Inhibitory Immune Checkpoint Molecules and Exhaustion of T cells in COVID-19

COVID-19 (Coronavirus Disease) is an infectious disease caused by the coronavirus SARS-CoV-2 (Severe acute respiratory syndrome Coronavirus 2), which belongs to the genus Betacoronavirus. It was first identified in patients with severe respiratory disease in December 2019 in Wuhan, China. It mainly affects the respiratory system, and in severe cases causes serious lung infection or pneumonia, which can lead to the death of the patient. Clinical studies show that SARS-CoV-2 infection in critical cases causes acute tissue damage due to a pathological immune response. The immune response to a new coronavirus is complex and involves many processes of specific and non-specific immunity. Analysis of available studies has shown various changes, especially in the area of specific cellular immunity, including lymphopenia, decreased T cells (CD3+, CD4+ and CD8+), changes in the T cell compartment associated with symptom progression, deterioration of the condition and development of lung damage. We provide a detailed review of the analyses of immune checkpoint molecules PD-1, TIM-3, LAG-3 CTLA-4, TIGIT, BTLA, CD223, IDO-1 and VISTA on exhausted T cells in patients with asymptomatic to symptomatic stages of COVID-19 infection. Furthermore, this review may help to better understand the pathological T cell immune response and improve the design of therapeutic strategies for patients with SARS-CoV-2 infection.

Association between inflammatory cytokines and immune–checkpoint molecule in rheumatoid arthritis

Background Anti-citrullinated peptide antibodies (ACPA) and inflammatory cytokines play important roles in the development of rheumatoid arthritis (RA). T cell immunoglobulin and mucin–domain containing–3 (TIM–3) is an immune-checkpoint molecule involved in inhibitory signaling. Galectin–9 (Gal–9) mediated ligation of TIM–3 induces the amelioration of autoimmune diseases. TIM–3 is expressed in synovial osteoclasts and involved in the rheumatoid bone destruction. The aim of this study was to investigate the relationships between inflammatory cytokines and immune–checkpoint molecules in RA patients. Methods Serum levels of interleukin–6 (IL–6), tumor necrosis factor–α (TNF–α), soluble TIM–3 (sTIM–3) and Gal–9 were determined by ELISA. Patients were stratified into two groups based on ACPA titers: low-medium ACPA (ACPA <200 U/mL) and high ACPA (ACPA ≥200 U/mL). Serum levels of cytokines or immune-checkpoint molecules were evaluated between RA patients with low-medium ACPA titers and high ACPA titers. Results Elevated serum levels of inflammatory cytokines were correlated with DAS28–ESR in RA patients. Although serum levels of sTIM–3 were elevated in RA patients, significant correlations between sTIM–3 and cytokines (IL–6 or TNF–α) were observed exclusively in RA patients with low-medium ACPA titers (<200 U/mL). Serum levels of IL–6 and TNF–α levels were significantly correlated with elevated Gal–9 levels regardless of ACPA status. A significant correlation between IL–6 and Gal–9 was observed in RA patients without advanced joint damage. Conversely, a significant correlation between TNF–α and Gal–9 was observed in RA patients with advanced joint damage. Conclusions Our data indicated that there are positive correlations between circulating inflammatory cytokines and checkpoint molecules in RA patients and these interactions can be modulated by ACPA status or joint damage stage.

High Bacterial Abundances of Dorea and Pediococcus in the Gut Microbiome Linked to Expansion, Immune Checkpoint Expression and Efficacy of CD19-Directed CAR T-Cells in Patients with r/r DLBCL

