POLYFUNCTIONAL SARS-CoV-2-SPECIFIC T CELLS PERSIST IN TISSUE OF COVID-19 CONVALESCENTS

Background: T cells play an essential role in SARS-CoV-2 immunity, including in defense against severe COVID-19. However, most studies analyzing SARSCoV- 2-specific T cells have been limited to analysis of blood. Furthermore, the role of T cells in SARS-CoV-2 immunity in pregnant women, which are at disproportionately higher risk of severe COVID-19, is poorly understood. Method(s): Here, we quantitated and deeply phenotyped SARS-CoV-2-specific T cells from convalescent women (n=12) that had mild (non-hospitalized) COVID-19 during pregnancy. Endometrial, maternal blood, and fetal cord blood specimens were procured at term, which ranged from 3 days to 5 months post-infection. SARS-CoV-2-specific T cells were deeply analyzed by CyTOF using a tailored phenotyping panel designed to assess the effector functions, differentiation states, and homing properties of the cells. Result(s): SARS-CoV-2-specific T cells were more abundant in the endometrium than in maternal or fetal cord blood. In a particularly striking example, in one donor sampled 5 months after infection, SARS-CoV-2-specific CD8+ T cells comprised 4.8% of total endometrial CD8+ T cells, while it only reached 1.4% in blood. Endometrial SARS-CoV-2-specific T cells were more frequently of the memory phenotype relative to their counterparts in maternal and fetal cord blood, which harbored higher frequencies of naive T cells. Relative to their counterparts in blood, endometrial SARS-CoV-2-specific T cells exhibited unique phenotypic features, including preferential expression of the T resident memory marker CD69, inflammatory tissue-homing receptor CXCR4, and the activation marker 4-1BB. Endometrial T cells were highly polyfunctional, and could secrete IFNg, TNFa, MIP1b, IL2, and/or IL4 in response to spike peptide stimulation. By contrast, their counterparts in blood preferentially produced the cytolytic effectors perforin and granzyme B. Conclusion(s): Polyfunctional SARS-CoV-2-specific T cells primed by prior exposure to the virus are abundant and persist in endometrial tissue for months after infection. These cells exhibit unique phenotypic features including preferential expression of select chemokine receptors and activation molecules. Compared to their blood counterparts, the effector functions of these cells are more cytokine-driven and less cytolytic. The long-term persistence of these cells in the endometrium may help protect future pregnancies from SARS-CoV-2 re-infection.

