Multiple Inhibitory Receptors Research Articles

Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.

TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.

In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. We show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

PD1 and CD39 expression on tumor infiltrating lymphocytes in patients with gastric cancer.

377 Background: Activation and clonal expansion of tumor antigen-specific T cells in cancer patients are crucial for immunotherapies. Recent reports indicate that CD39 is a marker to distinguish tumor-reactive CD8 T cell populations from bystander populations. In gastric cancer (GC), anti-CD39 antibody, anti-PD-1 antibody, and FOLFOX combination therapy may be effective therapy at AACR 2022. Methods: We retrospectively reviewed the data of 62 GC patients who underwent gastrectomy at our hospital (2017 - 2018) and examined their rejected specimen’s immune-microenvironment using immunohistochemistry. Then we prospectively enrolled 16 GC patients. Their tumor tissue and tumor-infiltrating lymphocytes (TILs) were extracted from surgical specimens, these fractions were subjected to the RNA-seq analysis ( n = 3), CD107a degranulation assay ( n = 7) and 3H-Thymidine incorporation assay ( n = 7). Results: The densities of CD39+CD8 TILs positively correlated with the densities of CD4 T cells and B-cells in GC tissues ( P = 0.01, < 0.01, respectively). Furthermore, higher densities of CD39+CD8 TILs in GC patients were associated with better overall survival ( P < 0.01). We classified TILs into four groups based on expression levels of the PD-1 and CD39. The proportion of CD39+CD8 T cells was significantly higher in GC tissues than their PBMCs ( n = 13) and non-cancerous tissues ( n = 4). CD39+PD-1+CD8 (DP) TILs in primary tumors were elevated expression of multiple inhibitory receptors such as LAG3, TIGIT and reduced expression of IL-2 by RNA-seq analysis. T cells expanded in vitro from DP-TILs ( n = 5) efficiently recognized autologous tumor cells in CD107a assay compared with other populations, and considered to be tumor-reactive T cells fraction. However, since these DP TILs in primary tumors show exhausted phenotypes, capacities to kill tumor are expected to be reduced in the tumor-microenvironment (TME). By adding ATP to the DP-TILs in vitro at the concentration reported in the TME, we observed reduction of cell proliferation by 3H-thymidene incorporation assay and of degranulation by CD107a assay, and we observed recovery of these changes by adding anti-CD39 inhibitory antibody. Conclusions: DP-TILs may have superior T cell receptors recognizing tumor-antigens and contribute to tumor eradication, especially when administrated anti-CD39 antibody.

NK Cell Dysfunction in CLL Is Mediated through SHP-1 Signaling and Is Associated with Poor Prognosis

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, presenting with a highly variable clinical course, outcome and response to therapy. This variability has been linked to a complex tumor microenvironment and genetic/epigenetic modifications that lead to immunosuppression. Indeed, the clinical hallmark of this disease is the susceptibility to infections and secondary malignancies. While T cell dysfunction in CLL has been well described, conflicting information exists on NK cell changes in CLL and their role in CLL immunosurveillance. Here, we analyzed samples from 75 untreated (or completed treatment >2 years ago) CLL patients and 30 healthy controls. We discovered a substantial variation in NK cell function compared with healthy controls, which we further classified into hyperfunctional, normofunctional and dysfunctional based on their ability to degranulate, and produce IFN- γ in response to K562 targets ( Figure 1). NK cells from ‘dysfunctional’ CLL samples also displayed impaired cytotoxicity against K562 targets, weaker synapse formation and Syk/Zap70 signaling in response to antibody-dependent cellular cytotoxicity (ADCC) compared to those from ‘functional’ samples. The NK functional status was strongly associated with multiple prognostic markers, including cytogenetics, IgVH mutation status, ZAP-70 expression and disease stage, with patients with dysfunctional NK cells having significantly higher rates of secondary malignancies and viral infections ( Table 1). Using multispectral flow cytometry we discovered high phenotypic heterogeneity with unique NK cell clusters present in the dysfunctional samples. Those clusters express a pattern of activation-induced exhaustion, with upregulation of multiple checkpoint molecules such as PD-1, TIGIT, LAG-3, KLRG-1, TIM-3 but not evidence of terminal differentiation (CD57 negative). Those clusters also showed concomitant expression of activating molecules such as NKG2D and DNAM-1, suggesting reversibility of their dysfunctional status. We subsequently found significantly higher (p<0.0001) constitutive phosphorylation of SHP-1 protein, a phosphatase downstream of multiple inhibitory NK cell receptors, in NK cells from dysfunctional samples. Treatment with a SHP-1 inhibitor NSC-87877 (EMD Millipore) or use of siRNA targeting SHP-1 (ThermoFisher scientific), resulted in reversal of NK dysfunction with full restoration of their functional capacity. Taken together, our data suggest that NK cells functional status plays an important role in CLL disease progression and secondary complications. The novel mechanism of NK cell immune dysfunction in CLL described here has the potential to improve outcomes with novel immunotherapies such as chimeric antigen receptors, checkpoint inhibitors and bi-specific antibody therapy that have shown disappointing results in CLL to date when compared with other B cell malignancies.

