Persistent Autoimmune Activation and Proinflammatory State in Post-COVID Syndrome

Background The immunopathological pathways enabling post-COVID syndrome (PCS) development are not entirely known. We underwent a longitudinal analysis of patients with COVID-19 who developed PCS aiming to evaluate the autoimmune and immunological status associated with this condition. Methods Thirty-three patients were included for longitudinal clinical and autoantibody analyses of whom 12 patients were assessed for cytokines and lymphocyte populations. Patients were followed during 7-11 months after acute COVID-19. Autoimmune profile and immunological status were evaluated mainly by enzyme-linked-immunosorbent assays and flow cytometry. Results Latent autoimmunity and overt autoimmunity persisted over time. A proinflammatory state was observed in patients with PCS characterized by upregulated IFN-α, TNF-α, G-CSF, IL-17A, IL-6, IL-1β, and IL-13, whereas IP-10 was decreased. In addition, PCS was characterized by increased levels of Th9, CD8+ effector T cells, naive B cells, and CD4+ effector memory T cells. Total levels of IgG S1-SARS-CoV-2 antibodies remained elevated over time. Discussion The clinical manifestations of PCS are associated with the persistence of a proinflammatory, and effector phenotype induced by SARS-CoV-2 infection. This long-term persistent immune activation may contribute to the development of latent and overt autoimmunity. Results suggest the need to evaluate the role of immunomodulation in the treatment of PCS.

Interferon-driven brain phenotype in a mouse model of RNaseT2 deficient leukoencephalopathy

Infantile-onset RNaseT2 deficient leukoencephalopathy is characterised by cystic brain lesions, multifocal white matter alterations, cerebral atrophy, and severe psychomotor impairment. The phenotype is similar to congenital cytomegalovirus brain infection and overlaps with type I interferonopathies, suggesting a role for innate immunity in its pathophysiology. To date, pathophysiological studies have been hindered by the lack of mouse models recapitulating the neuroinflammatory encephalopathy found in patients. In this study, we generated Rnaset2−/− mice using CRISPR/Cas9-mediated genome editing. Rnaset2−/− mice demonstrate upregulation of interferon-stimulated genes and concurrent IFNAR1-dependent neuroinflammation, with infiltration of CD8+ effector memory T cells and inflammatory monocytes into the grey and white matter. Single nuclei RNA sequencing reveals homeostatic dysfunctions in glial cells and neurons and provide important insights into the mechanisms of hippocampal-accentuated brain atrophy and cognitive impairment. The Rnaset2−/− mice may allow the study of CNS damage associated with RNaseT2 deficiency and may be used for the investigation of potential therapies.

Deep Phenotyping Reveals Expansion of T Follicular Regulatory Cells and Triple Positive (CTLA4, TIGIT, PD1) Exhausted Effector Memory T Cells Characterizing the Immunosuppressive Microenvironment in Follicular Lymphoma