HIGH RNAemia ASSOCIATES WITH SKEWED T-CELL RESPONSE IN PLWH HOSPITALIZED FOR COVID-19

Background: Severe COVID-19 outcomes have been reported in people living with HIV (PLWH). High SARS-CoV-2 RNAemia has emerged as a hallmark of severe COVID-19, yet its pathogenic role in the context of COVID-19 in PLWH is currently unknown. We hereby measured SARS-CoV-2 RNAemia and explored its association with T-cell/humoral responses and clinical severity in PLWH. Method(s): Unvaccinated PLWH and age/sex-matched people living without HIV (PLWOH) hospitalized for radiologically-confirmed COVID-19 pneumonia were consecutively enrolled (March 2020-January 2021). We measured: SARS-CoV-2 RNAemia (RT-qPCR);T-cell activation (HLA-DR+CD-38+), cytotoxic T-cells [granzyme-B(GRZB)+perforin(PRF)+], GRZB/PRF production (MFI) by cytotoxic T-cells (flow cytometry);SARS-CoV-2-specific cytokines (IFN-gamma/ TNF-alpha/IL-2/IL-4/IL-17A)-producing T-cells, after SARS-CoV-2 spike peptides challenge (flow cytometry);anti-RBD antibodies (ELISA), Spike-ACE2 binding inhibition (receptor binding inhibition assay). Statistics: Mann-Whitney test and Spearman's correlation. Result(s): 18 PLWH (16 on cART;median CD4 361.5/mL;HIV-RNA< 50 cp/ mL in 15/18) and 18 PLWOH were included at a median of 10 days from symptoms onset (Fig.1A). PLWH had lower PaO2/FiO2 [140 (122-151.5) vs. 207 (156.3-309.3);P=0.0005] and higher SARS-CoV-2 RNAemia (Fig.1B). While humoral responses were comparable between groups ( Fig.1C-D), as was T-cell activation, PLWH showed skewed T-cell responses: higher perforin production by cytotoxic T-cells (Fig.1E);fewer SARS-CoV-2-specific IFN-gamma+ and IL-4+ CD4 T-cells (Fig.1F);lower Th1 tri-functional (IFN-gamma+TNF-alpha+IL-2+) and bi-functional (IFN-gamma+TNF-alpha+) CD4 T-cells (Fig.1G);reduced TNF-alpha+ CD8 T-cells (Fig.1H). Interestingly, SARS-CoV-2 RNAemia correlated negatively with PaO2/FiO2 nadir and SARS-CoV-2-specific T-cells, yet positively with perforin production by cytotoxic T-cells (Fig.1I-M). No correlations between RNAemia and humoral responses were found. Conclusion(s): As compared to HIV-uninfected patients, PLWH hospitalized for COVID-19 pneumonia feature high SARS-CoV-2 RNAemia which is linked to respiratory failure and skewed T-cell responses, with higher perforin production by cytotoxic T-cells, and yet fewer polyfunctional SARS-CoV-2-specific T-cells. Our data suggest a link between HIV-related T-cell dysfunction and poor control over circulating SARS-CoV-2 that may in turn influence COVID-19 severity in PLWH. (Figure Presented).

Severely ill and high-risk COVID-19 patients exhibit increased peripheral circulation of CD62L+ and perforin+ T cells.

Introduction: The emergence of SARS-CoV-2, which causes COVID-19, has led to over 400 million reported cases worldwide. COVID-19 disease ranges from asymptomatic infection to severe disease and may be impacted by individual immune differences. Methods: We used multiparameter flow cytometry to compare CD4+ and CD8+ T cell responses in severe (ICU admitted) and non-severe (admitted to observational unit) hospitalized COVID-19 patients. Results: We found that patients with severe COVID- 19 had greater frequencies of CD4+ T cells expressing CD62L compared to non-severe patients and greater frequencies of perforin+ CD8+ T cells compared to recovered patients. Furthermore, greater frequencies of CD62L+ CD4+ and CD8+ T cells were seen in severely ill diabetic patients compared to non-severe and non-diabetic patients, and increased CD62L+ CD4+ T cells were also seen in severely ill patients with hypertension. Discussion: This is the first report to show that CD62L+ T cells and perforin+ T cells are associated with severe COVID-19 illness and are significantly increased in patients with high-risk pre-existing conditions including older age and diabetes. These data provide a potential biological marker for severe COVID-19.

Prolonged fever and inflammation in a young child in the midst of the pandemic - it's not always MIS-C

Case Report: Prolonged fever in children is a symptom that is seen in many different diseases, infections, malignancies, and autoimmune conditions. This can, at times, make the correct diagnosis challenging. A previously healthy 10-year-old male was transferred to our institution with one week history of fever, fatigue, abdominal pain, and vomiting. Laboratory studies demonstrated pancytopenia, transaminitis, electrolyte abnormalities, elevated pro-inflammatory markers & D-Dimer, and hypoalbuminemia. COVID-19 IgG was reactive. Due to the severity in presentation the patient was transferred to the ICU with a presumptive diagnosis of MIS-C. Hewas started on IVIG as well as a five-day course of high-dose methylprednisolone per protocol. Aspirin was added, but later discontinued, due to worsening thrombocytopenia. CT imaging with contrast showed small bilateral pleural effusions & periportal edema, mild splenomegaly, and echocardiogram showed diffuse dilation of the left main and left anterior descending arteries. Given the laboratory findings the differential diagnosis was expanded, Ehrlichia caffeensis serology was sent and empiric Doxycycline started. EBV Nuclear Antigen IgG antibody and EBV Viral Capsid Antigen IgM Antibody resulted as positive suggesting recent or reactivated infection. Respiratory viral PCR with COVID-19, Cytomegalovirus and Parvovirus PCR were negative. Despite initial treatment, the patient continued to have persistent fever, severe pancytopenia, and high ferritin up to 24 426 ng/mL, raising suspicion for Haemophagocytic Lymphohistiocytosis (HLH). Soluble interleukin-2 level was elevated & his presentation was then considered to be more consistent with HLH given that he met 6/8 criteria. Screening for primary HLH including CD107a, perforin and granzyme B, SAP, and XIAP resulted in the latter three being normal but CD107a was abnormal. Next generation sequencing for primary criteria was negative. E. Chaffeensis resulted positive: IgM 1:80, IgG 1:256. MIS-C and HLH have overlapping features but differ in some clinical manifestations. Timely recognition and management is paramount as the management differs. This case illustrates the importance of performing a broad search for potential causes, allowing for appropriate and timely treatment. COVID-19 serology alone should not be the basis for diagnosis of MIS-C in a patient with fever and inflammation. This is important as SARS-CoV2 becomes endemic. Infections such as EBV and Ehrlichiosis should be on the differential particularly in endemic areas and during seasons of higher prevalence for the latter, as these have been well documented to cause HLH. Copyright © 2023 Southern Society for Clinical Investigation.

