Immune Checkpoint Receptors PD-1 Research Articles

PD-1 instructs a tumor-suppressive metabolic program that restricts glycolysis and restrains AP-1 activity in T cell lymphoma

The PDCD1-encoded immune checkpoint receptor PD-1 is a key tumor suppressor in T cells that is recurrently inactivated in T cell non-Hodgkin lymphomas (T-NHLs). The highest frequencies of PDCD1 deletions are detected in advanced disease, predicting inferior prognosis. However, the tumor-suppressive mechanisms of PD-1 signaling remain unknown. Here, using tractable mouse models for T-NHL and primary patient samples, we demonstrate that PD-1 signaling suppresses T cell malignancy by restricting glycolytic energy and acetyl coenzyme A (CoA) production. In addition, PD-1 inactivation enforces ATP citrate lyase (ACLY) activity, which generates extramitochondrial acetyl-CoA for histone acetylation to enable hyperactivity of activating protein 1 (AP-1) transcription factors. Conversely, pharmacological ACLY inhibition impedes aberrant AP-1 signaling in PD-1-deficient T-NHLs and is toxic to these cancers. Our data uncover genotype-specific vulnerabilities in PDCD1-mutated T-NHL and identify PD-1 as regulator of AP-1 activity.

Multiplex serum immune profiling reveals circulating LAG-3 is associated with improved patient survival in high grade serous ovarian cancer

ObjectiveHigh grade serous ovarian cancer (HGSOC) exhibits low response rates to clinically available immunotherapies. Nevertheless, emerging research has demonstrated that certain immune factors are predictive for HGSOC patient clinical outcomes, with our own groups previous work demonstrating that intratumoral levels of the immune checkpoint receptor LAG-3 is associated with improved patient survival. In this current study we sought to uncover non-invasive circulating immune prognostic and predictive signatures in HGSOC. MethodsA multiplex approach was employed that examined circulating levels of immune checkpoint receptors LAG-3 and PD-1 along with 48 common cytokine and chemokines in a cohort of 75 HGSOC treatment naïve patient serum samples. ResultsElevated serum LAG-3 was significantly associated with improved progression-free survival (PFS) and overall survival (OS) in HGSOC, while circulating PD-1 levels were largely unrelated with patient clinical outcomes. Cytokine and chemokine analysis revealed lower IL-15 expression correlated with improved PFS and OS, while increased IL-1α, IL-1Ra, IL-6, IL8 and VEGF were significantly associated with preoperative CA-125 levels. ROC analysis demonstrated that serum LAG-3 levels exhibited consistent reasonable predictability as a single agent. ConclusionsSerum-derived LAG-3 was identified out of a diverse array of chemokine and cytokines as the immune-based factor most significantly associated with improved HGSOC survival. These findings suggest that LAG-3 could be implemented as a non-invasive patient predictive marker for improved HGSOC clinical outcomes.

Assessment of neutrophil subsets and immune checkpoint inhibitor expressions on T lymphocytes in liver transplantation: A preliminary study beyond the neutrophil-lymphocyte ratio.

Background: Advanced stages of cirrhosis are characterized by the occurrence of progressive immune alterations known as CAID (Cirrhosis Associated Immune Dysfunction). In advanced cirrhosis, liver transplantation (LT) remains the only curative treatment. Sepsis, shares many similarities with decompensated cirrhosis in terms of immuno-inflammatory response. In both conditions, the neutrophil-lymphocyte ratio (NLR) is associated with poor outcomes. Based on alterations in sepsis, we hypothesized that we could observe in cirrhotic and LT patients more detailed neutrophil and lymphocyte phenotypes. To this end, along with leukocyte count, we assessed immature neutrophils, LOX-1+ MDSC and PD-1 and TIM-3 lymphocyte expressions in cirrhotic patients before transplantation in association with liver disease severity and during the first month after transplantation. Methods: We conducted a prospective monocentric study including cirrhotic patients registered on LT waiting-list. Blood samples were collected at enrolment before LT and for 1month post-LT. In addition to NLR, we assessed by whole blood flow cytometry the absolute count of immature neutrophils and LOX-1+ MDSC as well as the expressions of immune checkpoint receptors PD-1 and TIM-3 on T lymphocytes. Results: We included 15 healthy volunteers (HV) and 28 patients. LT was performed for 13 patients. Pre-LT patients presented with a higher NLR compared to HV and NLR was associated with cirrhosis severity. Increased immature neutrophils and LOX-1+ MDSC counts were observed in the most severe patients. These alterations were mainly associated with acute decompensation of cirrhosis. PD-1 and TIM-3 expressions on T lymphocytes were not different between patients and HV. Post-LT immune alterations were dominated by a transitory but tremendous increase of NLR and immature neutrophils during the first days post-LT. Then, immune checkpoint receptors and LOX-1+ MDSC tended to be overexpressed by the second week after surgery. Conclusion: The present study showed that NLR, immature neutrophils and LOX-1+ MDSC counts along with T lymphocyte count and checkpoint inhibitor expression were altered in cirrhotic patients before and after LT. These data illustrate the potential interest of immune monitoring of cirrhotic patients in the context of LT in order to better define risk of sepsis. For this purpose, larger cohorts of patients are now necessary in order to move forward a more personalised care of LT patients.

