Single-Cell RNAseq Profiling of Human γδ T Lymphocytes in Virus-Related Cancers and COVID-19 Disease

Abstract

The detailed characterization of human γδ T lymphocyte differentiation at the single-cell transcriptomic (scRNAseq) level in tumors and patients with coronavirus disease 2019 (COVID-19) requires both a reference differentiation trajectory of γδ T cells and a robust mapping method for additional γδ T lymphocytes. Here, we incepted such a method to characterize thousands of γδ T lymphocytes from (n = 95) patients with cancer or adult and pediatric COVID-19 disease. We found that cancer patients with human papillomavirus-positive head and neck squamous cell carcinoma and Epstein-Barr virus-positive Hodgkin's lymphoma have γδ tumor-infiltrating T lymphocytes that are more prone to recirculate from the tumor and avoid exhaustion. In COVID-19, both TCRVγ9 and TCRVγnon9 subsets of γδ T lymphocytes relocalize from peripheral blood mononuclear cells (PBMC) to the infected lung tissue, where their advanced differentiation, tissue residency, and exhaustion reflect T cell activation. Although severe COVID-19 disease increases both recruitment and exhaustion of γδ T lymphocytes in infected lung lesions but not blood, the anti-IL6R therapy with Tocilizumab promotes γδ T lymphocyte differentiation in patients with COVID-19. PBMC from pediatric patients with acute COVID-19 disease display similar γδ T cell lymphopenia to that seen in adult patients. However, blood γδ T cells from children with the COVID-19-related multisystem inflammatory syndrome are not lymphodepleted, but they are differentiated as in healthy PBMC. These findings suggest that some virus-induced memory γδ T lymphocytes durably persist in the blood of adults and could subsequently infiltrate and recirculate in tumors.

Keywords: COVID-19; differentiation; gammadelta; human; lymphocyte; single cell; trajectory; transcriptome; tumor.

Figure 1

Differentiation and functional hallmarks of γδ T lymphocytes infiltrating HNSCC and HL tumors according to viral status of cancer patients. (a) Examples of γδ TILs extracted from representative HNSCC and HL tumors with viral status, overlaid on the public trajectory of γδ T lymphocytes reference (grey). Key: TCRVγ9 cells (light blue), Tex TCRVγ9 cells (dark blue), TCRVγnon9 cells (orange), Tex TCRVγnon9 cells (dark red), recirculating (non-Ttrm) cells (open circles), Ttrm (cross). Differentiation pseudotime scale: 0–5: Tn, 5–20: Tcm, 20–50: Tem; > 50: Temra. Together all the EBV-negative HL tumors totalized 53 mostly functional (43/53, 81%) γδ TILs, including 13 Ttrm with 39% of exhausted cells (5/13 Ttrm), while all the EBV-positive HL tumors totalized 60 mostly functional (56/60, 93%) γδ TILs, including 2 non-exhausted Ttrm γδ TILs. The HPV-negative HNSCC tumors totalized 698 mostly functional (489/698, 70%) γδ TILs, including 326 Ttrm with 39% of exhausted cells (127/326 Ttrm), while the HPV-positive HNSCC tumors totalized 357 mostly functional (310/357, 87%) γδ TILs, including 81 Ttrm with 31% of exhausted cells (25/81 Ttrm). (b) Right: rates of recirculating and Ttrm among γδ TILs (both subsets) per viral status of patients (pooled); left: rates of exhausted cells among the recirculating and Ttrm γδ TILs.

Figure 2

Differentiation and functional hallmarks of γδ T lymphocytes from control donor’s PBMC and BALF. (a) γδ T lymphocytes extracted from PBMC and BALF of healthy individuals and COVID-19 patients. No γδ T lymphocyte was found in any of the healthy control’s BALF (representative examples, cells are shown overlaid on the public trajectory of γδ T lymphocyte differentiation, same legend key as in Figure 1). (b) Rates of cells at each differentiation stage among the TCRVγ9 and TCRVγnon9 γδ T lymphocytes (group means are shown).

Figure 3

Differentiation and functional hallmarks of γδ T lymphocytes from adult COVID-19 patients. (a) BALF-derived γδ T lymphocytes from adult patients with mild or severe COVID-19 disease. (b) PBMC-derived γδ T lymphocytes from adult COVID-19 patients untreated or treated with Tocilizumab. (Representative examples overlaid on the public trajectory of γδ T lymphocyte differentiation, same legend key as in Figure 1).

Figure 4

Differentiation and functional hallmarks of γδ T lymphocytes from pediatric COVID-19 patients. (a) PBMC-derived γδ T lymphocytes from adult and pediatric controls. (b) PBMC-derived γδ T lymphocytes from pediatric COVID-19 patients with either MIS-C or acute disease. (Representative examples overlaid on the public trajectory of γδ T lymphocyte differentiation, same legend key as in Figure 1).