Immune Response Gaps Linked to SARS-CoV-2 Infection: Cellular Exhaustion, Senescence, or Both?

The COVID-19 pandemic, promoted by the SARS-CoV-2 respiratory virus, has resulted in widespread global morbidity and mortality. The immune response against this pathogen has shown a thin line between protective effects and pathological reactions resulting from the massive release of cytokines and poor viral clearance. The latter is possibly caused by exhaustion, senescence, or both of TCD8+ cells and reduced activity of natural killer (NK) cells. The imbalance between innate and adaptive responses during the early stages of infection caused by SARS-CoV-2 contributes to the ineffective control of viral spread. The present study evaluated the tissue immunoexpression of the tissue biomarkers (Arginase-1, CCR4, CD3, CD4, CD8, CD20, CD57, CD68, CD138, IL-4, INF-α, INF-γ, iNOS, PD-1, Perforin and Sphingosine-1) to understand the cellular immune response triggered in patients who died of COVID-19. We evaluated twenty-four paraffin-embedded lung tissue samples from patients who died of COVID-19 (COVID-19 group) and compared them with ten lung tissue samples from patients who died of H1N1pdm09 (H1N1 group) with the immunohistochemical markers mentioned above. In addition, polymorphisms in the Perforin gene were genotyped through Real-Time PCR. Significantly increased tissue immunoexpression of Arginase, CD4, CD68, CD138, Perforin, Sphingosine-1, and IL-4 markers were observed in the COVID-19 group. A significantly lower immunoexpression of CD8 and CD57 was also found in this group. It is suggested that patients who died from COVID-19 had a poor cellular response concerning viral clearance and adaptive response going through tissue repair.

Low perforin expression in CD8+ T lymphocytes during the acute phase of severe SARS-CoV-2 infection predicts long COVID.

T cell cytotoxicity plays a major role in antiviral immunity. Anti-SARS-CoV-2 immunity may determine acute disease severity, but also the potential persistence of symptoms (long COVID). We therefore measured the expression of perforin, a cytotoxic mediator, in T cells of patients recently hospitalized for SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to Intensive Care Units (ICUs) or non-ICU, and 29 age- and sex-matched healthy controls (HCs). Amounts of intracellular perforin and granzyme-B, as well as cell surface expression of the degranulation marker CD107A were determined by flow cytometry. The levels of 15 cytokines in plasma were measured by Luminex. The frequency of perforin-positive T4 cells and T8 cells was higher in patients than in HCs (9.9 ± 10.1% versus 4.6 ± 6.4%, p = 0.006 and 46.7 ± 20.6% vs 33.3 ± 18.8%, p = 0.004, respectively). Perforin expression was neither correlated with clinical and biological markers of disease severity nor predictive of death. By contrast, the percentage of perforin-positive T8 cells in the acute phase of the disease predicted the onset of long COVID one year later. A low T8 cytotoxicity in the first days of SARS-CoV-2 infection might favor virus replication and persistence, autoimmunity, and/or reactivation of other viruses such as Epstein-Barr virus or cytomegalovirus, paving the way for long COVID. Under this hypothesis, boosting T cell cytotoxicity during the acute phase of the infection could prevent delayed sequelae.

