Multiple Inhibitory Receptors Research Articles

Med1 Controls Effector CD8+ T Cell Differentiation and Survival through C/EBPβ-Mediated Transcriptional Control of T-bet.

Effector CD8+ T cells are crucial players in adaptive immunity for effective protection against invading pathogens. The regulatory mechanisms underlying CD8+ T cell effector differentiation are incompletely understood. In this study, we defined a critical role of mediator complex subunit 1 (Med1) in controlling effector CD8+ T cell differentiation and survival during acute bacterial infection. Mice with Med1-deficient CD8+ T cells exhibited significantly impaired expansion with evidently reduced killer cell lectin-like receptor G1+ terminally differentiated and Ly6c+ effector cell populations. Moreover, Med1 deficiency led to enhanced cell apoptosis and expression of multiple inhibitory receptors (programmed cell death 1, T cell Ig and mucin domain-containing-3, and T cell immunoreceptor with Ig and ITIM domains). RNA-sequencing analysis revealed that T-bet- and Zeb2-mediated transcriptional programs were impaired in Med1-deficient CD8+ T cells. Overexpression of T-bet could rescue the differentiation and survival of Med1-deficient CD8+ effector T cells. Mechanistically, the transcription factor C/EBPβ promoted T-bet expression through interacting with Med1 in effector T cells. Collectively, our findings revealed a novel role of Med1 in regulating effector CD8+ T cell differentiation and survival in response to bacterial infection.

Effector Th1 cells under PD-1 and CTLA-4 checkpoint blockade abrogate the upregulation of multiple inhibitory receptors and by-pass exhaustion.

Immune checkpoint inhibitor (ICI) immunotherapy relies on the restoration of T-cell functions. The ICI receptors are not only found on exhausted T cells but also upregulated upon activation and reach high levels on effector T cells. In an ex vivo model, this study explored the consequences of PD-1 and cytotoxic T-lymphocyte antigen (CTLA-4) blockade applied during specific time frames of T-cell stimulation that coincide with distinct functional phases in type 1 helper T (Th1) cells. When applied at an early stimulation stage, the checkpoint blockade interfered with the upregulation of multiple inhibitory receptors such as PD-1, LAG3, TIM-3 and CTLA-4. Moreover, extension of the blockade period restricted the hyporesponsiveness in T cells. Alternatively, a short-term ICI treatment was advantageous when applied at late time frames of Th1 cell stimulation. Here, a transition phase from effector to exhausted state, which coincided with the late time frames of Th1 stimulation, was clearly determined together with the transcriptomics data demonstrating the initiation of significant alterations in metabolic pathways, genetic information processes, effector and exhaustion specific pathways. Applied in this transition phase, PD-1 and/or CTLA-4 blockade downregulated the inhibitory receptors which were already present on the effector Th1 cells, potentially through endocytic pathways. Therefore, the efficacy of ICI therapy was modulated by the functional status of T cells and can be improved by modifying the timing and duration of PD-1 and CTLA-4 blockade. In conclusion, the ICI therapy not only supports the reactivation of T cells but can also constrain de novo exhaustion.

DISSECTION OF T CELL ACTIVATION PHASES FOR CHECK POINT BLOCKADE IMMUNOTHERAPY

Checkpoint blockade immunotherapy relies on the restoration of effector responses in precursor-exhausted T cells. Nevertheless, many inhibitory receptors are upregulated soon after the activation and reach high levels at effector phases. In addition to the functional status of type 1 helper T (Th1) cells, the timing and duration of PD-1 and CTLA-4 blockade are considered as critical factors for retaining T cell responsiveness. Here, we used a previously established ex-vivo model to display the decline in Th1 responses and to determine the state-of-function which may critically modulate the success of checkpoint blockade. In the absence of PD-1 and/or CTLA-4 blockade, proliferation and IFN-γ, TNF-α, IL-2 secretion capacities of Th1 cells were gradually abolished as the stimulation was continued. When applied at an early activation stage, the extension of blockade period interfered with the upregulation of multiple inhibitory receptors (PD-1, LAG3, TIM-3, and CTLA-4) and restricted Th1 hyporesponsiveness. The inhibitory receptors that were already expressed prior to the blockade were also downregulated potentially through an endocytic pathway. A time point representing the late stages of effector Th1 responses, which preceded the exhaustion, was determined as a therapeutic window for a short-term check point blockade. In conclusion, PD-1 and CTLA-4 checkpoint inhibitor therapy not only adjust the reactivation of hyporesponsive T cells but can also restrict the induction of de novo T cell exhaustion.