INTRODUCTION Lymphoma cells are dependent on the tumor microenvironment (TME) for their survival and proliferation. Dissection of TME composition provides insight into lymphomagenesis, better prognostication, and enhancement of therapeutic options. Flow cytometry provides a robust approach for single-cell analysis. Nonetheless lack of well-defined comparator groups and/or a robust, reproducible approaches for analyzing the multidimensional data often limited such studies. In the current study, we analyzed the T cell background on a large reference set of follicular hyperplasia (FH) lymph node samples utilizing a robust standardized high dimensionality flow cytometry and a novel reproducible analytical pipeline. We then compared this baseline reference set with tumor infiltrating T cell subsets in follicular lymphoma (FL; in treatment naïve FL-NA and relapsed/refractory FL-RR sets). METHODS On behalf of the imCORE Network we analyzed 44 FH and 81 FL (35 FL-NA and 44 FL-RR) with 2 standardized flow cytometry panels (23 antigen/18-color) (Table 1). We evaluated the T cell subset distribution as well as immune checkpoint expression (TIGIT, TIM3, PD-1, CD96, LAG3, CTLA4, CD73) within analytically defined T cell clusters. Analysis was performed using semiautomated multi-step analytical pipeline as outlined in Figure 1. Using standardized instrument settings and an R-based algorithms (gaussNorm) to minimize technical variations in sample acquisition we analyzed the T-cells subpopulations using a dimensionality reduction technique (UMAP), combined with an unsupervised clustering algorithm (FlowSOM). Statistical analysis was performed using non-parametric Wilcoxon test. RESULTS In the 3 cohorts, several marked differences in the composition T cells subsets were observed. Compared to FH the FL lymph nodes were depleted for CD4 & CD8 naïve subsets (Table 2) and were characterized by an immune suppressive microenvironment enriched in specific subsets of activated T regulatory cells and exhausted memory effector cells. The pool of CD8 naïve cells was restored in FL-RR cases (Table 2). FL-NA nodes showed enrichment of T follicular regulatory cells (Tfr; Table 2) (0.9% vs 2.7%, p<0.0001) expressing FoxP3, dim to negative CD25, CD45RO, TIGIT, PD1, CTLA and CXCR5 (Table 2) and activated regulatory T cells (T-Reg) characterized by a highly suppressive phenotype CD25+, CTLA4+, TIGIT+ and PD1+ (Fig. 2B). Moreover, Tfr compartment was further expanded in relapsed/refractory FL cases (2.7% vs 4.5%, p=0.02). In association with the increase of Tfr cells, the concentration of CTLA4+, TIGIT+, PD1bright T follicular helper cells (Tfh) was reduced in FL-RR compared to FL-NA (Fig. 2C). The Tfr/Treg expansion was also associated with a marked increase in exhausted memory T cells in both CD4 and CD8 compartments (CD4 EM TP and CD8 EM DP, Table 2). In FL isolates the CD4 compartment was characterized by the expression of triple positive (CTLA4, TIGIT, PD1) phenotype in EM cells while the memory CD8 cells overexpressed TIGIT and PD1 (Fig. 2D). A smaller subpopulation of memory CD8 cells, almost undetectable in FH samples, characterized by the expression of CTLA4, PD1, TIGIT and TIM3 is expanded in FL isolates (Fig. 2D). CONCLUSION Our data suggest that change in balance between TFH and Tfr may lead to more aggressive therapy resistant disease in FL. The interplay between TFH and Tfr, has been postulated to shape the immune response in FL with TFH promoting germinal center formation and Tfr inhibiting TFH and follicular effector T cells. While both TFH and Tfr compartments are expanded in FL, in the relapsed/ refractory FL cases, the Tfr compartment is further expanded at the expense of TFH leading to more immunosuppressive background. Furthermore our study suggests a rational way of designing immune checkpoint inhibitor studies in FL. Effector memory T-cells in FL isolates show an exhausted phenotype characterized by the expression of the inhibitory receptors CTLA4, TIGIT, PD1 in the CD4 compartment, and TIGIT, PD1 in the CD8. In addition, the noteworthy expansion of TIM3+ memory CD8 cells in FL. Targeting these most highly expressed checkpoints in FL alone or in combination may provide an avenue for rational trial design. Figure 1 Figure 1. Disclosures Roshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Dogan: Physicians' Education Resource: Honoraria; Peer View: Honoraria; Roche: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Seattle Genetics: Consultancy; EUSA Pharma: Consultancy.

P09.04 Impact of major oncologic surgery on immune responses in the immediate post-operative setting in oesophageal adenocarcinoma patients; a guide to harnessing the double-edged sword of cancer surgery

BackgroundImmune checkpoint inhibitors (ICIs) are being investigated for their role as an adjunct in the multimodal treatment of oesophageal adenocarcinoma (OAC). The most appropriate time to incorporate ICIs remains unknown. Our study profiles systemic anti-tumour immunity perioperatively to help inform the optimal timing of ICIs into current standards of care for OAC patients.MethodsSystemic immunity in 11 OAC patients was phenotyped prior to oesophagectomy and on post-operative days (POD) 0, 1, 3, 7 and week 6 using flow cytometry. Longitudinal serological profiling was conducted by 54-plex-ELISA. The frequency of circulating lymphocytes, T cells, T helper cells and cytotoxic T lymphocytes was profiled longitudinally. The activation status of T cells was also assessed using CD69, CD27, CD62L and CD45RA as well as the proportion of T cell subsets in circulation, which included: naïve, central memory, effector memory and terminally differentiated effector memory T cells. This study also profiled the longitudinal alteration of immune checkpoint expression on circulating T cells, which included: PD-1, CTLA-4, TIGIT, TIM-3, LAG-3, PD-L1 and PD-L2. Damage-associated molecular patterns (calreticulin, HMGB1 and MIC-A/B) were also assessed.ResultsThe frequency of naïve T cells increased in circulation post-oesophagectomy from POD-0 to POD-7 (p<0.01) but returned to baseline at week 6. Effector memory T cells had decreased by POD7 but increased substantially by week 6 (p<0.05). A steady increase in activated circulating CD27+ T cells was observed from POD-0 to POD-7 (p<0.05). The percentage of PD-1+ and CTLA-4+ T cells peaked on POD-1 and was substantially decreased by week 6 (p<0.01). Th1 cytokines were decreased in the immediate post-operative setting with a reduction in IFN-Y, IL-12p40, CD28, CD40L and TNF Alpha. In addition to this IP-10 aka cxcl-10 which is an important chemokine ligand in recruiting anti-tumour TH1 cells and polarising the immune response to a Th1 phenotype is significantly reduced perioperatively. There is a simultaneous increase in Th2 cytokines in the immediate post-operative setting with a significant increase in IL4, IL10, IL16, IL1RA and MCP1 before returning to preoperative levels at week 6.ConclusionOur study highlights the prevailing immunophenotype and responses to surgery with a switch in balance towards a Th2 and potentially M2 phenotype and consequently, an immunosuppressive milieu. Therefore, orchestrating M2 reprogramming toward an M1 phenotype and similarly shifting the balance in favour of a Th1 phenotype would offer a potent therapeutic approach for augmenting tumourigenesis and promoting cancer regression. Consequently, this study paves the way for further studies and appropriate trial design are needed to interrogate the use of ICB as a trimodal approach with chemoradiotherapy and chemotherapy alone for locally advanced disease in the neoadjuvant and adjuvant setting to determine the optimal timing and subset of patients for their use in the era of precision targeted therapies.Disclosure InformationN.E. Donlon: None. M. Davern: None. A. Sheppard: None. F. O’Connell: None. S. Ramjit: None. C. Hayes: None. M. Mc Clean: None. H. Temperley: None. C. Butler: None. N. Ravi: None. C. Donohoe: None. J. O’ Sullivan: None. M.R. Dunne: None. J.V. Reynolds: None. J. Lysaght: None.