Delayed Initiation of Calcineurin Inhibitor Is Critical for Tolerance Induction By Posttransplant Cyclophosphamide

[Introduction] We have previously shown that T-cell exhaustion of donor alloreactive T cells plays a critical role in tolerance induction after mouse allogeneic hematopoietic cell transplantation (allo-HCT) (Asakura S: JCI 2010). Emerging evidence indicates that T-cell exhaustion is a multistep process, including precursors of exhausted T cells (pTex) with stem cell-like properties, pTex-derived transitory exhausted T cells (transitory-Tex) with potent effector-like functions, and terminally exhausted T cells (terminal-Tex) with the expression of multiple inhibitory receptors and severely impaired functions. We have demonstrated, for the first time, that calcineurin inhibitors (CNIs) inhibit tolerance induction by suppressing the terminal exhaustion of donor T cells while inducing transitory-Tex after allo-HCT in mice (Senjo H: Blood 2023). In contrast, post-transplantation cyclophosphamide (PTCy) efficiently induces tolerance with low incidence of chronic GVHD. In the current study, we evaluated the mechanisms by which PTCy could induce tolerance even with the use of CNIs. [Methods] In mouse haploidentical HCT model,B6D2F1 recipients (H-2 b/d) were lethally irradiated and transplanted with 2 × 10 6 T cells together with T-cell-depleted bone marrow cells from C57BL/6 (H-2 b) donors on day 0. CSP (25 mg/kg) was administered from either day 0 (early-CSP) or day 5 (late-CSP), and PTCy (50 mg/kg) was i.p. injected on day 3 after HCT. In clinical study, peripheral blood mononuclear cells were prospectively collected from patients who underwent PTCy-based haplo-PBSCT at our institutions after approval of institutional review. [Results] We have shown that Ly6C is a specific marker for transitory-Tex in mice (Senjo H: Blood 2023). In mice, early-CSP followed by PTCy (CSP→PTCy) significantly expanded Ly6C +PD-1 +TOX low CD8 + T cells and Ly6C +PD-1 +GZMB + CD4 + T cells on day 14, while late-CSP following PTCy (PTCy→CSP) induced T-cell differentiation towards Ly6C -PD-1 +TOX high terminally exhausted T cells. When CSP→PTCy recipients were injected with anti-PD-L1 monoclonal antibodies, transitory-Tex demonstrated vigorous proliferation, while terminal-Tex did not respond to PD-1 blockade. These findings indicate that CSP administration after PTCy promotes tolerance induction, whereas CSP administered before PTCy inhibits tolerance induction. Next, we explored the specific markers for human transitory-Tex using scRNA-seq of T cells from the patients who underwent PTCy haplo-PBSCT, in which tacrolimus (TAC) was started either from day -1 (early-TAC) or day 5 (late-TAC). UMAP analysis showed that early-TAC expanded cluster 5 (C5) out of six CD8 + clusters (C1-C6) and C8 out of three CD4 + clusters (C7-C9) (Fig.1). C5 highly expressed exhaustion markers such as Tox and Pdcd1, together with effector molecules such as Prf1, Gzmb and Cx3cr1, while C8 was characterized with high expression of Gzmb, indicating that C5 and C8 are the human counterparts of transitory-Tex. Analysis of differentially expressed genes showed that Fcgr3a (CD16A) was selectively expressed in C5, while Gzmb and Cd74 were selectively expressed in C8. These two populations showed robust proliferative responses to TCR stimulation in vitro. ScTCR-seq demonstrated that C5 and C8 clusters showed less TCR diversity compared to other clusters, suggesting that C5 and C8 were enriched with alloreactive donor T cells that clonally expanded post-transplant. FCM confirmed that both CX3CR1 +CD16 + CD8 + T cells and GZMB +CD74 +CD4 + T cells were significantly increased in the early-TAC group compared to the late-TAC group on day 28 (Fig. 2). In the early-TAC group, 2/9 patients developed moderate and severe chronic GVHD, and only 1/9 patient was off-immunosuppressants (Fig 2). In sharp contrasts, in the late-TAC group, no patient developed chronic GVHD, and 7/9 patients were off-immunosuppressants. Importantly, two patients who developed chronic GVHD had a particularly high proportions of CX3CR1 +CD16 +CD8 + and GZMB +CD74 +CD4 + transitory-Tex on day 28 after HCT (Fig.2). [Conclusion] Our study demonstrated that early initiation of CNIs before PTCy induced transitory-Tex and inhibited tolerance induction after PTCy haplo-PBSCT. Administration of CNIs after PTCy is critical for tolerance reduction without chronic GVHD.

Five Inhibitory Receptors Display Distinct Vesicular Distributions in Murine T Cells.

