Development of Exhaustion and Acquisition of Regulatory Function by Infiltrating CD8+CD28- T Lymphocytes Dictate Clinical Outcome in Head and Neck Cancer

Abstract

Head and neck squamous cell carcinoma (HNSCC) has a poor clinical outcome despite the presence of a rich CD8+ T cell tumor infiltrate in the majority of patients. This may be due to alterations of tumor infiltrating CD8+ T cells. Here, we performed a characterization of HNSCC infiltrating CD8+ T cells in a cohort of 30 patients. The results showed that differential intratumoral frequency of CD8+CD28+ T cells, CD8+CD28- T cells, and CD8+CD28-CD127-CD39+ Treg distinguished between HNSCC patients who did or did not respond to treatment. Moreover, high PD1 expression identified a CD8+CD28- T cell subpopulation, phenotypically/functionally corresponding to CD8+CD28-CD127-CD39+ Treg, which showed a high expression of markers of exhaustion. This observation suggests that development of exhaustion and acquisition of regulatory properties may configure the late differentiation stage for intratumoral effector T cells, a phenomenon we define as effector-to-regulatory T cell transition.

Keywords: CD8+ T lymphocytes; exhaustion; head-neck cancer; regulatory T cells.

Figure 1

Frequency of tumor infiltrating CD8+CD28+ T cells, CD8+CD28− T cells and CD8+CD28−CD127−CD39+ Treg from HNSCC patients. (A–C): Comparative analysis of the frequencies of CD8+CD28+ T cells (Panel A), CD8+CD28− T cells (Panel B) and CD8+CD28−CD127−CD39+ Treg (Panel C) between Group 1 and Group 2 patients. (D–F): Survival of HNSCC patients divided into two groups based on the median (calculated in the overall population) of frequency of either CD8+CD28+ T lymphocytes (> or <47.5%) (Panel D), CD8+CD28− T lymphocytes (> or <52.5%) (Panel E) or CD8+CD28−CD127−CD39+ Treg (> or <13.5%) (Panel F).

Figure 2

Phenotypic characterization of tumor infiltrating CD8+CD28− T cells based on the levels of PD-1 expression. A and B. CD28− T cells were gated and tumor infiltrating CD8+CD28− T cells from representative patients #13 (A) and #19 (B) were divided in three subpopulations (CD8+CD28−PD-1^hi, CD8+CD28−PD1^int and CD8+CD28−PD-1^lo/−, respectively) based on the mean fluorescent intensity (MFI) of PD-1 expression (left panels). CD8+CD28−PD-1^hi, CD8+CD28−PD-1^int and CD8+CD28−PD-1^lo were considered cells with PD-1 MFI > 10³, >10² and <10³, or <10², respectively. The frequencies of CD127−CD39+ T cells (middle panels) and of CD39+TIM-3+ T cells (right panels) are separately shown for CD8+CD28−PD-1^hi, CD8+CD28−PD-1^int and CD8+CD28−PD-1^lo. (C) MFI mean values of PD-1 (left panel), CD39 (middle panel) and TIM-3 (right panel) in CD8+CD28−PD-1^hi, CD8+CD28−PD-1^int and CD8+CD28−PD-1^lo/− tumor infiltrating T cells from our series of HNSCC patients.

Figure 3

Multiparametric phenotypic characterization of tumor infiltrating CD8+ T cells from HNSCC patients. (A): tSNE algorithm was applied to the analysis of tumor infiltrating CD8+ T cells from patient #13. A red circle identifies a map area where cell clusters, negative for CD28, CD127, CD45RA and CCR7 expression and highly positive for PD-1, CD39 and TIM-3 expression, segregate. The colorimetric scale of expression is shown below each graph (blue clusters indicate absent expression, red clusters represent high expression). (B): t-SNE algorithm was applied to the analysis of tumor infiltrating CD8+ T cells from patient #27. A red circle identifies a map area where cell clusters, negative for CD28 and CD127 expression, highly positive for PD-1 and CD39 expression, and partly positive for TIM-3 and EOMES expression, segregate. The colorimetric scale of expression is shown below each graph (blue clusters indicate absent expression, red clusters represent high expression). (C): Frequencies of CD39-PD-1- T cells among tumor infiltrating CD8+CD28− T lymphocytes in Group 1 and 2 patients.

Figure 4

Relationship between tumor infiltrating CD8+CD28−PD1^hi T cells and CD8+CD28−CD127−CD39+ Treg in HNSCC patients. (A): Mean frequencies of CD8+CD28−CD127−CD39+ Treg among CD8+CD28−PD-1^lo, CD8+CD28−PD-1^int and CD8+CD28−PD-1^hi T cell subpopulations. Statistical significant differences are evidenced. (B): Statistical correlation between the frequency of tumor infiltrating CD8+CD28−PD-1^hi T cells and that of tumor infiltrating CD8+CD28−CD127−CD39+ Treg in our cohort of HNSCC patients. (C): Proliferation suppression assay performed with the CD8+ T cell infiltrate from the representative patient #5 containing comparable frequencies of CD8+CD28−PD-1^hi (66%) and CD8+CD28−CD127−CD39+ Treg (60%). The percentages of T cell proliferation under unstimulated conditions (left panel) or anti-CD3 UCHT1 mAb–stimulated conditions in absence (middle panel) or presence (right panel) of tumor infiltrating CD8+CD28−CD127−CD39+ Treg (CD8+ Treg in the Figure) are shown; the percentage of proliferation inhibition by CD8+CD28−CD127−CD39+Treg is shown in parentheses in the right panel. One out of two concordant experiments performed with cells of different patients.

Figure 5

Further phenotypic characterization of tumor infiltrating CD8+CD28−PD-1^hi/CD8+CD28−CD127−CD39+ T cells. (A): t-SNE algorithm was applied to the analysis of tumor infiltrating CD8+ T cells from patient #28. A red frame identifies a map area where cell clusters, negative for CD28 and CD127 expression and highly positive for CD39, PD-1, TIM-3 and CD103 expression, segregate. The colorimetric scale of expression is shown below each graph (blue clusters indicate absent expression, red clusters represent high expression). (B): t-SNE algorithm was applied to the analysis of tumor infiltrating CD8+ T cells from patient #28. A red circle identifies a map area where cell clusters, negative for CD28 and CD127 expression and highly positive for CD39, PD-1, CD137 and ki-67 expression, segregate. The colorimetric scale of expression is shown below each graph (blue clusters indicate absent expression, red clusters represent high expression). (C): Staining of tumor infiltrating CD8+ T cells (upper row), CD8+CD28−CD127−CD39+ Treg (middle row) and CD8+CD28−PD-1^hi T cells (lower row) from patient #30 with a G250 HLSTAFARV_217–225 loaded HLA-A2 pentamer. An unrelated, hTert ILAKFLHWL_540–548 loaded HLA-A2 pentamer was used as negative control (upper, right panel).

Figure 6

Schematic representation of the “effector-to-regulatory CD8+ T cell transition” at the tumor site.