Proximity proteome mapping reveals PD-L1-dependent pathways disrupted by anti-PD-L1 antibody specifically in EGFR-mutant lung cancer cells

BackgroundPD-L1, a transmembrane ligand for immune checkpoint receptor PD1, has been successfully targeted to activate an anti-tumor immune response in a variety of solid tumors, including non-small cell lung cancer (NSCLC). Despite the success of targeting PD-L1, only about 20% of patients achieve a durable response. The reasons for the heterogeneity in response are not understood, although some molecular subtypes (e.g., mutant EGF receptor tumors) are generally poor responders. Although PD-L1 is best characterized as a transmembrane PD1 ligand, the emerging view is that PD-L1 has functions independent of activating PD1 signaling. It is not known whether these cell-intrinsic functions of PD-L1 are shared among non-transformed and transformed cells, if they vary among cancer molecular subtypes, or if they are impacted by anti-PD-L1 therapy.MethodsHere we use quantitative microscopy techniques and APEX2 proximity mapping to describe the behavior of PD-L1 and to identify PD-L1's proximal proteome in human lung epithelial cells.ResultsOur data reveal growth factor control of PD-L1 recycling as a mechanism for acute and reversible regulation of PD-L1 density on the plasma membrane. In addition, we describe novel PD-L1 biology restricted to mutant EGFR cells. Anti-PD-L1 antibody treatment of mutant EGFR cells perturbs cell intrinsic PD-L1 functions, leading to reduced cell migration, increased half-life of EGFR and increased extracellular vesicle biogenesis, whereas anti-PD-L1 antibody does not induce these changes in wild type EGFR cells.ConclusionsGrowth factor acute regulation of PD-L1 trafficking, by contributing to the control of plasma membrane density, might contribute to the regulation of PD-L1's immune checkpoint activity, whereas the specific effects of anti-PD-L1 on mutant EGFR cells might contribute to the poor anti-PD-L1 response of mutant EGFR tumors.5zzQ_mo5e1oawe5DznU6q-Video

NMR Methods to Study the Dynamics of SH2 Domain-Phosphopeptide Complexes.

Nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying the dynamics of biological macromolecules in solution. By exploiting the intricate interplay between the effects of protein motion (both overall rotational diffusion and internal mobility) and nuclear spin relaxation, NMR allows molecular motion to be probed at atomic resolution over a wide range of timescales, including picosecond (bond vibrations and methyl-group rotations), nanosecond (loop motions and rotational diffusion), and microsecond-millisecond (ligand binding, allostery). In this chapter, we describe different NMR pulse schemes (R1, R1ρ, heteronuclear NOE, and CPMG relaxation dispersion) to characterize the dynamics of SH2 domains. As an example, we use the N-SH2 domain of protein tyrosine phosphatase SHP2 in complex with two phosphopeptides derived from immune checkpoint receptor PD-1 (ITIM and ITSM).

Deletion of PD-1 destabilizes the lineage identity and metabolic fitness of tumor-infiltrating regulatory T cells.

Regulatory T (Treg) cells have an immunosuppressive function and highly express the immune checkpoint receptor PD-1 in the tumor microenvironment; however, the function of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Here, we showed that conditional deletion of PD-1 in Treg cells delayed tumor progression. In Pdcd1fl/flFoxp3eGFP-Cre-ERT2(+/-) mice, in which both PD-1-expressing and PD-1-deficient Treg cells coexisted in the same tissue environment, conditional deletion of PD-1 in Treg cells resulted in impairment of the proliferative and suppressive capacity of TI Treg cells. PD-1 antibody therapy reduced the TI Treg cell numbers, but did not directly restore the cytokine production of TI CD8+ T cells in TC-1 lung cancer. Single-cell analysis indicated that PD-1 signaling promoted lipid metabolism, proliferation and suppressive pathways in TI Treg cells. These results suggest that PD-1 ablation or inhibition can enhance antitumor immunity by weakening Treg cell lineage stability and metabolic fitness in the tumor microenvironment.