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to a persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3, and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct, with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

Single-cell transcriptome analysis reveals T population heterogeneity and functions in tumor microenvironment of colorectal cancer metastases

Cell mediated immune escape, a microenvironment factor, induces tumorigenesis and metastasis. The purpose of this study was to display the characteristics of T cell populations in immune microenvironments for colorectal cancer (CRC) metastasis. Unsupervised cluster analysis was conducted to identify functionally distinct T cell clusters from 3,003 cells in peripheral blood and 4,656 cells in tissues. Subsequently, a total of 8 and 4 distinct T cell population clusters were identified from tumor tissue and peripheral blood, respectively. High levels of CD8+TEX, CD4+TRM, TH1-like T cells, CD8+TEM, tumor-Treg from tissues, and CD4+TN from peripheral blood are essential components of immune microenvironment for the prediction of CRC metastasis. Moreover, exhausted T cells are characterized by higher expression of multiple inhibitory receptors, including PDCD1 and LAG3. Some genes such as PFKFB3, GNLY, circDCUN1D4, TXNIP and NR4A2 in T cells of cluster were statistically different between CRC metastasis and non-metastasis. The ligand-receptor interactions identified between different cluster cells and metastases-related DEGs identified from each cluster revealed that the communications of cells, alterations of functions, and numbers of T subsets may contribute to the metastasis of CRC. The mutation frequency of KiAA1551, ATP8B4 and LNPEP in T cells from tissues and SOR1 from peripheral blood were higher in metastatic CRC than that in non-metastatic CRC. In conclusion, the discovery of differential genes in T cells may provide potential targets for immunotherapy of CRC metastasis and relevant insights into the clinical prediction and prognosis of CRC metastasis.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells

ObjectiveColorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from...

Hierarchical transcriptional network governing heterogeneous T cell exhaustion and its implications for immune checkpoint blockade.

The fundamental principle of immune checkpoint blockade (ICB) is to protect tumor-infiltrating T cells from being exhausted. Despite the remarkable success achieved by ICB treatment, only a small group of patients benefit from it. Characterized by a hypofunctional state with the expression of multiple inhibitory receptors, exhausted T (Tex) cells are a major obstacle in improving ICB. T cell exhaustion is a progressive process which adapts to persistent antigen stimulation in chronic infections and cancers. In this review, we elucidate the heterogeneity of Tex cells and offer new insights into the hierarchical transcriptional regulation of T cell exhaustion. Factors and signaling pathways that induce and promote exhaustion are also summarized. Moreover, we review the epigenetic and metabolic alterations of Tex cells and discuss how PD-1 signaling affects the balance between T cell activation and exhaustion, aiming to provide more therapeutic targets for applications of combinational immunotherapies.

T-cell dysfunction in natural killer/T-cell lymphoma

ABSTRACT Natural killer/T-cell lymphoma (NKTCL) is an incurable aggressive T-cell lymphoma closely correlated with Epstein‒Barr virus (EBV) infection. Chronic and consistent viral infection induces T-cell exhaustion. Herein, we describe T-cell dysfunction in NKTCL patients for the first time. Peripheral blood mononuclear cells (PBMCs) from age-matched healthy donors (HDs) and NKTCL patients were collected, and lymphocyte distributions, multiple surface inhibitory receptors (IRs), effector cytokine production and cell proliferation were determined by flow cytometry. PBMCs from HDs were cocultured with NKTCL cell lines to verify the clinical findings. IR expression was further assessed in NKTCL tumor biopsies using multiplex immunohistochemistry (mIHC). NKTCL patients have higher frequencies than HDs of inhibitory T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs). T-cell distribution also varies between NKTCL patients and HDs. T cells from NKTCL patients demonstrated higher expression levels of multiple IRs than HDs. Meanwhile, T-cell proliferation and interferon-γ production was significantly reduced in NKTCL patients. More importantly, the number of EBV-specific cytotoxic cells was lower in NTKCL patients, and these cells demonstrated upregulation of multiple IRs and secreted fewer effector cytokines. Interestingly, NKTCL cells caused normal PBMCs to acquire T-cell exhaustion phenotypes and induced generation of Tregs and MDSCs. In line with ex vivo finding, mIHC results showed that CD8+ T cells from NKTCL tumor biopsies expressed much higher level of IRs compared with reactive lymphoid hyperplasia individuals. The immune microenvironment of NKTCL patients exhibited T-cell dysfunction and accumulation of inhibitory cell components, which may contribute to impaired antitumor immunity.

