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Background & Aim: Adoptive T cell immunotherapy holds great promise for the treatment of viral complications. Our group has been developing and trialling virus-specific T cell therapies for more than 20 years. Recently, we have generated a repository of multi-virus-specific T cells for our clinical trials. Unfortunately, for many patients with viral complications, there is no suitable trial through which to access these therapies. In Australia, the Therapeutic Goods Administration has a Special Access Scheme (SAS) to enable provision of unapproved therapies for compassionate use. Our research group is now a leading Australian provider of "off-the-shelf" and custom-grown allogeneic virus-specific T cells to hospitals for patients with no other treatment options. Methods, Results & Conclusion(s): We have generated a repository of multi-virus-specific T cells from 20 healthy donors, with up to 150 doses of T cells per donor generated from a single blood sample. Each product batch is thoroughly characterised in terms of viral antigen specificity, HLA restriction and alloreactivity. These T cells target a combination of Epstein-Barr virus, cytomegalovirus, BK polyomavirus, John Cunningham virus and adenovirus epitopes. We have also generated a repository of SARS-CoV-2-specific T cells and occasionally grow custom patient-specific batches of T cells from nominated donors, on request. Since 2008, we have provided virus-specific T cells to 15 hospitals across Australia, and the volume of supply requests has significantly increased in recent years, as clinicians have gained interest in adoptive immunotherapy. In 2022, we provided T cells for 26 patients via the SAS. The majority were experiencing post-transplant complications, including cytomegalovirus disease, BK virus-associated haemorrhagic cystitis and post-transplant lymphoproliferative disorder. Through our clinical trials, we have developed rigorous processes for T cell therapy manufacture and characterisation, in addition to a computer-based selection algorithm, which we apply to SAS cases. As these cases are not part of a clinical trial, concomitant therapy varies, and monitoring is not uniform. However, we have received reports of clinical benefit from adoptive T cell therapy. These include cases of reduction in viral load, improvement in symptoms, and complete resolution of infection. We believe that these promising T cell therapies should be available to hospitals through a nationally funded centre for cellular therapies for critically ill patients.Copyright © 2023 International Society for Cell & Gene Therapy
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Background & Aim: Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer of SARS-CoV-2 specific T cells. Methods, Results & Conclusion(s): To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. Our results show that SARSCoV- 2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. In conclusion, our findings show that SARSCoV- 2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients.Copyright © 2023 International Society for Cell & Gene Therapy
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The emerging virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2 virus), agent of COVID-19, appeared in December 2019 in Wuhan, China, and became a serious threat to global health and public safety. Many COVID-19 vaccines have been approved and licensed around the world. Most of the developed vaccines include S protein and induce an antibody-based immune response. Additionally, T-cell response to the SARS-CoV-2 antigens could be beneficial for combating the infection. The type of immune response is greatly dependent not only on the antigen, but also on adjuvants used in vaccine formulation. Here, we compared the effect of four different adjuvants (AddaS03, Alhydrogel/MPLA, Alhydrogel/ODN2395, Quil A) on the immunogenicity of a mixture of recombinant RBD and N SARS-CoV-2 proteins. We have analyzed the antibody and T-cell response specific to RBD and N proteins and assessed the impact of adjuvants on virus neutralization. Our results clearly indicated that Alhydrogel/MPLA and Alhydrogel/ODN2395 adjuvants elicited the higher titers of specific and cross-reactive antibodies to S protein variants from various SARS-CoV-2 and SARS-CoV-1 strains. Moreover, Alhydrogel/ODN2395 stimulated high cellular response to both antigens, as assessed by IFN-γ production. Importantly, sera collected from mice immunized with RBD/N cocktail in combination with these adjuvants exhibited neutralizing activity against the authentic SARS-CoV-2 virus as well as particles pseudotyped with S protein from various virus variants. The results from our study demonstrate the immunogenic potential of RBD and N antigens and point out the importance of adjuvants selection in vaccine formulation in order to enhance the immunological response. IMPORTANCE Although several COVID-19 vaccines have been approved worldwide, continuous emergence of new SARS-CoV-2 variants calls for new efficient vaccines against them, providing long-lasting immunity. As the immune response after vaccination is dependent not only on antigen used, but also on other vaccine components, e.g., adjuvants, the purpose of this work was to study the effect of different adjuvants on the immunogenicity of RBD/N SARS-CoV-2 cocktail proteins. In this work, it has been shown that immunization with both antigens plus the different adjuvants studied elicited higher Th1 and Th2 responses against RBD and N, which contributed to higher neutralization of the virus. The obtained results can be used for design of new vaccines, not only against SARS-CoV-2, but also against other important viral pathogens.
