ABSTRACT
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.
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Background: mRNA vaccines have proven useful in protecting vulnerable populations against SARS-CoV-2 infection. However, certain therapeutics, specifically those used in cancer treatment, reduce mRNA vaccine-induced humoral responses against SARS-CoV-2. The effects on T cell responses are not well characterized. Here, we evaluate SARS-CoV-2 spike-specific T cell responses over the course of one year in solid tumor patients in BC, Canada. Method(s): 18 female, solid-tumor patients from the BC Cancer Agency were enrolled in this prospective, cohort study, with 7 patients receiving cytotoxic chemotherapy and 11 patients receiving non-cytotoxic treatments. Whole blood was collected 1-month (T1) and one-year +/- 1-month (T2) post series completion (2 mRNA doses). Antigen-induced marker assays (AIM assays) were used to quantify CD4+ and CD8+ T cell responses, where whole blood was stimulated with ancestral or omicron SARS-CoV2 Spike peptide pools or unstimulated for 48 hours at 37degree C, fluorescently stained for activation markers CD25 and OX40 (CD4+ T cells) or CD69 and CD137 (CD8+ T cells), and analyzed using a 5-laser flow cytometer. Phenotyping of antigen-specific CD4+ T cells was done in parallel to assess the frequency of spike-specific Tregs, Th1, Th2, Th9, Th17, and Th17.1 cells. Result(s): All individuals had detectable levels of spike-specific CD4+ T cells at T2, while only 72.2% of individuals had detectable levels of spike-specific CD8+ T cells. Treatment type did not significantly impact the magnitude or phenotype of T cell responses, including those to Omicron. However, increased age was associated with decreased ancestral CD8+ T cell responses at T2. Further, ancestral and omicron responses were significantly different at T2, with decreased magnitude and altered phenotype of omicron-specific CD4+ T cells. Conclusion(s): Here, we report that solid tumor patients, treated with either chemotherapy or biologics, mount robust T cell immunity to SARS-CoV-2 following vaccination. Additional data is needed to determine if these responses correlate with antibody levels and clinical illness.
ABSTRACT
Background: T cells play a critical role in the adaptive immune response to SARS-CoV-2 in both infection and vaccination. Identifying T cell epitopes and understanding how T cells recognize these epitopes can help inform future vaccine design and provide insight into T cell recognition of newly emerging variants. Here, we identified SARS-CoV-2 specific T cell epitopes, analyzed epitope-specific T cell repertoires, and characterized the potency and cross-reactivity of T cell clones across different common human coronaviruses (HCoVs). Method(s): SARS-CoV-2-specific T cell epitopes were determined by IFNgamma ELISpot using PBMC from convalescent individuals with mild/moderate disease (n=25 for Spike (S), Nucleocapsid (N) and Membrane (M)), and in vaccinated individuals (n=27 for S). Epitope-specific T cells were isolated based on activation markers following a 6-hour peptide stimulation, and scRNAseq was performed for TCR repertoire analysis. T cell lines were generated by expressing recombinant TCRs in Jurkat cells and activation was measured by CD69 upregulation. Result(s): We identified multiple immunodominant T cell epitopes across S, N and M proteins in convalescent individuals. In vaccinated individuals, we detected many of the same dominant S-specific epitopes at similar frequencies as compared to convalescent individuals. T cell responses to peptide S205 (amino acids 817-831) were observed in 56% and 59% of individuals following infection and vaccination, respectively, while 20% and 19% of individuals responded to S302 (a.a. 1205-1219) following infection and vaccination, respectively. For S205, a CD4+ T cell response, we confirmed 8 unique TCRs and determined the minimal epitope to be a 9mer (IEDLLFNKV). While TCR genes TRAV8-6*01 and TRBV30*01 were commonly utilized across the TCRs, we did identify TCRs with unique immunogenetic properties with different potencies of cross-reactivity to other HCoVs. For S302, a CD8+ T cell response, we identified two unique TCRs with different immunogenetic properties that recognized the same 9mer (YIKWPWYIW) and cross-reacted with different HCoV peptides (Figure 1). Conclusion(s): These data identify immunodominant T cell epitopes following SARS-CoV-2 infection and vaccination and provide a detailed analysis of epitope-specific TCR repertoires. The prospect of developing a vaccine that broadly protects against multiple human coronaviruses is bolstered by the identification of conserved immunodominant SARS-CoV-2 T cell epitopes that cross react with multiple other HCoVs.