Impaired immunity in elderly, vaccinated residents of an long-term care facility affected by a COVID-19 outbreak

The ongoing COVID-19 pandemic has hit long-term care facilities (LTCF), with outbreaks affecting both residents and health care workers (HCWs). Elderly persons have been prioritized in the implementation of vaccination programs. Here we investigated a COVID-19 outbreak, caused by the Beta variant (B.1.351) in a LTCF where residents and HCWs had received 2 doses of Comirnaty vaccine (Pfizer/BioNTech) until one month before the outbreak. Samples from 14 residents (SARS-CoV-2 PCR-negative: n = 8, PCR-positive: n = 6) and 10 HCWs (PCR-negative: n = 10) were collected at a median of 54 days following the second vaccine dose. IgG antibodies to SARS-CoV-2 spike glycoprotein and neutralizing antibody (NAb) titers were measured. Additionally, functional responses of PBMCs to SARS-CoV-2 spike and nucleocapsid proteins were investigated. We observed that Comirnaty induced higher IgG concentrations and NAb titers in HCWs compared to residents. PBMCs of HCWs responded vigorously to stimulation with SARS-CoV-2 spike glycoprotein, with the secretion of interferon gamma, granzyme B and perforin-1 into supernatants. In comparison, only 3 of 9 samples from residents showed positive cellular responses to spike glycoprotein. Group-level cellular responses directed at SARS-CoV-2 nucleoprotein remained low both in HCWs and in residents. Only 2 of 2 PCR-positive residents showed a positive response consistent with exposure to SARS-CoV-2 breakthrough infection. Our results show that elderly persons are at increased risk for breakthrough infection after vaccination. Weak vaccine-directed responses in the elderly need to be addressed in vaccination protocols.

Faster cytotoxicity with age: Increased perforin and granzyme levels in cytotoxic CD8+ T cells boost cancer cell elimination.

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM ) and central memory (TCM ) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.

Refractory Acute Disseminated Encephalomyelitis-like Illness Found to be CNS-isolated Hemophagocytic Lymphohistiocytosis due to a Novel PRF1 Mutation

Objective: 5-year-old boy with refractory acute disseminated encephalomyelitis (ADEM) found to have CNS-isolated Hemophagocytic Lymphohistiocytosis (HLH) with PRF1 mutation, c.4422G>A variant, not previously known to be pathogenic. Background: HLH is a hyperinflammatory condition that presents with fever, hepatosplenomegaly and characteristic laboratory findings. HLH can be familial (fHLH) or secondary (sHLH). Mutations in the perforin gene PRF1 have been identified as pathogenic for fHLH and they can also cause isolated CNS-HLH in the absence of systemic HLH. HLH can be triggered by malignancy, infections, or autoimmunity. Viruses are a common trigger and there have been cases of SARS-CoV-2 triggering HLH. Design/Methods: Clinical course, evaluation, management. Results: A 5-year-old boy presented to the emergency department with 3 weeks of headaches, right blurry vision, and emesis. He underwent an extensive evaluation and was diagnosed with ADEM that was thought to be triggered by SARS-CoV-2 after a positive nasal swab. Myelin oligodendrocyte glycoprotein (MOG) antibodies were negative. Patient completed a 5-day course of IV pulse steroids and plasma exchange. In the subsequent months, he was admitted twice due to worsening neuroinflammation and after several courses of IV pulse steroids, PLEX, and IVIG, the CNS inflammation stabilized with rituximab and monthly IVIG. A few months later, his younger brother presented to the emergency department with a similar syndrome. It was found that his parents were cousins, leading to concern for a genetic disorder. Genetic testing revealed a homozygous mutation for PRF1 in both siblings (variant c.4422G>A). Conclusions: This is the first presentation of CNS-isolated HLH triggered by SARS-CoV-2 in the pediatric population. Furthermore, thisis the first report of PRF1 mutation, the variant c.4422G>A, shown to be pathogenic. It highlights the relevance of genetics in pediatric neuroinflammatory disorders. It is possible that as our knowledge in neurogenetics develops, certain genes will be identified as predisposing factors to presentations such as ADEM.