Subject(s)
COVID-19 , Viral Vaccines , Animals , Mice , Humans , SARS-CoV-2 , COVID-19 Vaccines , COVID-19/prevention & control , Aluminum Hydroxide , Antibodies, Viral , Antibodies, Neutralizing , Immunogenicity, VaccineABSTRACT
Background: SARS-CoV-2 infection typically causes self-limited disease, but a subset of individuals experience more severe disease associated with respiratory manifestations, hospitalization and mortality. People living with HIV (PLWH) have been shown to have chronic immune activation and inflammation despite effective antiretroviral therapy. During the COVID pandemic, PLWH were found to have an increased risk of hospitalization and mortality with acute COVID-19. The immune response driving these worsened outcomes in PLWH is not defined. We analyzed immune activation and exhaustion markers, as well as antigen specific T cell responses during acute COVID-19 in PLWH versus HIV-seronegative controls to determine the impact of chronic HIV infection and inflammation on acute COVID-19. Method(s): We performed flow cytometric analyses on peripheral blood mononuclear cells from: 1) PLWH with acute COVID-19 (HIV+COVID), 2) HIVseronegative individuals with acute COVID-19 (COVID) and 3) pre-COVID-19 pandemic PLWH (HIV). COVID(+) samples were collected at an average of 4.7 (range 0-16) and 5.5 (range 0-20) days post-symptom onset for the COVID and HIV+COVID cohorts, respectively. Cells were stained for surface markers of activation/exhaustion and intracellular cytokines (with and without SARS-CoV- 2-specific antigen stimulation). Observed immune responses were correlated with disease severity. Result(s): PLWH with acute COVID-19 had increased classical (CD14+) monocytes compared to HIV-seronegative individuals with acute COVID-19. The HIV+COVID cohort also had higher expression of activation (OX40, CD137) and exhaustion (PD1, TIGIT) markers on CD4+ and CD8+ T cells compared to HIV-seronegative individuals. SARS-CoV-2 antigen stimulation resulted in similar response frequencies between the HIV+COVID and COVID cohorts. Conclusion(s): PLWH had increased activation and exhaustion and increased classical monocytes compared to HIV-seronegative presentations of COVID-19, highlighting the persistent immune dysregulation associated with chronic HIV infection. Our findings aid in further characterization of how chronic immune dysregulation impacts the immune response to acute SARS-CoV-2 infection. Future studies include characterizing the impact of acute SARS-CoV-2 infection duration, as well as how chronic immune dysregulation impacts the development of long COVID. (Table Presented).
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Background: The potential aerosol spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) has been suggested. While indoor air sampling for SARS-CoV- 2 has demonstrated detectable viral RNA and has been related to virus transmission, the contribution of outdoor air to the spread of the viral infection is not yet known. We aimed at developing a methodology to detect the virus in outdoor air. Method(s): T he s ampling w as p erformed u sing a C HEMVOL v olumetric impactor (Butraco) equipped with 2 stages (PM > 10 & 2.5 > PM > 10um). Filters were collected and preserved at -80 degreeC. Total RNA extraction was performed directly from the collected filters with the Phenol-Chloroform method using TRItidy GTM reagent according to the manufacturer's instructions. For total RNA purification samples were purified with the commercial kit E.Z.N.A. Total RNA Kit I. Real-Time Reverse Transcription PCR was executed to detect the N gene from the Sarbecovirus family and RdRp gene from SARS-CoV- 2 using the ViroReal Kit SARS-CoV- 2 Multiplex. A protein-rich fraction was obtained with ammonium bicarbonate buffer extraction followed by lyophilization. SARS-CoV- 2 spike protein was assessed by specific immunological detection (SARS-CoV- 2 Antigen Test Kit). Result(s): RT-PCR for N gene results, identifying Sarbecovirus family, were positive and Cq > 33, in the samples from the last week of December 2020 and the first and second weeks of January 2021, in both PM > 10 and 2.5 > PM > 10. The RdRp gene was undetectable, probably due to low virus concentration. The protein samples from the same days tested positive for the specific antigen spike protein. All results combined confirm the detection of SARS-CoV- 2 in outdoor air. Conclusion(s): Airborne SARS-CoV- 2 was detected in ambient air. These results will contribute to an early detection of SARS-Cov- 2 in ambient air, thus eventually providing the base for early alert systems allowing the implementation of preventive measures to control outbreaks.