Hemophagocytic Lymphohistiocytosis Susceptibility (HLH) Factors IL-18 and Cytotoxic Impairment Converge on Pathologic CD8 T-cell Hyperactivation

Excess IL-18 and graded defects in cytotoxicity are fundamental susceptibility factors for HLH and MAS. In mice, neither excess IL-18 (18tg mice) nor perforin-deficiency individually cause immunopathology without inflammatory challenge. However, 18tg mice lacking perforin (18tg; Prf1-/-) develop spontaneous lethal hyperinflammation, while subclinical MAS was found in 18tg;Prf1+/- mice. HLH and MAS patients demonstrate CD8 T-cell activation in tissue & peripheral blood. We sought to understand how IL-18 and cytotoxic impairment may interact in driving spontaneous hyperinflammation.We examined mice bearing single- and dual-susceptibility factors for “clinical” features of hyperinflammation, cytokine levels, and flow cytometric, transcriptional, functional, and TCR-seq phenotypes.Flow cytometric analysis of dual susceptibility mice showed spontaneous expansion of CD8 T-cells (and not CD4 T- or NK-cells), which expressed the IL-18 receptor and multiple inhibitory receptors (PD-1, Lag-3, CD39), yet overproduced IFNg. Such “hyperinflammatory CD8 T-cells” were present in reticuloendothelial organs (spleen, bone marrow, and liver) but not lymph nodes, peripheral blood, or thymus. Transcriptional analysis demonstrated hyperactivated CD8 T cells showed features of both terminal effector function and exhaustion when compared to well-described datasets. This analysis also revealed a strong signature for recent antigen activation. BulkCD8 TCR sequencing showed dramatic oligoclonal hyper-expansion of TCR sequences from dual susceptibility mice with no significant clonal overlap between biologic replicate samples. In vitro, TCR stimulation was necessary for IL-18 to promote IFNg production and restimulation-induced cell death (RICD), whereas perforin-deficiency prevented RICD. Broad-spectrum antibiotics did not rescue spontaneous hyperinflammation in dual-susceptibility mice, suggesting T-cell responses were not directed at commensal bacteria. Attempts to circumvent CD8 T-cell activation, using inducible CD8 T-cell-specific deletion of Il18r1 or by promoting allelic exclusion by fixing the T-cell receptor, showed breakthrough expansion of hyperactivated CD8 T-cells.Thus, IL-18 and cytotoxicity drive progressive, post-thymic, oligoclonal CD8 T-cell expansion that may underly life-threatening immunopathology in HLH and MAS. Expansion of hyperactivated CD8T cells in reticuloendothelial organs aligns with sites of hemophagocytosis. These data together implicate HLH/MAS susceptibility factors interact with TCR stimulation, whether by infectious triggers or as-yet undetermined self antigens, to drive potentially-targetable mechanisms of T-cell activation. [Display omitted] Supporting figures.

ITK inhibition improves the response to immune checkpoint blockade in solid tumors

Abstract Cytotoxic CD8+ T cell (CTL) exhaustion is driven by persistent T cell receptor (TCR) stimulation in chronic infections and tumors. The exhausted state of T cells is characterized by impaired effector function and proliferative capacity, upregulation of multiple inhibitory receptors and altered transcriptional profiles. The phenotypic and epigenetic changes of these cells hinder effective antitumor immunity and immunotherapy. Therefore, reinvigorating exhausted T cells may provide significant clinical benefits to patients with cancer. Our approach is to reduce chronic TCR stimulation by inhibiting IL2-inducible T-cell kinase (ITK), a pivotal component of TCR signaling. In our study, we found that intermittent ITK inhibitor treatment significantly improved ICB response in three immune checkpoint blockade (ICB) -resistant solid tumors (melanoma, mesothelioma or pancreatic cancer) in vivo. In vitro, we demonstrated that specific ITK inhibitor and ibrutinib, an inhibitor of Bruton’s tyrosine kinase (BTK) and ITK, directly reversed the exhaustion-related phenotypes in exhausted CTLs. The changes included enhanced cytokine production, decreased inhibitory receptor expression and changes in transcription factors. Subsequently, RNA-seq data revealed that exhaustion-related transcriptional profile of in vitro CTLs is reduced by ibrutinib treatment. Ibrutinib mediated reversal of CTL exhaustion was BTK independent. To conclude, our study demonstrates that ITK inhibition can be used to directly ameliorate CTLs exhaustion and overcomes immune checkpoint blockade resistance in solid tumors. This work was supported by the department of Immunology, KWF Grant 128371 (PDK), in part from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779295 (PDK), and by the China Scholarship Council for funding PhD fellowships (No. 201506160120 for MZ and 201906210055 for LL).

Blockade of CCR4 breaks immune tolerance in chronic hepatitis B patients by modulating regulatory pathways