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Background: Natural killer (NK) cells play a critical role in control of viral infections. However, empirical evidence thus far has been unclear on the role of NK cells in pathogenesis and control of SARS-CoV-2 infection with some research suggesting NK cell accumulation as beneficial while others indicate it as deleterious. To address this crucial deficit in understanding, we employed a non-human primate infection model with a validated experimental NK cell depletion technique. Method(s): A total of 12 experimentally naive (75% female) cynomolgus macaques (CM) of Cambodian origin were used in this study. Six CM were NK cell-depleted using an anti-IL-15 neutralizing antibody, while six controls received placebo, prior to intranasal and intratracheal challenge with the SARS-CoV-2 Delta variant at a TCID50 of 1X105. The cohort was monitored for five weeks with scheduled blood, colorectal (CR) biopsies, and lymph node (LN) collections. Total envelope and sub-genomic viral loads (VL) were measured in the nasal cavity, throat, and bronchoalveolar lavage (BAL). 23-color flow cytometry, pathology, and 27-plex inflammatory analyte Luminex analyses were conducted. Statistical tests used were Mann-Whitney U and Spearman's Correlation. Result(s): Control CM exhibited an increase in the frequency of circulating NK cells, reaching a peak at 10 days post-infection (DPI) and returning to baseline by 22DPI. Simultaneously, NK cells expressing activation and tissue retention marker, CD69, also significantly increased. Cytotoxic NK cells were positively associated with VL (r=0.66;p=0.02), suggestive of a virus-induced mobilization. Total experimental NK cell ablation was verified in blood, CR, and LN of NK celldepleted CM, which had higher VL compared to controls in all tissues evaluated, reaching significance at 10DPI (p=0.01) and demonstrated a longer duration of viremia. Although Luminex measures were similar in plasma, BAL samples from NK cell-depleted CM had universally higher concentrations of inflammatory mediators, most notably a 25-fold higher concentration of IFN-alpha compared to controls. Lung pathology scores were also higher in NK cell-depleted CM with increased evidence of fibrosis, syncytia, pneumocyte hyperplasia, and endothelialitis. Conclusion(s): Overall, we find significant and conclusive evidence for NK cell-mediated control of SARS-CoV-2 virus replication and disease pathology. These data suggest adjunct therapies for infection could largely benefit from NK cell-targeted approaches.
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The decline in vaccine efficacy and the risk of reinfection by SARS-CoV-2 make new studies important to better characterize the immune response against the virus and its components. Here, we investigated the pattern of activation of T-cells and the expression of inflammatory factors by PBMCs obtained from naive and previously infected subjects following COVID-19 vaccination, after PBMCs stimulation with S1, RBD, and N-RBD SARS-CoV-2 proteins. PBMCs showed low levels of ACE2 and TMPRSS2 transcripts, which were not modulated by the exposure of these cells to SARS-CoV-2 proteins. Compared to S1 and RBD, N-RBD stimulation showed a greater ability to stimulate T-cell reactivity, according to CD25 and CD69 markers. Interestingly, T-cell reactivity was more pronounced in vaccinated subjects with prior SARS-CoV-2 infection than in vaccinated donors who never had been diagnosed with COVID-19. Finally, N-RBD stimulation promoted greater expression of IL-6 and IFN-gamma in PBMCs, which reinforces the greater immunogenic potential of this protein in the vaccinated subjects. These data suggest that PBMCs from previously infected and vaccinated subjects are more reactive than those derived from just vaccinated donors. Moreover, the N-RBD together viral proteins showed a greater stimulatory capacity than S1 and RBD viral proteins.Copyright © 2022
ABSTRACT