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The pathogenesis of severe coronavirus infection COVID-19 is associated with activation of immune system, cytokine storm, impaired blood clotting, microvascular thrombosis, organ ischemia and multiple organ dysfunction syndrome. The role of various lymphocyte subpopulations in COVID-19 is still debated. The aim of our study was to analyze the subpopulational profile of peripheral blood lymphocytes in COVID-19 patients as compared with healthy donors. The study included 20 COVID-19 patients (11 males and 9 females,) and 26 healthy donors. Average age of the patients was 52 and 56 years, respectively. Clinical examinations were performed by standard laboratory methods. Peripheral blood lymphocytes were isolated in the Ficoll gradient. The cells were stained with antibodies to specific antigens of main lymphocyte populations, endothelial cells, and apoptotic cell markers. The analysis was performed by flow cytometry. The results showed that all patients had elevated C-reactive protein (14- to 35-fold), ferritin (1.2- to 13-fold), D-dimers (1.2- to 90-fold). 55% of men had a decrease in the absolute number of lymphocytes, in women this index was at the low normal limit. Cytometric analysis showed that, among peripheral blood lymphocytes, the proportion of functional cells expressing the CD45 marker ranged from 2 to 12% in 70% of patients, as compared with 80-99% among the donors. The proportion of CD45+ lymphocytes significantly correlated with the level of hemoglobin, but not with the levels of inflammatory biochemical markers. Among the functional lymphocytes of patients, there was a decrease in the proportion of CD3+, CD4+, CD8+T cells, increased proportion of natural killer CD56+ and the apoptotic (AnnexinV+) cell contents, but the proportion of CD19 and HLA-DR+B cells was not changed. Analysis of the lymphocyte (LC) subpopulations that did not express CD45 marker showed that this fraction contained different lymphocyte subsets with reduced expression of CD4, CD8, CD19, CD56 etc. in the blood of patients and donors. Higher percentage of endothelial cells expressing CD62P marker made the difference between patients and donors. Laboratory determination of lymphocyte subsets in blood samples of COVID-19 patients does not reflect the real severity pattern of the disease, thus requiring studies of the CD45-expressing functional cell populations.Copyright © Svirshchevskaya E.V. et al., 2023 The article can be used under the Creative Commons Attribution 4.0 License.
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Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNγ in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of low-frequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations.
Subject(s)
CD8-Positive T-Lymphocytes , COVID-19 , Humans , CD4-Positive T-Lymphocytes , Integrins/metabolism , COVID-19 Vaccines/metabolism , COVID-19/metabolism , SARS-CoV-2 , Antigens/metabolism , CD40 Ligand , Cytokines/metabolismABSTRACT
Background The adaptive immune system identifies foreign antigens based on a series of highly specific interactions involving multiple immune cell types. Identifying the exact mechanisms of said interactions can be difficult to achieve using bulk sequencing methods due to poor resolution. Single cell sequencing offers the ability to match a specific antigen to an immune cell receptor sequence at the cellular level. Methods We used Barcode Enabled Antigen Mapping (BEAM) and Single Cell Immune Profiling technology to profile hundreds of thousands of human peripheral blood mononuclear cells (PBMCs) from a donor following their recovery from COVID-19. These cells were screened for potential binding interactions with multiple antigens from SARS-CoV-2 and other viral pathogens. Sequencing data were also generated for gene expression and paired sequences for both BCRs and TCRs. Results The combination of these two techniques allowed us to identify a number of antigen-specific clonotypes of T cells and B cells. The high throughput of the experiment allowed us to gain understanding on a global scale of the state of the immune system following recovery from a COVID-19 infection, as well as to identify potentially rare clonotypes that may not have been discerned from a smaller sample size. Conclusions This experiment demonstrates the ability of BEAM to both profile the entire immune system at the cellular level at a given point in time as well as distinguish specific antigen-receptor interactions with the same resolution. Insights provided by similar experiments could be invaluable in the creation of precision cell therapies for use in cancer treatment, as well as the development of vaccines and analysis of allergic and autoimmune responses.