High co-expression of immune checkpoint receptors PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT on tumor-infiltrating lymphocytes in early-stage breast cancer

High expression of immune checkpoint receptors (ICRs) in the tumor microenvironment regulates the anti-tumor response. In this study, the differential expressions of ICRs on tumor-infiltrating lymphocytes (TILs) in patients with early-stage breast cancer were investigated.The study included 32 patients who underwent surgery with a diagnosis of early-stage breast cancer between September 2018 and March 2020. TIL isolation was performed using a MACS tumor separation device and tumor separation kit. PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT expression of cytotoxic T and natural killer (NK) cells on TILs and peripheral blood lymphocytes (PBLs) were determined by flow cytometry.Patients with a high Ki-67 index, high TIL density, and HER-2 positivity were more likely to have increased CD16+CD56dim NK cells on TILs. Patients with T2 tumors were more likely to have increased expression of PD-1, LAG-3, and TIGIT on tumor-infiltrating CD8+ cytotoxic T cells than those with T1 tumors. PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3 expression of CD8+ T and CD16-CD56bright NK cells in TILs showed significant positive correlations with each other. PD1+CD8+, TIGIT+CD16+, and CTLA-4+CD56+ cells in PBLs and TILs were found to be negatively correlated, whereas only TIM-3+ expression of CD8+ T and CD16+CD56dim cells in PBLs and TILs showed positive correlations.Our results suggest that CD16+CD56dim NK cells on TILs may play a major role in the immune response against HER2-positive or highly proliferating breast tumors in patients with early-stage breast cancer. Furthermore, various ICRs were found to be highly co-expressed with each other on TILs, including PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT. These receptors may synergistically suppress the response to the tumor, which may trigger immune escape mechanisms in the early stage of carcinogenesis. However, ICR expressions other than TIM3 on PBLs were not found to accompany their counterparts on TILs.

Natural killer cell profiles in recurrent pregnancy loss: Increased expression and positive associations with TACTILE and LILRB1.

NK cells are important for healthy pregnancy and aberrant phenotypes or effector functions have been associated with RPL. We compared expression of a broad panel of NK cell receptors, including immune checkpoint receptors, and investigated their clinical association with RPL as this might improve patient stratification and prediction of RPL. Peripheral blood mononuclear cells were isolated from 52 women with RPL and from 2 women with an uncomplicated pregnancy for flowcytometric analysis and plasma was used to determine anti-CMV IgG antibodies. Between RPL and controls, we observed no difference in frequencies of T-, NKT or NK cells, in CD56dimCD16+ or CD56brightCD16- NK cell subsets or in the expression of KIRs, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46 or DNAM1. NK cells from women with RPL had a higher expression of LILRB1 and TACTILE and this was associated with the number of losses. The immune checkpoint receptors PD1, TIM3 and LAG3 were not expressed on peripheral blood NK cells. In RPL patients, there was a large variation in NKG2C expression and higher levels could be explained by CMV seropositivity. Our study identified LILRB1 and TACTILE as NK cell receptors associated with RPL. Moreover, we provide first support for the potential role of CMV in RPL via its impact on the NK cell compartment. Thereby our study could guide future studies to confirm the clinical association of LILRB1, TACTILE and NKG2C with RPL in a larger cohort and to explore their functional relevance in reproductive success.

Effect of new strains of rotaviruses on PD1 and PD-L1 expression on peripheral blood T cells.

e14539 Background: Promising oncolytic viruses are evaluated, as a rule, by their direct cytotoxic effect on cancer cells. However, the positive effect of cancer virotherapy may be associated with an effect on the immune system, in particular, on the expression of PD-1 and PD-L1 on lymphocytes. The aim of the study was to evaluate the effect of unclassified non-pathogenic strains of rotaviruses RVK100 and RVK228 on the expression of PD-1 and PD-L1 on peripheral blood T-cells of breast cancer patients. Methods: PBMC were cultured at 106 cells/ml cell density in RPMI 1640 medium (Gibco, USA) without the addition of serum at 37°C in an atmosphere containing 5.0% CO2. Four experimental variants were established - 1) negative control without the addition of viruses, 2) positive control of activation with phytohemagglutinin (PHA) addition, 3) addition of RVK100 strain in concentration 107 particles/ml, 4) addition of RVK228 strain in concentration 107 particles/ml. After 24 and 72 hours of incubation, the expression of PD-1 (CD279) and PD-L1 (CD274) on T cells was determined by flow cytometry. Results: After 24 hours of cultivation, an increase in PD-1 expression on CD4+ cells was observed in samples with addition of PHA - 40.5%, RVK100 - 42.3% and RVK228 - 37.5% compared with the control (18.1%). A similar, although less pronounced increase in PD-1 expression, was observed on CD8+ cells: PHA - 41.7%, RVK100 - 46.4%, RVK228 - 42.6% compared with 27.7% in the control. Expression of PD-L1 on CD4+ cells increased to 67.0% and 58.6% in samples with addition of RVK100 and RVK228 strains, respectively, while under the influence of PHA it increased to 75.1% compared with the control (44.8%). A similar trend was also found on CD8+ cells (control - 46.2%, RVK100 - 63.4%, RVK228 - 58.4%, PHA - 52.8%). After 72 hours, an increase in PD-1 expression on CD4+ cells was observed only in the control (up to 41.2%), while in the experimental variant with RVK100 addition there was a 2-fold decrease (up to 21.6%) and no significant changes were found in samples with addition of PHA and RVK228 compared to 24 h. At the same time, cultivation with RVK100 caused a decrease in PD-1 expression on CD8+ cells by 2.7 times (up to 17.4%) compared with 24 h, without significant changes in other samples (control - 33.1%, PHA - 48.1%, RVK228 - 38.4%). The expression of PD-L1 on CD4+ cells generally remained unchanged compared to 24 h, while proportion of CD8+CD279+ cells increased in all variants and reached 67-79% the in experimental and control samples. Conclusions: Both strains, like the nonspecific T-mitogen PHA, stimulated the expression of immune checkpoint receptors PD-1 and PD-L1 on T-helpers and CTLs after 24 hours of cultivation. After 72 hours of cultivation, RVK100, in contrast to RVK228, was able to reduce the expression of PD-1 on these cells.