BackgroundImmunotargets including checkpoint inhibitors and toll-like receptor 8 agonists have recently gained attention for the recovery of hepatitis B virus (HBV)-specific T cell exhaustion in chronic hepatitis B(CHB). Chemokine receptors have a similar significant role during viral infections; however, their role in CHB remains poorly understood. Therefore, in this study we evaluated the role of chemokine receptor 4 (CCR4) in deriving immunosuppression during CHB.MethodsWe characterized CCR4+CD8+ T cells in CHB and identified their involvement in immunosuppression. Further, we examined if CCR4 blockade with mogamulizumab antibody can recover the functional exhaustion in HBsAg-specific T cells.ResultsCHB patients exhibit higher frequency of CCR4+CD8+ T cells that increase with higher HBsAg levels and fibrosis scores. In vitro, HBs antigen triggers CCR4 expression. These cells express multiple inhibitory receptors and exhibit immunosuppressive functions by producing excessive immunoregulatory cytokines IL-4, IL-5, IL-10 and TGF-β1. CCR4 Blockade significantly boosted HBsAg-specific antiviral-cytokine production(IFN-γ, TNF-α and IL-21) in T cells through enhancing their proliferation capacity and polarizing these cells towards T helper 1(Th1) and T follicular helper cells(TFH) in case of CD4 cells, and cytotoxic T cell 1(TC1) and cytotoxic T follicular(TCF) cells in case of CD8. Cytotoxic potential was improved, while no induction of immunosuppressive-cytokines was seen after anti-CCR4 treatment thereby eliminating the risk of treatment-induced immunosuppression. CCR4 blockade inhibited the development and effector function of Tregs by controlling their expansion and TGF-β1 production preventing Tregs-induced immunotolearance.ConclusionsCCR4 blockade reconstitutes antiviral immune response in T cells and limits the immunosuppressive functions of Tregs, representing them as a promising immunotherapeutic target for functional cure of CHB.

Abstract 1259: Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor microenvironment

Abstract Background: Overexpression of sialic acids on glycans, called hypersialylation is a common alteration in cancer that drives immune evasion via interaction with Sialic acid-binding immunoglobulin-like lectin (Siglec) immunoregulatory receptors on tumor-infiltrating immune cells. Myeloid derived suppressor cells (MDSCs) generated by cancer-induced aberrant myelopoiesis are highly immunosuppressive cells inhibiting immune cells via direct interaction or secretion of suppressive cytokines. Targeting the complex suppressive tumor microenvironment (TME) remains a challenge. Methods: Siglec expression of murine and human MDSCs in healthy conditions and tumor setting was assessed by flow cytometry. Functional analysis of Siglec-E knockout on MDSCs in mice was evaluated using SigE-LysMCre mice and suppressive capacity was tested in vitro in combination with SiglecE blocking and cleavage of Sialoglycans using Sialidase. Results were confirmed in the human setting using an in vitro assay to generate MDSC-like cells including RNA-Sequencing and a MDSC suppression assay with cancer-derived MDSCs. Results: MDSCs express multiple inhibitory Siglec receptors in humans and mice. Knockout of SiglecE in the myeloid compartment in vivo resulted in prolonged survival and increased T cell infiltration compared to litter mates expressing Siglec-E. Of note, desialylation of MDSCs and Siglec-E blocking in vitro reduced MDSCs suppressive function against T cells. Similarly, reducing Sialoglycans by Sialidase in human MDSC generated in vitro resulted in increased suppressive capacity and downregulation of functional MDSC markers on RNA level. Findings were validated with lung cancer patient derived MDSCs. Conclusion: Our results provide first insights into the importance of the Siglec-Sialoglycans axis to modulate MDSCs in the TME. Targeting glycosylation of MDSCs with Sialidase reduces their suppressive capacity making it a powerful approach to improve cancer immunotherapy. Further studies are needed to reveal the underlying mechanisms. Citation Format: Ronja Wieboldt, Andreas Zingg, Emanuele Carlini, Anastasiya Börsch, Heinz Läubli, Natalia Rodrigues Manutano. Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1259.

Abstract 2909: Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma

Abstract In this study we test if knockout of DNAM-1 competing inhibitory receptors on Natural Killer (NK) cells improves cytotoxic response against neuroblastoma cell lines. Neuroblastoma and other peripheral nervous cell tumors account for 6% of all pediatric cancer. While the 5-year survival rate has greatly improved, the prognosis of children with high-risk neuroblastoma at diagnosis is less than 50%. Immune checkpoint therapy is a promising new application for pediatric oncology; however, neuroblastoma is often immunologically “cold” and current immune checkpoint targets are not abundant in pediatric solid tumors. To improve response to checkpoint blockade and directly target tumor cells for elimination, this study evaluated the use of NK cells. NK cells prime the immune system for successful response to cancer immunotherapy. NK cells activated with particle technology (PM21-NK cells) are highly cytotoxic, produce IFNγ and induce PD-L1 on cancer cells. Although highly cytotoxic, PM21-NK cells express inhibitory receptors that compete for ligands that trigger NK cell lysis, such as PVR, a ligand highly expressed on neuroblastoma cells. We knocked out expression of multiple PVR inhibitory receptors in PM21-NK cells to further enhance cytotoxicity and prevent exhaustion. Three inhibitory receptors on NK cells that bind PVR - TIGIT, CD96, and PVRIG were knocked out alone or in combination and effector functions of these edited NK cells tested. Cytotoxicity against 3D spheroids of the neuroblastoma cell line SK-NA-S cells was assessed and compared to wild type PM21-NK cells. Knockout (KO) of TIGIT enhanced NK cell killing compared to wildtype NK cells (72%±10 vs 55%±3% killing) as well as PVIRG KO (84±3% vs 46±6% killing) and CD96 KO (62%±9% vs 46±6% killing) at a 1:3 E:T ratio at 72 h. Double or triple KO of these receptors resulted in further increased killing of cancer cells. The knockouts of each of the receptors could affect cytokine production and cytokine responsiveness and this will be examined. Overall, PM21-NK cells show promise for therapeutic use against neuroblastoma and preventing the PVR-mediated inhibitory axis signaling enhances their function. Citation Format: Tayler J. Croom-Perez, Jeremiah L. Oyer, Md Faqrul Hasan, Liza D. Robles-Carrillo, Brian P. Tullius, Alicja J. Copik. Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2909.