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Introduction: Some MS DMTs, such as anti-CD20 antibodies and sphingosine-1-phosphate (S1P) receptor modulators, decrease post-infection and post-vaccination SARS-Cov-2 humoral responses. However, humoral immunity is only one component of the adaptive response, and T cell responses, which may be preserved in anti-CD20 treated patients, play an important role. Objective(s): To characterize SARS-Cov-2 spike specific memory T-cell receptor repertoires in patients with MS and related conditions post-vaccination with mRNA vaccines. Aim(s): To characterize SARS-Cov-2 vaccine-mediated responses for people using MS DMTs. Method(s): Patients without prior COVID-19 infection provided a whole blood sample >3 weeks and <6 months after vaccination with two doses of Pfizer-BioNTech or Moderna mRNA vaccines. Sequencing of the complementary determining region within T-cell receptors (TCRs) was performed. Antigen recognition activates unique TCRs and results in expansion of antigen-specific T-cell clones. Sample TCR sequences were cross-matched with sequences known to react to SARS-CoV-2 using "Multiplex Identification of T-cell Receptor Antigen Specificity (MIRA)", allowing for characterization of SARS-CoV-2-spike-specific TCR frequency (clonal depth) and diversity (clonal breadth). Humoral responses were compared. Result(s): 39 patients were recruited: age 25-77;27 female;37 with MS, 2 with NMO, and 1 with another neuroimmune condition. DMTs included anti-CD20 (N=13), natalizumab (N=9), fumarates (N=8), S1P receptor modulators (N=3), and controls (2 glatiramer acetate, 4 no DMT). Mean time interval between 2nd vaccination dose and TCR testing was 13.3+6.0 weeks. Humoral responses (Roche) were absent in all anti-CD20 and S1P treated patients but preserved in all others. SARS-CoV-2-spikespecific clonal depth and breadth did not differ across all treatment classes except S1P modulators. Despite lack of antibody production, patients treated with anti-CD20 therapies demonstrated comparable TCR depth and breadth to all other groups in univariable assessment. No spike-specific TCRs were found in patients treated with S1P modulators. TCR breadth and depth did not vary with time since vaccination even up to 24 weeks following vaccination. Conclusion(s): TCR repertoires were preserved except for in those treated with S1P receptor modulators.Humoral responses were diminished with both anti-CD20 and S1P DMTs. These findings may help guide counseling of patients with regards to DMT choice.
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Although the global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, there are currently no specific and highly efficient drugs for COVID-19 available, particularly in severe cases. Recent findings demonstrate that severe COVID-19 disease that requires hospitalization is associated with the hyperactivation of CD4+ and CD8+ T cell subsets. In this study, we aimed to counteract this high inflammatory state by inducing T-cell hyporesponsiveness in a SARS-CoV-2-specific manner using tolerogenic dendritic cells (tolDC). In vitro-activated SARS-CoV-2-specific T cells were isolated and stimulated with SARS-CoV-2 peptide-loaded monocyte-derived tolDC or with SARS-CoV-2 peptide-loaded conventional (conv) DC. We demonstrate a significant decrease in the number of interferon (IFN)-γ spot-forming cells when SARS-CoV-2-specific T cells were stimulated with tolDC as compared to stimulation with convDC. Importantly, this IFN-γ downmodulation in SARS-CoV-2-specific T cells was antigen-specific, since T cells retain their capacity to respond to an unrelated antigen and are not mediated by T cell deletion. Altogether, we have demonstrated that SARS-CoV-2 peptide-pulsed tolDC induces SARS-CoV-2-specific T cell hyporesponsiveness in an antigen-specific manner as compared to stimulation with SARS-CoV-2-specific convDC. These observations underline the clinical potential of tolDC to correct the immunological imbalance in the critically ill.