TGF-β-mediated silencing of genomic organizer SATB1 promotes Tfh cell differentiation and formation of intra-tumoral tertiary lymphoid structures

The immune checkpoint receptor PD-1 on T follicular helper (Tfh) cells promotes Tfh:B cell interactions and appropriate positioning within tissues. Here, we examined the impact of regulation of PD-1 expression by the genomic organizer SATB1 on Tfh cell differentiation. Vaccination of CD4CreSatb1f/f mice enriched for antigen-specific Tfh cells, and TGF-β-mediated repression of SATB1 enhanced Tfh differentiation of human Tcells. Mechanistically, high Icos expression in Satb1-/- CD4+ Tcells promoted Tfh cell differentiation by preventing T follicular regulatory cell skewing and resulted in increased isotype-switched B cell responses invivo. Ovarian tumors in CD4CreSatb1f/f mice accumulated tumor antigen-specific, LIGHT+CXCL13+IL-21+ Tfh cells and tertiary lymphoid structures (TLS). TLS formation decreased tumor growth in a CD4+ Tcell and CXCL13-dependent manner. The transfer of Tfh cells, but not naive CD4+ Tcells, induced TLS at tumor beds and decreased tumor growth. Thus, TGF-β-mediated silencing of Satb1 licenses Tfh cell differentiation, providing insight into the genesis of TLS within tumors.

Siglec Signaling in the Tumor Microenvironment.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of receptors that recognize sialoglycans – sialic acid containing glycans that are abundantly present on cell membranes. Siglecs are expressed on most immune cells and can modulate their activity and function. The majority of Siglecs contains immune inhibitory motifs comparable to the immune checkpoint receptor PD-1. In the tumor microenvironment (TME), signaling through the Siglec-sialoglycan axis appears to be enhanced through multiple mechanisms favoring tumor immune evasion similar to the PD-1/PD-L1 signaling pathway. Siglec expression on tumor-infiltrating immune cells appears increased in the immune suppressive microenvironment. At the same time, enhanced Siglec ligand expression has been reported for several tumor types as a result of aberrant glycosylation, glycan modifications, and the increased expression of sialoglycans on proteins and lipids. Siglec signaling has been identified as important regulator of anti-tumor immunity in the TME, but the key factors contributing to Siglec activation by tumor-associated sialoglycans are diverse and poorly defined. Among others, Siglec activation and signaling are co-determined by their expression levels, cell surface distribution, and their binding preferences for cis- and trans-ligands in the TME. Siglec binding preference are co-determined by the nature of the proteins/lipids to which the sialoglycans are attached and the multivalency of the interaction. Here, we review the current understanding and emerging conditions and factors involved in Siglec signaling in the TME and identify current knowledge gaps that exist in the field.

Molecular features underlying differential SHP1/SHP2 binding of immune checkpoint receptors.

A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.

The Upregulation of Molecules Related to Tumor Immune Escape in Human Pituitary Adenomas.

Human pituitary adenomas are one of the most common intracranial neoplasms. Although most of these tumors are benign and can be treated medically or by transsphenoidal surgery, a subset of these tumors are fast-growing, aggressive, recur, and remain a therapeutic dilemma. Because antibodies against immune checkpoint receptors PD-1 and CLTA-4 are now routinely used for cancer treatment, we quantified the expression of mRNA coding for PD-1, CLTA-4, and their ligands, PD-L1, PD-L2, CD80, and CD86 in human pituitary adenomas and normal pituitary glands, with the ultimate goal of exploiting immune checkpoint therapy in aggressive pituitary adenomas. Aggressive pituitary adenomas demonstrated an increased expression of PD-L2, CD80, and CD86 in compared to that of normal human pituitary glands. Furthermore, aggressive pituitary tumors demonstrated significantly higher levels of CD80 and CD86 compared to non-aggressive tumors. Our results establish a rationale for studying a potential role for immune checkpoint inhibition therapy in the treatment of pituitary adenomas.