T-CELL RESPONSE QUALITY to ACUTE SARS-CoV-2 INFECTION, IMMUNE MEMORY, and HCoV

Background: SARS-CoV-2 specific T-cell response has been associated with disease severity, immune memory and heterologous response to endemic coronaviruses (HCoV). However, an integrative approach combining a comprehensive analysis of the quality of SARS-CoV-2 specific T-cell response and antibody levels is needed. Methods: We assayed SARS-CoV-2 specific T-cell response in 103 participants. Thirty-seven (18 mild and 19 severe) were hospitalized during acute COVID-19 and 33 were recruited seven months after SARS-CoV-2 infection (19 previously hospitalized (H) and 14 non-hospitalized (NH) during acute infection). Pre-COVID-19 healthy donors (HD, n=33) were included. PBMCs were stimulated with Spike (S) and Nucleocapside (N) SARS-CoV-2 peptide pools. Likewise, an optimized peptide pool of HCoV S protein was used in HD. T-cell polyfunctionality by intracellular cytokine staining (IFN-γ, IL-2, TNF-α, CD107a and perforin (PRF)) was assayed by multiparametric flow cytometry together with measurements of T cell subsets, activation, exhaustion and senescence. Anti-S SARS-CoV-2 and HCoV IgG titers and pro-inflammatory markers were measured in plasma. Non-parametric statistic was used for the analysis. Results: Mild disease was associated with high T-cell polyfunctionality biased to IL-2 production and inversely correlated with anti-S IgG levels (eg, N-specific EM CD4+ IL-2+ T-cell, r=-0.594, p=0.004). However, only IFN-γ combinations without PRF production was mostly observed for severe disease (eg, S-specific TEMRA CD4+ CD107a-IFN-γ+IL-2-PRF-TNF-α-T-cells, p=0.008). Moreover, this response was long-lasting seven months after SARS-CoV-2 infection. Both NH and H individuals presented robust anti-S IgG levels and SARS-CoV-2 specific T-cell response. In addition, only H individuals showed a T-cell exhaustion profile (eg, TEMRA CD4+ TIGIT+ T cells, p=0.0004). Combinations including IL-2, but not IFN-γ, in response to HCoV S protein, were associated with SARS-CoV-2 S-specific T-cell response in HD (eg, S-specific CM CD8+ CD107a-IFN-γ-IL-2+PRF-TNF-α-T-cells, r=5414, p=0.001). Conclusion: T-cell polyfunctionality features were associated with disease severity. Moreover, T-cell response was robust seven months after infection, although previously hospitalized patients showed signs of exhaustion. SARS-CoV-2 and HCoV immune cross-reactivity have implications for protective immunity against SARS-CoV-2 to design new prototypes of vaccines in order to achieve of broader long-lasting protection against COVID-19.