Abstract 3155: "Super NK cells" - natural killer cells derived from engineered hiPSC with enhanced NK receptor expression demonstrate better anti-tumor effects for solid tumors

Abstract CAR-T cells expressing chimeric antigen receptors (CAR) have shown clinical success for treating hematologic malignancies, but their efficacy in solid tumors has been limited, largely due to tumor heterogeneity and immunosuppressive tumor microenvironment (TME). Natural Killer (NK) cells derived from human induced pluripotent stem cells (hiPSC) hold great potential to become the next-generation cell therapy products because of their established safety profile and their unique biological activities, especially their potential effectiveness for targeting solid tumor. In contrast to T cells, NK cells exert anti-tumor activity without requiring antigen recognition through MHC complex; they are instead activated by calculated signaling through ligation of multiple activating and inhibitory receptors. As various solid tumors highly express activation ligands for NK cells, we reason that enhanced expression of activation receptors on NK cells could improve their killing activity towards solid tumors. Here, we report that “super NK cells” with more than 10 folds higher expression of NK cell receptors such as NKG2D and NCRs derived from engineered hiPSC show improved cytotoxicity against solid tumors compared with non-engineered ones. The “super NK cells” can achieve more than ten rounds of serial killing against both K562 and THP-1 cell lines in vitro. More importantly, the “super NK cells” show improved cytotoxicity to multiple solid tumors in both in vitro system and in vivo xenograft mouse models. In summary, we have engineered hiPSCs which can be differentiated into "super NK cells" as a potential cell therapy product for improved efficacy against solid tumors. Citation Format: Yiyun Wang, Xiangjun He, Linqin Wang, Mi Shao, Yanan Yue, Yangbin Gao, George Church, He Huang, Luhan Yang. "Super NK cells" - natural killer cells derived from engineered hiPSC with enhanced NK receptor expression demonstrate better anti-tumor effects for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3155.

Cytokine storm promoting T cell exhaustion in severe COVID-19 revealed by single cell sequencing data analysis.

BackgroundEvidence has suggested that cytokine storms may be associated with T cell exhaustion (TEX) in COVID-19. However, the interaction mechanism between cytokine storms and TEX remains unclear.MethodsWith the aim of dissecting the molecular relationship of cytokine storms and TEX through single-cell RNA sequencing data analysis, we identified 14 cell types from bronchoalveolar lavage fluid of COVID-19 patients and healthy people. We observed a novel subset of severely exhausted CD8 T cells (Exh T_CD8) that co-expressed multiple inhibitory receptors, and two macrophage subclasses that were the main source of cytokine storms in bronchoalveolar.ResultsCorrelation analysis between cytokine storm level and TEX level suggested that cytokine storms likely promoted TEX in severe COVID-19. Cell–cell communication analysis indicated that cytokines (e.g. CXCL10, CXCL11, CXCL2, CCL2, and CCL3) released by macrophages acted as ligands and significantly interacted with inhibitory receptors (e.g. CXCR3, DPP4, CCR1, CCR2, and CCR5) expressed by Exh T_CD8. These interactions formed the cytokine–receptor axes, which were also verified to be significantly correlated with cytokine storms and TEX in lung squamous cell carcinoma.ConclusionsCytokine storms may promote TEX through cytokine-receptor axes and be associated with poor prognosis in COVID-19. Blocking cytokine-receptor axes may reverse TEX. Our finding provides novel insights into TEX in COVID-19 and new clues for cytokine-targeted immunotherapy development.

