Strong Expression of the Immune Checkpoint Regulators LAG3 and Tim3 in Hodgkin Lymphoma

Abstract Background: Recent results of immune checkpoint blockade trials have provided a proof of concept for immunotherapy in classical Hodgkin lymphoma (cHL) with more than two third of relapsed/refractory patients responding to blockade of the PD1/PDL1 axis. Unfortunately, there is still a proportion of patients who will present primary or secondary resistance to immunotherapy. Besides the PD1/PDL1 axis, several other molecules are critical regulators of the immune response and may be the target of therapeutic intervention. Combined immune checkpoint targeting has shown interesting results in preclinical and clinical trials in several types of tumors. Methods: Patients with initially diagnosed or relapsed cHL for whom formalin fixed paraffin embedded (FFPE) tissue was available at our institution were identified. Fifty-seven cases were selected depending solely on the availability and the quality of the FFPE blocks. Expression of the following immune checkpoints PD1, PDL1, LAG3, TIM3 was assessed using immunohistochemical methods with a threshold of 5% set for positivity. Results: Complete results for 25 cases were available at the time the abstract was written. Hodgkin and Reed Sternberg cells (HRS) were identified morphologically upon microscopic examination. Consistently with data published in the literature, HRS stained positively and intensely for PDL1 in 100% of the cases (25/25). HRS were positive for Tim3 in 36% (9/25) of cases but with more varying intensities. No PD1 or LAG3 expression was found on HRS cells except for a single case where 5% of HRS stained weakly for LAG3. In the tumor microenvironment, PD1 expression was detected in 65% of cases (15/23) and PDL1 in 60% of cases (15/25). Impressively, LAG3 and TIM3 stained positively in 96% (23/24) and 92% (24/25) of cases respectively. Lymphocyte-rosetting was present in 9/25 cases. These CD4+ FoxP3- T cells surrounding HRS were positive for PD1 in 5 cases, for LAG3 in 2 cases and for both PD1 and LAG3 in 2 cases, suggesting they represented exhausted T-cells. Concomitant expression of PD1 and PDL1 in the tumor microenvironment was present in 43% of cases (10/23). Conclusion: LAG3 and TIM3 are nearly universally expressed in the tumor microenvironment of cHL. These findings provide a strong rationale for their blockade alone or in combination in relapsed/refractory patients with cHL. The role of TIM3 expression by HRS remains unclear. Correlation of these findings with clinical data and survival outcome of the patients will be done for the whole sample. Disclosures Ribrag: NanoString: Membership on an entity's Board of Directors or advisory committees; Esai: Membership on an entity's Board of Directors or advisory committees; ArgenX: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

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The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

Blood ◽

10.1182/blood-2021-145840 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3513-3513

Author(s):

Jay Gunawardana ◽

Muhammed B. Sabdia ◽

Karolina Bednarska ◽

Soi C. Law ◽

Sandra Brosda ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Immune Checkpoint ◽