Background & Aim: Due to its immunomodulatory potential, therapy based on the transfer of regulatory T cells (Tregs) has acquired great interest in the treatment of diseases in which it is necessary to restore immune homeostasis. Until now, autologous Treg cell therapy has proven to be safe, but the employment of blood as the source of Treg presents several limitations in terms of Treg recovery and the quality of the employed Tregs. Our group has developed a new technology to produce massive amounts of GMP Treg derived from the pediatric thymic tissue discarded in pediatric cardiac surgeries (thyTreg) that could overcome the main obstacles. Indeed, we are employing thyTreg cells with success in a clinical trial as autologous cell therapy in transplanted children. Given the large amounts of thyTreg that can be obtained from a single thymus, the main objective of this work is to evaluate the immunogenicity of thyTreg and confirm that its immature phenotype makes possible the allogeneic use of this cellular therapy in order to treat a range of immune diseases and patients. Methods, Results & Conclusion: The thyTreg obtained in the laboratory using the protocol developed by our group exhibit high viability (>90%) and high purity (>80%) in terms of CD25+FoxP3+ expression. ThyTreg have been observed to express low levels of immunogenicity markers (CD40L, CD80, CD86) by flow cytometry. Moreover, in vitro models of thyTreg co-culture with allogeneic peripheral blood mononuclear cells (PBMC) from healthy donors have been performed to i) determine if thyTreg generate an immunogenic response on PBMC, and ii) evaluate the capacity of thyTreg to suppress the proliferation of allogeneic PBMC. Even that the HLA disparity in the allogeneic cocultures between thyTreg and PBMC was high (13 of the 21 typed pairs had HLA <4/12 concordance), thyTreg did not induce the expression of activation markers (CD25, CD69) nor the proliferation or the production of pro-inflammatory cytokines (IFN-g) by allogeneic PBMCs. Moreover, thyTreg greatly inhibit the proliferation of allogeneic CD4 and CD8 T cells, reaching levels of around 70% inhibition of proliferation at a 1: 1 ratio. The results suggest that allogenic thyTreg are not immunogenic and are capable of exerting their suppressive function in an allogeneic context, indicating their possible off-the-shelf use as a treatment for transplant rejection, graft-versus- host disease, autoimmune diseases or the cytokine release syndrome characteristic of severe COVID-19 patients.
ABSTRACT
Introduction: Patients with hematological malignancies exhibit inferior response to SARS-CoV2 vaccination, compared to healthy individuals, however little is known about patients with precursor hematological malignancies and the cellular underpinnings of vaccination response. Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Myeloma (SMM) are plasma cell premalignancies that precede Multiple Myeloma (MM) and exhibit signs of immune dysregulation;they affect approximately 5% of the population over 50 years of age, who remain largely undiagnosed, due to lack of screening. In November 2019, we launched the IMPACT study to characterize the immune response to SARS-CoV2 vaccination in patients with plasma cell dyscrasias and healthy individuals. Methods: We performed single-cell RNA-sequencing on 224 peripheral blood mononuclear cell samples drawn from 118 IMPACT (IRB #20-332) participants with MGUS (n=20), SMM (n=48), or MM (n=24), as well as healthy individuals (n=26). Samples were collected before vaccination and after 2 doses of BNT162b2 (Pfizer-BioNtech) (n=123), mRNA-1273 (Moderna) (n=83) or 1 dose of Ad26.COV2.S (Janssen) (n=14). Results: Overall, we sequenced 2,025,611 cells from 224 samples of 118 patients with MGUS, SMM, MM and healthy individuals pre- and post-vaccination for SARS-CoV2, and profiled 553,082 T-cells, 95,392 B-cells, 74,394 NK cells, 195,371 Monocytes, and 35,236 Dendritic cells (DC). We identified activated clusters of B-cells, NK cells and DCs expressing genes such as CD83, CD69, CXCR4, and genes related to the NF-kB and AP-1 pathways. We compared cell type abundances pre- and post-vaccination within each participant population and found that activated CD83+ cells significantly increased post-vaccination in healthy individuals and patients with MGUS (paired t-test, q < 0.1), but not in patients with SMM or overt MM. At baseline, patients with SMM and MM had significantly fewer memory B-cells and significantly more cytotoxic T-cells and NK cells, compared to healthy individuals (Wilcoxon, q < 0.1), which could partly explain the differences observed post-vaccination. Patients with MM also had significantly higher levels of tolerogenic IL-10-expressing DCs (DC10) at baseline (Wilcoxon, q < 0.1), which could be dampening antigen-specific T-cell responses. Conclusion: We identified a significant expansion of activated B-cell, NK cell and DC subpopulations expressing CD83, CD69 and CXCR4, following vaccination in healthy individuals and patients with MGUS, but less so in patients with SMM and overt MM. Our results provide insight into the cellular mechanisms of immune response to SARS-CoV2 vaccination in healthy individuals and patients with precursor plasma cell malignancies and suggest that asymptomatic individuals with SMM may exhibit inferior response to vaccination.