Interrogating mechanisms of CD8 T cell dysfunction in obese asthma

Abstract Pediatric obese atopic asthma (OA) is a complex and poorly understood disease at the intersection of two of the most common chronic inflammatory diseases of childhood. Pediatric OA patients often experience severe asthma exacerbations caused by respiratory viral infections. However, the immunological mechanisms by which obesity modifies immune function in asthma remain poorly understood. Using high dimensional immune profiling, we have demonstrated CD8 T cells from pediatric OA patients have an exhausted-like immunophenotype compared to non-obese asthmatics. To more deeply characterize the role of CD8 T cell dysfunction in OA, we developed a mouse model of OA using high fat diet (HFD) to induce obesity and house dust mite (HDM) to induce asthma. We found that non-naïve CD8 T cells isolated from the lungs of OA mice demonstrate increased expression of multiple activation and inhibitory receptors (e.g., PD-1, CD39 and Tim3) as well as TOX, all consistent with T cell exhaustion, compared to healthy controls. We hypothesize the exhausted-like state of CD8 T cells in OA impairs anti-viral responses, thereby increasing severity of asthma exacerbation during respiratory viral infections. Ongoing work is testing antigen specific responses to influenza infection. Overall, this reveals a previously unrecognized role for exhausted-like CD8 T cell responses in mediating the immunopathogenesis of pediatric obese asthma. This work was supported by the Translational Research Training Program in Environmental Health Sciences Pre-Doctoral Fellowship to C.A.H. and K08-AI135091 to S.E.H.

Simultaneous Genetic Ablation of PD-1, LAG-3, and TIM-3 in CD8 T Cells Delays Tumor Growth and Improves Survival Outcome.

Immune checkpoint inhibitors (ICI) represented a step forward in improving the outcome of patients with various refractory solid tumors and several therapeutic regimens incorporating ICI have already been approved for a variety of tumor entities. However, besides remarkable long-term responses, checkpoint inhibition can trigger severe immune-related adverse events in some patients. In order to improve safety of ICI as well as T cell therapy, we tested the feasibility of combining T cell-based immunotherapy with genetic disruption of checkpoint molecule expression. Therefore, we generated H-Y and ovalbumin antigen-specific CD8+ T cells with abolished PD-1, LAG-3, and TIM-3 expression through CRISPR/Cas9 technology. CD8+ T cells, subjected to PD-1, LAG-3, and TIM-3 genetic editing, showed a strong reduction in immune checkpoint molecule expression after in vitro activation, while no relevant reduction in responsiveness to in vitro stimulation was observed. At the same time, in B16-OVA tumor model, transferred genetically edited OT-1 CD8+ T cells promoted longer survival compared to control T cells and showed enhanced expansion without associated toxicity. Our study supports the notion that antigen-specific adoptive T cell therapy with concomitant genetic disruption of multiple checkpoint inhibitory receptors could represent an effective antitumor immunotherapy approach with improved tolerability profile.

Differential expression of inhibitory receptor NKG2A distinguishes disease-specific exhausted CD8+ T cells.

Exhausted CD8+ T (Tex) cells are caused by persistent antigenic stimulation during chronic viral infection or tumorigenesis. Tex cells upregulate and sustain the expressions of multiple immune inhibitory receptors (IRs). Blocking IRs of Tex cells, exemplified by PD‐1, can partially restore their effector functions and thus lead to viral suppression or tumor remission. Tex cells derived from chronic viral infections share the expression spectrum of IRs with Tex cells derived from tumors; however, whether any IRs are selectively expressed by tumor‐derived Tex cells or virus‐derived Tex cells remains to be learnt. In the study, we found that Tex cells upregulate IR natural killer cell lectin‐like receptor isoform A (NKG2A) specifically in the context of tumor but not chronic viral infection. Moreover, the NKG2A expression is attributed to tumor antigen recognition and thus bias expressed by tumor‐specific Tex cells in the tumor microenvironment instead of their counterparts in the periphery. Such dichotomous NKG2A expression further dictates the differential responsiveness of Tex cells to NKG2A immune checkpoint blockade. Therefore, our study highlighted NKG2A as a disease‐dependent IR and provided novel insights into the distinct regulatory mechanisms underlying T cell exhaustion between tumor and chronic viral infection.

Perspective of HLA-G Induced Immunosuppression in SARS-CoV-2 Infection.

COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has threatened public health worldwide. Host antiviral immune responses are essential for viral clearance and disease control, however, remarkably decreased immune cell numbers and exhaustion of host cellular immune responses are commonly observed in patients with COVID-19. This is of concern as it is closely associated with disease severity and poor outcomes. Human leukocyte antigen-G (HLA-G) is a ligand for multiple immune inhibitory receptors, whose expression can be upregulated by viral infections. HLA-G/receptor signalling, such as engagement with immunoglobulin-like transcript 2 (ILT-2) or ILT-4, not only inhibit T and natural killer (NK) cell immune responses, dendritic cell (DC) maturation, and B cell antibody production. It also induces regulatory cells such as myeloid-derived suppressive cells (MDSCs), or M2 type macrophages. Moreover, HLA-G interaction with CD8 and killer inhibitory receptor (KIR) 2DL4 can provoke T cell apoptosis and NK cell senescence. In this context, HLA-G can induce profound immune suppression, which favours the escape of SARS-CoV-2 from immune attack. Although detailed knowledge on the clinical relevance of HLA-G in SARS-CoV-2 infection is limited, we herein review the immunopathological aspects of HLA-G/receptor signalling in SARS-CoV-2 infection, which could provide a better understanding of COVID-19 disease progression and identify potential immunointerventions to counteract SARS-CoV-2 infection.

Selinexor Enhances NK Cell Activation Against Malignant B Cells via Downregulation of HLA-E.

Selinexor is an FDA approved selective inhibitor of the nuclear export protein exportin-1 (XPO1) and causes specific cancer cell death via nuclear accumulation of tumor suppressor proteins. Design of rational studies for the use of selinexor in combination with other therapeutic agents, such as immunotherapies, requires a fundamental understanding of the effects of selinexor on the immune system. One important emerging area of immunotherapy are natural killer (NK) cell based therapeutics. NK cell function is tightly regulated by a balance of signals derived from multiple activating and inhibitory receptors. Thus in cancer, up-regulation of stress ligands recognised by activating receptors or down-regulation of HLA class I recognised by inhibitory receptors can result in an anti-cancer NK cell response. Changes in XPO1 function therefore have the potential to affect NK cell function through shifting this balance. We therefore sought to investigate how selinexor may affect NK cell function. Selinexor pre-treatment of lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4, JeKo-1 and Ramos cells, concurrent with increased CD107a and IFNγ expression on NK cells. In addition, selinexor enhanced ADCC against lymphoma cells coated with the anti-CD20 antibodies rituximab and obinutuzumab. In probing the likely mechanism, we identified that XPO1 inhibition significantly reduced the surface expression of HLA-E on lymphoma cell lines and on primary chronic lymphocytic leukemia cells. HLA-E binds the inhibitory receptor NKG2A and in accordance with this, selinexor selectively increased activation of NKG2A+ NK cells. Our data reveals that selinexor, in addition to its direct cytotoxic activity, also activates an anti-cancer immune response via disruption of the inhibitory NKG2A:HLA-E axis.

Neutrophil Extracellular Traps Promote T Cell Exhaustion in the Tumor Microenvironment.

While neutrophil extracellular traps (NETs) are important for directly promoting cancer growth, little is known about their impact on immune cells within the tumor microenvironment (TME). We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. Herein, to induce a NET-rich TME, we performed liver Ischemia/Reperfusion (I/R) in an established cancer metastasis model or directly injected NETs in subcutaneous tumors. In this NET-rich TME, the majority of CD4+ and CD8+ tumor infiltrating lymphocytes expressed multiple inhibitory receptors, in addition these cells showed a functional and metabolic exhausted phenotype. Targeting of NETs in vivo by treating mice with DNAse lead to decreased tumor growth, decreased NET formation and higher levels of functioning T cells. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion and dysfunction; an effect abrogated by using PD-L1 KO NETs or culturing NETs with PD-1 KO T cells. Furthermore, we found elevated levels of sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. Neutrophils isolated from patients after surgery were primed to form NETs and induced exhaustion and dysfunction of human CD4+ and CD8+ T cells. We next targeted PD-L1 in vivo by injecting a blocking antibody during liver I/R. A single dose of anti-PD-L1 during surgery lead to diminished tumors at 3 weeks and functional T cells in the TME. Our data thus reveal that NETs have the capability of suppressing T cell responses through metabolic and functional exhaustion and thereby promote tumor growth. Furthermore, targeting of PD-L1 containing NETs at time of surgery with DNAse or anti-PD-L1 lead to diminished tumor growth, which represents a novel and viable strategy for sustaining immune competence within the TME.