Research Funding ◽

Immune Checkpoint Blockade ◽

Checkpoint Blockade ◽

Immune Checkpoints ◽

Advisory Committees ◽

Tcr Repertoire

Abstract Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) comprises 5% of all Hodgkin lymphomas (HL). Its biology remains poorly characterized. Like classical HL (cHL), it contains minimal malignant cells embedded within a T cell rich intra-tumoral microenvironment (TME). Unlike cHL, it can transform to diffuse large B cell lymphoma (DLBCL). Immune-checkpoint blockade is effective in cHL but has minimal activity in DLBCL. No data is currently available regarding the potential to reactivate host anti-tumoral activity via immune-checkpoint blockade in NLPHL. Diagnostic FFPE samples from 49 NLPHL patients retrospectively collected from 4 Australian centres were interrogated. Inclusion criteria were sample availability and centrally confirmed histological NLPHL. Characteristics were in line with the literature: median age 45 years, range 13-82 years; F:M 1:3.5; stage I/II 55%, III/IV 35% (10% stage unknown) with the majority of cases were of immuno-architectural types A or C. RNA was digitally quantified using the NanoString 770-gene PanCancer Immune panel. Multi-spectral immunofluorescent (mIF) microscopy, plasma soluble PD-1 quantification, cell sorting, T cell receptor (TCR) repertoire analysis and functional immuno-assays were also performed. Results were compared with samples from 38 cHL and 35 DLBCL patients. We initially compared gene expression of NLPHL and cHL, looking for molecular similarities and differences. Ten non-lymphomatous nodes (NLN) were included as controls. Unsupervised clustering showed all but 3 NLPHL cases segregated from the cHL cluster. All NLN congregated in a discrete sub-cluster. As expected, RNA analysis showed significant enrichment for CD20 in NLPHL and CD30 in HL. Volcano plots (Fig. 1a), corrected for false-discovery showed marked variation in gene expression. For NLPHL (vs. cHL) there were 105 upregulated and 337 down regulated genes. Strikingly, the most significantly differentially over-expressed genes in NLPHL were all T cell related (CD247: CD3 zeta chain; CD3D: CD3 delta chain; GZMK: granzyme K; EOMES: marker of CD8 + T cell tolerance; and the immune checkpoints PDCD1: encodes for PD-1; and TIGIT). CD8B expression was increased in NLPHL. For cHL, the most over-expressed genes included macrophage-derived chemokines CCL17 and CCL22. Gene set enrichment analysis revealed activation of the PD-L1 expression and PD-1 checkpoint pathway and 9 of the top 10 Gene Ontology (GO) term enrichment scores involved lymphocyte signalling in NLPHL (Fig. 1b). To better appreciate the impact of the relevant immune checkpoints on their signalling axis, we compared gene ratios for PD-1 and TIGIT receptors with their ligands (PD-L1/L2 and PVR, respectively). NLPHL showed the highest enrichment ratios of these signalling pathways vs. cHL, DLBCL and NLN (Fig. 1c). Although it is known that CD4 +PD-1 +T cells form rosettes around NLPHL cells, the differential cellular localization of immune proteins has not been compared between HL entities. Using mIF, the proportion of intra-tumoral PD-1 + was markedly higher for CD4 + (~7-fold; p<0.0001) and CD8 + (~5-fold; p<0.001) T cells in NLPHL. However, the proportion of T cells expressing LAG3 was similar. Soluble PD-1 was elevated for both NLPHL and cHL, indicating circulating blood is influenced by the TME. For both HL entities over 80% of circulating CD4 + and CD8 + T cells expressed PD-1 alone or in combination with TIGIT. TCR repertoire analysis of sorted T cell subsets showed large intra-tumoral clonal T cell expansions were also detectable in circulating T cells. T cell clones were predominantly PD1 +CD4 + T cells in both HL types. Finally, we developed a functional assay using PD-L1/PD-L2 expressing NLPHL and cHL cell lines. These were co-cultured with genetically engineered PD-1 +CD4 + T cells that express a luciferase reporter. Similar levels of heightened T cell activation were seen with immune-checkpoint blockade for both HL entities, indicating that immune-checkpoint inhibition may also be of benefit in NLPHL. In conclusion, our multi-faceted analysis of the immunobiological features of the TME in NLPHL, provides a compelling rationale for early phase clinical studies that incorporate immune-checkpoint blockade in NLPHL. Figure 1 Figure 1. Disclosures Hawkes: Bristol Myers Squib/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Specialised Therapeutics: Consultancy; Merck KgA: Research Funding; Merck Sharpe Dohme: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Antigene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Speakers Bureau; Janssen: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accommodation expenses, Research Funding, Speakers Bureau. Swain: Janssen: Other: Travel expenses paid; Novartis: Other: Travel expenses paid. Keane: BMS: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Karyopharm: Consultancy; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Talaulikar: Takeda: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Jansenn: Honoraria, Research Funding; Roche: Honoraria, Research Funding; EUSA Pharma: Honoraria, Research Funding. Gandhi: janssen: Research Funding; novartis: Honoraria.

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The Tumor Microenvironment of Nodular Lymphocyte Predominant Hodgkin Lymphoma Is a Unique Immunobiological Entity Distinct from Classical Hodgkin Lymphoma

Blood ◽

10.1182/blood-2018-99-115836 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4123-4123

Author(s):

Jay Gunawardana ◽

Karolina Bednarska ◽

Soi C Law ◽

Justina Lee ◽

Muhammed Bilal Sabdia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Hodgkin Lymphoma ◽

