Spatial distribution of FoxP3+ and CD8+ tumour infiltrating T cells reflects their functional activity

Rebecca Posselt, Katharina Erlenbach-Wünsch, Matthias Haas, Jonas Jeßberger, Maike Büttner-Herold, Marlen Haderlein, Markus Hecht, Arndt Hartmann, Rainer Fietkau, Luitpold V Distel, Rebecca Posselt, Katharina Erlenbach-Wünsch, Matthias Haas, Jonas Jeßberger, Maike Büttner-Herold, Marlen Haderlein, Markus Hecht, Arndt Hartmann, Rainer Fietkau, Luitpold V Distel

Abstract

Background: Regulatory and cytotoxic T cells are key players in the host's anticancer immune response. We studied the spatial distribution of FoxP+ and CD8+ cells to identify potential interactions.

Methods: In 202 patients 103 pre-radiochemotherapy biopsies and 153 post-radiochemotherapy tumour specimens of advanced rectal cancer were available and an immunohistochemical double staining of FoxP3+ and CD8+ tumour-infiltrating lymphocytes was performed to investigate cell density and cell-to-cell distances.

Results: FoxP3+ cells decreased after radiochemotherapy by a factor of 3 while CD8+ cells remained nearly unchanged. High epithelial (p=0.033) and stromal (p=0.009) FoxP3+ cell density was associated with an improved overall survival. Cell-to-cell distances of randomly distributed cells were simulated and compared to observed cell-to-cell distances. Observed distances shorter than the simulated, random distances were hypothesized to represent FoxP3+ cells actively interacting with CD8+ cells. Epithelial short distances were associated with a favourable prognosis while the opposite was true for the stromal compartment.

Conclusion: The analysis of cell-to-cell distances may offer a tool to predict outcome, maybe by identifying functionally active, interacting infiltrating inflammatory cells in different tumour compartments.

Keywords: CD8+; FoxP3+; cytotoxic T cells; regulatory T cells; tumour-infiltrating lymphocytes.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there is no conflicts of interest.

Figures

Figure 1. CD8+ and FoxP3+ cell densities…
Figure 1. CD8+ and FoxP3+ cell densities in rectal adenocarcinoma
Kaplan Meier plots for overall survival, metastasis free survival and no evidence of disease in the complete cohort A. Enlarged section (1:200) of tumour stroma with immunohistochemical double staining for FoxP3 (red nucleic staining) and CD8 (blue predominantly membranous staining) B. Tissue samples were processed into tissue microarrays using a core diameter of 2 mm C. Epithelial and stromal compartments were separately analysed D. Lymphocyte densities (cells/mm2) in the biopsy, the tumour centre and the invasive front E. FoxP3+/CD8+ ratio in stromal and epithelial compartment F. Stromal/epithelial ratio of FoxP3+ and CD8+ cells G. Ratio of pre-RCT biopsies, central tumour and invasive front of epithelial and stromal FoxP3+ and CD8+ cells H.
Figure 2. Kaplan-Meier plots for the densities…
Figure 2. Kaplan-Meier plots for the densities of FoxP3+ cells in the stromal and epithelial compartment in pre-RCT biopsies and post-RCT central tumour (overall survival)
FoxP3+cell density in epithelial pre-RCT biopsies A., FoxP3+ cell density in the stromal pre-RCT biopsies B., FoxP3+ cell density in the epithelial post-RCT central tumour C., FoxP3+ cell density in the stroma post-RCT central tumour D.
Figure 3. CD8+ to FoxP3+ cell distances…
Figure 3. CD8+ to FoxP3+ cell distances in rectal adenocarcinoma
Double staining for FoxP3+ and CD8+ in tumour stroma with highlighted direct cell-to-cell interactions (arrows, 200x original magnification) and a direct FoxP3+ to CD8+ interactions at higher magnification, bottom right A. Example for the calculation of the shortest distance between FoxP3+ and CD8+ cells (orange arrows) and CD8+ to FoxP3+ (green arrows) B. Cell-to-cell distances (μm) in the biopsy, the central tumour area and the invasive front C. CD8+-FoxP3+/FoxP3+-CD8+ ratio in preRCT biopsies and post RCT central tumour and invasive front D. Epithelial/stromal ratio of FoxP3+-CD8+ and CD8+-FoxP3+ distances E. Ratio of preRCT biopsies, central tumour and invasive front of epithelial and stromal FoxP3+-CD8+ and CD8+-FoxP3+ distances F.
Figure 4. Kaplan-Meier plots for the cell-to-cell…
Figure 4. Kaplan-Meier plots for the cell-to-cell distances of FoxP3+-CD8+ distances in the stromal and epithelial compartment of preRCT biopsies and post-RCT central tumour specimens (overall survival)
FoxP3+-CD8+ cell distances in the epithelial pre-RCT biopsies A., FoxP3+-CD8+ cell distances in the stromal pre-RCT biopsies B., FoxP3+-CD8+ cell distances in the epithelial post-RCT central tumour C., FoxP3+-CD8+ cell distances in the stromal post-RCT central tumour D.
Figure 5. Observed compared to expected cell-to-cell…
Figure 5. Observed compared to expected cell-to-cell distances
Observed cell-to-cell distances in dependence of cell densities (green open squares and blue diamonds) were compared to simulated values (red plus and orange crosses). The solid line indicates 10% shorter distance than the simulated values and was used as cut off for defining observed distances shorter than the expected values. Cell-to-cell distances compared to cell densities in pre-RCT biopsies in the epithelial compartment A. and in the stromal compartment B. as well as in post-RCT central tumour in the epithelial C. and stromal compartment D. Kaplan-Meier plots using the above identified samples with shorter distances than expected compared to longer distances for OS. FoxP3+-CD8+ cell distances in the epithelial pre- RCT biopsies E.; CD8+-FoxP3+ cell distances in the epithelial pre-RCT biopsies F.; FoxP3+-CD8+ cell distances in the epithelial post-RCT central tumour G.; CD8+-FoxP3+ cell distances in the epithelial post-RCT central tumour H. and FoxP3+-CD8+ cell distances in the stromal pre-RCT biopsies J.; CD8+-FoxP3+ cell distances in the stromal pre-RCT biopsies K.; FoxP3+-CD8+ cell distances in the stromal post-RCT central tumour L.; CD8+-FoxP3+ cell distances in the stromal post-RCT central tumour M.
Figure 6. Kaplan-Meier plots for the combination…
Figure 6. Kaplan-Meier plots for the combination of cell-to-cell distances
Kaplan-Meier plots using the ratio of short epithelial distances with the respective stromal distances compared to long epithelial distances with the respective stromal distances. NED survival in the post-RCT samples for FoxP3+-CD8+ cell distances A. and CD8+-FoxP3+ cell distances B. and OS for FoxP3+-CD8+ cell distances C. and CD8+-FoxP3+ cell distances D. Model for epithelial and stromal cell-to-cell interactions and their prognostic values E.

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