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

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

PD-1 and TIGIT downregulation distinctly affect the effector and early memory phenotypes of CD19-targeting CAR T cells

CD19-targeting chimeric antigen receptor (CAR) Tcells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short hairpin RNA cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR Tcells in the context of four different checkpoint combinations-PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4, and PD-1/TIGIT-and found that CAR Tcells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects. Importantly, functional and phenotypic analyses suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT-downregulated CAR Tcells generated from diffuse large B cell lymphoma patient-derived Tcells also showed robust antitumor activity and significantly improved persistence invivo. The efficacy and safety of PD-1/TIGIT-downregulated CD19-targeting CAR Tcells are currently being evaluated in adult patients with relapsed or refractory large B cell lymphoma (ClinicalTrials.gov: NCT04836507).

Abstract MP49: Single Cell Multiplex Immunophenotyping Using Mass Cytometry And CITE-Seq Reveals Decreases In Circulating PD-1 + CD8 + Memory T Cells With Features Of Exhaustion In Human Hypertension

Emerging evidence from animal models has demonstrated the importance of multiple innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets are altered in human hypertension. To test this, we performed high dimensional single cell profiling of human peripheral blood mononuclear cells using mass cytometry. Unsupervised computational analysis revealed a 40% decrease in CD8 + memory T cells in hypertensive individuals. Using Phenograph to identify subsets of these cells revealed a selective 60% decrease in PD-1 + CD8 + memory T cells in hypertension. This observation was confirmed in a validation cohort using flow cytometry in which PD-1 + CD8 + memory T cells were significantly decreased 44% in hypertensive compared to control individuals. To determine the phenotype of these PD-1 + CD8 + memory T cells, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) on four control and four hypertensive individuals. Using antibodies to identify PD-1 + and PD-1 - CD8 + memory T cells, gene set enrichment analysis of the coordinate single cell transcriptomic data revealed that PD-1 + cells exhibit over-representation of features of both immunologically active effector T cells and hypofunctional exhausted T cells. Thus, clustering analysis of PD-1 + CD8 + memory T cells was performed which demonstrated 4 distinct subclusters. One of these subclusters was decreased in hypertension and exhibited selective expression of multiple inhibitory receptors characteristic of exhausted T cells. At the protein level, this subcluster was marked by expression of the inhibitory receptor LAG3 and low levels of CD57. Combining these markers to identify PD-1 + LAG3 + CD57 - CD8 + memory T cells permitted identification of exhausted cells which demonstrated a significant 35% decrease in hypertensive compared to control individuals using flow cytometry. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1 + CD8 + memory T cells with features of exhaustion in human hypertension. These findings provide new insights into the pathogenesis of human hypertension including loss and/or re-invigoration of exhausted T cells.

Schrödinger's T Cells: Molecular Insights Into Stemness and Exhaustion.

T cell stemness and exhaustion coexist as two key contrasting phenomena during chronic antigen stimulation, such as infection, transplant, cancer, and autoimmunity. T cell exhaustion refers to the progressive loss of effector function caused by chronic antigen exposure. Exhausted T (TEX) cells highly express multiple inhibitory receptors and exhibit severe defects in cell proliferation and cytokine production. The term T cell stemness describes the stem cell-like behaviors of T cells, including self-renewal, multipotency, and functional persistence. It is well accepted that naïve and some memory T cell subsets have stem cell-like properties. When investigating the exhaustive differentiation of T cells in chronic infection and cancer, recent studies highlighted the stemness of “precursors of exhausted” T (TPEX) cells prior to their terminal differentiation to TEX cells. Clinically successful checkpoint blockades for cancer treatment appear to invigorate antitumor TPEX cells but not TEX cells. Here we discuss the transcriptional and epigenetic regulations of T cell stemness and exhaustion, with a focus on how systems immunology was and will be utilized to define the molecular basis underlying the transition of TPEX to TEX cells. We suggest a “stepwise model” of T cell stemness and exhaustion, in which loss of stemness and exhaustion progression are gradual multi-step processes. We provide perspectives on the research needed to define T cell stemness and exhaustion in the transplantation setting, in which allogenic T cells are also chronically exposed to alloantigens. A better understanding of T cell stemness and exhaustion will shed light on developing novel strategies for immunotherapies.

