Supplementary Figure S1: Alignment of CD28H. (a) Alignment of human CD28H with other known B7 receptors. (b) Alignment of CD28H orthologs.

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1 Supplementary Figure S1: Alignment of CD28H. (a) Alignment of human CD28H with other known B7 receptors. (b) Alignment of CD28H orthologs. Predicted CD28H protein sequences from human, chimpanzee (pan troglodytes), cow (bos taurus), guinea pig (cavia porcellus) and zebrafish were aligned using MacVector 6.5 program. The shaded box refers to shared amino acids among CD28H orthologs.

2 Supplementary Figure S2: Verifying the binding specificity of mabs for CD28H and B7-H5. (a) HEK293T were transiently transfected with full-length pcdna from CD28H or other CD28 family members. 18 hours later cells were harvested and stained with control (filled), respective antibody indicated (open), or anti-cd28h mab (open) as indicated. (b) Stable CHO-B7-H5 cell line or control CHO cells were stained with control (filled) or anti-b7-h5 mab (clone 2D3) (open).

3 Supplementary Figure S3: CD28H expression in thymocytes from humanized mice. Three months following injection of NSG recipients with human HSCs, mice were euthanized by CO2 asphyxiation and thymus was harvested. Thymocytes were stained for human CD45, CD8, CD4 and CD28H, and human cells were distinguished from mouse cells based on human CD45 expression. Gated thymocytes were further divided into four populations based on human CD4 and CD8 surface expression (left). The percentage of CD28H-positive cells in each population was shown (right).

4 Supplementary Figure S4: CD28H expression in T cells from human cord blood. Leucocytes from human cord blood were stained for CD3 together with control (left) or CD28H mab (right).

5 Supplementary Figure S5: Characterizing CD28H-negative T cells. (a) CD28H expression on different CD4+ T cell subsets. CD4+ T cells were stained with anti-cd45ra, anti- CCR7, and anti-cd28h mabs. Upon analysis of CD45RA and CCR7 expression, three subsets of CD4+ T cells were identified: T naive (CD45RA+CCR7+), T CM (CD45RA-CCR7+), T EM (CD45RA-CCR7-). Numbers indicated were percentages of CD28H-positive cells in each population. (b) Human T cells were sorted by flow cytometry from fresh hpbmcs into two populations, based on surface CD28H expression. Human CD28H transcripts in these two populations were examined by PCR. (c) Phenotypes of CD28H-positive T cell v.s. CD28H-negative cell. Human T cells were divided into two subpopulation based on CD28H staining. Different surface molecules expressed on two populations were indicated. (d) (e)upon stimulation for five hours by PMA plus Ionomysin, hpbmcs were stained with CD4/CD8, CD3 and CD28H, followed with CD69 or intracellular staining of different cytokines as indicated. Data shown were gated on CD4+CD3+ (c) or CD8+CD3+ (d).

6 Supplementary Figure S6: Regulation of CD28H expression on T cells upon activation. (a) Purified naïve or CD28Hnegative human T cells were labeled with CFSE. Cells were activated by anti-hcd3 mab together with anti-cd28 mab and recombinant human IL-2. On day 3, T cells were harvested and stained for CD28H. The expression of CD28H on T cells together with CFSE staining was examined by flow cytometry. (b) Naïve human T cells were repeated activated in vitro for 20 days. Live cells were sorted by flow cytometry into two populations, based on surface CD28H expression. Human CD28H transcripts in these two populations were examined by PCR. (c) Sorted human T cells as shown in Figure S5b were stained for intracellular CD28H.

7 Supplementary Figure S7: CD28H-negative T cells exhibit senescent phenotype. (a) Co-staining of CD28H with different cell surface molecules as indicated in CD8+ T cell subset. (b) Untouched CD4+ naive or H7CR-negative T cells were negatively isolated by MACS beads, labeled with CFSE, and stimulated with coated anti-human CD3 mab. Cell division was measured by dilution of CFSE on day 7. Data (left) represent one from five independent experiments (right) with different donor. (c) Human PBMCs labeled with CFSE were stimulated with human CD3 mab for 7 days. Live cells were stained for CD3, CD8 and CD28H. Data shown were gated on CD8-CD3+ (left) or CD8+CD3+ (right). (d) Purified naïve CD4+ T cells were repeatedly activated in vitro for 14 days as Figure 2c. Cells were re-stimulated by PMA plus Ionomysin for intracellular cytokines as indicated.

8 Supplementary Figure S8: CD28H does not interact with any known B7 family member. HEK293T cells were transfected with expression vector for individual known human B7 ligand. About 16 hours later, cells were stained with control (shaded), respective mab indicated (solid line) or CD28H.Ig (solid line).

9 Supplementary Figure S9: Homology of B7-H5 to other B7 members. Alignment of human B7-H5 with other known B7 family members via MacVector 6.5.

10 Supplementary Figure S10: Identifying CD28H as the sole counter-receptor for B7-H5 by CDS screening. CDSscreening of membrane-bound molecule library with CD28H-mIg fusion protein. Human CD28H (J18) was identified as a positive hit besides positive controls, FcR (O5) and OLN (P1).

11 Supplementary Figure S11: CD28H binds human cell lines expressing B7-H5, but not B7-H5-negative cells. The staining of CD28H fusion protein or control protein to human monocytes (a) and monocyte-derived dendritic cells (b). Cells were co-stained for CD14 (monocytes) or DC-SIGN and was analyzed by flow cytometry. (c) Human cell lines, M12 and HT29, were stained for B7-H5 by B7-H5 mab (solid line) or control mab (shade). (d) The binding of CD28H fusion protein to these two cell lines was measured by flow cytometry in the presence or absence of a B7-H5 blocking mab (clone 2D3), as indicated.

12 Cell line B7-H5 CD28H-Ig Cell line B7-H5 CD28H-Ig expression binding expression binding SK-MEL-28 no - HT29 no - SK-MEL-526 no - SW620 no - SK-MEL-1359 no - SW403 yes + SK-MEL-1558 no - OSE10 no - Mel-624 no - SK-OV-3 no - M12 yes +++ Jurkat T cell no - Du145 no - U937 no - 22RV1 no - Raji no - LNCaP no - A204 yes ++ SK-BR-3 no - A431 no - HBL100 no - HEP-2 no - Supplementary Table S1: B7-H5 expression in human cell lines