Research Funding ◽

Host Immunity ◽

Immune Checkpoints ◽

Classical Hodgkin Lymphoma ◽

Advisory Committees ◽

Immune Effector

Abstract There is proven pre-clinical and clinical efficacy of mono or combinatorial immune strategies to boost host anti-lymphoma immunity, with classical Hodgkin Lymphoma (cHL) seen as the 'poster child'. Approaches include blockade of immune-checkpoints on exhausted tumor-specific T-cells (via mAb blockade of PD-1, TIM3, LAG3, TIGIT or their ligands), activation of T-cells via mAbs agonistic to CD137, and finally modulation of FOXP3, CTLA-4 and/or LAG3 regulatory T-cells (Tregs) or immunosuppressive tumor-associated macrophages (TAMs). In contrast, studies characterizing the circulating and intra-tumoral microenvironment (TME) of the distinct but rare CD20+ Hodgkin Lymphoma entity (5-8% of HL), Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL), are minimal. Furthermore, to our knowledge no functional profiling studies comparing the host immunity of NLPHL with cHL has been performed. We compared host immunity in 29 NLPHL patients, 30 cHL patients and 10 healthy individuals, with a focus on pertinent and clinically actionable immune parameters. Paraffin-embedded tissue and paired (pre- and post-therapy) peripheral blood mononuclear cells samples were interrogated by digital multiplex hybridization (Nanostring Cancer Immune Profiling Panel) and flow cytometry. Although cytotoxic T-cell gene counts (CD8a, CD8b) were similar, compared to cHL there were higher levels of the immune effector activation marker CD137 (gene counts 439 vs. 287; P<0.01). Consistent with this, CD4 and the Treg markers LAG3, FOXP3 and CTLA-4 were lower in NLPHL (2-4 fold lower, all P<0.05), with no difference in T-helper cell activation markers CD40L and CD30L seen between tumors. TAMs and dendritic cell markers MARCO, CD36, CD68, CD163, COLEC12 and CD11b were all lower in NLPHL than cHL (all P<0.05). In line with the known 'rossette' formed around LP cells by PD-1+ T-lymphocytes, we observed strikingly elevated PD-1 and the other T-cell checkpoints TIM3 and TIGIT in NLPHL (all 2-3 fold, P<0.001). However, in line with the known gene amplification of PD-L1 on HRS cells and its presence on TAMs, gene counts of this checkpoint ligand were 2-fold higher in cHL (P<0.001). Flow cytometry profiling of immune subsets in peripheral blood showed findings consistent with findings in the TME. Specifically, there was elevation of multiple exhaustion markers within CD4, CD8, and NK immune effector cells, with a striking proportion of highly anergic dual-LAG3/PD-1 positive CD8+ T-cells. Also there was elevation of immune-suppressive monocyte/macrophages in cHL relative to NLPHL. Relative to healthy lymph nodes, there was prominent up-regulation of a range of T-cell associated exhaustion markers in both NLPHL and cHL, indicating dysregulated priming of effector immune responses and host immune homeostasis. Comparison between NLPHL and cHL illustrated that NLPHL had a myriad of features that marked its intratumoral TME as a unique immunobiological entity typified by elevated immune checkpoint markers and T-cells with a highly anergic phenotype. Put together, these findings indicate that distinct immune evasion mechanisms are operative within the TME of NLPHL, including markedly higher levels of multiple immune-checkpoints relative to cHL. In contrast, Treg subsets and immune-suppressive monocyte/macrophages were relatively lower than that seen in cHL. T-cells frequently had dual immune-checkpoint expression. The findings from this study provides a compelling pre-clinical rationale for targeting PD-1 or combinatory checkpoint inhibition in NLPHL and sets the basis for future 'chemo-free' rituximab + checkpoint inhibitor clinical trials. Disclosures Tobin: Amgen: Other: Educational Travel; Celgene: Research Funding. Birch:Medadvance: Equity Ownership. Keane:Takeda: Other: Educational Meeting; BMS: Research Funding; Roche: Other: Education Support, Speakers Bureau; Celgene: Consultancy, Research Funding; Merck: Consultancy. Gandhi:BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Takeda: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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A Complicated Neighborhood: Insights into the Hodgkin Lymphoma Microenvironment

Blood ◽

10.1182/blood-2019-121284 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. SCI-8-SCI-8

Author(s):

Maher K. Gandhi

Keyword(s):

