Expression Of Immune Checkpoint Receptors Research Articles

Influence of chemoradiation on the immune microenvironment of cervical cancer patients

PurposeCervical cancer remains a leading cause of cancer death in women. While immunotherapy has shown great success in combating cancer, the value of immunotherapy in cervical cancer is still only beginning to be explored. Thus, we performed a prospective analysis of patient blood and tumor samples at the beginning and end of conventional chemoradiation to assess changes in the immune cell and immunoreceptor compartments, and investigate if and when the addition of immunotherapy could be beneficial.MethodsPatients with FIGO II–III cervical cancer receiving standard chemoradiation between January 2020 and December 2021 were included. We collected tumor and blood samples from patients before and at the end of therapy and analyzed immune cell composition and immune checkpoint receptor expression on both immune and tumor cells using multicolor flow cytometry.ResultsIn all, 34 patients were eligible in the study period; 22 could be included and analyzed in this study. We found that chemoradiation significantly reduces T cell numbers in both tumors and blood, but increases macrophage and neutrophil numbers in tumors. Furthermore, we found that the percentage of immune checkpoint receptor PD‑1 and TIGIT-expressing cells in tumors was significantly reduced at the end of therapy and that CD4 and CD8 memory T cell populations were altered by chemoradiation. In addition, we observed that while PD-L1 expression intensity was upregulated by chemoradiation on blood CD8 cells, PD-L1 expression frequency and the expression intensity of antigen-presenting molecule MHC‑I were significantly reduced on tumor cells.ConclusionOur data demonstrate that chemoradiation significantly alters the immune cell composition of human cervical tumors and the expression of immune checkpoint receptors on both lymphocytes and tumor cells. As our results reveal that the percentage of PD‑1+ CD8 cells in the tumor as well as the frequency of PD-L1-expressing tumor cells were reduced at the end of therapy, neoadjuvant or simultaneous anti-PD‑1 or anti-PD-L1 treatment might provide better treatment efficiency in upcoming clinical studies.

Major pathways involved in macrophage polarization in cancer.

Macrophages play an important role in tissue homeostasis, tissue remodeling, immune response, and progression of cancer. Consequently, macrophages exhibit significant plasticity and change their transcriptional profile and function in response to environmental, tissue, and inflammatory stimuli resulting in pro- and anti-tumor effects. Furthermore, the categorization of tissue macrophages in inflammatory situations remains difficult; however, there is an agreement that macrophages are predominantly polarized into two different subtypes with pro- and anti-inflammatory properties, the so-called M1-like and M2-like macrophages, respectively. These two macrophage classes can be considered as the extreme borders of a continuum of many intermediate subsets. On one end, M1 are pro-inflammatory macrophages that initiate an immunological response, damage tissue integrity, and dampen tumor progression by fostering robust T and natural killer (NK) cell anti-tumoral responses. On the other end, M2 are anti-inflammatory macrophages involved in tissue remodeling and tumor growth, that promote cancer cell proliferation, invasion, tumor metastasis, angiogenesis and that participate to immune suppression. These decisive roles in tumor progression occur through the secretion of cytokines, chemokines, growth factors, and matrix metalloproteases, as well as by the expression of immune checkpoint receptors in the case of M2 macrophages. Moreover, macrophage plasticity is supported by stimuli from the Tumor Microenvironment (TME) that are relayed to the nucleus through membrane receptors and signaling pathways that result in gene expression reprogramming in macrophages, thus giving rise to different macrophage polarization outcomes. In this review, we will focus on the main signaling pathways involved in macrophage polarization that are activated upon ligand-receptor recognition and in the presence of other immunomodulatory molecules in cancer.

Tumor Budding Is an Independent Prognostic Factor in Pancreatic Adenocarcinoma and It Positively Correlates with PD-L1 Expression on Tumor Cells.