ABSTRACT
During the first period of the Covid-19 pandemic, most of the immunological studies on SARS-CoV-2 were based on hospitalized-and intensive care unit patients. In this study, a healthy population continuously exposed to the virus, Swedish primary health care workers (n = 156), were monitored for 6 months and the development of antibody patterns and T-cell responses to SARS-CoV-2 were evaluated. In addition to blood sampling, demographic-and clinical information such as PCR-tests, self-reported symptoms, underlying medical conditions, and medications were collected. Multivariate statistical analysis using OPLS-DA showed that Covid-19 infection was associated with SARS-CoV-2 specific IgG antibodies, T-cell responses, male sex, hypertension, and higher BMI and contrary, that not contracting the infection, was associated with female sex, no-or only SARS-CoV-2 specific IgA antibodies, smoking and airborne allergy. Analysis with Cytometry by Time-of-flight (CyTOF) revealed a unique cytotoxic CD4+ T cell population in participants with IgG-dominated antibody responses which expressed CD25, CD38, CD69, CD194, CD279, CTLA-4 and granzyme B. 10% of the study participants had only SARS-CoV-2 specific IgA antibodies with no detectable SARS-CoV-2 specific IgG antibodies. These IgA antibodies could partially neutralize the virus in vitro and none of the participants with this antibody pattern contracted Covid-19 during the study period. These results have the potential to further help us understand the immunological responses to SARS-CoV-2 infection.
ABSTRACT
Background: As part of a combined HIV CURE immuno-therapy strategy, we transduced primary human NK cells with the high affinity CD64 Fc receptor and pre-loaded them with HIV-specific bNAbs. We named these chimeric NK cells "NuKES" (NK Enhancement Strategy) for their augmented capacity to mediate ADCC and their potential clinical application as an autologous primary NK cell immuno-therapy against HIV. Methods: We transduced primary NK cells from control donors with a lentivirus expressing human CD64 in the presence or absence of irradiated K562 feeder cells expressing co-stimulatory molecules (CD40, 4-1BB) and/or cytokine pre-stimulation (IL-2, IL-21, IL-15). CD64 expressing NK cells were CFSE labeled and expanded ex vivo or FACS sorted at various times post transduction to high purity. CD64 expressing NK cells were then pre-loaded with HIV-specific bNAbs and tested in a functional ADCC CD107a degranulation assay against HIV-1 infected autologous CD4+ primary T cells. Results: After pre-stimulation with cytokines and/or irradiated K562 Feeder Cells, we could routinely achieve (n=5) greater than 40% CD64 expression in primary human NK cells (Day 14 post-transduction shown in Figure 1A). NK cells maintained strong proliferation potential with greater than 6 cells divisions beyond 10 days post transduction as determined by CFSE dilution (Day 10 post-transduction shown in Figure 1B). Phenotypically, CD64 transduced NK cells were similar to control NK cells and possessed strong expression of CD56, CD16, CD69 with intermediate levels of the NK maturation marker CD57. CD64 transduced NK cells could be successfully pre-loaded with HIV-specific bNAbs and possessed an enhanced capacity (GMFI of 2,014 versus 276) to retain 10-1074 for several hours as compared to control NK cells (Figure 1C). Functionally, CD64 transduced NK cells showed a significant two-fold increase in ADCC-triggered degranulation capacity against autologous HIV-1 infected CD4+ primary T cells compared to control NK cells after pre-loading with HIV-specific bNAbs (27.6% versus 13.2% CD107a). Conclusion: Primary human NK cells can be successfully transduced with CD64 and expanded ex vivo to high purity. Preparation of bNAbs specific NuKES represent a viable autologous NK immuno-therapy approach against HIV-1 with potential adaptation for added disease targets (i.e., COVID, Cancer) moving forward.