Abstract 902: Harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells

Abstract Colorectal cancer (CRC) is the 2nd cause of cancer-related death. Standard therapies often fail, with more than 50% of patients experiencing relapse, eventually with metastatic disease. Colorectal tumors are densely infiltrated by immune cells that have a role in surveillance and modulation of tumor progression. However, exhaustion mechanisms acting within the tumor microenvironment impede their functional capacity against tumor cells. We paired high-dimensional flow cytometry, RNA sequencing, immunohistochemistry and immunofluorescence to describe the T cell functional landscape in tumor and peritumoral tissues from primary colorectal cancers and liver metastases. Analysis of the healthy, peritumoral and neoplastic tissues of treatment-naïve primary CRCs and of the peritumoral and tumoral tissues of CRC patients undergoing surgery for liver metastasis, revealed extensive transcriptional and spatial remodeling across tumors, being metabolic pathways among the major drivers of this variance. Regarding the immune infiltrate, we found that T cells are mainly localized at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors. Unsupervised analysis of flow cytometry data performed by an advanced pipeline of data handling by dimensionality reduction and clustering algorithms allowed the definition of a peculiar inhibitory receptors signature in TILs enriched both in primary CRCs and liver metastases. Among the highly co-expressed inhibitory receptors, CD39 was found to represent the major driver of exhaustion in both primary and metastatic colorectal tumors. CD39 is a diphosphohydrolase converting ATP into AMP that is emerging as exhaustion marker for tumor-specific T cells, thus highlighting its relevance as molecular target for T cells engineering. We leveraged on these findings to generate a novel cellular product for the adoptive cell therapy of CRC. By CRISPR/Cas9 genome editing tools, we simultaneously redirected T cell specificity by disrupting the alpha and beta genes of the endogenous T cell receptor with 90% efficiency for both genes, and disrupted CD39 with 100% efficiency, generating triple-knockout engineered lymphocytes. By lentiviral transduction, we redirected the specificity of our engineered T cell product employing a novel T-cell receptor targeting the HER-2 antigen. Triple-edited, HER2-redirected T cells were challenged in vitro against HER2+ patient-derived organoids from liver metastases (mPDOs). Measurement of Cas3/7 displayed a functional advantage for CD39-disrupted, HER2-redirected T cells in recognizing and killing mPDOs. We then evaluated the efficacy of our T cell product in two different in vivo models: subcutaneous injection of mPDOs, and intra-hepatic injection of mPDOs. In both models, CD39-disrupted, HER2-redirected T cells displayed a superior capacity of controlling tumor outgrowth long term. Citation Format: Alessia Potenza, Chiara Balestrieri, Luca Albarello, Federica Pedica, Martina Spiga, Francesco Manfredi, Beatrice C. Cianciotti, Claudia De Lalla, Lorena Stasi, Elena Tassi, Silvia Bonfiglio, Giulia M. Scotti, Miriam Redegalli, Donatella Biancolini, Danilo Abbati, Fabio Simeoni, Dejan Lazarevic, Ugo Elmore, Guido Fiorentini, Giulia Di Lullo, Giulia Casorati, Claudio Doglioni, Giovanni Tonon, Paolo Dellabona, Riccardo Rosati, Luca Aldrighetti, Eliana Ruggiero, Chiara Bonini. Harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 902.

Increased co-expression of TIM-3 with TIGIT or 2B4 on CD8+ T cells is associated with poor prognosis in locally advanced nasopharyngeal carcinoma.

The use of immune checkpoint inhibitors in malignant tumors improves patient outcomes. Because single-agent immune checkpoint blockade has a low objective response rate, it is meaningful to explore combined blockade of immune checkpoint receptors. We aimed to investigate the co-expression of TIM-3 with TIGIT or 2B4 on peripheral blood CD8+ T cells from patients with locally advanced nasopharyngeal carcinoma. The correlation between co-expression level and clinical characteristics and prognosis was studied to provide a basis for immunotherapy for nasopharyngeal carcinoma. Flow cytometry was used to detect TIM-3/TIGIT and TIM-3/2B4 co-expression on CD8+ T cells. The differences in co-expression between patients and healthy controls were analyzed. The correlation between co-expression of TIM-3/TIGIT or TIM-3/2B4 and the patient clinical characteristics and prognosis was examined. Also, the correlation between the TIM-3/TIGIT or 2B4 co-expression and other common inhibitory receptors was analyzed. We further validated our results using mRNA data from the Gene Expression Omnibus (GEO) database. TIM-3/TIGIT and TIM-3/2B4 co-expression was upregulated on peripheral blood CD8+ T cells from patients with nasopharyngeal carcinoma. They were both correlated with poor prognosis. There was a correlation between TIM-3/TIGIT co-expression and patient age and pathological stage, whereas TIM-3/2B4 co-expression correlated with age and sex. CD8+ T cells with elevated mRNA levels of TIM3/TIGIT and TIM3/2B4 also showed increased expression of multiple inhibitory receptors, indicating T cell exhaustion in locally advanced nasopharyngeal carcinoma. TIM-3/TIGIT or TIM-3/2B4 can be used as potential targets for combination immunotherapy in locally advanced nasopharyngeal carcinoma.