Pancreatic adenocarcinoma is one of the leading causes of cancer-related death in developed countries. Only 15% of patients are candidates for radical surgery, and adequate prognostication may guide proper postsurgical management. We aimed to retrospectively assess the prognostic significance of the immunohistochemical expression of immune checkpoint receptors (PD-L1 and VISTA), markers of systemic inflammation, thrombosis in the tumor area, and the tumor budding in the group of 107 patients diagnosed with pancreatic adenocarcinoma in a single center. The high expression of PD-L1 on tumor cells (TCs) was associated with worse overall survival (OS, p = 0.041, log-rank). On the contrary, high PD-L1 or VISTA on tumor-associated immune cells (TAICs) was correlated with better OS (p = 0.006 and p = 0.008, respectively, log-rank). The joint status of PD-L1 on TCs and TAICs stratified patients into three prognostic groups. The cases with high-grade budding were characterized by higher PD-L1 expression on TCs (p = 0.008) and elevated systemic inflammatory markers. Moreover, budding was identified as the independent prognostic factor in multivariate Cox regression analysis (HR = 2.87; 95% CI = 1.75–4.68; p < 0.001). To conclude, the pattern of PD-L1 and VISTA expression was associated with survival in univariate analysis. Tumor budding accurately predicts outcomes in pancreatic cancer and should be incorporated into routine histopathological practice.

Abstract 1935: Interplay between TIM3+ immune cells and other immune checkpoints in more than 40 different human carcinoma entities using 18+1 BLEACH&amp;STAIN mfIHC

Abstract An increasing number of therapy regimens using a combination of different immune checkpoint inhibitors (ICIs) have shown remarkable results in several different tumor entities. However, the likelihood of a positive response rate to combined ICIs is poor in most tumor entities and depends on several parameters including the tumor microenvironment. Particularly little is known about the spatial orchestration and spatial interplay between different immune checkpoint expressing cells. Given that the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is expressed on both immune cells as well as tumor cells and that several phase I/II studies are currently evaluating anti-TIM3 drugs, the interplay between these immune checkpoints in human cancers is of topical interest. To study the spatial orchestration and interplay between TIM3, CTLA-4, PD-1, and PD-L1 expression on T-cell subsets, macrophage subsets, CD11c+ dendritic cells, CD20+B-cells in relation to panCK+ malignant cells, CD31+ vessels and other structural tumor compartments, a multiplex fluorescence immunohistochemistry approach was used to stain 18 different antibodies on a set of tissue microarrays containing samples from more than 3000 carcinoma samples. In addition, a deep learning-based framework for cell type identification was developed and validated in this study. TIM3, PD-1, PD-L1, and CTLA-4 expression was measured on tumor cells (panCK+), cytotoxic T-cells (CD3+CD8+), T-helper cells (CD3+CD4+), regulatory T-cells (CD3+CD4+FOXP3+), subsets of macrophages (CD68+CD163+/CD68+iNOS+) and dendritic cells (CD11c+). Interestingly, TIM3 as well as CTLA-4 expression on CD3+CD8+ cytotoxic T-cells and CD3+CD4+FOXP3+ regulatory T-cells showed a spatially more diverse expression pattern - particularly inverse expression profile - compared to PD-1 expression on all analyzed T-cells subsets that was consistently accompanied by PD-L1 expression on immune and tumor cells (p&amp;lt;0.001). Combined analysis of cell densities, expression patterns, intensity measurements, interaction and distance analysis between immune cells and tumor cells revealed distinct changes in the immune cell infiltration pattern that was linked to several major immune checkpoint receptor expression profiles. Previously uncharacterized immune cell-composition dynamics in clustered tumor phenotypes, according to the immune checkpoint expression, were detected. This included for instance, a significant inverse association between CTLA-4 expression on T-cells and high expression levels of the PD-1/PD-L1 axis. In conclusion, deep profiling of 18 biomarkers in more than 40 different carcinoma entities revealed complex changes in the spatial orchestration of a wide range of immune cell subsets that was driven by the expression profile and composition of TIM3, PD-1, PD-L1, and CTLA-4. Citation Format: Nicolaus F. Debatin, Elena Bady, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Niclas C. Blessin. Interplay between TIM3+ immune cells and other immune checkpoints in more than 40 different human carcinoma entities using 18+1 BLEACH&amp;STAIN mfIHC [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 1935.

Epigenetic regulation of T cell exhaustion.

Chronic antigen stimulation during viral infections and cancer can lead to T cell exhaustion, which is characterized by reduced effector function and proliferation, and the expression of inhibitory immune checkpoint receptors. Recent studies have demonstrated that T cell exhaustion results in wholescale epigenetic remodeling that confers phenotypic stability to these cells and prevents T cell reinvigoration by checkpoint blockade. Here, we review foundational technologies to profile the epigenome at multiple scales, including mapping the locations of transcription factors and histone modifications, DNA methylation and three-dimensional genome conformation. We discuss how these technologies have elucidated the development and epigenetic regulation of exhausted T cells and functional implications across viral infection, cancer, autoimmunity and engineered T cell therapies. Finally, we cover emerging multi-omic and genome engineering technologies, current and upcoming opportunities to apply these to T cell exhaustion, and therapeutic opportunities for T cell engineering in the clinic.

