao H. Prabhala ◽

Andreea Negroiu ◽

Saem Lee ◽

Mariateresa Fulciniti ◽

Puru Nanjappa ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Advisory Committees ◽

Healthy Donors ◽

Ifn Γ ◽

B Cell Subsets

Abstract Abstract 3986 B cell-malignancies exhibit considerable immune dysfunction particularly in multiple myeloma (MM). We have previously demonstrated that in T cell-compartment, regulatory T helper cells are dysfunctional in multiple myeloma (MM) while Th17 cells are significantly elevated and IL-17 produced by them is associated with MM cell growth and survival as well as suppressed immune responses and bone disease. We have here investigated the B cell-subsets and their ability to re-program anti-tumor immunity in MM. We have first characterised four different B cell-subsets (B1a, B1b, B2 and regulatory B cells) using 10-color flow cytometric analysis in both peripheral blood and bone-marrow (BM) samples from MM patients compared with normal healthy donors. We observe that CD5+ B1a-B cells are significantly elevated in peripheral blood of MM patients (N=7) compared to healthy donor (N=15) (42±8% vs 13±3%, respectively, p<0.05); while normal B cells (B2 cells) are significantly reduced in peripheral blood (29.8±6.5, p<0.05) and in the BM samples (11±4.8, N=4, p<0.05) of MM patients compared to healthy donors (59±3, and 60.2±2, N=10, respectively). We also observed that both B1b (47.9±18 vs. 22.8±4) and regulatory B cells (7.1±4.5 vs. 1.54±0.3) are elevated in BM samples of MM compared to healthy donors, however there were no differences in B1b and regulatory B cells in the peripheral blood of MM compared to healthy donor samples. Interestingly, in myeloma we observe higher levels of activated B cell subsets but lower levels of memory B cell subsets compared to healthy donors. These results, particularly very low levels of normal B cells in MM patients, may explain the decreased levels of uninvolved immunoglobulin in MM. As removal of B cell population has been shown to re-program T helper cell populations, we next investigated impact of B cell population on T cell activation. We activated normal PBMC via the anti-CD3 antibody, in the presence or absence of B or CD25+ cells and measured intra-cellular IFN-γ levels in CD69+ cells. We found that the absence of B cells significantly inhibited interferon-producing T cells compared to PBMC (by 43%; p<0.05). Importantly, following removal of CD25+ cells, which consists of both Tregs and activated memory T cells, with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells are influencing memory T cells rather than naïve T cells for the production of IFN-γ. This prompted us to identify the phenotypic signature of regulatory T cell populations when purified memory T cells are polarized with the regulatory T cell cocktail in presence or absence of B cells. We observed that B cells reduce FOXP3 expression by 18 %(N=5) and establish cognitive interactions with T cells. This occurred by increasing the expression of GITR (154%) and CTLA4 (54%); while reducing PD1 (−24%) and OX40 (−21%) expression on T cells without affecting HLA expression. We have also observed these improvements by B cell modulation on T cells in MM. Our results indicate that targeting these re-programmable capabilities of B cells to modulate T helper cell populations may enable us to improve T cell function in MM; and may improve immune function in MM and also allow us to enhance responses to vaccinations. Disclosures: Ghobrial: Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Download Full-text

Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽

10.1182/blood-2020-138601 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-43

Author(s):

Prajish Iyer ◽

Lu Yang ◽

Zhi-Zhang Yang ◽

Charla R. Secreto ◽

Sutapa Sinha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Gene Signature ◽

T Cell Subsets ◽

Rna Seq ◽

Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

Download Full-text

Bleeding Complications in Patients with Cancer and COVID 19- Analysis from the COVID 19and Cancer Consortium (CCC19) Registry

Blood ◽

10.1182/blood-2021-154314 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4997-4997

Author(s):

Surbhi Shah ◽

Shuchi Gulati ◽

Ang Li ◽

Julie Fu ◽

Vaibhav Kumar ◽

...

