T cell factor 1 (TCF-1) defines T cell differentiation in colorectal cancer

Abstract

The presence of precursor to exhausted (T_pex) CD8⁺ T cells is important to maintain robust immunity following treatment with immune checkpoint inhibition (ICI). Impressive responses to ICI are emerging in patients with stage II-III mismatch repair (MMR)-deficient (dMMR) colorectal cancer (CRC). We found 64% of dMMR and 15% of mismatch repair-proficient (pMMR) stage III CRCs had a high frequency of tumor infiltrating lymphocytes (TIL-hi). Furthermore, expression of TCF-1 (Tcf7) by CD8⁺ T cells predicted improved patient prognosis and T_pex cells (CD3⁺CD8⁺TCF-1⁺PD-1⁺) were abundant within lymphoid aggregates of stage III CRCs. In contrast, CD3⁺CD8⁺TCF-1^-PD-1⁺ cells were more abundant at the invasive front and tumor core, while γδ T cells were equally abundant in all tumor areas. Interestingly, no differences in the frequency of T_pex cells were observed between TIL-hi dMMR and TIL-hi pMMR CRCs. Therefore, T_pex cell function and ICI response rates in TIL-hi CRC warrants further investigation.

Keywords: Cancer; Components of the immune system; Immunology.

Graphical abstract

Figure 1

TIL frequency correlates with MMR status in stage III CRC (A and B) Stage III CRCs were analyzed and stratified as A) TIL-hi or TIL-lo infiltration and for B) MMR status. Data are presented as number of patients and the frequency on each graph represents the percent of patients in each subgroup. (C) Frequency of TIL-hi and TIL-lo tumors shown for dMMR and pMMR subgroups in stage III CRC. (D) Number and frequency of dMMR or pMMR patients among TIL-hi stage III CRCs. A total of 411 stage III CRCs were analyzed.

Figure 2

Differences in CD8⁺ T cell and γδ T cell spatial distribution in stage III CRC (A) Representative hematoxylin and eosin (H&E) staining of a stage III CRC tumor whole tissue section. Areas of interest for analysis include tumor stroma, tumor core, normal-adjacent tissue, lymphoid aggregates and invasive front. Scale bar: 2000 μm. (B–F) Representative images of mIHC staining of the five compartments of interest, i.e., B) tumor core (T), C) invasive front (IF), D) lymphoid aggregate (LA), E) tumor stroma (S) and F) normal-adjacent tissue (N). Staining represented as merged and single channel staining for CD3, CD8, TCRδ and PanCK and are shown with DAPI staining. White square boxes are magnified to the right (middle panel). Scale bar: 50 μm. (G–I) Densities (mm², upper panel) and frequencies (lower panel) of G) total CD3⁺, H) CD3⁺CD8⁺TCRδ⁻ and I) CD3⁺CD8⁻TCRδ⁺ cells across the five tumor areas of interest. Orange data points show dMMR cases, blue data points show pMMR cases. Each point represents the average of five multispectral images per patient. Error bars represent SEMs. p-values calculated using one-way ANOVA test and only p-values < 0.05 are displayed.

Figure 3

TCF-1-expressing CD8⁺ T cells predict improved survival in stage III CRC (A) Flowchart depicting overview of experiment setup. (B and C) Percentage disease-free and C) overall survival of stage III CRC patients with high or low CD3⁺CD8⁺TCF-1⁺, total CD3⁺ or total CD3⁺CD8⁺ T cell infiltration. Staining and analysis is performed on 3 replicate tissue microarray samples per tumor from 63 stage III CRC patients. p-values calculated using Log rank Mantel-Cox test.

Figure 4

CD8⁺TCF-1⁺PD-1⁺ T_pex cells are more abundant in lymphoid aggregates in stage III CRC (A) Representative CD8⁺TCF-1⁺PD-1⁺ T_pex (upper panel) and CD8⁺TCF-1⁻PD-1⁺ T cells (lower panel) mIHC staining in a lymphoid aggregate and tumor core of stage III CRC whole tumor section. Merged images display overlay of CD3, CD8, TCRδ, PD-1, TCF-1, PanCK and DAPI staining. Single stains of CD3, CD8, TCRδ, PD-1, TCF-1 and PanCK are shown with DAPI staining. White square boxes are magnified to the right (middle panel). Scale bar: 50 μm. (B–E) Percentage frequencies of B) T_pex (CD8⁺TCF-1⁺PD-1⁺), C) CD8⁺TCF-1⁻PD-1⁺, D) CD8⁺TCF-1⁻PD-1^- and E) CD8⁺TCF-1⁺PD-1^- T cell subsets across the five tumor areas of interest; tumor core (T), invasive front (IF), lymphoid aggregates (LA), tumor stroma (S) and normal-adjacent tissue (N). Percentage frequencies for each CD8⁺ T cell subset is calculated based on the total CD3⁺ T cell count. Orange data points represent dMMR cases, blue data points represent pMMR cases. Each point represents the average of five multispectral images per patient. Error bars represent SEMs. p-values calculated using one-way ANOVA test and only p-values < 0.05 are shown.