Evaluation of PD-L1 (E1L3N, 22C3) expression in venous tumor thrombus is superior to its assessment in renal tumor in predicting overall survival in renal cell carcinoma.

Renal cell carcinoma (RCC) frequently invades renal vein forming neoplastic thrombus. The expression of immune checkpoint receptors in RCC was addressed in multiple studies, but little is known about the expression and prognostic significance of programmed death ligand-1 in tumor thrombus. The study aimed to evaluate the expression of PD-L1 within venous tumor thrombus and primary tumor using 2 independent antibody clones (22c3 and E1L3N) and to assess its value in predicting overall survival (OS) in the subgroup of patients with RCC and renal vein thrombus. Eighty-two patients with RCC and venous tumor thrombus that underwent nephrectomy were enrolled. The expression of PD-L1 was assessed utilizing tissue microarrays separately on tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs). The frequency of PD-L1 expression on TCs and TILs varied between tumor and thrombus compartments and was dependent on the antibody clone used. The expression of PD-L1 on TCs and/or TILs was associated with worse OS irrespectively of the antibody and the analyzed compartment. Nevertheless, the best prognostic performance was noted for the combined assessment of PD-L1 expression on TCs and TILs in venous tumor thrombus with the use of 22c3 antibody. The multivariable Cox regression model predicting OS incorporated PD-L1 22c3 in venous tumor thrombus (hazards ratio [HR] = 3.64, 95% confidence interval [CI] = 1.63-8.14, P = 0.002) and nodal status (HR = 2.88, 95% CI = 1.18-7.03, P = 0.02). PD-L1 may become a valuable tool for prognostic purposes in this specific subgroup of patients and be incorporated into the respective models qualifying for adjuvant treatment.

Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity

BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK)...

Toll-like receptor agonist combinations augment mouse T-cell anti-tumor immunity via IL-12- and interferon ß-mediated suppression of immune checkpoint receptor expression

ABSTRACT We previously found that activated CD8+ T-cells increase expression of PD-1, which can be attenuated in the presence of specific Toll-like receptor (TLR) agonists, mediated by IL-12 secreted by professional antigen-presenting cells. While these CD8+ T-cells had greater anti-tumor activity, T-cells stimulated by different TLR had different gene expression profiles. Consequently, we sought to determine whether combinations of TLR agonists might further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity. Activation of CD8+ T-cells in the presence of specific TLR ligands resulted in decreased expression of PD-1, LAG-3, and CD160, notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of E.G7-OVA or TRAMP-C1 tumor-bearing mice with peptide or DNA vaccines, co-administered with combination of TLR3 and TLR9 agonists, showed greater suppression of tumor growth. The anti-tumor effect of TLR1/2 and/or TLR9, but not TLR3, was abrogated in IL-12KO mice. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and type 1 IFN pathway activation following TLR3 stimulation. Our results provide a mechanistic rationale for the choice of optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.

CTLA4Ig/VISTAIg combination therapy selectively induces CD4+ T cell-mediated immune tolerance by targeting the SOCS1 signaling pathway in porcine islet xenotransplantation.

T cell inhibitory receptors can regulate the proliferation or function of T cells by binding to their ligands and present a unique opportunity to manage destructive immune responses during porcine islet xenotransplantation. We applied ex vivo porcine islet xenotransplantation and in vitro mixed lymphocyte-islet reaction models to assess immune checkpoint receptor expression profiles in recipient T cells, investigated whether CTLA4 or VISTA immunoglobulin (Ig) combination therapy alone could suppress porcine islet xenograft rejection and further analyzed its potential immune tolerance mechanism. Recipient T cells expressed moderate to high levels of CTLA4, PD-1, TIGIT and VISTA, and the frequency of CTLA4+ CD4+ , TIGIT+ CD4+ , VISTA+ CD4+ and VISTA+ CD8+ T cells was positively correlated with porcine islet xenograft survival time in xenotransplant recipients. Combined treatment with CTLA4Ig and VISTAIg selectively inhibited recipient CD4+ T cell hyper-responsiveness and proinflammatory cytokine production and significantly delayed xenograft rejection. SOCS1 deficiency in CD4+ T cells stimulated by xenogeneic islets facilitated hyper-responsiveness and abolished the suppressive effect of combination therapy on recipient T cell-mediated porcine islet damage in vivo and in vitro. Further mechanistic studies revealed that combined treatment significantly induced SOCS1 expression and inhibited the Jak-STAT signalling pathway in wild-type recipient CD4+ T cells stimulated by xenogeneic islets, whereas SOCS1 deficiency resulted in Jak-STAT signalling pathway activation in recipient CD4+ T cells. We demonstrated a major role for CTLA4 and VISTA as key targets in CD4+ T cell hyper-responsiveness and porcine islet xenograft rejection. The selective inhibition of CD4+ T cell immunity by CTLA4Ig/VISTAIg is based on SOCS1-dependent signalling.