Board Of Directors ◽

Hodgkin Lymphoma ◽

Immune Checkpoint ◽

Research Funding ◽

Immune Checkpoints ◽

Biological Entity ◽

Advisory Committees ◽

Term Outcome ◽

Viral Oncoprotein ◽

Long Term Outcome

Classical Hodgkin Lymphoma (cHL) is a heterogenous and complex biological entity. Whereas early studies focussed on characterization of the Reed-Sternberg (RS) cell, there is now increasing recognition of the importance of the tumor microenvironment (TME). This critical but only partially understood component of cHL biology is likely to impact pathogenesis, chemo-sensitivity and long-term outcome. Here, the non-malignant infiltrate is variably comprised of macrophages, regulatory T cells, and stroma, with the relative composition varying by histological sub-type. Notably, relative to other B cell lymphomas, cHL expresses high levels of immune checkpoint receptors such as PD-1 and LAG3, and immune checkpoint ligands such as PD-L1, PD-L2, the latter of which are frequently the subject of genetic amplifications. Hence the bi-directional relationship between the microenvironment and the malignant cell is now a valid target. Breakthroughs in our understanding of the TME in cHL have contributed to its status as the 'poster-child' for how checkpoint blockade can de-activate tumor-tolerance to induce meaningful clinical benefit. A further layer of complexity within the TME is the potential aetiological relationship between cHL and the Epstein-Barr virus, a ubiquitous virus found to reside within the RS cells in 40% of cases, particularly in those cases that have mixed cellularity. Unlike benign EBV-infected B cells, within RS cells the virus is in a highly aberrant latency state with expression of the viral oncoprotein LMP1, a virus that is known to induce immunosuppression and drive PD-L1 expression through the NFkB and the JAK-STAT pathways. But numerous unanswered mechanistic questions remain, not least in the light of the frequent genetically driven deficiencies in antigen presentation present in RS cells, particularly in cases of nodular sclerosing disease. This has reignited debate about the relative roles of adaptive and innate immunity in this disease. Remaining questions that need to be addressed include the evolution of the interaction between the TME and the malignant compartment during the course of cHL, the role of other immune checkpoints and their impact on combinatorial immune based strategies, and the contribution to immune-evasion played by stromal cells. Further understanding will assist the rational development of new immune-based strategies, and potentially one day a chemo-free regimen that is as clinically efficacious but less toxic than current chemotherapies. Disclosures Gandhi: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

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836 Releasing the restraints of Vγ9Vδ2 T-cells in cancer immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0836 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A888-A888

Author(s):

Laura Ridgley ◽

Angus Dalgleish ◽

Mark Bodman-Smith

Keyword(s):

T Cells ◽

Cancer Immunotherapy ◽

Tumour Cell ◽

Immune Checkpoint ◽

Expansion Method ◽

Immune Checkpoint Blockade ◽

Immune Checkpoints ◽

Inhibitory Receptors ◽

Healthy Donors ◽

Vγ9vδ2 T Cells

BackgroundVγ9Vδ2 T-cells are a subset of cells with a crucial role in immunosurveillance which can be activated and expanded by multiple means to stimulate effector responses, often exploited in cancer immunotherapy. Little is known about the expression of checkpoint molecules on this cell population and whether the ligation of these molecules can regulate their activity. The aim of this study was to assess the expression of activatory and inhibitory markers on Vγ9Vδ2 T-cells to assess potential avenues of regulation to target with immunotherapy.MethodsPBMCs were isolated from healthy donors and the expression of activatory and inhibitory receptors was assessed on Vγ9Vδ2 T-cells by flow cytometry at baseline, following 24 hours activation and 14 days expansion using zoledronic acid (ZA) and Bacillus Calmette-Guerin (BCG), both with IL-2. Activation and expansion of Vδ2 cells was assessed by expression of CD69 and by frequency of Vδ2 cells, respectively. Production of effector molecules was also assessed following coculture with various tumour cell targets. The effect of immune checkpoint blockade on Vγ9Vδ2 T-cells was also assessed.ResultsVγ9Vδ2 T-cells constitutively expressed high levels of NK-associated activatory markers NKG2D and DNAM1 which remained high following stimulation with ZA and BCG. Vγ9Vδ2 T-cells expressed variable levels of checkpoint inhibitor molecules at baseline with high levels of BTLA, KLRG1 and NKG2A and intermediate levels of PD1, TIGIT and VISTA. Expression of checkpoint receptors were modulated following activation and expansion with ZA and BCG with decreased expression of BTLA and upregulation of numerous markers including PD1, TIGIT, TIM3, LAG3 and VISTA. Expression of these markers is further modulated upon coculture with tumour cell lines with changes reflecting activation of these cells with Vγ9Vδ2 T-cells expressing inhibitory receptors PD1 and NKG2A producing the highest level of TNF.ConclusionsOur data reveals unique characteristics of Vδ2 in terms of their expression of immune checkpoints, which provide a mechanism which may be utilised by tumour cells to subvert Vγ9Vδ2 T-cell cytotoxicity. Our work suggests different profiles of immune checkpoints dependent on the method of stimulation. This highlights importance of expansion method in the function of Vγ9Vδ2 T-cells. Furthermore, this work suggests important candidates for blockade by immune checkpoint therapy in order to increase the successful use of Vγ9Vδ2 T-cells in cancer immunotherapy.

