Re-Evaluation of PD-1 Expression by T Cells as a Marker for Immune Exhaustion during SIV Infection

<div><p>PD-1 expression is generally associated with exhaustion of T cells during chronic viral infections based on the finding that PD-1 expressing cells respond poorly to antigen activation and blockade of PD-1/PD-ligand interaction restores such antigen specific responses in vitro. We tested this hypothesis by examining PD-1 expression on virus-specific CD8 T cells and total T cells in vivo to determine whether PD-1 expression constitutes a reliable marker of immune exhaustion during SIV infection. The expression of PD-1 and Ki67 was monitored longitudinally on T cell subsets in peripheral blood, bone marrow, lymph node and rectal biopsy specimens from rhesus macaques prior to and post infection with pathogenic SIVmac239. During the course of infection, a progressive negative correlation was noted between PD-1 density and Ki67 expression in p11CM⁺ CD8⁺ T cells, as seen in other studies. However, for total and memory CD4 and CD8 T cells, a positive correlation was observed between PD-1 and Ki67 expression. Thus, while the levels of non-proliferating PD-1⁺ p11CM⁺ CD8 T cells were markedly elevated with progressing infection, such an increase was not seen on total T cells. In addition, total memory PD1⁺ T cells exhibited higher levels of CCR5 than PD-1⁻ T cells. Interestingly, few PD-1⁺ CD8⁺ T cells expressed CCR7 compared to PD-1⁺ CD4 T cells and PD-1⁻ T cells. In conclusion, overall PD1⁺ T cells likely represent a particular differentiation stage or trafficking ability rather than exhaustion and in the context of chronic SIV infection, the level of PD-1 expression by T cells does not by itself serve as a reliable marker for immune exhaustion.</p> </div

Alteration in the frequencies of circulating Ki67⁺PD-1⁺, Ki67⁻PD-1⁺, Ki67⁺PD-1⁻ T cell subsets in ART treated animals during chronic SIVmac239 infection.

<p>Ten rhesus macaques received ART for 28 days initiated at 112 days post infection. (A) level of plasma viral load prior to (wk 16), during (wk 19) and after cessation of ART treatment (wk37) at chronic SIV infection. The MFI of PD-1, the frequencies of Ki67⁺PD-1⁺, Ki67⁻PD-1⁺, and Ki67⁺PD-1⁻ on CD4 (B, E, H and K), CD8 (C, F, I, and L), and p11CM⁺ CD8⁺ T cells (D, G, J, and M) before and after the ART treatment at chosen time points. The p-values shown are the result of paired t-test analysis. For purpose of control, values from the 10 non-ART treated control monkeys (CD4 and CD8 T cells) or the 3 non-ART treated controls (p11CM⁺CD8 T cells) were averaged and shown as a discontinuous line in panels B-M.</p

Longitudinal analysis of SIVgag-specific CD8 T cells in seven Mamu A*01 rhesus macaques infected with SIVmac239.

<p>The gating strategy and representative flow cytometry showing the percentage (Mean +/− S.D.) of PD-1 expressing p11CM⁺ CD8⁺ T cell (Ki67<b>⁺</b> vs Ki67⁻) in PBMC at 14 and 112 dpi is shown in (A). The frequency of p11CM⁺ CD8⁺T cells and their PD-1 expression in PBMC (B), lymph node (C) and colorectal tissues (D) at 14, 42, 112 dpi are shown. The frequency of Ki67⁻ PD-1<b>⁺</b>, Ki67⁺ PD-1<b>⁺</b> and Ki67⁺ PD-1⁻ cells in p11CM⁺ CD8⁺ T cell in PBMC (E), lymph node (F), and colorectal tissues (G) following SIVmac239 infection are shown. The relative density of PD-1 expression (MFI) by p11CM⁺ CD8⁺T cells is plotted as a function of proliferative capacity based on Ki67 expression in PBMC (H), lymph node (I), and colorectal tissues (J) at 14, 42, and 112 dpi. PBMC, lymph node, and colorectal cell samples from Seven 7 Mamu-A*001 animals were included in these analyses.</p

Effect of anti-CD200/CD200R/Mincle monoclonal antibodies on the in vitro replication of macrophage tropic SHIV.