In This Issue

See article, p. 95.Shree et al. find that up to a year after anti-CD20 treatment, most patients with lymphoma do not generate neutralizing antibody responses to COVID-19 vaccination. However, if patients are vaccinated and mount an antibody response prior to anti-CD20 treatment, they tend to maintain these antibody responses through subsequent anti-CD20 lymphoma therapy, suggesting a strategy of vaccinating first and treating later. These results also call to weigh the risks of maintenance therapy with rituximab or other B cell–depleting antibodies during the COVID-19 era.See article, p. 103.Downregulation of surface epitopes, including CD22, causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Zheng et al. report that CD22 mRNA is pervasively misspliced in B-ALL cell lines and primary samples. Some misspliced isoforms still encode plasma membrane–bound CD22 variants, while others lack the start codon in exon 2 and are noncoding. These exon 2–skipping isoforms are prevalent in high-risk B-ALL patients treated with the CD22-directed antibody–drug conjugate inotuzumab, where they correlate with low CD22 surface expression and are likely to account for treatment failure in two cases. Thus, aberrant CD22 splicing is an important mechanism of resistance to immunotherapy.See article, p. 116.Polycomb repressive epigenetic complexes are recurrently dysregulated in cancer. The mechanisms by which genetic alterations affect polycomb regulatory function to drive oncogenesis are poorly understood. By combining human genomics with biochemical and functional approaches, Schaefer et al. demonstrate that highly recurrent mutations in leukemia of the PRC1.1 subunits BCOR and BCORL1 dissociate DNA binding from enzymatic subunits. Uncoupling of the PRC1.1-repressive function from target genes alters epigenetic and signaling states, including FGFR derepression. BCOR deletion or truncation confers ibrutinib resistance, which is abrogated by FGFR inhibition. This epigenetic mechanism explains PRC1.1 tumor-suppressive activity and informs therapeutic approaches in PRC1.1-mutated cancer.See article, p. 136.IFNγ production has long been considered necessary for antitumor T-cell response. IFNγ also fuels macrophage activation in cytokine release syndrome (CRS), a significant toxicity of chimeric antigen receptor (CAR) T-cell therapy. Bailey et al. have discovered that either blockade or genetic knockout of IFNγ does not impair the anticancer activity of CAR T cells in preclinical models of hematologic malignancies. Further, there is reduced expression of immune checkpoint receptors on the CAR T cells and less activation of macrophages. Thus, it may be possible to separate the toxicity from the efficacy of CAR T cells for safer yet effective use.See article, p. 154.Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with very poor clinical outcomes. Mundy-Bosse et al. demonstrate that ENKTL cells are arrested at early stages of NK-cell maturation, characterized by broad epigenetic silencing of genes involved in normal NK-cell development. Treatment with the DNA hypomethylating agent 5-azacytidine led to a reduction of DNA hypermethylation, reexpression of normal NK-cell developmental genes, phenotypic NK-cell differentiation, and significant prolongation of survival in recipient mice. These studies lay the foundation for epigenetic-directed therapy targeting the developmental blockade in ENKTL.

The prognostic significance of immune checkpoint receptor expression in patients with lymphoma: Association with disease status and clinical outcomes.