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Phase 1/2 Trial of Durvalumab and Lenalidomide in Patients with Cutaneous T Cell Lymphoma (CTCL): Preliminary Results of Phase I Results and Correlative Studies

Blood ◽

10.1182/blood-2018-99-114001 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2931-2931

Author(s):

Christiane Querfeld ◽

Jasmine M. Zain ◽

Devin L. Wakefield ◽

Tijana Jovanovic-Talisman ◽

Sung Hee Kil ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

Board Of Directors ◽

Dose Escalation ◽

Immune Checkpoint ◽

Phase 1 ◽

Super Resolution ◽

Advisory Committees ◽

Skin Biopsies ◽

Super Resolution Microscopy

Abstract Background: In CTCL, intratumoral T cells are functionally exhausted and are characterized by the expression of immune inhibitory molecules such as PD1 and PD-L1 (Cancer Immunol Res 6; 2018). These findings justify the evaluation of immune checkpoint inhibition to reverse T cell exhaustion in CTCL. To this end, we initiated a phase 1/2 clinical trial of lenalidomide and durvalumab to determine the safety and efficacy of this regimen. Durvalumab is a human monoclonal antibody with high affinity and selectivity for PD-L1, with mechanisms of action that target the exhausted T cells and distinct cells within their environment. Lenalidomide, an oral immunomodulatory drug and analog of thalidomide, has previously shown activity in CTCL (Blood 123; 2014). Durvalumab may restore an anti-tumor immune response, and the combination of durvalumab and lenalidomide may enhance immune checkpoint blockade-induced immune responses. Methods: A Phase 1 portion is ongoing to characterize the safety and tolerability of durvalumab and lenalidomide combination. Patients (pts) are enrolled in sequential cohorts to receive durvalumab (fixed dose at 1500 mg) and dose escalation of lenalidomide (cohort 1 = 10 mg; cohort 2 = 15 mg; subsequent planned dose increments of 5 mg) to evaluate safety, efficacy and antitumor activity. Serial skin and blood samples were collected to assess the impact on the tumor micro-environment. We examined the correlation between clinical response and resistance and the following biological factors: PD1 clustering at the single molecule level using super-resolution microscopy, and expression of PD-L1 and ICOS at the tissue level by means of multiplex immunohistochemistry on pre-treatment primary cells (migrated from skin explants), and skin tissue (formalin-fixed and paraffin-embedded) from clinical trial subjects. Results: Six patients (5 males/1female, age 32-57 years) with refractory/advanced CTCL (mycosis fungoides/Sezary syndrome subtype), clinical stage IB (1), IIA (1), IIB (3), IIIA (1) have been enrolled as of July 2018. Duration time on treatment was 4 to 13+ months. Four patients showed improvement of skin disease with 2 patients achieved partial response with > 90% improvement of skin disease by mSWAT. Two patients developed progressive disease. No serious adverse events (AEs) were observed. The most frequently reported AEs were fatigue (n=6), skin pain (n=4), anemia (n=3) chills (n=4), and decreased appetite (n=3). All treatment-related AEs were Grade 1 or 2 in severity. One grade 3 fatigue occurred in one patient. No dose limiting toxicity has been observed to date. Using multispectral microscopy, we analyzed expression panels of several checkpoints: PD1, PD-L1, and ICOS on lesional skin biopsies at baseline. Strong PD-L1 and ICOS expression is observed from non-responders. Detectable levels of PD-L1, but low levels of ICOS is observed in responding patients. Quantitative super-resolution microscopy detected nanoscale clusters of PD1 in T cells from responders and no PD1 clustering was observed in T cells from non-responders. Conclusions: Durvalumab/lenalidomide has significant clinical activity in patients with refractory/advanced CTCL, which will be formally evaluated in the Phase 2 portion of this trial. Responses were durable and ongoing, and treatment was well tolerated with a low toxicity profile. Dose escalation is planned up to lenalidomide 20 mg daily. Our preliminary results from patients on trial demonstrated that immune signatures on skin biopsies at baseline may be predictive of response to checkpoint blockade and yield insights into mechanisms of therapeutic resistance. Disclosures Querfeld: Acelion: Membership on an entity's Board of Directors or advisory committees; Kyowa: Membership on an entity's Board of Directors or advisory committees; Bioniz: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Single Cell Transcriptomic Characterization of the Immune Microenvironment in Naturally Progressing Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood-2018-99-110931 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3112-3112