Cover story: Eur. J. Immunol. 8'21

European Journal of ImmunologyVolume 51, Issue 8 Cover PictureFree Access Cover story: Eur. J. Immunol. 8'21 First published: 04 August 2021 https://doi.org/10.1002/eji.202170080AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Our front cover features a high-dimensional BH-SNE representation of the CD4 T cell phenotype in patients affected by COVID-19. The image is taken from Manfredi and Abbati et al., and developed through cytoChain, an innovative application for the analysis of high-dimensional flow cytometry data. CytoChain refined the COVID-19 dataset analysis published by Neumann et al. (DOI 10.1002/cti2.1204), underlining previously missed differences among COVID-19 patients. A novel set of surface markers was thus uncovered, indicating how a T cell subset expressing multiple inhibitory receptors is more abundant in severe COVID-19 cases. Volume51, Issue8August 2021 RelatedInformation

Abstract 115: Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment

Abstract Neutrophil extracellular traps (NETs) have recently been implicated in the growth of tumors, however their impact on immune cells within the tumor microenvironment (TME) remain incompletely understood. We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. We performed liver Ischemia/Reperfusion (I/R) in an established colorectal cancer metastasis model to induce NET formation in the TME. In this NET-rich TME we discovered elevated levels of phenotypically and functionally exhausted T cells. We demonstrated that the majority of CD4 and CD8 T cells are not effector cells, but rather express multiple inhibitory receptors and are highly dysfunctional, with reduced cytokine production and proliferative capacity. This effect was limited to the T cells present the the NET-rich TME in comparison to T cells isolated from the spleen. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion; an effect abrogated by using PD-L1 KO NETs. This exhausted state was directly linked to mitochondrial dysfunction in the T cells. Furthermore, NETs were targeted in vivo by treating mice with DNAse or anti-PD-L1 at time of I/R. Treatment caused decreased metastatic burden, decreased NET formation and higher levels of functioning non-exhausted T cells. Conversely, injecting NETs in tumors led to acceleration of tumor growth and T cell exhaustion in the TME. In addition, we measured sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. High levels of both markers were significantly associated with decreased disease free survival. Neutrophils isolated from patients after surgery were primed to form NETs that caused a similar exhaustion and dysfunction of human CD4 and CD8 T cells. Our data thus reveal that NETs have the capability of suppressing T cell responses and promoting tumor growth. This mechanism of T cell suppression by NETs represents a viable target for sustaining immune competence within the TME. Citation Format: Christof Kaltenmeier, Hamza Yazdani, Samer Tohme. Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 115.

NKG2A Represents an Important Immune Checkpoint for Human Cytokine-Induced Memory-like NK Cells in Patients with AML

Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell anti-leukemia potential is unclear. Paradigm-shifting reports have shown that NK cells exhibit “memory-like” properties following hapten exposure, virus infection, or combined cytokine pre-activation. We and others have established that human cytokine-induced memory-like NK cells display enhanced re-stimulation responses to numerous activating stimuli, including tumor target cells. We have translated this finding into a potential cellular therapy for rel/ref AML patients, and have completed the dose escalation of a phase 1/2 study. We demonstrated that donor memory-like NK cells expand in patients' blood and bone marrow and retain enhanced functionality ex vivo. Further, of the 11 evaluable AML patients, we have observed 7 clinical responses, including 6 patients with CR/CRi. Since NK cell recognition depends on signals from multiple activating and inhibitory receptors, we developed mass cytometry panels to immunophenotype and track the diversity and effector functions of these human in vivo-differentiated memory-like NK cells.Mass cytometry revealed that in vivo-differentiated memory-like (ML) NK cells were distinct from baseline (BL) NK cells from the same donor, as well as NK cells from normal donor PBMC. Multidimensional analyses revealed a memory-like phenotype: CD56hi CD11blo CD62L+ NKG2Ahi NKp30hi Ki-67+ (baseline: 2.7% ± 0.45% v memory-like: 87.4% ± 5.3%, mean ± SEM within memory-like gate). Furthermore, Citrus analyses revealed that higher NKG2A expression was significantly correlated with treatment failure [TF median NKG2A= 89.2 ± 25.0; responder median NKG2A= 8.1 ± 2.9 (mean ± SEM); p = 0.007, n = 5 each group; SAM FDR <0.1]. NKG2A is a C-type lectin receptor with two immunoreceptor tyrosine-based inhibitory motifs. Signaling through NKG2A is achieved when it engages its ligand, HLA-E. HLA-E is a non-classical major histocompatibility complex class I molecule that is expressed abundantly on many normal tissue types as well as tumors, including AML. Based on these findings that NKG2A is upregulated on memory-like NK cells and the intensity of NKG2A on memory-like NK cells correlates with patient responses, we hypothesized that NKG2A/HLA-E interactions in vivo represent a major barrier to memory-like NK cell responses.In order to interrogate the role NKG2A may play in limiting cytokine-induced memory-like NK cell responses, we stimulated in vitro-differentiated ML cells with the K562 AML tumor line displaying forced expression of HLA-E (K562-E) in the presence of isotype or anti-NKG2A blocking antibody. Increased IFN-γ (p = 0.02) and TNF (p = 0.05) production by NKG2A-blocked ML NK cells was detected, compared to isotype-treated ML NK cells. We performed a similar stimulation assay using primary AML blasts as targets, which express a variable level of HLA-E. Again, ML NK cells treated with NKG2A blockade produced significantly more IFN-γ than isotype-treated ML NK cells (p = 0.001). Studies examining NKG2A checkpoint blockade on in vivo-differentiated ML NK cells from AML patients are ongoing. In conclusion, in vivo-differentiated cytokine-induced memory-like NK cells are phenotypically distinct from conventional NK cells. Further, targeting NKG2A expressed on ML NK cells in vivo has the potential to enhance the clinical efficacy of memory-like NK cell therapy for patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Dominant Negative SHP-2 Which Abrogates PD-1 Signalling Pathways and Restores Function of Cytotoxic CAR T Cells