Single-Cell Multi-Omics Reveals Immune Microenvironment Alterations in T-Cell Acute Lymphoblastic Leukemia

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that predominantly occurs in children and young adults. Although intensified multi-agent chemotherapy improved overall survival rates, relapsed/refractory T-ALL remains difficult to treat. In addition, these intensive chemotherapies carry a risk for short- and long-term side effects, especially in children. There is an urgent need for novel therapeutic strategies to improve patient outcome. Targeting the tumor microenvironment has been recognized as a promising strategy in many adult tumors with high neoantigen load, but it is thought to be less effective in pediatric and hematologic malignancies as these carry few genetic mutations. However, comprehensive studies of the immune microenvironment are lacking in many pediatric malignancies, including T-ALL, but are urgently required to identify novel therapeutic targets. The aim of this study is to unravel mechanisms of immune evasion in T-ALL and to establish novel immunotherapeutic targets for the treatment of T-ALL. Methods We performed 5’ single cell transcriptome, surface proteome of ~200 proteins and immune repertoire analyses of ~54,000 cells from 10 primary T-ALL pediatric patient samples at initial diagnosis and 4 normal donors using the 10x Genomics platform. Sequencing data was processed using Cell Ranger (reference genome hg38). Quality control was performed using Scrublet (doublet removal), DropletQC (quality control damaged cells) and Seurat (percentage mtDNA < 10% and genes expressed > 200). RNA counts were normalized with sctransform and surface protein antibody counts were centered log-ratio normalized using Seurat. Non-linear dimensional reduction (UMAP) and differential gene/protein expression was done using Seurat and cell type annotation was done using Azimuth in combination with known marker genes. In addition, we analyzed differential cell type abundance (MiloR), the immune repertoire (Immunarch), transcription factor activity (pyScenic) and ligand-receptor activity (NicheNet). Results Our multi-omics analysis revealed changes in the immune cell type composition in T-ALL associated with an immunosuppressive microenvironment. Specifically, we identified a significant enrichment of non-classical monocytes, atypical memory B-cells and double-negative T-cells (CD3+, CD4-, CD8-) in the T-ALL immune microenvironment compared to the normal immune microenvironment. These CD21-, CD11c+ atypical memory B-cells showed high expression of multiple inhibitory receptors (including PD-1, FCRL3/5 and ILT2), suggesting muted BCR-signaling. Transcription factor analysis showed transcriptional activity of known B-cell regulators SOX5, TBX21 and POU2F2, of which SOX5 is known to be highly expressed during late B-cell differentiation stages including atypical memory B-cells. Double-negative (DN) T-cells, which were oligoclonal, were also overrepresented in the T-ALL microenvironment. Correspondingly, immune repertoire analysis of normal T-cells showed decreased TCR diversity (Chao1 index, p = 0.016, Wilcoxon rank sum test) compared to normal donors. Surprisingly, these DN T-cells displayed features of CD8+ T-cell dysfunction, such as high expression of PD-1, TIM3 and LAG3, as well as features of regulatory T-cells, such as high expression of IL10, ICOS and CTLA4. These cells also demonstrated high transcription factor activity of EOMES, NFATC1 and ETV1, which are all associated with a CD8+ T-cell dysfunctional transcriptional network, as well as FOXP3 transcription factor activity, the master transcription factor in regulatory T-cells. Conclusions Multi-omics profiling of the immune microenvironment in T-ALL revealed the presence of several populations of dysfunctional cells associated with an immunosuppressive microenvironment. Studying ligand-receptor interactions analysis between malignant and immune cells will provide the basis for future (pre)clinical studies leading to the development of novel immunotherapeutic strategies for the treatment of T-ALL.

