Furthermore, studies investigating vaccine responses predominantly focused on the production of specific antibodies while very few studies examined the T cell response evaluated by SARS-CoV-2-specific tetramer-positive CD8 T cells and functional CTLs [21C30]. with SARS-CoV-2 spike proteinCspecific tetramers, and for functional CTLs (CD8+ CD107a+ granzyme B+ perforin+), with monoclonal antibodies and isotype controls and analyzed by circulation cytometry. Results SARS-CoV-2-specific tetramer?+?CD8 T cells and functional CTLs in the patient with XLA following COVID-19 infection were higher, as compared to healthy control subject following COVID-19 infection. On the other hand, SARS-CoV2-tetramer?+?CD8 T cells and functional CTLs were lower in CVID patient following COVID19 infection as compared to healthy control following COVID-19 infection. SARS-CoV2-tetramer?+?CD8 T cells and Rhein-8-O-beta-D-glucopyranoside functional CTLs were significantly lower in SARS-CoV2-naive CVID patients (gene. He also had bronchiectasis. He developed COVID-19 disease in early 2021 manifested as moderate pneumonia and recovered completely without MGC129647 any specific treatment or hospitalization. The patients with CVID were diagnosed according to European Society for Immunodeficiencies and Pan-American Group for Main Immunodeficiency criteria of low IgG and low IgA/IgM, and impaired response to vaccine [32]. No gene mutation studies were performed. One of the patients with CVID, a 67-year-old woman with infection-only phenotype and untested genotype, developed a moderate COVID-19 disease presenting as severe fatigue and moderate pneumonia that also resolved without any complications or hospitalization. Blood samples were collected 29?days following COVID-19 contamination in XLA patient, and 32?days following COVID-19 contamination in CVID patient, and prior to next immunoglobulin infusion. Blood samples from healthy control were drawn 30?days following COVID-19 contamination. Blood samples were drawn from 10 SARS-CoV-2-naive CVID patients 4C5?weeks (mean?=?31.8?days; median 31.5?days) and 10 SARS-CoV-2-naive healthy controls 4C6?weeks (mean?=?32.4?days, median 31.0?days) following 2nd dose Pfizer-BioNTech COVID-19 vaccine. Blood samples from healthy controls were obtained from UCI Institute for Clinical and Translational Science (ICTS). Patients with PAD were from UCI Immunology clinics. Demographic data on study patients are shown in Table ?Table1.1. CD3+, CD4+, and CD8+ T cells in COVID-19-infected patients were within normal ranges for healthy laboratory controls of institutional CLIA-certified laboratory. Table 1 Clinical and immunological characteristics of antibody-deficient patients immunoglobulin replacement therapy, enzyme-facilitated immunoglobulin, subcutaneous immunoglobulin, healthy controls, idiopathic thrombocytopenia, not relevant *XLA was diagnosed with BTK mutation (BTK c.1085A?>?G (p.His362Arg) hemizygous pathogenic) Antibodies and Reagents Antibodies used included CD8 PerCP (clone SK1), granzyme B Alexa647 (clone GB11), Perforin FITC (clone dG9), and CD107a PE (clone H4A3) all from BioLegend (San Diego), and HLA-A*02:01 SARS-CoV-2 Spike Glycoprotein Tetramer PE (YLQPRTFLL) from MBL International (Woburn, MA). Methods SARS-CoV-2-Specific Tetramer-Positive CD8 T Cells Cells were analyzed by the following technique: 200 L blood was mixed with 5 L CD8PerCP monoclonal antibody and 10 L HLA-A*0201 spike Tetramer PE (HLA-A*02:01 SARS-CoV-2 Spike Glycoprotein Tetramer YLQPRTFLL), vortexed softly and incubated for 30?min at room heat protected from light. Red blood cells were lysed using 1?mL of Lyse Reagent supplemented with 0.2% formaldehyde fixative reagent per tube. Tubes were centrifuged at 150??for 5?min and supernatants were removed. Three milliliters of FACS buffer was added, and tubes were Rhein-8-O-beta-D-glucopyranoside centrifuged at 150??for another 5?min. Cell pellets were resuspended in 500 L of phosphate-buffered saline (PBS) and 0.1% formaldehyde and stored at 4?C for 1?h in the dark prior to analysis by circulation cytometry. Functional Cytotoxic CD8 T Cells Functional cytotoxic CD8 T cells (CTLs) were analyzed by the following technique: 200 blood samples were incubated for 30?min with Rhein-8-O-beta-D-glucopyranoside CD8PerCP and CD107a PE (a degranulation marker) for surface staining; lysed, fixed, and permeablized by Fix Perm buffer (BD biosciences, San Diego, CA); and then incubated with granzyme B AL647 Rhein-8-O-beta-D-glucopyranoside and Perforin FITC monoclonal antibodies (MLB International, Woburn, MA) and appropriate isotype control. All fluorescent minus one (FMO) controls and isotype controls were stained and fixed by 2% paraformaldehyde for circulation cytometry. Cells were acquired by BD FACS Celesta (Becton-Dickenson, San Jose, CA) equipped with BVR laser. Forward and side scatters and singlets were used to gate and exclude cellular debris. Thirty thousand cells were acquired and analyzed using FLOWJO software (Ashland, OR). Gating Strategy Representative pseudocolor plot was utilized for gating strategy; gated lymphocytes were analyzed for singlet, live, and CD8 expressing cells. These CD8+ cells were further analyzed for granzyme B and perforin expression, and for SARS-CoV-2 spike proteinCspecific tetramer-positive cells (Fig.?1). Open in a separate windows Fig. 1 Gating strategy. Representative pseudocolor plot shows gating strategy. Gated lymphocytes were analyzed for singlet, live, and CD8 expressing cells. These CD8+ cells were further analyzed for granzyme B and perforin expression, and for SARS-CoV-2 spike proteinCspecific tetramer-positive cells Data are expressed as percent of total CD8+-gated (100%) T cells. Total numbers of SARS-CoV-2 spike proteinCspecific tetramer-positive cells and functional CTLs were calculated by multiplying percentage with complete CD8+ T cell.