Little is known about the expression of immune checkpoint receptors in the peripheral blood of lymphoma patients. Herein, we assessed the expression of inhibitory checkpoint receptors, including CTLA-4, PD-1/PDL-1, LAG-3, and TIM-3 in the peripheral blood of lymphoma patients and its correlation with the clinical outcomes of patients. Therefore, 47 classical Hodgkin lymphoma (cHL), 48 non-Hodgkin lymphoma patients with diffuse large B-cell lymphoma (DLBCL) subtype, and 30 healthy controls were recruited. The expression of inhibitory receptors was evaluated using SYBR Green real-time PCR method. CTLA-4, LAG-3, and TIM-3 genes were significantly upregulated in both cHL and DLBCL patients compared to the healthy controls. In addition, the level of these molecules was differentially expressed in cHL and DLBCL patients at different disease phases compared to the healthy controls. The CTLA-4 gene was highly expressed in newly diagnosed (ND) cHL patients compared to the relapsed ones. Relapsed DLBCL patients had significantly increased LAG-3 expression compared to patients at remission, as well as ND patients. Regarding cHL patients, high CTLA-4 expression was correlated with low lactate dehydrogenase level and better performance status, whereas the level of LAG-3 was significantly elevated in patients with poor performance status. Lower initial PD-1 expression was associated with improved disease-free survival in cHL patients. Inhibitory immune checkpoint receptors are aberrantly expressed in the peripheral blood of cHL and DLBCL patients in which high LAG-3 in DLBCL patients and PD-1/LAG-3 in cHL patients are associated with relapse occurrence and worse prognosis, respectively.

Mediastinal lymph node removal ameliorates cytotoxic T-lymphocyte functions in patients with non-small cell lung cancer.

Mediastinal lymph node (MLN) removal by video-assisted mediastinoscopic lymphadenectomy (VAMLA) for preoperative cancer staging was reported to be associated with increased survival. The aim of this study was to evaluate the immunologic effects of complete MLN removal by VAMLA on cytotoxic T lymphocyte (CTL) phenotype and function. Seventeen patients with non-small cell lung cancer (NSCLC) (stage cT1-4N0-3M0-1A) and 20 healthy participants were included in this study. Blood samples were collected before and 4 weeks after the procedure. Lymphocytes were isolated from the removed MLNs. CTL phenotypes and functions were evaluated by flow cytometry. Plasma levels of soluble programmed cell death protein 1 (sPD-1), soluble programmed cell death protein 1 ligand, and soluble CTL antigen 4 (sCTLA-4) were measured with enzyme-linked immunosorbent assay. The ratio of the immunosenescent CTLs (CD3+CD8+CD28-) was increased in peripheral blood and MLNs of the patients with NSCLC compared to controls (p = 0.037), and MLN removal did not change this ratio. PD-1 and CTL antigen 4 expressions were significantly reduced in peripheral blood CTLs after MLN removal by VAMLA (p = 0.01 and p = 0.01, respectively). Granzyme A expression was significantly reduced in the peripheral blood CTLs of the patients compared to controls (p = 0.006) and MLN removal by VAMLA significantly improved Granzyme A expression in CTLs (p = 0.003). Plasma concentrations of sPD-1 and sCTLA-4 remained unchanged after VAMLA. CTLs in the MLNs and peripheral blood of the patients with NSCLC had an immunosenescent phenotype, increased immune checkpoint receptor expression, and impaired cytotoxicity. MLN removal by VAMLA improved these phenotypic and functional characteristics of CTLs. These changes may explain the potential contribution of VAMLA to improved survival.

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).

The Expression of Immune Checkpoint Receptors and Ligands in the Colorectal Cancer Tumor Microenvironment.

The limited efficacy of immune checkpoint inhibitors in colorectal cancer (CRC) is likely due to immunosuppressive mechanisms including T cell exhaustion caused by inhibitory immune checkpoints in the tumor microenvironment. We investigated the expression status of the inhibitory immune checkpoint receptors on tumor-infiltrating T cells and their ligands on tumor cells by flow cytometry and immunohistochemistry, using surgically-resected specimens of CRC. Flow cytometry analysis indicated that TIM-3, TIGIT, and PD-1 were expressed on tumor-infiltrating CD4+ (8.3%, 56.0%, 26.1%) and CD8+ T cells (8.2%, 51.6%, 23.5%), and CRC cells abundantly expressed PD-L1, CEACAM-1, and CD155 (2.2%, 77.0%, 46.8%). Immunohistochemical analysis revealed that the tumor proportional score of PD-L1, CEACAM-1, and CD155 was 42.4%, 54.2%, and 52.1%, respectively. PD-1, TIM-3, and TIGIT axes may reduce T cell function in the CRC tumor microenvironment.