IntroductionRecent successes in the clinic with monoclonal antibodies against cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed-death 1 (PD-1) have highlighted the importance of developing strategies to overcome T cell inhibition when investigating anti-tumour immunotherapies. Strategies to avoid inhibitory signals by the tumour microenvironment have recently been incorporated into CAR T cells with the most exploited strategy being the conversion of the inhibitory signal from one receptor into an activating one. Many of these inhibitory receptors signal through either the SHP-1/SHP-2 phosphatases. Here we present a method to block these phosphatases using a dominant negative protein. Unlike previous methods that focus on blocking one inhibitory receptor at a time, this approach enables the disarmament of multiple inhibitory receptors at once.Methods and resultsWe generated dominant negative forms of SHP-2 (dnSHP2) that block the function of this endogenous inhibitory phosphatase. This was transduced into T cells coupled with an anti-CD19 CAR and CAR T function was assessed in the presence of PD-1/PD-L1 ligation. In the absence of dnSHP-2, CAR T cell killing, proliferation and cytokine release was significantly impaired when co-cultured with target cells that ligated both the CAR and PD-1. In contrast, CAR T cells that expressed dnSHP-2 observed no defect in killing response in the presence of PD1 ligation. Furthermore, dnSHP2 was shown to impair PD-1 mediated inhibition of cytokine release and proliferation in CAR T cells. This ability to block PD-1 mediated inhibition in CAR T cells was independent of the CAR generation, with similar results in first and second-generation CARs with either a 41BB or CD28 co-stimulatory domain.ConclusionsWe have successfully engineered a protein (dnSHP2) that is capable of abrogating the function of the inhibitory phosphatase SHP-2. The expression of dnSHP2 impaired PD-1 mediated inhibition of CAR T cells and could theoretically impair any inhibitory receptor that signals through SHP2, making this a universal approach to nullify inhibitory receptors on CAR T cells. DisclosuresBaldan:Autolus Ltd.: Employment, Equity Ownership. Ghongane:Autolus Ltd.: Employment, Equity Ownership. Kokalaki:Autolus Ltd.: Employment, Equity Ownership. Lim:Autolus Ltd.: Employment, Equity Ownership. Onuoha:Autolus Ltd.: Employment, Equity Ownership. Cordoba:Autolus Ltd.: Employment, Equity Ownership. Thomas:Autolus Ltd.: Employment, Equity Ownership. Pule:Autolus Ltd.: Employment, Equity Ownership.