Introduction: The crosstalk of the gut microbiome and the human host can influence T-cell immune responses and has emerged as a modulator in cancer immunotherapy (Gopalakrishnan et al., 2018). Individual bacteria isolated from human fecal specimen were also shown to shape systemic and gut mucosal T cell repertoires (Geva-Zatorsky et al., 2017), and several commensal members of the human gut microbiome were recently associated with the kinetics of the reconstitution of peripheral immune cells after allo-HCT (Schluter et al., 2020). However, in patients treated with CAR T-cells a link between gut microbiome configurations and T-cell characteristics has not been described yet. Recently, we and others could associate low CAR T-cell expansion and dysfunction with treatment failure. Here, we hypothesize that certain gut microbes or even intestinal monodomination of facultative pathogens may correlate with CAR T-cell expansion and expression profile of immune checkpoint molecules which might impact treatment outcome. Methods: Patients with relapsed / refractory Diffuse-Large B-Cell Lymphoma (DLBCL) were treated with the CD19 specific CAR T-cell products Axicabtagene-Ciloleucel or Tisagenlecleucel at our institution from April 2019 to May 2021. Within this time-period, peripheral blood and fecal biospecimens from 23 patients were collected sequentially before, during and after CAR T-cell transfusion (specific time points: before lymphodepleting chemotherapy, day of CAR T-cell transfusion, day 7, day 14). CAR T-cells and the expression profile of immune checkpoint molecules (PD-1, TIM-3, LAG-3, 2B4) were studied by multiparameter flow cytometry. CAR T-cell peak expansion was assessed relatively (% CAR + T-cells of CD3+ T-cells) and absolutely (/ul). In addition, effector : target (E:T) ratios were estimated as absolute peak expansion of CAR T-cells (/ul) per tumor volume (as sum of the product of diameters based on Lugano criteria with up to 6 target lesions in cm 3). 16S rRNA gene sequencing was performed on 83 stool samples. Responder (R, complete or partial remission) were retrospectively assessed compared to Non-Responder (NR, stable or progressive disease) according to response assessment with PET-CT three months after CAR T-cell transfusion. Results: Higher alpha diversity (i.e., within-sample diversity) prior to CAR T-cell transfusion correlated positively with a favorable E:T ratio (CAR peak expansion/ul : tumor volume in cm 3, p=0.04, r=0.608; Fig. 1). Interestingly, the abundances of certain taxa prior to CAR T-cell transfusion correlated with the expansion of CAR T-cells in vivo. In particular we found a positive association with Pediococcus (p=0.018, r=0.751, FDR corrected) and with Anaerovoracaceae (E:T ratio, p=0.027, r=0.84, FDR corrected). Next, we studied the expression profile of immune checkpoint molecules on CAR T-cells in the context of microbial taxa in R vs NR patients. Notably, we observed a positive association between the frequency of CD8+ CAR T-cells devoid of inhibitory molecules (PD-1, TIM-3, LAG-3 and 2B4) and the relative abundance of the intestinal microbial genus Dorea (p=0.039, r=0.416: see Fig. 1). Furthermore, we found a higher relative abundance of Dorea (p=0.043) prior to transfusion in R compared to NR patients as well as significant positive associations of Dorea (p=0.0.008, r=0.54, not FDR corrected) and Pediococcus (p=0.047, r=0.462, not FDR corrected) with progression free survival . Conclusion: For the first time, we describe associations between the compositional changes of the gut microbiome and kinetics as well as immune characteristics of CAR T-cell therapy in patients with relapsed / refractory DLBCL. In the future, our findings need to be validated in a larger patient cohort and might serve as a predictive biomarker for microbiome-based patient assessment and microbiome-targeted therapeutic approaches to identify benefiting patients and to improve outcome. Figure 1 Figure 1. Disclosures Blumenberg: Kite/Gilead: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS/Celgene: Research Funding; Janssen: Research Funding. von Bergwelt: MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau. Buecklein: Amgen: Consultancy, Honoraria; BMS / Celgene: Consultancy, Research Funding; Kite / Gilead: Consultancy, Honoraria, Other: Congress and travel support , Research Funding; Janssen: Consultancy; Miltenyi: Research Funding; Novartis: Consultancy, Other: Congress and travel support , Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Miltenyi: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau.

Increased Intrahepatic Expression of Immune Checkpoint Molecules in Autoimmune Liver Disease

Immune checkpoint molecules (ICM) are critical in maintaining immunologic homeostasis and participate in preventing or promoting autoimmune disease development. Exploring a large panel of intrahepatic inhibitory and stimulatory ICM is necessary for drawing a general picture of the immune alterations in autoimmune hepatitis (AIH). Here, we performed a multiparametric analysis of ICM, including PD-1, TIM3, LAG3, CTLA-4, OX40 and 4-1BB, and we determined their expression on intrahepatic lymphocyte subsets in untreated and in treated patients with AIH in comparison to normal liver tissue. AIH patient-derived liver tissue revealed the overexpression of ICM, mainly PD-1 and 4-1BB, as well as the strong correlation between PD-1+ CD8+ T-cell abundance and severity of AIH (alanine transaminase and aspartate transaminase levels). Our results show that the ICM play an important role in the loss of immune homeostasis in the liver, providing an attractive approach to investigate their role as targets for effective therapeutic interventions.