PD-1, CTLA-4, LAG-3, and TIGIT: The roles of immune checkpoint receptors on the regulation of human NK cell phenotype and functions

The roles of immune checkpoint receptors were defined in many cancers and autoimmune diseases, while there is limited information on their functional roles in the NK cells of healthy individuals. Immune checkpoint receptor expression of NK cell subsets and their association with NK cell functions (cytotoxic capacity and cytokine production) in healthy population were investigated. PD-1, CTLA-4, LAG-3 and TIGIT expression of peripheral blood NK cells, cytokine levels (TNF-α, IFN-γ, IL-10) and cytotoxic functions (granzyme A, perforin, CD107a; with/without K562 target cell stimulation) were evaluated by flow cytometry. CD56dimCD16dim NK cells had the highest expression of TIGIT, while CD56dimCD16− NK cells had highest expression of PD-1, CTLA-4 and LAG-3. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had increased amount of IL-10 however, reduced IFN-γ and TNF-α levels. Cytotoxic granule expressions (perforin and granzyme A) were reduced in PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells. However, TIGIT expression did not alter perforin and granzyme A expressions. Degranulation capacity was reduced in three groups of NK cells (PD-1+ or LAG-3+ or TIGIT+). TIGIT+ NK cells responded strongly to target cell stimulation, while NK cells in the other groups (PD-1+ or CTLA-4+ or LAG-3+) were resistant. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had a regulatory phenotype, impaired cytotoxic functions, and response to target cell stimulation. In contrast, TIGIT+ NK cells had strong baseline cytotoxic activity that further increased in response to target cell stimulation.

Expression of Immune Checkpoint Receptors in Placentae With Infectious and Non-Infectious Chronic Villitis.

Pregnancy is an immunological paradox whereby maternal immunity accepts a genetically unique fetus (or fetuses), while maintaining protective innate and adaptive responses to infectious pathogens. This close contact between the genetically diverse mother and fetus requires numerous mechanisms of immune tolerance initiated by trophoblast cell signals. However, in a placental condition known as villitis of unknown etiology (VUE), there appears to be a breakdown in this tolerance allowing maternal cytotoxic T-cells to traffic into the placenta to destroy fetal villi. VUE is associated with several gestational complications and an increased risk of recurrence in a subsequent pregnancy, making it a significant obstetrical diagnosis. The cause of VUE remains unclear, but dysfunctional signaling through immune checkpoint pathways, which have a critical role in blunting immune responses, may play an important role. Therefore, using placental tissue from normal pregnancy (n=8), VUE (n=8) and cytomegalovirus (CMV) infected placentae (n=4), we aimed to identify differences in programmed cell death 1 (PD-1), programmed death ligand-1 (PD-L1), LAG3 and CTLA4 expression between these etiologies by immunohistochemistry (IHC). Results demonstrated significantly lower expression of PD-L1 on trophoblast cells from VUE placentae compared to control and CMV infection. Additionally, we observed significantly higher counts of PD-1+ (>100 cells/image) and LAG3+ (0-120 cells/image) cells infiltrating into the villi during VUE compared to infection and control. Minimal CTLA4 staining was observed in all placentae, with only a few Hofbauer cells staining positive. Together, this suggests that a loss of tolerance through immune checkpoint signaling may be an important mechanism leading to the activation and trafficking of maternal cells into fetal villi during VUE. Further mechanistic studies are warranted to understand possible allograft rejection more clearly and in developing effective strategies to prevent this condition from occurring in utero.

Baseline Immune Dysregulation in Autologous Stem Cell Transplant Recipients Is Associated with a ‘Graft Versus Host‘-like Syndrome and Results in Poor Outcomes

