Clustering analysis of flow cytometry data was done using FlowJo (v10.5.3). First, we excluded dead cells, doublets, and non-lymphocyte cells (based on viability staining and FSC/SSC parameters). CD4+ cells were used for further analysis. Data were sampled using the “down sampling” function to obtain 40,000 representative cells from each sample. Then, we applied t-SNE algorithm with the following parameters: Iteration = 1000, Perplexity = 40, and Learning rate (eta) = 2800. Mean fluorescence intensity projected on the t-SNE plots for each protein to infer the cluster’s identity.

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