Phenotype and tissue distribution of CD28H⁺ immune cell subsets

Figures & data

Figure 1. CD28H is expressed on immune cell subsets in healthy human peripheral blood. (A) Representative flow cytometric plots showing staining against CD28H or isotype control on FSC/SSC^SINGLETSCD45⁺CD3⁻CD19⁻ granulocytes (CD33⁺CD14⁻CD15⁺), monocytes (CD33⁺CD14⁻CD15⁺), myeloid dendritic cells (MDCs) (CD33⁺CD14⁻CD15⁻CD11 c⁺HLA-DR⁺) and plasmacytoid cells (PDCs) (CD33⁻HLA-DR⁺CD123⁺). (B) Representative flow cytometric plots showing staining against CD28H or isotype control on FSC/SSC^LOWCD45⁺CD3⁻ B cells (CD19⁺), NK cells (CD7⁺CD19⁻CD16⁺NKp46⁺), innate lymphoid cells (ILCs) (lin⁻CD7⁺CD19⁻CD16⁻CD127⁺), and FSC/SSC^LOWCD45⁺CD19⁻ T cells (CD3⁺). (C) Bar graphs summarize CD28H expression from (A) and (B). (D) Representative flow cytometric plots showing staining against CD28H or isotype control on FSC/SSC^LOWCD45⁺CD33⁻CD19⁻CD3⁺ naïve (CD45RA⁺CD45RO⁻CCR7⁺CD62 L⁺) and memory (CD45RA⁻CD45RO⁺ CCR7⁻CD62 L⁻) CD4⁺ and CD8⁺ T cells. (E) Bar graphs summarize CD28H expression from (D). *, P < 0.05. 6–10 donors.

Figure 2. CD28H⁺ naïve T cells show increased naïve characteristics. (A) Expression of CD31 on naïve T cells. Representative plots and the mean percentages + SEM showing CD31 expression on CD28H⁺ and CD28H⁻ naïve T cells. (B) Expression of T-bet on naïve T cells. Representative plots and the mean percentages + SEM showing T-bet expression on CD28H⁺ and CD28H⁻ naïve T cells. (C) Expression of IFNγ on naïve T cells. Representative plots and the mean percentages + SEM showing IFNγ expression on CD28H⁺ and CD28H⁻ naïve T cells. (D) Expression of TNFα on naïve T cells. Representative plots and the mean percentages + SEM showing TNFα expression on CD28H⁺ and CD28H⁻ naïve T cells. (E) Expression of CD28 on naïve T cells. Representative plots and the mean percentages + SEM showing CD28 expression on CD28H⁺ and CD28H⁻ naïve T cells. 6–8 donors, *, P < 0.05. (F) Kinetic CD28H expression in in vitro cultured enriched naïve T cells. Enriched peripheral blood CD4⁺ naïve T cells were stimulated with anti-CD3 and anti-CD28 every 3 d for up to 20 d as described. CD28H surface expression was measured by FACS. 3 donors with repeats.

Figure 3. CD28H⁺ memory T cells show less effector function marks. (A, B) Expression of IFNγ on memory T cells. Representative plots and the mean percentages + SEM showing IFNγ expression on CD28H⁺ and CD28H⁻ memory T cells. (C, D) Expression of CD57 on memory T cells. Representative plots and the mean percentages + SEM showing CD57 expression on CD28H⁺ and CD28H⁻ memory T cells. 6–8 donors, *, P < 0.05.

Figure 4. CD28H expression on tissue T cell subsets. (A, B) Representative flow cytometric plots for isotype and anti-CD28H staining (A) and the percentages + SEM of CD28H expression (D) on secondary lymphoid tissue T cells. 4–6 donors. (C, D) Representative flow cytometric plots for isotype and anti-CD28H staining (C) and the percentages + SEM of CD28H expression (D) on pathological tissue T cells. 4–6 donors. (E, F) The mean percentages + SEM of CD57 (E) and IFNγ (F) expressing CD8⁺ T cells in CD28H⁺ and CD28H⁻ subsets from pathological tissue T cells. 4–6 donors, *, P < 0.05. OCB, blood from ovarian cancer patient; Ca, Cancer.

Figure 5. CD28H expression on NK cells and ILCs. (A-C) NK cells were gated on CD45⁺CD3⁻CD7⁺CD16⁺CD19⁻CD33⁻CD34⁻CD127⁻. (A) Representative flow cytometric plots for isotype and anti-CD28H staining in NK cells. (B) The mean percentage + SEM of CD28H positive cells in NK cells in peripheral blood of healthy donors (n = 8), tonsil (n = 5) and spleen (n = 4). (C) The mean percentage + SEM of CD28H positive cells in NK cells in ovarian cancer patient blood (n = 5) and cancer tissues from patients with ovarian cancer (n = 5), colon cancer (n = 5), and lung cancer (n = 4). *, P < 0.05. (D-F) ILCs were gated on CD45⁺ CD3⁻ CD7⁺ CD16⁻ CD19⁻ CD33⁻ CD34⁻ CD127⁺. (D) Representative flow cytometric plots of ILCs in secondary lymphoid organs and cancer tissues for isotype and anti-CD28H staining. (E) The mean percentage + SEM of CD28H positive cells in ILCs in peripheral blood of healthy donors (n = 8), tonsil (n = 5) and spleen (n = 4). (F) The mean percentage + SEM of CD28H positive cells in ILCs in ovarian cancer patient blood (n = 5) and cancer tissues from patients with ovarian cancer (n = 5), colon cancer (n = 5), lung cancer (n = 4), and breast cancer (n = 3).