Introduction: Acute Graft Versus Host Disease (GVHD) is caused by the recognition of recipient antigens by donor T lymphocytes following allogeneic stem cell transplantation (SCT). The development of clinical and histo-pathological changes similar to GVHD after autologous SCT (ASCT) is a poorly understood and controversial phenomenon. Given the controversy around this topic, we aimed to: 1) analyze outcomes and clinical correlatives for the development of GVHD in ASCT recipients and 2) analyze baseline T and Natural Killer (NK) cell functions in patients that developed GVHD after ASCT, compared to matched controls.Methods: This is a retrospective analysis of patients that received ASCT at Mayo Clinic between January 2006 and December 2016. The study was approved by the Institutional Review Board. ‘Autologous GVHD’ was defined as the development of clinical and histo-pathological findings indicative of GVHD in ASCT recipients, as confirmed by pathology review. Data regarding baseline characteristics, prior treatments, presentation, treatment and outcomes were collected and analyzed. Patients and controls with available cryopreserved PBMCs at baseline and at time of GVHD development were identified. Controls were matched for age, disease status, prior therapies and conditioning regimen. Samples were analyzed for T cell functions, expression of activation and inhibitory receptors, T cell subsets and natural killer (NK) cell functions. Unpaired t-test was used to compare T-cell subset frequencies. Survival was estimated using the Kaplan Meier and Log Rank tests.Results: Between 2006 and 2016, 3,891 consecutive patients underwent ASCT. Of these, 35 (0.9%) developed symptoms suggestive of GVHD warranting biopsies. In 19 of these 35 patients (54%), the histopathological changes were consistent with GI and/or skin GVHD. The most common underlying disease was multiple myeloma (14 patients, 73.7%) and the most common conditioning regimen was melphalan (16 patients, 84.2%). The median age at ASCT was 62 (range 49-73) years. Most patients (14, 73.7%) had grade 3 or 4 GVHD. Delay in steroid initiation beyond 1 week led to lower response rates (30.8% vs 69.2%, p=0.03), longer duration of symptoms (median 28 vs 4 days, p=0.02) and a trend towards worse 1-year OS (64.5% vs. 83.3%, p=0.1). Patients that received at least 1 mg/kg of prednisone had better response to treatment (76.9% vs. 23.1%, p=0.5), although the difference was not statistically significant. The median overall survival (OS) from the time of ASCT was not reached and 53% of patients were alive at 3 years. Five of the 19 patients (26.3%) died due to complications of this immunological phenomenon.We then performed immune profiling of the available cryopreserved samples in the Mayo Clinic transplant biobank. We identified 4 patients with ‘autologous GVHD’ and 7 ASCT recipients as controls, matched for age and underlying disease. The percentage of NK cells (defined as CD3−CD56+)/total live cells was higher in patients compared to controls (34.2% vs 4.3%, p=0.009). T-cell subset analysis revealed higher levels of central memory T-cells (Tcm, defined as CCR7+CD45RA-, 27.4% in patients vs 2% in controls, p=0.02). This is consistent with what has been reported in murine models of allogeneic GVHD where Tcm induce GVHD, due to their TCR's ability of recognizing more alloantigens and their better capacity of undergoing clonal expansion. Furthermore, expression of immune checkpoint receptors was higher in patients compared to controls (CD3+PD1+/CD3+ 15.7% vs 5.7%, p=0.048 and CD3+TIM3+/CD3+ 25.4% vs 2.3% p=0.0003 respectively). This is in line with studies showing upregulation of PD1 and TIM3 on T-cells of allogeneic SCT recipients that develop GVHD and linking higher TIM3 levels with more severe gut GVHD.Conclusions:Our analyses indicate that the development of GVHD-like syndrome after ASCT is associated with baseline immune dysregulation, similar to what is reported in allogeneic GVHD models. This is associated with poor prognosis if prompt steroid treatment is not initiated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TIM3 expression on TILs is associated with poor response to neoadjuvant chemotherapy in patients with locally advanced triple-negative breast cancer

BackgroundThe expression of immune checkpoint receptors (ICRs) on tumor-infiltrating lymphocytes (TILs) is associated with better response to immunotherapies via immune checkpoint inhibitors. Therefore, we investigated various ICR expressions on TILs in patients with locally advanced triple-negative breast cancer (TNBC) after neoadjuvant chemotherapy (NAC).MethodsExpressions of ICRs were examined immunohistochemically in surgical specimens (n = 61) using monoclonal antibodies for PDL-1, PD-1, TIM-3, LAG-3, and CTLA-4. Positivity was defined as staining > 1% on TILs.ResultsThe median age was 49 (24–76) years. The majority of patients were clinically T3–4 (n = 31, 50.8%) and clinically N1–3 (n = 58, 95.1%) before NAC. Of those, 82% were found to have CTLA-4 positivity, whereas PD1, PDL-1, LAG3, and TIM-3 expressions on TILs were 62.3, 50.9, 26.2, and 68.9%. A high expression of CTLA-4 was found to be associated with a better chemotherapy response (OR = 7.94, 95% CI: 0.9–70.12, p = 0.06), whereas TIM-3 positivity was contrarily associated with a worse chemotherapy response (OR = 0.253, 95% CI: 0.066–0.974, p = 0.047) as measured by the MDACC Residual Cancer Burden Index. At a 47-month follow-up, ypN0 (DFS; HR = 0.31, 95% CI: 0.12–0.83, p = 0.02 and DSS; HR = 0.21, 95% CI: 0.07–0.62, p = 0.005) and CTLA-4 high expression on TILs (DFS; HR = 0.38, 95% CI: 0.17–0.85, p = 0.019 and DSS; HR = 0.34, 95% CI: 0.15–0.78, p = 0.01) were found to be associated with improved survival.ConclusionsThese findings demonstrate that CTLA-4, PD-1, PDL-1, and TIM-3 were highly expressed in TNBC. Based on these high expression patterns, further studies directed towards combined therapies are warranted in advanced TNBC in future.