T cell exhaustion drives osteosarcoma pathogenesis.

BackgroundOsteosarcoma (OS) is a rare cancer with a bimodal age distribution that peaks in children and young adults. It has been shown that the expression of programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) on tumor-infiltrating immune cells negatively correlates with prognosis of OS patients. However, a comprehensive assessment of the tumor-infiltrating immune cells in OS and their function has not been performed.MethodsCD8+ T cells were isolated from biopsy tissue samples collected from OS patients and control subjects. Mass cytometry, Treg suppression assay, mixed lymphocyte reaction assay, and effector T cell functional assay were performed to analyze the function of tumor-infiltrating T cells. A xenograft metastasis model was established in BALB/c nude mice.ResultsMacrophages and CD3+ T cells comprised most of the tumor-infiltrating immune cells in OS, with a disproportionately higher number of helper CD4+ T cells than effector CD8+ T cells. Whereas the tumor-infiltrating regulatory T cells were functionally intact, the CD8+ T cells showed increased expression of the immune checkpoint receptor (ICR) PD-1 and T cell immunoglobulin and mucin-domain containing 3 (TIM3) and were functionally inactive. TIM3 blockade using a monoclonal antibody restored the T cell alloreactive function of the CD8+ T cells ex vivo. TIM3 blockade in a xenograft model of OS impaired tumor growth in vivo. TIM3 blockade decreased the number of tumor-infiltrating CD4+ T cells while increasing the numbers and functional activation of tumor-infiltrating CD8+ T cells in vivo.ConclusionsThese results highlight that TIM3 blockade might be a viable therapeutic option and should be tested in additional preclinical models.

T Cell Versus T Cell; A Study of the Immune Checkpoint Landscape in Cutaneous T Cell Lymphoma

Introduction.Tumor infiltrating T cells (TILs) are capable of recognising tumor-specific mutations presented as peptides on MHC class I. Overexpression of immune checkpoint ligands on cancer cells, and the corresponding expression of the receptors on T cells, appears to be an important mechanism of immune evasion, exploited with success in immune checkpoint therapy. However, when the cancer cell is itself a T cell, the microenvironment interaction is likely to be more complex. To investigate this further we utilized the clonal nature of mycosis fungoides (MF), a primarily CD4 T cell cancer, by targeting the TCR receptor, differentiating between TIL and tumor in the microenvironment.Methods.43 patients with mycosis fungoides (MF) were prospectively consented to the study, and a skin biopsy and blood sample were obtained. The skin biopsy was macerated and digested with collagenase D, and a TCR V-beta clonogram was used to identify the tumor population. Flow cytometry was used to isolate five cell populations: peripheral blood CD4 and CD8, TIL CD4 and CD8, and tumor cells. The expression of a panel of immune checkpoint receptors (PD-1, TIM-3, LAG-3, Fas & TIGIT) and ligands (PD-L1, PD-L2, Galectin 9, Fas ligand) were measured using conjugated antibodies.Results.46 biopsies from MF patients with stage IA(n=6), IB(n=18), IIA(n=1), IIB(N=14), IIA(n=1) & IVA(n=3) were analysed with paired blood. The technique was effective in distinguishing tumour from TIL in 20 cases, isolating a population with CD7 downregulation, high forward and side scatter (indicative of larger and more complex cells) and a unique immune checkpoint profile (figure 1a & 1b). Tumour infiltrating lymphocytes of both CD4 and CD8 phenotype demonstrated significant overexpression of immune checkpoint receptors PD-1 (p < 0.0001), TIGIT (p < 0.0001) & fas (p < 0.0001), with an increased population of T regulatory cells in the CD4 cells (p < 0.001) and CD8 expressing high TIM-3 (p < 0.0001) compared to peripheral blood. While the TIL population was similar across the demographic, the tumour population demonstrated a significantly varied phenotype, with up and down-regulation of immune checkpoints and HLA-DR (figure 1c). Principal component analysis (figure 1d) revealed three tumour subtypes:1. High PD-L1, Fas, HLA-DR (n=4)2. High PD-1. Low PD-L1 (n=12)3. Low PD-1, PD-L1, Fas (n=3)These subtypes did not relate to disease type, stage, presence of large cell transformation or folliculotropic disease, though those with advanced disease had tumor phenotypes which differed more from the TILs. Tumor cells not only expressed MHC class I and HLA-DR, but upregulated these compared to peripheral blood (p < 0.001).Discussion.The TIL phenotype in MF is surprisingly homogeneous across the demographic, demonstrating upregulation of immune checkpoint receptors and an exhaustion phenotype seen in other cancers. However, the tumor cells did not show consistent upregulation of immune checkpoint ligands, instead their phenotype across patients was heterogeneous with both up and down-regulation. Expression of immune checkpoint ligands by the tumor cells may have negative growth consequences for both tumor cells as well as the immune system, particular since tumor T cells often express immune checkpoint receptors. We did not see concurrent high expression of PD-L1 and PD-1 on tumor cells, which would be expected to be detrimental to tumor survival. Immune checkpoint ligand over-expression is unlikely to be the main method of immune evasion in mycosis fungoides, and as a result, immune checkpoint inhibition may have unexpected responses in this disease.Legends.Figure 1a. TCR V-beta antibody used to separate three CD4 populations (red: peripheral blood CD4, green: CD4 TIL, blue: tumor) and compare HLA-DR expression.Figure 1b. tSNE clustering of cells in the tumour microenvironment, demonstrating clear separation of three populations - tumor, CD4 TIL and CD8 TIL. Coloring by expression of PD-L1 (red: high, blue: low).Figure 1c. Heatmap of immune checkpoint markers (red: high, blue: low) in the tumor cell population only. This demonstrates the heterogeneity, particularly in PD-1, Fas, PD-L1 and HLA-DR expression.Figure 1d. Principal component analysis demonstrating the observation that TIL expression of immune checkpoints are similar between patients (green and blue), whereas tumor expression of these markers (red) can vary markedly and cluster. [Display omitted] DisclosuresScarisbrick:NHS: Employment; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Mallinckrodt: Consultancy, Honoraria; Innate Pharma: Consultancy; Actelion: Consultancy.

