Fig 2: kNN latent cells from different donors are phenotypically similar.(A) The atlas (gray) and kNN latent cells (red) for four donors are depicted within the same tSNE space. In all donors, the center region of the tSNE is devoid of kNN latent cells. The kNN latent cells depicted here are those identified in Figure 2. (B) Expression levels of select antigens on kNN latent cells as compared to the total population of memory CD4+ T cells. Top plots show the antigen expression levels as a heatmap within the tSNE space depicted in panel A. Bottom graphs show the mean signal intensity (MSI) of each antigen in the total population of memory CD4+ T cells (Total) compared to the kNN latent cells (Latent). Proteins consistently expressed at higher levels on kNN latent cells than on the total population included immune checkpoint molecules (PD1, CTLA4) and the HIV co-receptors CCR5 and CXCR4. Some activation markers (CD69, CD25, HLADR) but not others (CD38) were also upregulated, as were markers of Th1, Th2, and Th17 cells (Tbet, CRTH2, and CCR6, respectively). Some markers of Tfh (PD1, ICOS) but not others (CXCR5) were consistently elevated. By contrast, the Tcm marker CD27 was expressed at low levels on kNN latent cells. *p<0.05, **p<0.01 as determined by a Student’s paired t-test. n.s.: not significant.

Fig 3: Subset distribution of total and SARS-CoV-2-specific T cells during mild, moderate, and severe COVID-19 by manual gating(A and B) Distribution of baseline and SARS-CoV-2-specific CD4+ (A) and CD8+ (B) T cells from individuals with COVID-19 among memory (CD45RO+CD45RA−) and a mixture of Tn, Temra, and Tscm (CD45RO−CD45RA+) cells. Breakdown into further subsets was achieved by manually gating on subset marker combinations. Downstream of the CD45RO and CD45RA gates, we defined the subsets as follows: Tcm (CD27+CCR7+), Tem (CD27−CCR7−), Ttm (CD27+CCR7−), Tn (CCR7+CD95−), Temra (CCR7−), Tscm (CCR7+CD95+).(C) Proportions of Treg (CD45RO+CD45RA−CD25+CD127low) and cTfh (PD1+CXCR5+) cells among baseline and SARS-CoV-2-specific CD4+ T cells from individuals with COVID-19.(D) The proportion of apoptosis-prone (PD1+CD95+) cells among CD4+ and CD8+ T cells is increased in severe COVID-19 cases for total and SARS-CoV-2-specific T cells.(E) The proportion of exhausted cells (PD1+TIGIT+) among total and SARS-CoV-2-specific CD8+ T is increased in severe COVID-19. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, as determined by one-way analysis of variance with a Bonferroni post-test.Each datapoint corresponds to a different specimen (biological replicates).