IMMUNE PARAMETERS ASSOCIATED with LOWER SARS-CoV-2 VACCINATION RESPONSE in OLD PEOPLE

Background: The number of cases of SARS-CoV-2 infection after BNT162b2 mRNA vaccination is significantly higher in elderly people, which has been associated to lower frequencies of SARS-CoV-2 neutralizing antibodies. Our objective was to investigate the differences in the cellular response in old and young people after the SARS-CoV-2 vaccination. Methods: Young (24-53 years, n=20) and old (70-76 years, n=20) healthy subjects vaccinated with BNT162b2 SARS-CoV-2 mRNA vaccine were studied before vaccination, two weeks after the first dose and two months after the second dose. SARS-CoV-2 (spike) specific T cell response, TLR-4 dependent monocyte response and TLR-3 dependent myeloid dendritic cell (DC) response and DC, monocyte and T-cell immunophenotype, were studied by multiparametric flow cytometry. TLR-9 dependent interferon-α (IFNα) production by PBMCs was measured by ELISA and thymic function assayed by sj/β TREC ratio using droplet digital PCR. Results: The SARS-CoV-2 specific T cell response was lower and less polyfunctional in old people. Most of the differences in CD4+ and CD8+ T cell subsets were found in degranulation (CD107a), cytokine (IFN-γ) and cytotoxic (perforin) profile (eg, Memory CD8+ perforin+;p=0.0016). The lower SARS-CoV-2 specific T cell response was associated with lower thymic function levels (eg, Memory CD4+ perforin+, r=0.631;p=0.0001). The vaccination induced a higher activation and proliferation (eg, CM CD4 HLA-DR+ p=0.002, Ki67+ p=0.019) of T cells in young people than in old ones, in addition to a higher level of homing makers to different tissues and inflammatory sites (eg, CD1c mDC integrin β7+ p=0.001, intermediate monocytes CCR2+ p=0.0003) in DCs and monocytes. Moreover, after the vaccination, old subjects showed a higher production of proinflammatory cytokines by monocytes in response to LPS (eg, IL6+;p=0.015), while young people showed a higher production of IFNα by plasmacytoid DCs after CpG-A stimulation (p=0.0009). Conclusion: The magnitude and polyfunctionality of SARS-CoV-2 specific T cell response is lower in old people, associated to a lower thymic function. In old people, the vaccination induced less immune activation and homing and the myeloid TLR-dependent response is directed towards a proinflammatory response, while in young people prevails IFNα production, related to a more effective antiviral response. These results support the additional boosting strategies in this vulnerable population.

Pharmacologic inhibition of SUMOactivating enzyme potentiates interferon response and T cell-mediated anti-tumor immunity in chronic lymphocytic leukemia (CLL) and lymphoma models

Introduction: CLL is characterized by deficient immunity which clinically manifests as an increased predisposition toward malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with a predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Unlike chemotherapy, novel targeted agents may have beneficial immunomodulatory effects, which may be particularly relevant in the COVID-19 era. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription, and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFjB signaling, IFN response, and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in the context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. Activation, proliferation, and apoptosis of CD3+ T cells were studied following T-cell receptor engagement (TCR;aCD3/CD28) with/without 0-1 lM TAK-981. Cytokines were measured after in vitro stimulation. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP;Clariom S), RNA was harvested after 3 and 24 h of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 mg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days before spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1×106 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement. Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels, yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFjB, and NFAT activation detected by immunoblotting, immunocytochemistry, and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4+ T cells following 72 and 96 h of TCR stimulation vs. control. Meanwhile, the expression of CD25, HLA-DR, and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced 2-fold in TAK-981-treated cells after 24 h and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 lM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL- 2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNc and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift toward Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4+ naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNc, and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in express on of FoxP3 and an increased production of IFNc. In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNc secretion in CD8+ TILs. Conclusion: Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies.

A rare disease in the community hospital: Hemophagocytic lymphohistiocytosis (HLH) needs immediate local attention for early recognition and individualized treatment