Epigenetic Features of HIV-Induced T-Cell Exhaustion Persist Despite Early Antiretroviral Therapy.

T cell dysfunction occurs early following HIV infection, impacting the emergence of non-AIDS morbidities and limiting curative efforts. ART initiated during primary HIV infection (PHI) can reverse this dysfunction, but the extent of recovery is unknown. We studied 66 HIV-infected individuals treated from early PHI with up to three years of ART. Compared with HIV-uninfected controls, CD4 and CD8 T cells from early HIV infection were characterised by T cell activation and increased expression of the immune checkpoint receptors (ICRs) PD1, Tim-3 and TIGIT. Three years of ART lead to partial – but not complete – normalisation of ICR expression, the dynamics of which varied for individual ICRs. For HIV-specific cells, epigenetic profiling of tetramer-sorted CD8 T cells revealed that epigenetic features of exhaustion typically seen in chronic HIV infection were already present early in PHI, and that ART initiation during PHI resulted in only a partial shift of the epigenome to one with more favourable memory characteristics. These findings suggest that although ART initiation during PHI results in significant immune reconstitution, there may be only partial resolution of HIV-related phenotypic and epigenetic changes.

Expression of the immune checkpoint receptors PD-1, LAG3, and TIM3 in the immune context of stage II and III gastric cancer by using single and chromogenic multiplex immunohistochemistry

ABSTRACT We sought to determine the clinicopathological significance of PD-1, LAG3, and TIM3 in gastric cancer (GC) by examining their expression and immune context. Immunohistochemistry (IHC) for PD-1, TIM3, LAG3, and tumor-infiltrating immune cell (TIIC) markers was performed in 385 stage II/III GCs. Epstein-Barr virus (EBV) and microsatellite stability (MSI) testing were performed for molecular classification. Chromogenic multiplex IHC (mIHC) for PD1, TIM3, LAG3, CD3, CD8, FOXP3, CD68, and cytokeratin was performed in 58 of the total samples. PD-1, LAG3, and TIM3 expression in TIICs was observed in 91 (23.6%), 193 (50.1%), and 257 (66.8%) GCs by single IHC, respectively. The expression was associated with EBV+ and MSI-H molecular subtypes (p ≤ 0.001). A positive expression of LAG3 in the invasive margin of the tumor was associated with better prognosis in univariate (p = .020) and multivariate (p = .026) survival analyses. The expression of different immune checkpoint receptors (ICRs) was significantly positively correlated. Dual or triple ICR expression was more frequent in high PD-1 and TIM3 density groups than in low-density groups by mIHC (all p ≤ 0.05). ICRs were mainly expressed in CD3+CD8+ and CD3+CD8− T cells. Fifty-eight GCs were classified into three groups by clustering analysis based on mIHC, and the group with the highest ICR expression in TIICs showed significantly better outcomes in progression-free survival (p = .020). In GC, PD-1, LAG3, and TIM3 expression is positively correlated and associated with better prognosis. Our study provides information for the application of effective immune checkpoint inhibitors against GC.

Pembrolizumab Induces an Unexpected Conformational Change in the CC'-loop of PD-1.

