Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry.

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25+CD69+CD40L+TNF-α- T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25+CD69+TNF-α- T cells were identified. Within CD8α+ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs.

Studying antigen-specific T cells through a streamlined, whole blood-based extracellular approach.

Techniques currently used for the study of antigen-specific T-cell responses are either poorly informative or require a heavy workload. Consequently, many perspectives associated with the broader study of such approaches remain mostly unexplored in translational research. However, these could benefit many fields including but not limited to infectious diseases, oncology, and vaccination. Herein, the main objective of this work was to develop a standardized flow cytometry-based approach that would combine ease of use together with a relevant study of antigen-specific T-cell responses so that they could be more often included in clinical research. To this extent, a streamlined approach relying on 1/ the use of whole blood instead of peripheral blood mononuclear cells and 2/ solely based on the expression of extracellular activation-induced markers (AIMs), called whole blood AIM (WAIM), was developed and further compared to more conventional techniques such as enzyme-linked immunospot (ELISpot) and flow cytometry-based intracellular cytokine staining (ICS). Based on a cohort of 20 individuals receiving the COVID-19 mRNA vaccine and focusing on SARS-CoV-2 and cytomegalovirus (CMV)-derived antigen T-cell-specific responses, a significant level of correlation between the three techniques was found. Based on the use of whole blood and on the expression of extracellular activation-induced markers (CD154, CD137, and CD107a), the WAIM technique appears to be very simple to implement and yet allows interesting patient stratification capabilities as the chosen combination of extracellular markers exhibited higher orthogonality than cytokines that are commonly considered in ICS (IFN-γ, TNF-α, and IL-2).

Activation-induced marker assays for identification of Trypanosoma cruzi-specific CD4 or CD8 T cells in chronic Chagas disease patients.

Antigen-specific T cells are central to the adaptive immune response against T. cruzi infection and underpin the efficacy of on-going vaccine strategies. In this context, the present study focuses on T-cell assays that define the parasite-specificity on the basis of upregulation of TCR stimulation-induced surface markers. For this purpose, we tested different dual marker combinations (OX40, CD25, CD40L, CD137, CD69, PD-L1, CD11a, CD49d, HLA-DR, CD38) to reliably identify activated CD4+ and CD8+ T-cell populations from PBMCs of chronic Chagas disease (CCD) patients after 12 or 24 h of stimulation with T. cruzi lysate. Results demonstrated that activation-induced markers (AIM) assays combining the expression of OX40, CD25, CD40L, CD137, CD69 and/or PD-L1 surface markers are efficient at detecting T. cruzi-specific CD4+ T cells in CCD patients, in comparison to non-infected donors, after both stimulation times. For CD8+ T cells, only PD-L1/OX40 after 24 h of antigen exposure resulted to be useful to track a parasite-specific response. We also demonstrated that the agnostic activation is mediated by different T. cruzi strains, such as Dm28c, CL Brener or Sylvio. Additionally, we successfully used this approach to identify the phenotype of activated T lymphocytes based on the expression of CD45RA and CCR7. Overall, our results show that different combinations of AIM markers represent an effective and simple tool for the detection of T. cruzi-specific CD4+ and CD8+ T cells.

Activation-induced markers for evaluation of specific CD4 + T cell immune response in HIV-1-infected patients / 中华微生物学和免疫学杂志

Objective:To establish a method using activation-induced markers (AIM) to detect the function of HIV-1-specific CD4 + T cell subsets for evaluating the immune response of HIV-1-specific CD4 + T cells more effectively. Methods:Twelve chronically HIV-1-infected patients without antiviral therapy and six healthy people without HIV-1 infection were enrolled in this study. The function of HIV-1-specific T lymphocytes was detected by AIM and ICS based on polychromatic flow cytometry. The performance of the two methods in assessing HIV-1-specific CD4 + T cell immune response in HIV-1-infected patients was evaluated. Results:The positive rates of HIV-1-specific PD-1 + CD25 + CD4 + T, CD69 + CD200 + CD4 + T, CD69 + ICOS + CD4 + T, CD69 + ICOS + CD8 + T、CD137 + CD69 + CD8 + T、PD-1 + CD25 + CD8 + T and OX40 + PD-1 + CD8 + T cells in all of the HIV-1 patients were 11/12, 8/12, 7/12, 8/12, 8/12, 7/12 and 7/12 using AIM method. ICS results showed that the positive rates of HIV-1-specific IL-2 + CD4 + T, IFN-γ + CD4 + T, TNF-α + CD4 + T, IFN-γ + CD8 + T, TNF-α + CD8 + T and IL-2 + CD8 + T cells were 2/12, 2/12, 0, 12/12, 10/12 and 5/12, respectively. Conclusions:AIM method was more sensitive in antigen-specific CD4 + T cell detection, and could be used as a complementary method to ICS in assessing antigen-specific T cell response.

Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination.

SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses on healthy SARS-CoV-2-naive and recovered individuals prior to and following mRNA prime and boost vaccination. Vaccination induced rapid antigen-specific CD4+ T cell responses in naive subjects after the first dose, whereas CD8+ T cell responses developed gradually and were variable in magnitude. Vaccine-induced Th1 and Tfh cell responses following the first dose correlated with post-boost CD8+ T cells and neutralizing antibodies, respectively. Integrated analysis revealed coordinated immune responses with distinct trajectories in SARS-CoV-2-naive and recovered individuals. Last, whereas booster vaccination improved T cell responses in SARS-CoV-2-naive subjects, the second dose had little effect in SARS-CoV-2-recovered individuals. These findings highlight the role of rapidly primed CD4+ T cells in coordinating responses to the second vaccine dose in SARS-CoV-2-naive individuals.

Simultaneous monitoring assay for T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface.

Isolation of antigen (Ag)-specific T cells is an important step in the investigation of T-cell immunity. Activation-induced markers (AIMs), such as CD154/tumor necrosis factor (TNF)/CD107A/CD134/CD137 enable the sorting of Ag-specific T cells without using human leukocyte antigen (HLA)-multimers. However, optimal conditions suitable for simultaneous detection of both Ag-specific CD4 and CD8 T cells have not been investigated. Here, conditions were optimized to simultaneously detect the maximum number of activated CD4 and CD8 T cells in a TCR-dependent manner. First, the frequency of total pools of AIM-positive cells induced by superantigen, staphylococcal enterotoxin B (SEB), stimulation in various culture conditions was monitored and compared side-by-side. The total amount of AIM-positive CD4 T cells, but not CD8 T cells, was significantly abrogated by addition of brefeldin A. TNF-alpha converting enzyme inhibitor treatment effectively increased the TNF-positive population, without affecting other markers' positivity. AIM-positive CD4 T cells and CD8 T cells were detected at least 3 h after stimulation. Furthermore, examination of the multiple combination of each marker revealed that minimum contribution of CD134 on the total pool of AIM-positive cells at this setting, suggesting the essential and non-essential AIMs to maximize the detected number of AIM-positive cells. Taken together, this optimized method will be a useful tool for the simultaneous monitoring the T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface including Ag-specific T cells.

Activation-induced Markers Detect Vaccine-Specific CD4⁺ T Cell Responses Not Measured by Assays Conventionally Used in Clinical Trials.

Immunogenicity of T cell-inducing vaccines, such as viral vectors or DNA vaccines and Bacillus Calmette-Guérin (BCG), are frequently assessed by cytokine-based approaches. While these are sensitive methods that have shown correlates of protection in various vaccine studies, they only identify a small proportion of the vaccine-specific T cell response. Responses to vaccination are likely to be heterogeneous, particularly when comparing prime and boost or assessing vaccine performance across diverse populations. Activation-induced markers (AIM) can provide a broader view of the total antigen-specific T cell response to enable a more comprehensive evaluation of vaccine immunogenicity. We tested an AIM assay for the detection of vaccine-specific CD4⁺ and CD8⁺ T cell responses in healthy UK adults vaccinated with viral vectored Ebola vaccine candidates, ChAd3-EBO-Z and MVA-EBO-Z. We used the markers, CD25, CD134 (OX40), CD274 (PDL1), and CD107a, to sensitively identify vaccine-responsive T cells. We compared the use of OX40⁺CD25⁺ and OX40⁺PDL1⁺ in CD4⁺ T cells and OX40⁺CD25⁺ and CD25⁺CD107a⁺ in CD8⁺ T cells for their sensitivity, specificity, and associations with other measures of vaccine immunogenicity. We show that activation-induced markers can be used as an additional method of demonstrating vaccine immunogenicity, providing a broader picture of the global T cell response to vaccination.