Keyword(s):

Mechanical Ventilation ◽

Board Of Directors ◽

Stock Options ◽

Research Funding ◽

Tumor Board ◽

Bleeding Complications ◽

Advisory Committees ◽

Icu Admission ◽

Patients With Cancer ◽

Privately Held

Abstract Background : Patients (pts) with COVID-19 are reported to have increased risk of venous thromboembolism yet bleeding has been an under recognized complication. Rates of bleeding remain unexamined in all patients especially in pts with cancer and COVID-19. Aim: To estimate the incidence of bleeding complication in patients with cancer and COVID 19 Methods: The CCC19 international registry (NCT04354701) aims to investigate complications of COVID-19 in pts with cancer. Our aim was to investigate the frequency of bleeding in hospitalized adult pts with cancer andCOVID-19, enrolled between March 16, 2020 and Feb 8, 2021. The incidence of bleeding complications was captured as defined by CCC19 and included both major and non major bleeding . Associated baseline clinic-pathologic prognostic factors and outcomes such as need for mechanical ventilation, intensive care unit (ICU) admission and mortality rates were assessed Results :3849 pts met analysis inclusion criteria. Bleeding was reported in 276 (7%) pts with median age of 70years; incidence was 6.6 % in females and 7.6 % in males, 6.5% in non-Hispanic white pts, 8.2 % in non-Hispanic Black pts, and 7.8 % in Hispanic pts. 74% had solid cancer and 29% had hematologic malignancies, 33% had received anti-cancer therapy in preceding 30 days, and 8% had surgery within 4weeks. In pts taking antiplatelet or anticoagulant medications at baseline, 7.2% developed bleeding. Need for mechanical ventilation, ICU admission, 30-day mortality, and total mortality were significantly higher in those with bleeding complications compared to those without, p<0.05 Conclusion : We describe the incidence of bleeding in a large cohort of pts with cancer and COVID-19. Bleeding events were observed in those with adverse outcomes including mechanical ventilation, ICU admission, and high mortality; the overall mortality of 43% in patients with bleeding complications is especially notable. This important complication may reflect underlying COVID-19 pathophysiology as well as iatrogenic causes. Figure 1 Figure 1. Disclosures Kumar: Diagnostica Stago: Honoraria. Zon: AMAGMA AND RLZ: Consultancy, Current holder of individual stocks in a privately-held company. Byeff: Pfizer, BMS, Takeda,Teva, Merck, United health: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Nagaraj: Novartis: Research Funding. Hwang: astrazaneca,Merck,bayer, Genentech: Consultancy, Research Funding. McKay: Myovant: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Calithera: Membership on an entity's Board of Directors or advisory committees; Tempus: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tempus: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Dendreon: Consultancy; Caris: Other: Serves as a molecular tumor board ; Vividion: Consultancy; Sorrento Therapeutics: Consultancy; Bayer: Research Funding. Warner: Westat, Hemonc.org: Consultancy, Current holder of stock options in a privately-held company. Connors: Pfizer: Honoraria; CSL Behring: Research Funding; Alnylam: Consultancy; Bristol-Myers Squibb: Honoraria; takeda: Honoraria; Abbott: Consultancy. Rosovsky: Janssen: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Inari: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽

10.1182/blood.v116.21.2431.2431 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2431-2431

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Candida Vitale ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Board Of Directors ◽

Stromal Cells ◽

Peripheral Blood ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

Download Full-text

Cellular Immune Responses To Vector In a Gene Therapy Trial For Hemophilia B Using An AAV8 Self-Complementary Factor IX Vector

Blood ◽

10.1182/blood.v122.21.717.717 ◽

2013 ◽

Vol 122 (21) ◽

pp. 717-717

Author(s):

Etiena Basner-Tschakarjan ◽

Federico Mingozzi ◽

Yifeng Chen ◽

Amit Nathwani ◽

Edward Tuddenham ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Factor Ix ◽