ABSTRACT
Background: T cells have been shown to play a role in the immune response to SARS-CoV-2. Identification of T cell epitopes and a better understanding of the T cell repertoire will provide important insights into how T cells impact antiviral immunity. Here, we identified T cell epitopes within the Spike (S), Nucleocapsid (N) and Membrane (M) proteins from SARS-CoV-2 convalescent individuals and performed TCR sequencing on epitope-specific T cells. Methods: Epitope mapping was performed by IFNγ ELISpot on PBMC from SARS-CoV-2 convalescent patients with mild/moderate disease (n = 19 for S;n=15 for N and M), and minimum epitopes were determined using truncated peptides and ICS. TCR sequence analysis was performed on a subset of individuals (n=9 donors;2-3 epitopes/donor), with longitudinal samples for 7 donors (2-3 time points/donor;33 to 236 days post-symptom onset). T cells were stimulated with individual peptides for 6 hours and sorted based on the expression of activation markers (CD4+: CD69, CD40L;CD8+: CD69, CD107a, surface TNF). scRNAseq was performed on sorted cells for TCR repertoire and transcriptome analysis. Results: We identified several peptides recognized by multiple individuals, including S42 (amino acids 165-179;7/19 donors), S302 (a.a. 1205-1219;6/19 donors), N27 (a.a. 106-120;6/14 donors) and M45 (a.a. 177-191;10/14 donors). S42 elicited both CD4+ (n=5) and CD8+ (n=1) T cell responses, with one individual having both a CD4+ and CD8+ response. The minimum epitope for S42 was determined to be a 9mer (FEYVSQPFL) for both CD4+ and CD8+ cells. TCR sequencing of S42-specific T cells identified a dominant gene pairing for TCRα across multiple donors (TRAV35;TRAJ42) and for both CD4+ and CD8+ T cells (Figure 1). In general, epitope-specific CD4+ responses (S42, M45) were more clonally diverse than CD8+ responses (S42, S302, N27). For both CD4+ and CD8+ T cells, conserved TCR gene usage and gene pairings could be identified within multiple donors responding to the same epitope. Conclusion: These data suggest that in SARS-CoV-2 convalescent people, epitope-specific CD4+ and CD8+ T cells can differ in their clonal diversity and that related TCRs can be identified across multiple donors. S42-specific T cell studies are ongoing to determine their transcriptional profile and pMHC presentation. Ongoing longitudinal analysis will provide a better understanding of different epitope-specific TCR repertoires and T cell transcriptional profiles, and how they evolve after infection.
ABSTRACT
Many patients with haematological cancers remain incompletely protected from SARS-CoV-2 following two doses of vaccine with Pfizer-BioNTech BNT162b2 nCoV-19 or ChAdOx1. Myelodysplastic syndrome (MDS) represents a spectrum of clonal bone marrow neoplasms. The response of patients with MDS to the COVID-19 vaccines remains unknown. Here, we report the humoral and T-cell responses of patients with low-and high-risk myelodysplastic syndrome (MDS), 2 weeks following completion of the second-dose schedules of ChAdOx1 or BNT162b2 nCoV-19 vaccines. Patients with MDS ( n = 38) followed up at Kings College Hospital, London were vaccinated with either BNT162b2 mRNA or ChAdOx1 nCoV-19 vaccine. Written informed consent was provided. Eligibility criteria included the diagnosis of MDS as per the WHO classification and age ≥18 years. Healthy volunteers (HV;n = 30) served as a reference group. Blood samples were collected 2 weeks after the second vaccine dose. Plasma samples were tested for SARS-CoV-2-specific antibody aimed at the SARS-CoV-2 spike (S) protein receptor-binding domain and neutralisation assays against pseudotypes with SARS-CoV-2 Wuhan strain (WT), VOC.B.1.1.7 (alpha) or VOC.B.1.617.2 (delta) Spike. Cellular responses were assessed using IFNγ ELISPOT and flow cytometry (CD25 and CD69 expression) after 24 h peptide stimulation. IFNγ ELISpot analysis was performed ex vivo for assessment of T-cell response. 32% of the MDS patients received BNT162b2 and 58% received ChAdOx1 nCoV-19 vaccines. All HV received BNT162b2. Overall serological responses were as follows: HV BNT162b2 100% (26/26);MDS BNT162b2 100% (15/15) and MDS ChAdOx1 76.2% (16/21). Notably, the MDS ChAdOx1 cohort demonstrated significantly decreased serological titres to the MDS BNT162b2 cohort. The functional implications of seroconversion were assessed by neutralisation assays for SARS-CoV-2 WT and VOC alpha and delta. All but four MDS patients could neutralise all variant strains, but MDS cohorts showed significantly reduced median neutralisations for all three variant strains compared to HV. Five MDS ChAdOx1 patients who did not have a serological response were able to mount T-cell responses. Additionally, treatment with either azacytidine or calcineurin inhibitor cyclosporin did not impair appropriate T-cell responses. The numbers of individuals who were both serological and T-cell responders were as follows: HV 95% (20/21), MDS BNT162b2 71.4% (10/14) and MDS ChAdOx1 52.9% (9/17). Overall serological responses in the MDS cohorts were 100% for those who had completed the two-dose BNT162b2 vaccine schedule compared to 76.2% of patients vaccinated with the ChAdOx1 vaccine. It may be advisable that MDS patients are boosted with an mRNA-based vaccine to promote enhanced immunity in this specific population. We observed that neutralisation in seroconverted patients was significantly weaker for both the ChAdOx-1 and BNT162b2 MDS cohorts compared to HV. This highlights the continued need for a third primary dose for this clinically vulnerable patient group and our further work will analyse the cohort's response to this.