Peripheral Blood Cytotoxic T Cells from Myelofibrosis Patients Show Early Exhausted Features Targetable By CTLA-4 Inhibition

Introduction: Myelofibrosis (MF), either primary or secondary, belongs to Philadelphia-negative myeloproliferative neoplasms (MPN) and is characterized by bone marrow fibrosis, hematopoietic stem cell mobilization and extramedullary hematopoiesis. Compelling evidence are suggesting that persistent antigen stimulation in the tumor microenvironment can differentiate T effector cells into terminally exhausted T cells, a functional state characterized by decrease in proliferation, cytotoxicity, and cytokine production. Innovative therapies aim at reversing the exhausted state through immune checkpoint inhibition to recover the immune response against the tumor. Even in myeloid neoplasms such as MPNs, T cells are showing dysfunctional features; thus we investigated T cell exhaustion in MF to assess the possibility of immune checkpoint inhibition as a new effective therapy. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 44 MF patients and 22 healthy donors (HD). CD3+CD8+ T cells were characterized by multicolor flowcytometry to assess surface expression of the following inhibitory receptors (IRs): PD-1, CD244, CD160, TIM-3, LAG-3, CTLA-4. Results were correlated with clinical features such as Dynamic International Prognostic Scoring System (DIPSS) classification, splenomegaly, high-molecular risk (HMR) mutation number and other negative prognostic factors. We performed intracellular flowcytometry staining to assess residual cytokine production in PBMCs stimulated overnight with anti-CD3, anti-CD28 and Brefeldin A. Total cellular RNA was isolated from granulocytes of 141 primary and secondary MF patients and 28 HDs, and we performed Gene expression profiling (GEPs) by microarray platform to evaluate IRs-ligands expression. To test the possible inhibitory effect of the neoplastic clone we cultured T cells either alone or in co-culture with the myeloid cells for 4 days in the presence of anti-CD3/CD28 stimulatory beads, SCF, TPO, FLT3l, IL6, IL7 and IL15 and then we tested the activation marker OX40 expression level. The co-culture system was also used to test the effect of CTLA-4 inhibition. Results: We observed increased expressions of PD-1, CD244, CTLA-4 and CD160 on cytotoxic T cells from MF patients together with the expansion of PD-1+CD57- cytotoxic T cell populations (Panel A), which also showed increased expression of all the IRs assessed, with CD160 and CTLA-4 being the more significantly upregulated. Higher levels of PD-1, CD244, CD160, CTLA-4, LAG-3 or TIM-3 on cytotoxic T cells correlate with detrimental features like splenomegaly, low hemoglobin levels, HMR mutation number and more severe DIPSS classification. Moreover, we observed decreased secretion of IFNɣ and TNFɑ by total CD3+CD8+ T cells and PD1+ subpopulation from MF patients (Panel B). A significant reduction of Granzyme B production was observed mainly in patients with expansion of PD1+CD57- exhausted T cells. GEPs analysis highlighted increased expression of IR-ligands like CD274, CEACAM1, CD48, CD80 on granulocytes from MF patients compared to HDs. While cytotoxic T cells from HDs were effectively activated either alone or in co-culture with myeloid cells, the activation of cytotoxic T cells from MF patients was damped in presence of myeloid cells and restored when T cells were cultured alone. Treatment with anti-CTLA-4 was able to partially restore the MF T cells activation potential in the presence of myeloid cells. Conclusions: Our data evidenced in MF patients the presence of an impaired population of peripheral blood cytotoxic T cells expressing multiple IRs and with reduced cytokine production after in vitro activation. Clinical correlations suggest a more severe disease in patients with higher IRs expression on cytotoxic T cells. Moreover, granulocytes from MF patients display higher level of IR-ligands transcripts suggesting an immunosuppressive interplay between the myeloid neoplastic clone and T cells. This was also suggested by reduced activation level of T cells in co-culture with myeloid cells that was restored by removing of myeloid cells or with anti-CTLA-4 treatment. Taken together this data highlights an exhausted state likely implicated in immune escape that could be targeted and potentially reverted by immune checkpoint inhibitions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel targets for immunotherapy associated with exhausted CD8 + T cells in cancer.

In response to prolonged stimulation by tumour antigens, T cells gradually become exhausted. There is growing evidence that exhausted T cells not only lose their potent effector functions but also express multiple inhibitory receptors. Checkpoint blockade (CPB) therapy can improve cancer by reactivating exhausted effector cell function, leading to durable clinical responses, but further improvements are needed given the limited number of patients who benefit from treatment, even with autoimmune complications. Here, we suggest, based on recent advances that tumour antigens are the primary culprits of exhaustion, followed by some immune cells and cytokines that also play an accomplice role in the exhaustion process, and we also propose that chronic stress-induced hypoxia and hormones also play an important role in promoting T-cell exhaustion. Understanding the classification of exhausted CD8+ T-cell subpopulations and their functions is important for the effectiveness of immune checkpoint blockade therapies. We mapped the differentiation of T-cell exhausted subpopulations by changes in transcription factors, indicating that T-cell exhaustion is a dynamic developmental process. Finally, we summarized the novel immune checkpoints associated with depletion in recent years and combined them with bioinformatics to construct a web of exhaustion-related immune checkpoints with the aim of finding novel therapeutic targets associated with T-cell exhaustion in malignant tumours, aiming to revive the killing ability of exhausted T cells and restore anti-tumour immunity through combined targeted immunotherapy.