Purpose: HLH is a severe hyperinflammatory syndrome characterized by highly active cytotoxic T-cells, NK-cells, and macrophages. If undiagnosed, HLH can lead to multiorgan damage and death. Conditions triggering HLH are infections, malignancies and autoimmune/-inflammatory (MAS-HLH) disorders. Immunosuppressive patients are prone to develop infection triggered HLH. The incidence in the European community hospital is unknown, as is the number of unrecorded cases. HLH-patients, diagnosed at a single communal hospital with an adjacent catchment area of 500,000 citizens, were reviewed in the context of national and international guidelines. Methods: From 08/2016 to 11/2020, 13 HLH patients were analysed retrospectively. Both HLH-2004 criteria and the web-based Hscore were used to diagnose HLH. The collected data depicts clinical presentation, underlying disease, laboratory findings, and treatment. Results: This Study includes 13 HLH-patients (10 male). Median age at diagnosis was 53, ranging from 27-80 years. Most common triggers in our cohort were infections (n=7) and malignancies(n=4). MAS-HLH (n=1) was seen in a Still's disease patient. HLH-related gene mutation was identified (n=1) with a heterozygote mutation in Perforin (PrfA91). Lymphomas of B-as well as T-cell origin (n=2) and AML (n=3) represented main cause in malignancy associated HLH. Viral infections i.e., COVID-19(n=1), RSV (n=1) and EBV (n=1), also bacterial infections like M. tuberculosis (n=1), and the attenuated strain BCG (n=2) were seen in infection associated HLH. Most patients presented with fever (n=9) and splenomegaly (n=4). HLH patients show pancytopenia, peak ferritin levels ranging 1352-185000 ng/ml (median=21600), peak soluble IL-2 receptor levels ranging 2571-21660 U/ml (median=6606), and peak triglyceride levels ranging 175-610 mg/ml (median=227). Hemophagocytosis in bone marrow was found in 6 patients. First line therapy was glucocorticoids (n=12) combined with polyvalent immunoglobulins. Etoposide (n=5) and chemotherapy (n=4) were given to malignancy triggered HLH. Rituximab was applied in EBV-triggered HLH. Anakinra (n=3) and Ruxolitinib (n=4) was given to selected patients. Two patients received cytokine-depletion using adsorption columns Cytosorb®. Multiorgan failure (n=5) was the most common cause of death. Conclusion: This data provides incidence estimation of HLH in adult patients. Institutional and national measures will be presented to prevent death due to HLH.

Covid-19 in a patient with Familial Hemophagocytic Lymphohistiocytosis in children.

BACKGROUND: Based on the information obtained so far, COVID- 19 is relatively mild in children. We will present a 6-month-old male patient infected with COVID -19 in April 2020, while receiving HLH 2004 chemotherapy protocol with the diagnosis of familial (Genetic / Primary) Hemophagocytic Lymphohistiocytosis (HLH). CASE: Herein we present a case accompanied by a defective perforin gene defect in the primary HLH pathogenesis, Covid-19 infection with the presence of fever and hyperferritinemia, which was evaluated in favor of reactivation and the patient was given both the HLH-2004 chemotherapy protocol treatment and COVID -19 therapy as recommended by the guidelines. Our patient improved clinically and in terms of laboratory test results at the end of the 15 < sup > th < /sup > day of hospitalization and was discharged. CONCLUSIONS: It should be remembered that COVID-19 can be seen with different clinical manifestations in the pediatric age group, and COVID-19 tests should be recommended, especially in children with immunosuppression and fever.

Early Response of CD8+ T Cells in COVID-19 Patients.

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

Perforin, COVID-19 and a possible pathogenic auto-inflammatory feedback loop.

During COVID-19 infection, reduced function of natural killer (NK) cells can lead to both compromised viral clearance and dysregulation of the immune response. Such dysregulation leads to overproduction of cytokines, a raised neutrophil/lymphocyte ratio and monocytosis. This in turn increases IL-6 expression, which promotes scar and thrombus formation. Excess IL-6 also leads to a further reduction in NK function through downregulation of perforin expression, therefore forming a pathogenic auto-inflammatory feedback loop. The perforin/granzyme system of cytotoxicity is the main mechanism through which NK cells and cytotoxic T lymphocytes eliminate virally infected host cells, as well as being central to their role in regulating immune responses to microbial infection. Here, we present epidemiological evidence suggesting an association between perforin expression and resistance to COVID-19. In addition, we outline the manner in which a pathogenic auto-inflammatory feedback loop could operate and the relationship of this loop to genes associated with severe COVID-19. Such an auto-inflammatory loop may be amenable to synergistic multimodal therapy.

Dose-Related Aberrant Inhibition of Intracellular Perforin Expression by S1 Subunit of Spike Glycoprotein That Contains Receptor-Binding Domain from SARS-CoV-2.