Simple SummaryCancer cells are normally destructed by killer T-cells. However, T-cells expose the PD-1 receptor on their surface, acting as a checkpoint: If it is activated through a special molecule, PD-L1, the T-cell kills itself, ending the attack. Cells often need to present PD-L1 to prevent T-cells from over-aggressive attacks which cause autoimmune disease. There are tumors which also present PD-L1, thereby evading natural clearing, allowing them to continue growing. New anticancer drugs (checkpoint inhibitors: nivolumab and pembrolizumab) disrupt this evasion: They competitively bind to PD-1, without activating it, and re-enable immune tumor destruction. We scrutinize the binding mechanisms via molecular dynamics simulation. We demonstrate that these drugs deform the CC′-loop of the PD-1 in ways differing from those seen with PD-L1 as a binding partner. Pembrolizumab induces a new conformation of the CC′-loop not known to date. These findings might pave the way for the development of new anti-cancer drugs.To improve cancer immunotherapy, a clearer understanding of key targets such as the immune checkpoint receptor PD-1 is essential. The PD-1 inhibitors nivolumab and pembrolizumab were recently approved by the FDA. The CC′-loop of PD-1 has been identified as a hotspot for drug targeting. Here, we investigate the influence of nivolumab and pembrolizumab on the molecular motion of the CC′-loop of PD-1. We performed molecular dynamics simulations on the complete extracellular domain of PD-1, in complex with PD-L1, and the blocking antibodies nivolumab and pembrolizumab. Conformations of the CC′-loop were analyzed unsupervised with the Daura et al. clustering algorithm and multidimensional scaling. Surprisingly, two conformations found were seen to correspond to the ‘open’ and ‘closed’ conformation of CC′-loop in apo-PD-1, already known from literature. Unsupervised clustering also surprisingly reproduced the natural ligand, PD-L1, exclusively stabilizing the ‘closed’ conformation, as also known from literature. Nivolumab, like PD-L1, was found to shift the equilibrium towards the ‘closed’ conformation, in accordance with the conformational selection model. Pembrolizumab, on the other hand, induced a third conformation of the CC′-loop which has not been described to date: Relative to the conformation ‘open’ the, CC′-loop turned 180° to form a new conformation which we called ‘overturned’. We show that the combination of clustering and multidimensional scaling is a fast, easy, and powerful method in analyzing structural changes in proteins. Possible refined antibodies or new small molecular compounds could utilize the flexibility of the CC′-loop to improve immunotherapy.

FLT3-CD3 Bispecific Antibody Specifically Eliminates Normal Hematopoietic Progenitors and AML in Humanized Mouse Models

Acute myeloid leukemia (AML) is a blood cancer arising from clonal proliferation of myeloid progenitors harboring oncogenic mutations. AML patients have poor clinical prognosis and limited therapeutic options, with myeloablation followed by hematopoietic stem cell transplantation (HSCT) as the only curative treatment. The conditioning regimens used indiscriminately kill all highly proliferative cell types, leading to life threatening side effects, and are also potentially ineffective against quiescent AML subpopulations. To address the challenges of efficacy and resistance in targeting AML, we developed a humanized bispecific antibody (CDX) that recruits T cells through CD3 to target and kill Fms-like Tyrosine Kinase 3 (FLT3) expressing cells. Normal FLT3 expression is mostly restricted to hematopoietic stem and progenitor cells (HSPCs) and its activation, through binding of FLT3 ligand (FLT3L), promotes normal differentiation of downstream blood lineages. FLT3 is also expressed on AML blasts in a majority of patients and is thought to promote survival and proliferation of AML. Although multiple promising tyrosine kinase inhibitors (TKIs) have been and are being developed to specifically target FLT3, secondary mutations leading to resistance against FLT3-TKIs remain a major obstacle. Surface expression of FLT3 in leukemic cells, as well as in HSCs, makes it an excellent target for T cell mediated conditioning that specifically eliminates both AML blasts and HSPCs, allowing for subsequent HSCT. In addition, HDX mass spectrometry revealed that our CDX binds an extracellular, membrane proximal FLT3 domain, which does not compete with FLT3L and is outside the regions commonly mutated in AML, therefore, unlike other therapies, targets all FLT3-expressing cells, regardless of mutations in the receptor. We found that in vitro treatment with CDX redirected primary human T cells to kill multiple AML-derived FLT3+ cell lines, some harboring common FLT3 mutations, at EC50 ranging from 0.3 pM to 15 pM. Additionally, the presence of FLT3L (10 nM) had no effect on the EC50 of CDX binding to FLT3+ target cells. To test CDX in vivo, we generated xenograft mice engrafted with primary human peripheral blood mononuclear cells (PBMCs) and MV4;11 cells (FLT3+ AML cell line), which were engineered to stably express EGFP. Mice were treated intravenously with 3 doses of CDX at a concentration of 0.1 mg/kg or left untreated over 1 week (n=5). Progression of AML was tracked by monitoring the frequency of EGFP-MV4;11 cells in peripheral blood (PB) of xenografted mice, which were euthanized if they presented with cachexia, hind-leg paralysis, and severe weight loss (symptoms of AML progression). Treatment with CDX significantly delayed the proliferation of MV4;11 cells, based on lower PB frequencies of EGFP-MV4;11, and increased median survival by 28 days relative to untreated mice. To more stringently model CDX efficacy in a clinical setting, we injected EGFP-MV4;11 cells into humanized mice that were generated by transplantation of human cord blood derived, CD34+ HSPCs and displayed stable multi-lineage engraftment (~20% human CD45+) with significant populations of T cells at 35 weeks. We observed that T cells in CDX-treated mice over-expressed the immune checkpoint receptor PD1, relative to untreated mice, and hypothesized that PD1 inhibition could enhance MV4;11 clearance via CDX treatment by preventing T cell exhaustion. Treatment regimens consisted of 3 doses of CDX (0.1 mg/kg), anti-PD1 antibody (100 μg), or a combination of CDX and anti-PD1 (n=5), followed by transplant with autologous mouse bone marrow cells. Treatment with CDX alone led to decreased PB frequencies of EGFP-MV4;11 cells, but only modestly improved median survival relative to anti-PD1 alone (9 days). In contrast, co-treatment with CDX and anti-PD1 antibody significantly increased median survival (16 days) and was associated with decreased PD1 expression on T cells. In addition, administration of CDX alone or in combination with PD1 in humanized mice resulted in efficient elimination of the human hematopoietic compartment from the mouse bone marrow. Based on our findings, CDX shows promise for treatment of AML with concurrent conditioning for HSCT with improved specificity compared to standard of care and even displays synergy with checkpoint inhibition of PD1. Disclosures Sirochinsky: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Shrestha:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