High Dose ◽

Advisory Committees ◽

Ifn Γ ◽

Cellular Immune

Abstract In a clinical study of gene transfer for hemophilia B an adeno-associated virus vector serotype 8 (AAV8) expressing a self-complementary liver-specific expression cassette for the factor IX (FIX) transgene was administered intravenously in ten affected subjects. The results of the first part of the study have been published (NEJM 365:2357-65, 2011). In this abstract we present the immunomonitoring data, using Interferon-gamma (IFN-γ) ELISpot and polyfunctional T cell analysis of peripheral blood mononuclear cells (PBMCs) to monitor cellular immune responses to vector capsid and to Factor IX. We have previously shown that the cellular immune response was directed solely towards AAV capsid epitopes, not FIX, and that the response was dose-dependent. Out of six subjects infused in the high dose cohort (2x1012vg/kg), 4/6 manifested a minor rise in liver enzyme levels and detection of capsid-specific T cell reactivitiy in the ELISpot assay at ∼7-10 weeks post vector infusion. Maximum results on IFN- γ ELISpots ranged from 200-500 sfu/million cells. In two of these cases a modest decline in FIX level also occurred. Prompt initiation of prednisolone reversed these effects and rescued FIX levels. The remaining two subjects infused at the high dose, showed no rise in liver enzyme levels at any time point. However capsid reactive T cells were detectable in one subject as early as one to two weeks after vector infusion in peripheral blood by IFN-γ ELISpot assay, while no activation at all was detected in the other subject, possibly due to low cell recovery and viability of the cells. A similar immune response profile, with early detection of activated T cells but no rise in liver enzymes, was also observed in both subjects in the intermediate dose cohort in the first part of this study. Polyfunctional T cell analysis revealed concurrent Interleukin-2, Tumor necrosis factor-alpha and CD107a positivity in activated T cells at the peak of activation. Furthermore it showed that capsid-specific early T cell responses were detectable in the CD4+ T cell and later in the CD8+T cell compartment. Long-term immune monitoring of all subjects is ongoing. Importantly in one of the first two subjects treated at the high dose, capsid reactive T cells were detected by ELISpot 1.5 years after gene transfer; these cells were not detected in the other subject in whom long-term follow-up samples are available. Of note, capsid-reactive T cells were also seen at late time points (>1 year after infusion) in a middle dose subject and a low dose subject. Despite detectable T cell reactivity towards the AAV capsid in the peripheral blood FIX expression remained stable, suggesting that there is a short window of time during which transduced hepatocytes present a target for cytotoxic T cells, and that T cell positivity after this window is without any clinical consequences. In conclusion, for this scAAV8 vector there appears to be a critical threshold vector dose for a clinically detectable immune response, starting at 2x1012 vg/kg. The clinically detectable response occurred in four out of six subjects so far, and was manifest within a critical time interval of 7-10 weeks post infusion. The capsid-specific response was polyfunctional and detected in CD4+ and CD8+T cells in peripheral blood. It is important to note that not all subjects treated at the high dose developed an immune response. However, given the limited dataset, it is not yet possible to define predictive parameters, e.g. HLA type of a subject, for an immune response. Continued monitoring and future studies with more subjects will be necessary to confirm the presented findings, in particular time and rate of occurrence of a cellular response as well as successful treatment with a short course of Prednisolon. Disclosures: Tuddenham: Pfizer: Consultancy. Reiss:Hemophilia of Georgia: Honoraria. High:BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Download Full-text

CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

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.

Download Full-text

Targeting BTN2A1 By a Unique Activating Mab Improves Vγ9Vδ2 T Cell Cytotoxicity Against Primary Acute Lymphoblastic Blasts

Blood ◽

10.1182/blood-2021-152577 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2302-2302

Author(s):

Anne-Charlotte Le Floch ◽

Caroline Imbert ◽

Aude De Gassart ◽

Florence Orlanducci ◽

Aude Le Roy ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Leukemic Cells ◽

Target Cells ◽

Advisory Committees ◽

Vγ9vδ2 T Cells ◽

Privately Held

Abstract Introduction Vγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study). Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1. Material and methods 5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays. We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14. We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts. DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR. Results We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells. We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells. Discussion Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. Disclosures De Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

Download Full-text