Immunotherapeutic Potential of T Memory Stem Cells

Memory T cells include T memory stem cells (TSCM) and central memory T cells (TCM). Compared with effector memory T cells (TEM) and effector T cells (TEFF), they have better durability and anti-tumor immunity. Recent studies have shown that although TSCM has excellent self-renewal ability and versatility, if it is often exposed to antigens and inflammatory signals, TSCM will behave as a variety of inhibitory receptors such as PD-1, TIM-3 and LAG-3 expression, and metabolic changes from oxidative phosphorylation to glycolysis. These changes can lead to the exhaustion of T cells. Cumulative evidence in animal experiments shows that it is the least differentiated cell in the memory T lymphocyte system and is a central participant in many physiological and pathological processes in humans. It has a good clinical application prospect, so it is more and more important to study the factors affecting the formation of TSCM. This article summarizes and prospects the phenotypic and functional characteristics of TSCM, the regulation mechanism of formation, and its application in treatment of clinical diseases.

Altered circulating CCR6+and CXCR3+ T cell subsets are associated with poor renal prognosis in MPO-ANCA-associated vasculitis

Background Effector memory T cells are pivotal effectors of adaptive immunity with enhanced migration characteristics and are involved in the pathogenesis of ANCA-associated vasculitis (AAV). The diversity of effector memory T cells in chemokine receptor expression has been well studied in proteinase 3 (PR3)-AAV. However, few studies have been conducted in myeloperoxidase (MPO)-AAV. Here, we characterized chemokine receptor expression on effector memory T cells from patients with active MPO-AAV. Methods Clinical data from newly diagnosed MPO-AAV patients and healthy subjects were collected and analyzed. Human peripheral blood mononuclear cells (PBMCs) isolated from patients with active MPO-AAV were analyzed by flow cytometry. The production of effector memory T cell-related chemokines in serum was assessed by ELISA. Results We observed decreased percentages of CD4+ and CD8+ T cells in the peripheral blood, accompanied by a significant decrease in CCR6-expressing T cells but an increase in CXCR3+ T cells, in active MPO-AAV. Furthermore, the decrease in CCR6 and increase in CXCR3 expression were mainly limited to effector memory T cells. Consistent with this finding, the serum level of CCL20 was increased. In addition, a decreasing trend in the TEM17 cell frequency, with concomitant increases in the frequencies of CD4+ TEM1 and CD4+ TEM17.1 cells, was observed when T cell functional subsets were defined by chemokine receptor expression. Moreover, the proportions of peripheral CD8+ T cells and CD4+ TEM subsets were correlated with renal prognosis and inflammatory markers. Conclusions Our data indicate that dysregulated chemokine receptor expression on CD4+ and CD8+ effector memory T cells and aberrant distribution of functional CD4+ T cell subsets in patients with active MPO-AAV have critical roles related to kidney survival.

Spatiotemporally specific roles of TLR4, TNF, and IL-17A in murine endotoxin-induced inflammation inferred from analysis of dynamic networks

Background Bacterial lipopolysaccharide (LPS) induces a multi-organ, Toll-like receptor 4 (TLR4)-dependent acute inflammatory response. Methods Using network analysis, we defined the spatiotemporal dynamics of 20, LPS-induced, protein-level inflammatory mediators over 0–48 h in the heart, gut, lung, liver, spleen, kidney, and systemic circulation, in both C57BL/6 (wild-type) and TLR4-null mice. Results Dynamic Network Analysis suggested that inflammation in the heart is most dependent on TLR4, followed by the liver, kidney, plasma, gut, lung, and spleen, and raises the possibility of non-TLR4 LPS signaling pathways at defined time points in the gut, lung, and spleen. Insights from computational analyses suggest an early role for TLR4-dependent tumor necrosis factor in coordinating multiple signaling pathways in the heart, giving way to later interleukin-17A—possibly derived from pathogenic Th17 cells and effector/memory T cells—in the spleen and blood. Conclusions We have derived novel, systems-level insights regarding the spatiotemporal evolution acute inflammation.