Author(s):

Noelia Purroy ◽

Jean Fan ◽

Shuqiang Li ◽

Laura Z. Rassenti ◽

James A Lederer ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

Immune Cell ◽

Immune Checkpoints ◽

Advisory Committees ◽

Slow Progressors ◽

Classical Monocytes ◽

Over Time

Abstract The genetic changes occurring during natural progression and following therapy in CLL have recently been characterized. However, the role of co-existing immune cell populations during disease evolution has not been systematically evaluated. New single cell transcriptome sequencing (scRNA-seq) technologies provide a powerful approach for dissecting the composition and state of complex heterogeneous cell populations such as immune cells. We hypothesized that changes of specific immune features underlie the kinetics of progression of CLL. To this end, we selected a discovery set of CLL samples from 6 patients previously genetically characterized by bulk whole-exome sequencing (WES). Three patients were "slow-progressors" (median time to first treatment [TTFT]: 10.7 years (yrs), range: 7.7-12.2), while the other three were "fast-progressors" (median TTFT: 1.9 yrs, range: 1.3-2.4). We processed paired samples obtained close to the time of diagnosis (T1; median yrs from diagnosis: 2.54, range 0.6-4.2) and close to first treatment (T2; median yrs to TTFT: 0.22, range 0-5.9). Non-CD19+CD5+ cells were isolated from peripheral blood mononuclear cells by flow cytometry for scRNA-seq using the 10X Genomics Chromium Controller platform. Replicates were available in 8 of 12 samples, for a total of 20 specimens. Dimensionality reduction and clustering was then performed by a novel pipeline called MUDAN (multi-sample unified discriminant analysis). We used a mixed-effects model to identify trends in cell type-specific gene expression and proportions over time across patients. We observed changes in the composition of immune cells over time across these patients. In total, 57,069 cells were analyzed (median number of cells per sample: 4,394, range 774-8657) and the mean number of genes per cell was 906 (SD: 116.7). We identified a total of 13 immune cell types, consistent with diverse subsets of T and NK cells, myeloid cells, and hematopoietic stem cells. At T1, fast-progressors had a higher proportion of myeloid cells (dendritic cells, classical and non-classical monocytes, p<0.0001, p=0.004 and p<0.0001, respectively) as compared to slow-progressors, which showed a higher proportion of naïve and memory CD4+ T cells (p=0.001 and p=0.016, respectively). Over the course of disease, both fast and slow-progressors converged into a decrease in the lymphoid:myeloid ratio (L:M ratio in T1: 10.1 vs. T2: 7.3, p=0.023), due in part to a proportional increase in non-classical monocytes (p=0.004) and a decrease in CD4+ T cells (p=0.003). Over time, we observed a broad upregulation of gene expression across all cell types in both fast and slow-progressors. Within the lymphoid compartment, we observed an upregulation of cell activation (CD3E, CD69, and GITR), chemotaxis (CCL3, CCL4, and XCL2), cytotoxicity (GZMK, GZMA, and NKG7) and pro-inflammatory (INFG, ISG15, and TNF) markers, especially in memory CD4+ T cells. In contrast, memory CD8+ T cells, which were marked by the inhibitory molecule KLRB1(CD161) in our cohort, showed the lowest increase in these gene signatures. TIGIT followed by LILRB2, CD160 and LAG3 were the most upregulated immune checkpoints across time, especially in memory CD4+ and naïve CD8+ T cells, and resting NK cells. LAG3 was revealed to be the only of these immune checkpoints associated with fast-progressors (p<0.05). No significant changes in the expression of PDCD1, HAVCR2 (TIM3) or TNFRSF14 (HVEM) were observed over time. Within the myeloid compartment, we observed a lack of upregulation of cell activation markers along with a marked downregulation of chemoattractants (XCL1, CXCR5 and CXCR4) and antigen presentation molecules (HLA-DOA, HLA-E, and PSME1) over time, especially in classical monocytes. Further analyses correlating CLL genetics to immune kinetics and validating these results in a larger patient cohort are ongoing. Altogether, these findings provide fresh insights of the evolving immune microenvironment during CLL progression. Despite differences in the immune composition at the time of diagnosis, fast and slow-progressors converged into an immune activation state, mostly dominated by CD4+ T cells, ineffective cytotoxic cells and poor immunogenic phagocytic cells that ultimately contributed to the progression of the disease. These results provide a rationale for new immunotherapeutic strategies in previously untreated CLL patients. Disclosures Kipps: Verastem: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Celgene: Consultancy; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding. Wu:Neon Therapeutics: Equity Ownership.