Studies had shown that severe cases of COVID-19 tend to have high viral loads and correlate with functional impairment of cytotoxic lymphocytes, and the features of cytokine storm syndrome are similar to manifestations of severe influenza that have been partially explained by suppressed perforin expression. To test the hypothesis that the spike glycoprotein from SARS-CoV-2 may inhibit the perforin expression, we determined the kinetics of immune responses of CD8+ T cells to low dose (LD) or high dose (HD) of S1 stimulation through an in vitro dendritic cell (DC)-T cell model over seven days of incubation. The cytotoxic activity and intracellular perforin expression of CD8+ T cells induced by HD-S1-presenting DCs were aberrantly lower than those induced by LD-S1-presenting DCs from day three of incubation. Discrepantly, the levels of lymphoproliferation and cytokine (interferon-γ and tumor necrosis factor-α) production induced by HD-S1-presenting DCs were significantly higher than those induced by LD-S1-presenting DCs from day four. The dose-related responses between doses of S1 and intracellular perforin expression showed a significant linear correlation with a negative slope. In conclusion, the S1 subunit may suppress the perforin expression in CD8+ T cells to decrease the cytotoxic capacity to kill spike-presenting cells in a dose-dependent manner; the persistence of antigen presentation may result in an overproduction of interferon-γ and subsequent proinflammatory cytokines. That may help explain the insufficient cytotoxicity against high quantities of viruses or highly replicated strains of SARS-CoV-2 in severe cases of COVID-19.

SARS-CoV-2 infection paralyzes cytotoxic and metabolic functions of the immune cells.

The SARS-CoV-2 virus is the causative agent of the global COVID-19 infectious disease outbreak, which can lead to acute respiratory distress syndrome (ARDS). However, it is still unclear how the virus interferes with immune cell and metabolic functions in the human body. In this study, we investigated the immune response in acute or convalescent COVID-19 patients. We characterized the peripheral blood mononuclear cells (PBMCs) using flow cytometry and found that CD8+ T cells were significantly subsided in moderate COVID-19 and convalescent patients. Furthermore, characterization of CD8+ T cells suggested that convalescent patients have significantly diminished expression of both perforin and granzyme A. Using 1H-NMR spectroscopy, we characterized the metabolic status of their autologous PBMCs. We found that fructose, lactate and taurine levels were elevated in infected (mild and moderate) patients compared with control and convalescent patients. Glucose, glutamate, formate and acetate levels were attenuated in COVID-19 (mild and moderate) patients. In summary, our report suggests that SARS-CoV-2 infection leads to disrupted CD8+ T cytotoxic functions and changes the overall metabolic functions of immune cells.

COVID-19 pneumonia: CD8+ T and NK cells are decreased in number but compensatory increased in cytotoxic potential.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is posing a huge threat to human health worldwide. We aim to investigate the immune status of CD8+ T and NK cells in COVID-19 patients. METHODS: The count and immune status of lymphocytes were detected by flow cytometry in 32 COVID-19 patients and 18 healthy individuals. RESULTS: As the disease progression in COVID-19 patients, CD8+ T and NK cells were significantly decreased in absolute number but highly activated. After patients' condition improved, the count and immune status of CD8+ T and NK cells restored to some extent. GrA+CD8+ T and perforin+ NK cells had good sensitivity and specificity for assisting diagnosis of COVID-19. CONCLUSIONS: As the disease progression, the declined lymphocytes in COVID-19 patients might lead to compensatory activation of CD8+ T and NK cells. GrA+CD8+ T and perforin+ NK cells might be used as meaningful indicators for assisting diagnosis of COVID-19.

[Lymphocytic myocarditis in patients with COVID-19 (4 autopsy cases)]. / Limfotsitarnyi miokardit u patsientov s COVID-19 (4 autopsiinykh nablyudeniya).

The paper describes 4 autopsy cases of myocarditis in elderly patients with confirmed coronavirus infection. It gives the data of a morphological study of heart specimens and a detailed characterization of a myocardial infiltrate. An immunohistochemical study with cellular infiltrate typing was performed. The fact that lymphocytic viral myocarditis can develop in COVID-19 was morphologically and immunohistochemically confirmed. The features of myocarditis in COVID-19 are the development of the former in the presence of coronaritis and the possibility of its concurrence with lymphocytic endo- and pericarditis.