Subject(s)
COVID-19 , T-Lymphocytes , Humans , SARS-CoV-2 , Immune Tolerance , Dendritic Cells , Antigens , Peptides , ApoptosisABSTRACT
Background & Aim: Background. Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer of SARS-CoV-2 specific T cells. Methods, Results & Conclusion: Methods. To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. Results. Our results show that SARS-CoV-2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. Conclusion. In conclusion, our findings show that SARS-CoV-2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients.
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Background: A positive direct antiglobulin test (DAT) is frequently observed for patients suffering from COVID-19 but its interpretation in transfusion dependent patients such as patients with solid and haematological malignancies is complex and multi-factorial. Aims: • To identify the incidence of DAT positivity in cancer patients hospitalized with COVID-19. • To explore the significance of DAT positivity in those patients by comparing with the DAT-negative COVID-19 patients (control group). Methods: This was a cross-sectional study done in India where samples were obtained from 88 cancer patients with confirmed COVID- 19. All patients were hospitalized. No patient had received COVID-19 convalescent plasm. IAT was negative in all patients. DAT was performed using column agglutination technology, and samples that were DAT positive were further investigated using monospecific DAT facility. If the strength of DAT was 2+ or above with IgG then the elution was performed with an elution kit. The eluates were then tested with a commercial three-cell panel of RBCs to establish whether the IgG antibodies had any RBC antigen specificity. In case the DAT was positive for C3d, the cold agglutination titre was estimated. A chi-square test was used for discrete variables, and a student t-test or a Wilcoxon-Mann-Whitney test was used with p-values below 0.05 considered as significant. Results: There was no significant difference between the patients with positive DAT and patients with negative DAT (Table 1). DAT was positive in 32 of 88 (36.36%) patients. Of the 32 cases, 28 (87.5%) were positive for IgG only, one (3.13%) was positive for both IgG and complement, and three (9.38%) were positive for C3d.More than 2+ agglutination was seen in 6 (18.8%) patients. Out for those six cases two were positive for C3d only (4+) with a cold agglutination titre of 2048. None of the eluted samples in rest of the four cases with IgG reactivity showed specificity for RBC antigens in the three-cell panel. Total bilirubin and LDH values were not different between two groups which suggest that the anaemia in DAT-positive group was not due to haemolysis rather it could be associated with severity of disease. The increase morality that was observed in DAT-positive group might be explained similarly. Summary/Conclusions: Results of this study show that a high percentage of cancer patients with COVID-19 are DAT positive, but majority of these patients do not have any evidence of haemolysis and do not require more blood transfusion compared to others. The clinical implications of this high DAT positivity in COVID-19 patients need further exploration. (Table Presented).
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Introduction: Since December 2019, COVID-19 has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as patients after allogeneic haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both shortlived neutralising antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Methods: Activation of CD4+ T cells from 18 COVID-19 patients was determined by flow cytometry, both ex vivo and after in vitro restimulation with SARS-CoV-2 Spike and Nucleocapsid antigens. Immunodominant, 15-mer peptides were identified using epitope mapping. T cells clones specific for these epitopes were further chararacterised for the sensitivity and polarisation of their cytokine responses after in vitro restimulation, by ELISA and cytometric assay. Next-generation sequencing revealed fulllength, paired T Cell Receptor (TCR) αβ sequences. Results: We identified three patients with strong CD4+ T cells to SARSCoV- 2 antigens. From these patients, 81 T cell clones specific for a selection of 9 immunodominant epitopes (7 Spike and 2 Nucleocapsid epitopes) were generated. Cytokine analysis showed that the sensitivity and polarisation of T cell responses varied depending on the specific epitope. Moreover, TCRαβ sequences revealed an epitope-dependent difference in the level of clonality. Conclusions: We provide detailed information on SARS-CoV-2-specific CD4+ T cells, including their antigen-specificity, the nature of their cytokine responses and the full sequence of their TCRαβ. These cells have the potential to direct an effective immune response in COVID-19 patients. Our results form a crucial first step towards T cell therapy. Efforts are underway to develop transgenic CD4+ T cells that express the SARS-CoV- 2-specific TCRs identified.
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We have previously shown that conformational change in the ß2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the ß2-integrin. The kinetics of ß2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4-6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining ß2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.