Molecular Pathways Responsible for Adaptive Resistance to Single-Checkpoint Inhibition May Reveal Biomarkers and Immunotherapy Targets in Multiple Myeloma Patients

Immune checkpoints blocking antibodies represent a promising strategy to harness anti-tumor immune responses and improve the clinical outcome of patients with hematological malignancies. Early studies based on single-agent PD-1 blockade have fallen short of clinical expectations in multiple myeloma patients. Thus, several strategies are under consideration to implement and optimize the clinical efficacy of immune checkpoint blockade in multiple myeloma patients. We have focused on Vγ9Vδ2 T cells because we believe that these cells are very sensitive tools to assess the immune competence of multiple myeloma (MM) patients in the tumor microenvironment. By interrogating the bone marrow (BM) Vγ9Vδ2 T-cell immune reactivity to phosphoantigens, we have revealed a very early and unexpected tumor microenvironment commitment to immune suppression which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted in clinical remission after autologous stem cell transplantation. Multiple cell subsets (myeloma cells, myeloid-derived suppressor cells, regulatory T cells, bone marrow stromal cells) are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the immune checkpoint circuity.We have shown that there is a multifaceted immune checkpoint expression in the tumor microenvironment which is dependent on the clinical stage and cannot be neutralized with single-agent PD-1 blockade. To test our hypothesis that adaptive resistance to PD-1 blockade in MM patients may be due to the compensatory up-regulation of other immune checkpoints, we analyzed the expression of other inhibitory receptors (TIM-3, LAG-3, TIGIT and CTLA-4) in PB and BM Vg9Vd2 T cells from healthy donors and MM patients and observed that also TIM-3 beyond PD-1 is significantly upregulated in BM Vγ9Vδ2 T cells from MM patients at diagnosis. Unlike healthy immune cells, the attempt to activate functionally exhausted immune cells can further compromise their anti-tumor functions. pAg stimulation of PD-1+ and TIM-3+ BM Vγ9Vδ2 T cells further increases their inhibitory receptor expression. Interestingly, TIM-3 up-regulation is even more pronounced than PD-1 up-regulation in BM Vγ9Vδ2 T cells and it is specular to Vγ9Vδ2 T -cell proliferation, confirming that any inappropriate attempt to activate these cells leads further worsen their immune competence status. Our observations about the compensatory up-regulation of multiple inhibitory receptors (PD-1 and TIM-3) on BM Vγ9Vδ2 T cells and the further up-regulation of TIM-3 after anti-PD1 treatment suggested us to assess the combination of both immune checkpoint inhibitors (anti-PD1 and anti-TIM3) to overcome the PD-1 single blocking resistance and recover anti-myeloma reactivity. TIM-3 blockade alone was able to partially recover pAg-induced Vγ9Vδ2 T-cell proliferation but the best recovery was obtained when pAg stimulation is carried out in the presence of concurrent PD-1 and TIM-3 blockade. To investigate the molecular mechanisms behind the up-regulation of multiple immune checkpoints responsible for PD-1-mediated hyporesponsiveness and the effector cells exhaustion, we analyzed the main PD-1-mediated (PI3K/AKT) and TIM-3-mediated (JAK/STAT) signal transduction pathways. After pAg stimulation, purified Vγ9Vδ2 T cells from peripheral blood (PB) CTRL, PB MM and BM MM has been tested by western blot analysis for the expression of several intracellular proteins: Ras, phospho-ERK1/2, ERK1/2, phospho-Akt, Akt, phospho- JAK1, JAK1, phospho-STAT1, STAT1, phospho-STAT3, STAT3. Vγ9Vδ2 T cells isolated from BM MM and PB MM showed a remarkable down-modulation of pAKT, a key player of PD-1-mediated signal transduction, and pJAK-1 and pSTAT1, both involved in TIM-3-mediated signaling pathway. Morever, we observed a significantly reduced expression of the transcription factor T-bet, the downstream target gene of pSTAT1, selectively in Vγ9Vδ2 T cells isolated from the tumor site, suggesting that low T-bet expression is a further hallmark of BM Vγ9Vδ2 T cells exhaustion.Altogether, our findings allow to identify potential molecular mechanisms responsible for the poor efficacy of single immune checkpoint blockade in the tumor microenvironment and to design rational combination therapeutic strategy to be applied to the different clinical settings. DisclosuresBoccadoro:Celgene: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Massaia:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Novartis: Research Funding.