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Modulation of SRSF2 expression reverses the exhaustion of TILs via the epigenetic regulation of immune checkpoint molecules

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03362-4 ◽

2019 ◽

Vol 77 (17) ◽

pp. 3441-3452 ◽

Cited By ~ 3

Author(s):

Ziqiang Wang ◽

Kun Li ◽

Wei Chen ◽

Xiaoxia Wang ◽

Yikun Huang ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Immune Checkpoint ◽

Tumor Infiltrating Lymphocytes ◽

Immune Checkpoints ◽

Gene Promoters ◽

Immune Checkpoint Molecules ◽

Elevated Expression ◽

Infiltrating Lymphocytes ◽

Checkpoint Genes

AbstractThe elevated expression of immune checkpoints by the tumor microenvironment is associated with poor prognosis in several cancers due to the exhaustion of tumor-infiltrating lymphocytes (TILs), and the effective suppression of the expression of these genes is key to reversing the exhaustion of TILs. Herein, we determined that serine/arginine-rich splicing factor 2 (SRSF2) is a target for blocking the tumor microenvironment-associated immunosuppressive effects. We found that the expression of SRSF2 was increased in exhausted T cells and that SRSF2 was involved in multiple immune checkpoint molecules mediating TILs’ exhaustion. Furthermore, SRSF2 was revealed to regulate the transcription of these immune checkpoint genes by associating with an acyl-transferases P300/CBP complex and altering the H3K27Ac level near these genes, thereafter influencing the recruitment of signal transducer and activator of transcription 3 (STAT3) to these gene promoters. Collectively, our data indicated that SRSF2 functions as a modulator of the anti-tumor response of T cells and may be a therapeutic target for reversing the exhaustion of TILs.

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The Role of CXCL12-Mediated Aberrant Methylation and Tumor Microenvironment Remodeling in Carcinogenesis and Prognosis of Bladder Cancer

10.21203/rs.3.rs-882682/v1 ◽

2021 ◽

Author(s):

Yiheng Du ◽

Jin Cao ◽

Xiang Jiang ◽

Xiaowei Cai ◽

Bo Wang ◽

...

Keyword(s):

Bladder Cancer ◽

T Cells ◽

Tumor Microenvironment ◽

Immune Checkpoint ◽

Bioinformatics Analysis ◽

Immune Checkpoint Blockade ◽

Checkpoint Blockade ◽

Specific Marker ◽

Cxcl12 Expression ◽

Potential Association

Abstract Background Bladder cancer (BLCA) is the most common genitourinary tumor but lacks specific diagnostic biomarkers. Recent years have seen significant advances in the use and approval of immune checkpoint blockade (ICB) therapy to manage bladder cancer at advanced stages when platinum-based therapy has failed. The tumor microenvironment (TME) in bladder cancer is an essential player in patient's responsiveness to ICB therapy. Therefore, this manuscript explored the TME and identified CXCL12, a specific marker for inflammatory cancer associated fibroblasts(iCAFs), as potential molecular markers and therapeutic targets for bladder cancer. Methods We examined the gene expression profiles in the TCGA and GEO datasets to reveal the potential association of CXCL12 with the carcinogenesis and prognosis of bladder cancer. Methylation analysis of CXCL12 was performed using the UALCAN and MethSurv databases. The MCP-COUNTER, ESTIMATE, and TIDE algorithms were applied to estimate the TME components and predict immunotherapy responsiveness. An iCAFs signature was constructed using the ssGSEA algorithm. Bioinformatics analysis results were validated through immunohistochemistry of clinical samples. IMvigor210 cohort was used to validate bioinformatic predictions of therapeutic responsiveness to immune checkpoint inhibitors Results Our analysis revealed the potential association between aberrant promoter methylation of CXCL12 and bladder cancer carcinogenesis. CpG sites methylation of the CXCL12 gene body was associated with bladder cancer prognosis. Moreover, the expression level of CXCL12 exhibited a significant correlation with patients' pathological features and prognosis. Through gene enrichment analysis, CXCL12 was demonstrated to be associated with immune modulation and tumor microenvironment remodeling. The MCP-COUNTER and ESTIMATE algorithms verified significant correlations between CXCL12 and TME components, particularly CAFs, macrophages, and T cells. The TIDE algorithm provided evidence that T-cell clearance and dysfunction were more pronounced in bladder cancers characterized by high CXCL12 expression and high iCAFs scores, contributing to inferior responsiveness to ICB therapy. Patients who expressed high CXCL12 levels and had high iCAFs scores were likely to have less frequent FGFR3 mutation and a stromal-rich molecular subtype. Immunohistochemistry revealed that the close association of CXCL12 with iCAFs in bladder cancer potentially influenced the intratumoral infiltration of CD8 + T cells. CXCL12 expression in MIBC was increased significantly in NMIBC, which supports the bioinformatics analysis results. The IMvigor210 cohort confirmed the iCAFs score to be significantly associated with the responsiveness to immune checkpoint blockade therapy. Conclusions This work explores carcinogenesis and cancer-promoting roles of CXCL12 in bladder cancer. As a specific marker gene of iCAFs, CXCL12 potentially promotes bladder cancer progression by regulating the tumor microenvironment. Further exploration of the association between CXCL12 and iCAFs may unravel potential therapeutic targets for bladder precision medicine and improve the responsiveness of immune checkpoint blockade therapy.