Abstract B18: YAP1 and TAZ mediate BET inhibitor-dependent immune regulation in NSCLC

Abstract Bromodomain and extra-terminal domain proteins inhibitors (BETi) are promising immunomodulatory agents, able to regulate gene expression in tumor microenvironment (TME), influencing immune cell differentiation and activation. However, immune-related molecular mechanisms underlying BETi activity need to be clarified. We demonstrated that BET protein BRD4 is an important regulator of YAP1/TAZ oncogene expression in several cancer cell lines, including non-small cell lung cancer (NSCLC) cells. YAP1 and TAZ are paralogue genes involved in tumor cell proliferation, migration, and survival. BETi affect YAP1/TAZ expression and impair their transcriptional program. As previously reported, YAP1 and TAZ oncogenes are also involved in tumor immune escape, through the impairment of lymphocyte activation and enhanced PD-L1 expression. We investigated whether BETi-dependent immune regulation depends on YAP1/TAZ activity in NSCLC patients. To this end, we isolated NSCLC patient-derived tissue-originated spheroids (CTOS) and tumor-infiltrating lymphocytes (TILs) and treated both populations with BETi (JQ1 or OTX-015). BETi treatment significantly increased the activation of TILs, which was also associated with decreased expression of the immune checkpoint receptors PD-1, LAG-3, TIM-3, BTLA, and CD160. Notably, BETi treatment also downregulated the expression of corresponding immune checkpoint ligands (PD-L1, LGALS9, and TNFRSF14). Intriguingly, patients displaying higher expression of immune checkpoint molecules exhibited also higher expression of YAP1 and TAZ, suggesting that these transcription factors may be involved in this regulatory network. Accordingly, YAP1 and TAZ were significantly downregulated after BETi treatment in both CTOS and TILs. To confirm specific YAP/TAZ activity in immune regulation, the knockout (KO) of YAP1 or TAZ was achieved through CRISPR/Cas9 approach in NSCLC cell lines. Of interest, YAP1 and TAZ display distinct regulatory effects, suggesting a not overlapping function. The KO of YAP1, but not of TAZ, was sufficient to downregulate PD-L1 expression. Conversely, KO of TAZ alone efficiently downregulated LGALS9, which was not affected by YAP1 KO. TNFRSF14 was significantly reduced in both TAZ and YAP1 KO. These preliminary data suggest that BETi exert a YAP/TAZ-dependent immunomodulatory activity, acting on both tumor cells and TME immune landscape. In this context, BETi may reactivate immune response against tumor cells, acting on both immune checkpoint inhibitor expression and TIL-specific activation. These findings further support the use of BETi as immunomodulatory agents in NSCLC patients. Citation Format: Francesca Reggiani, Giulia Gobbi, Filippo Lococo, Massimiliano Paci, Eleonora Zznetti, Simonetta Piana, Alessia Ciarrocchi, Valentina Sancisi. YAP1 and TAZ mediate BET inhibitor-dependent immune regulation in NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B18.