<p>Monocytes were isolated using a procedure outlined in the methods section. A 48 well plate was seeded with 10⁴ monocytes/well and cultured for 7 days and aliquots infected with either SHIV-Bo159N4 (A) or SIVmac251Desi#4 in the presence of normal mouse Ig (10 μg/ml) or 10 μg/ml of the monoclonal antibodies against either CD200, CD200R, or Mincle. Culture supernatants were collected at various time points as shown and the p27 levels quantitated using a commercial ELISA kit. NS = non-significant and the notation * reflects a ‘p’ value of <0.05 noted for data obtained with the addition of mAb against CD200 or CD200R.</p

Comparative analysis of the frequencies of CD200, CD200R, Mincle, Siglec-1 and Siglec-3 expressing cells by the gated population of CD14⁺ cells from the PBMC samples from normal adult rhesus macaques (n = 19, black bars) and sooty mangabeys (n = 18, grey bars) (A) and similar analysis on infant (< 3 months) rhesus macaques (n = 10) and sooty mangabeys (n = 4) (B).

<p>The notations * represents p < 0.05 and *** represents p < 0.001.</p

The net frequencies (background using isotype control Ig deducted) of CD200, CD200R, Mincle, Siglec-1 and Siglec-3 expressing Lin^-, HLA-DR⁺, CD123⁺ plasmacytoid dendritic cells (pDCs, panel A) and Lin^-, HLA-DR⁺, CD11c⁺ myeloid dendritic cells (mDCs, panel B) in the peripheral blood samples from 3 normal rhesus macaques and 3 normal sooty mangabeys are displayed.

<p>The absolute numbers of mDCs noted in the blood of rhesus macaques were 355 +/- 42 and pDCs were 48 +/- 17. The data reflect the Mean +/- SD values and * reflects p< 0.05, ** reflects p< 0.01.</p

Representative flow cytometric profiles of CD200, CD200R, Mincle, Siglec-1 and Siglec-3 expression by the U937 (panel a) and THP-1 (panel b) cell lines and by the gated population of CD14+ cells from PBMCs from a representative normal rhesus macaque (panel c) and for comparison the profile of the gated population of CD14+ cells from the human PBMCs stained with our panel of anti-CD200, CD200R and Mincle antibodies (panel d) and the commercially obtained monoclonal antibodies with specificity for human CD200, CD200R and Mincle (panel e).

<p>The profile in red reflects the use of anti-mouse Ig (control) and the one in blue reflects the profile noted with the appropriate anti-lectin monoclonal antibody (mAb). The numbers in red and blue are the MFI values noted for the anti-mouse Ig control and the anti-lectin mAb, respectively.</p

Longitudinal analysis of PD-1 and Ki67 expression on CD4⁺ and CD8⁺ T cells in blood and tissues of twenty SIVmac239-infected rhesus macaques.

<p>The gating strategy utilized and representative flow cytometry profiles showing the percentage (Mean +/- S.D.) of PD-1⁺ CD4 and CD8⁺ T cell (Ki67⁺ vs Ki67⁻) in whole blood at 0, 28, 112 dpi shown in (A). The frequency of PD-1⁺ Ki67⁺, PD-1⁺ Ki67⁻ and PD-1⁻ Ki67⁺ cells in CD4⁺ (B, D, F and H) and CD8⁺ T cells (C, E, G, and I) prior to and following SIVmac239 infection in whole blood (B and C), bone marrow (D and E), lymph node (F and G), and colorectal tissues (H and I). Whole blood, bone marrow, lymph node, and colorectal cell samples from twenty animals were used for the analyses, except for the lymph node at 0 dpi (n = 13).</p

Representative flow cytometric profile of CD200, CD200R, Mincle, Siglec-1 and Siglec-3 expression on the gated population of CD14⁺ cells isolated from the bone marrow (panel a), spleen (panel b), liver (panel c) and mononuclear cells isolated from rectal biopsy samples (panel d) from a representative healthy normal rhesus macaque.

<p>The profile in red reflects the use of anti-mouse Ig (control) and the one in blue reflects the profile noted with the appropriate anti-lectin monoclonal antibody (mAb). The numbers in red and blue are the MFI values noted for the anti-mouse Ig control and the anti-lectin mAb, respectively.</p

Longitudinal analysis of correlations between the density of PD-1 expression (MFI) by CD4 and CD8 T cells with the frequencies of Ki67 expressing cells in blood and tissues during SIV infection.

<p>PD1 expression positively correlated with Ki67 expression on CD4 (A, C, E and G) and CD8 T cells (B, D, F, and H) during the chronic infection, except for CD4 T cells from the colorectal tissues. Data of each individual animal pre- and post- SIV infection are shown. The correlation was assessed by Spearman's rank correlation test. A p-values of less than 0.05 were considered statistically significant. Whole blood, bone marrow, lymph node, and colorectal cell samples from twenty animals were used for the analyses, except for the lymph node at 0 dpi (n = 13).</p