Impact of immune checkpoint molecules on FoxP3+ Treg cells and related cytokines in patients with acute and chronic brucellosis

Background The immunoregulatory functions of regulatory T cells (Tregs) in the development and progression of some chronic infectious diseases are mediated by immune checkpoint molecules and immunosuppressive cytokines. However, little is known about the immunosuppressive functions of Tregs in human brucellosis, which is a major burden in low-income countries. In this study, expressions of immune checkpoint molecules and Treg-related cytokines in patients with acute and chronic Brucella infection were evaluated to explore their impact at different stages of infection. Methods Forty patients with acute brucellosis and 19 patients with chronic brucellosis admitted to the Third People’s Hospital of Linfen in Shanxi Province between August 2016 and November 2017 were enrolled. Serum and peripheral blood mononuclear cells were isolated from patients before antibiotic treatment and from 30 healthy subjects. The frequency of Tregs (CD4+ CD25+ FoxP3+ T cells) and expression of CTLA-4, GITR, and PD-1 on Treg cells were detected by flow cytometry. Levels of Treg-related cytokines, including IL-35, TGF-β1, and IL-10, were measured by customised multiplex cytokine assays using the Luminex platform. Results The frequency of Tregs was higher in chronic patients than in healthy controls (P = 0.026) and acute patients (P = 0.042); The frequency of CTLA-4+ Tregs in chronic patients was significantly higher than that in healthy controls (P = 0.011). The frequencies of GITR+ and PD-1+ Tregs were significantly higher in acute and chronic patients than in healthy controls (P < 0.05), with no significant difference between the acute and chronic groups (all P > 0.05). Serum TGF-β1 levels were higher in chronic patients (P = 0.029) and serum IL-10 levels were higher in acute patients (P = 0.033) than in healthy controls. We detected weak correlations between serum TGF-β1 levels and the frequencies of Tregs (R = 0.309, P = 0.031) and CTLA-4+ Tregs (R = 0.302, P = 0.035). Conclusions Treg cell immunity is involved in the chronicity of Brucella infection and indicates the implication of Tregs in the prognosis of brucellosis. CTLA-4 and TGF-β1 may contribute to Tregs-mediated immunosuppression in the chronic infection stage of a Brucella infection.

Imaging Changes and Immune-Checkpoint Expression on T Cells in Bronchoalveolar Lavage Fluid from Patients with Pulmonary Sarcoidosis

Sarcoidosis is a systemic, granulomatous disease caused by unknown immunological abnormalities. The organs most vulnerable to sarcoidosis are the lungs. Patients often resolve spontaneously, but the lungs can also be severely affected. Although details regarding prognostic factors in sarcoidosis patients with lung involvement remain unclear, several reports have suggested that immune checkpoint molecules are involved in the pathogenesis of sarcoidosis. In this study, we divided sarcoidosis patients into two groups based on chest computed tomography (CT) findings and compared immune checkpoint molecules expressed on T cells in bronchoalveolar lavage fluid (BALF) in the two groups, using flow cytometry. We found elevated programmed cell death 1 (PD-1) or T cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) expression on T cells in BALF in patients with spontaneous improvement in CT findings, compared with those in patients without improvement in CT findings. In conclusion, our study implies that PD-1 or TIM-3 expression on T cells in BALF may be a prognostic factor for pulmonary lesions in sarcoidosis.

Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells

While the treatment of squamous cell carcinoma of the head and neck (HNSCC) with radiotherapy (RT) is complemented more and more by immunotherapy in clinical trials, little is known about the impact of the human papillomavirus (HPV) status or the applied RT scheme on the immune phenotype of the tumor cells. Therefore, we aimed to examine the impact of the HPV status of four human HNSCC cell lines on cell death and the expression of immune checkpoint molecules (ICMs) after RT with either hypofractionation irradiation (5x3.0Gy) or a high single dose (1x19.3Gy) via multicolor flow cytometry and quantitative PCR at an early time point after therapy. In our study, 5x3.0Gy RT induced high numbers of early and late apoptotic cells independent of the HPV status, but necrosis was only increased in the HPV-positive UM-Scc-47 cells. Generally, the immune stimulatory ICMs (CD70, CD137-L, ICOS-L) were less affected by RT compared to the immune suppressive ones (PD-L1, PD-L2, and the herpesvirus entry mediator (HVEM)). A significant higher surface expression of the analyzed ICMs was found after hypofractionated RT compared to a single high dose; however, regardless of the HPV status, with the exception of ICOS-L. Here, HPV-positive HNSCC tumor cells showed a stronger response to 5x3.0Gy than HPV-negative ones. On the RNA level, only minor alterations of ICMs were observed following RT, with the exception of the HPV negative cell line CAL33 treated with 5x3.0Gy, where PD-L2, HVEM and CD70 were significantly increased. We conclude that the HPV status may not distinctly predict immunological responses following RT, and thus cannot be used as a single predictive marker for therapy responses in HNSCC. In contrast, the patient-specific individual expression of ICMs following RT is preferable for the targeted patient selection for immune therapy directed against distinct ICM.