The impact of selenium on regulatory T cell frequency and immune checkpoint receptor expression in patients withdiffuse large B cell lymphoma (DLBCL).

For many decades, selenium (Se) has been known as a potential anti-cancer agent that can also improve the function of immune cells in a variety of solid tumors. However, there is no report on the role of Se on CD4+ T cell subsets like CD4+CD25+FOXP3+ regulatory T cells (Tregs) in lymphoma patients. In this randomized clinical trial, we investigated the effect of 3-month Se consumption on the frequency of CD4+CD25+FOXP3+ Tregs and the expression of immune checkpoint receptors in thirty-two non-Hodgkin lymphoma (NHL) patients (16 patients with Se (Se+) and 16 without Se (Se-) consumption) with diffuse large B-cell lymphoma (DLBCL) subtype at stable remission. The change in the frequency of Tregs and expression of immune checkpoint receptors including CTLA-4, LAG-3, TIM-3, and PD-L1 genes were evaluated after 3months in both groups using flow cytometry and SYBR Green Real-time PCR method, respectively. The results showed that the frequency of CD4+CD25+FOXP3+ Tregs and expression of immune checkpoint receptors did not significantly change after 3-month Se consumption in DLBCL patients. However, alteration in the frequency of CD4+CD25-FOXP3+ Treg subsets was positively correlated with change in CTLA-4, LAG-3, and TIM-3 expression in the Se+ group. Three-month Se supplementation did not prevent relapse in Se+ group. Taken together, Se supplementation alone did not affect the frequency of CD4+CD25+FOXP3+ Tregs, expression of checkpoint receptors, and prevention of relapse in DLBCL patients at stable remission phase but might influence the functional properties of other Treg subsets like CD4+CD25-FOXP3+ Tregs.

Phenotypic and Functional Analyses Guiding Combination Immune Checkpoint Immunotherapeutic Strategies in HTLV-1 Infection.

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) develops in 1–5% of HTLV-1-infected individuals. Previous studies by us and others have shown that the expression of negative immune checkpoint receptors (NCRs) is significantly increased on CD8 T cells in various chronic viral infections and are associated with poor anti-viral immunity. We have previously identified the differential expression of NCRs on CD8 T cells in blood from patients with HAM/TSP and in central nervous system (CNS) tissues of HTLV-1 infected humanized mice and defined the association with neurological complications. In this study, we determined the co-expression patterns of several key NCRs (PD-1, TIGIT, TIM-3, and LAG-3) and their cognate ligands in HTLV-1 infection and assessed how combination strategies targeting these pathways would impact HTLV-1-specific CD8 T-cell effector functions as an approach to reduce CNS disease outcomes. We found that global CD8 T cells from HAM/TSP patients co-express multiple NCRs at significantly higher frequencies than asymptomatic carriers (AC). Moreover, NCR ligands (PVR and PD-LI) on both plasmacytoid and myeloid dendritic cells were also expressed at higher frequencies in HAM/TSP compared to AC. In both AC and HAM/TSP subjects, combination dual PD-L1/TIGIT or triple PD-L1/TIGIT/TIM-3 blockade with monoclonal antibodies resulted in increases in intracellular cytokine expression in CD8 T cells after virus stimulation, particularly CD107a, a marker of degranulation, and TNF-α, a key cytokine that can directly inhibit viral replication. Interestingly, almost all blockade combinations resulted in reduced IL-2+ HTLV-1-specific CD8 T cell frequencies in HAM/TSP subjects, but not in AC. These results define a novel combinatorial NCR immunotherapeutic blockade strategy to reduce HAM/TSP disease burden.