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Rejuvenated BCMA-Specific CD8 + Cytotoxic T Lymphocytes Derived from Antigen-Specific Induced Pluripotent Stem Cells : Immunotherapeutic Application in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153110 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 75-75

Author(s):

Jooeun Bae ◽

Shuichi Kitayama ◽

Laurence Daheron ◽

Zach Herbert ◽

Nikhil C. Munshi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

T Cell Differentiation ◽

Immune Checkpoints ◽

Publicly Traded ◽

Advisory Committees ◽

Induced Pluripotent

Abstract T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. Figure 1 Figure 1. Disclosures Munshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Combined PD-L1 and TIM-3 blockade improves the expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy

10.1101/2021.08.30.21262835 ◽

2021 ◽

Author(s):

Shirin Lak ◽

Valérie Janelle ◽

Anissa Djedid ◽

Gabrielle Boudreau ◽

Ann Brasey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Checkpoint ◽

Cell Expansion ◽

Ex Vivo ◽

Immune Checkpoint Blockade ◽

Checkpoint Blockade ◽

Immune Checkpoints ◽

T Cell Expansion ◽

And Function

AbstractBackgroundThe stimulation and expansion of antigen-specific T cells ex vivo enables the targeting of a multitude of cancer antigens. However, clinical scale T-cell expansion from rare precursors requires repeated stimulations ex vivo leading to T-cell terminal effector differentiation and exhaustion that adversely impact therapeutic potential. We leveraged immune checkpoint blockade relevant to antigen-specific CD8+ human T cells to improve the expansion and function of T cells targeting clinically relevant antigens.MethodsA clinically-compliant protocol relying on peptide-pulsed monocyte-derived dendritic cells and cytokines was used to expand antigen-specific CD8+ targeting the oncogenic Epstein-Barr virus (EBV) and the tumor associated antigen (TAA) Wilms Tumor 1 (WT1) protein. The effects of antibody-mediated blockade of immune checkpoints applied to the cultures (T-cell expansion, phenotypes and function) were assessed at various time points. Genomic studies including single cell RNA (scRNA) sequencing and T-cell receptor sequencing were performed on EBV-specific T cells to inform about the impact of immune checkpoint blockade on the clonal distribution and gene expression of the expanded T cells.ResultsSeveral immune checkpoints were expressed early by ex vivo expanded antigen-specific CD8+ T cells, including PD-1 and TIM-3 with co-expression matching evidence of T-cell dysfunction as the cultures progressed. The introduction of anti-PD-L1 (expressed by the dendritic cells) and anti-TIM-3 antibodies in combination (but not individually) to the culture led to markedly improved antigen-specific T-cell expansion based on cell counts, fluorescent multimer staining and functional tests. This was not associated with evidence of T-cell dysfunction when compared to T cells expanded without immune checkpoint blockade. Genomics studies largely confirmed these results, showing that double blockade does not impart specific transcriptional programs or patterns on TCR repertoires. However, our data indicate that combined blockade may nonetheless alter gene expression in a minority of clonotypes and have donor-specific impacts.ConclusionsThe manufacturing of antigen-specific CD8+ T cells can be improved in terms of yield and functionality using blockade of TIM-3 and the PD-L1/PD-1 axis in combination. Overcoming the deleterious effects of multiple antigenic stimulations through PD-L1/TIM-3 blockade is a readily applicable approach for several adoptive-immunotherapy strategies.

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