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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.
ABSTRACT
Myelodysplastic syndromes (MDS) represent a spectrum of clonal bone marrow neoplasms from low risk disease through to those transforming into acute myeloid leukaemia. The COVID-19 pandemic has presented a great risk to those with hematological malignancies who are at higher risk of severe disease and death than the general population. Previous studies looking at the immune response to influenza vaccination in those with MDS had shown promising results, with immune responses not differing from those of healthy family members. Whilst some data exist to reassure the MDS community that majority of patients show seroconversion following Covid-19 vaccination, little data exists on their neutralizing capacity or post vaccination T-cell responses in this cohort. In addition, the majority of patients in these studies received BNT162b2 and there is little published data on vaccine response to the ChAdOx1 nCoV-19 vaccine. We have investigated the humoral and T-cell response of 39 patients with MDS two to four weeks following Covid-19 booster vaccination with BNT162b2 or ChAdOx1 nCoV-19 through the SOAP study (Sars-cov-2 fOr cAncer Patients, IRAS project ID:282337). Plasma and PBMCs from MDS cases and healthy controls have been collected, and are being assessed for both humoral and cellular responses to SARS_CoV_2, the alpha (B.1.1.7) and delta (B.1.617.2) variants. Humoral responses will be assessed using ELISA (peptide binding) and functional viral neutralization assays. Cellular responses will be assessed using IFNy ELISPOT and flow cytometry (CD25 and CD69 expression) after 24h peptide stimulation. All data at time point 1 (2 - 4 weeks following booster vaccination) have been collected and will subsequently be collected at 6 months and 12 months post-vaccination. We also report on the safety data for these vaccines within this patient population. Of this cohort 64% were male with a median age of 65 years (range 21-84). 54% received vaccination with ChAdOx1 nCoV-19 and 44% received BNT162b2 (2% unrecorded). The vaccines were well tolerated with no serious adverse events to date. The mean interval between doses was 70.7 days (range 50 - 90 days). 71% of the cohort were receiving no disease modifying therapy at the time of vaccination, half of whom were receiving supportive therapy and the other half no intervention for their MDS. Of those receiving disease modifying therapy;5 were receiving azacitidine, (1 in conjunction with low-dose cytarabine) and 3 ciclosporin. We will report the largest study of the humoral and T-cell mediated response to the Covid-19 vaccine in MDS patients to date. This will include cellular response to the delta variant and immunogenicity of both the BNT162b2 and ChAdOx1 nCoV-19 vaccines. Given the vulnerability of these patients to severe disease, investigating the immune response to the vaccines begins to build an evidence base for advising MDS patients on their ongoing risk of infection during the pandemic and going forward. The SOAP study will reassess the immune response at 6 and 12 months post-vaccination to continue to investigate post-vaccine immunity in this cohort. Disclosures: Kulasekararaj: F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Speakers Bureau;Apellis: Consultancy;Akari: Consultancy, Honoraria, Speakers Bureau;Biocryst: Consultancy, Honoraria, Speakers Bureau;Achilleon: Consultancy, Honoraria, Speakers Bureau;Alexion: Consultancy, Honoraria, Speakers Bureau;Ra Pharma: Consultancy, Honoraria, Speakers Bureau;Amgen: Consultancy, Honoraria, Speakers Bureau;Novartis: Consultancy, Honoraria, Speakers Bureau;Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau;Alexion, AstraZeneca Rare Disease Inc.: Consultancy, Honoraria, Other: Travel support. Patten: JANSSEN: Honoraria;NOVARTIS: Honoraria;GILEAD SCIENCES: Honoraria, Research Funding;ROCHE: Research Funding;ASTRA ZENECA: Honoraria;ABBVIE: Honoraria.