Canine tissue-associated CD4+CD8α+ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential

Abstract

Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4+ or CD8α+ single-positive (sp) T cells. In this study, we investigated CD4+CD8α+ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer's patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a-CD4+CD8α+ dp T cell population in all analyzed organs, with highest frequencies within Peyer's patches. Constitutive expression of the activation marker CD25 is a feature of many CD4+CD8α+ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4+CD8α+ dp T cells is FoxP3+ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4+CD8α+ dp intraepithelial lymphocytes. In addition, a fraction of CD4+CD8α+ dp intraepithelial lymphocytes and of mesenteric lymph node CD4+CD8α+ dp T cells is TCRγδ+. However, the main T cell receptor of all tissue-associated CD4+CD8α+ dp T cells could be identified as TCRαβ. Interestingly, the majority of the CD4+CD8α+ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α+ sp T cells, and CD4+CD8α+ dp thymocytes which are mainly CD8αβ+. The presented data provide the basis for a functional analysis of tissue-specific CD4+CD8α+ dp T cells to elucidate their role in health and disease of dogs.

Fig 1. Mature CD4⁺CD8α⁺ double-positive (dp) T cells are present in secondary lymphatic organs as well as in non-lymphatic tissues of healthy dogs with highest frequencies in Peyer’s patches.

(A) The frequency of canine CD4⁺ single-positive (sp), CD8α⁺ sp, and CD4⁺CD8α⁺ dp T cells in different organs was analyzed by flow cytometry. Shown are representative pseudocolor plots with numbers indicating percentages. Organs of 6-10 dogs in total have been analyzed in two independent experiments. Thymus was used as control (grey background). Only living non-doublet CD5⁺ T cells were included in the analysis. The gating strategy is shown in supporting S2 Fig. (B) Quantification of CD4⁺ sp (green hexagons), CD8α⁺ sp (blue diamonds), and CD4⁺CD8α⁺ dp (red triangles) T cells in canine tracheobronchial lymph nodes (tLN), mesenteric lymph nodes (mLN), spleen, Peyer’s patches (PP), intraepithelial lymphocytes of the small intestine (IEL), and lung is depicted. Thymus served as control (grey background). Pooled data of two independent experiments are shown. Each dot represents one individual dog, the horizontal bars indicate mean values. For clarity reasons, a statistical analysis (One-way ANOVA with Bonferroni’s Multiple Comparison Test, * p < 0.05, *** p < 0.001, n. s.: not significant) is shown in (C). (D) Tissue-associated canine CD4⁺CD8α⁺ dp T cells are mature T cells lacking the thymic marker CD1a (red histograms). As control, thymus (grey histogram) was analyzed for CD1a expression with the appropriate fluorescence minus one (FMO) control (white histogram). Results of one representative dog are depicted. Organs of 4–10 dogs in total have been analyzed in two independent experiments.

Fig 2. The majority of canine CD4⁺CD8α⁺ double-positive (dp) T cells in secondary lymphatic and non-lymphatic organs is characterized by expression of the CD8αα homodimer.

(A) CD4⁺CD8α⁺ dp T cells were analyzed by flow cytometry for the expression of CD8αα and CD8αβ in comparison to CD8α⁺ single-positive (sp) T cells. The pseudocolor plots of Peyer’s patches stand representative for all secondary lymphatic and non-lymphatic organs in this study. Thymocytes are shown for control. The numbers in the pseudocolor plots imply percentages. Organs of 4–10 dogs in total have been analyzed in two independent experiments. The CD8α⁺CD8β^- population indicates a CD8αα homodimer, the CD8α⁺CD8β⁺ population a CD8αβ heterodimer. (B) Proportions of CD8αα (dark blue dots) vs. CD8αβ (light blue dots) expression of CD8α⁺ sp (white background) and mature CD4⁺CD8α⁺ dp T cells (red background) in tracheobronchial lymph node (tLN), mesenteric lymph node (mLN), spleen, Peyer’s patches (PP), intraepithelial lymphocytes of the small intestine (IEL), and lung with thymus as control were quantified. Pooled data of two independent experiments are shown. Each dot represents one individual dog, the horizontal bars indicate mean values. Differences in percentages of CD8αα⁺ cells among CD4⁺CD8α⁺ dp and CD8α⁺ sp T cells were analyzed by unpaired Student’s t-test (two-tailed; *** p < 0.001) and are summarized in (C).

Fig 3. Canine CD4⁺CD8α⁺ double-positive (dp) T cells in secondary lymphatic and non-lymphatic organs are mainly TCRγδ-negative.

(A) Shown are representative results of T cell receptor γδ (TCRγδ) expression in CD4⁺CD8α⁺ dp, CD4⁺ sp, and CD8α⁺ sp T cells among intraepithelial lymphocytes of the small intestine (IEL). The numbers in the flow cytometry plots indicate percentages. (B) Quantification of the frequency of TCRγδ⁺ cells among CD4⁺ sp (green hexagons), CD8α⁺ sp (blue diamonds), and CD4⁺CD8α⁺ dp (red triangles) T cells in mesenteric lymph node (mLN), spleen, Peyer’s patches (PP), IEL, and lung is shown. Thymus is depicted for control. Pooled data of two independent experiments are shown. Each dot represents one individual dog, the horizontal bars indicate median values. Statistical analysis was performed by One-way ANOVA with Dunn’s Multiple Comparison Test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 4. Canine CD4⁺CD8α⁺ double-positive (dp) T cells of secondary lymphatic and non-lymphatic organs constitutively express CD25 in contrast to CD4⁺CD8α⁺ dp thymocytes.

(A) The expression of CD25 on CD4⁺ single-positive (sp), CD8α⁺ sp and CD4⁺CD8α⁺ dp T cells was analyzed by flow cytometry. Shown are representative data of Peyer’s patches in comparison to thymus. The numbers in the pseudocolor plots indicate percentages. Two independent experiments have been performed including organs of 4–10 dogs in total. (B + C) Pooled data of CD25 expression on CD4⁺ sp (green hexagons), CD8α⁺ sp (blue diamonds), and CD4⁺CD8α⁺ dp (red triangles) T cells in indicated organs are depicted. (B) The graphs include normally distributed data values, each symbol represents one individual dog. The horizontal bars indicate mean values. Statistical analysis was performed by One-way ANOVA with Bonferroni’s Multiple Comparison Test (** p < 0.01, *** p < 0.001). (C) The graphs show non-normally distributed data values, each symbol represents one individual dog. The horizontal bars indicate median values. Statistical analysis was performed by One-way ANOVA with Dunn’s Multiple Comparison Test (* p < 0.05, *** p < 0.001).

Fig 5. A proportion of lymph node CD4⁺CD8α⁺ double-positive (dp) T cells comprises FoxP3⁺ regulatory T cells.

(A) Representative results of FoxP3 expression in CD4⁺CD8α⁺ dp T cells of secondary lymphatic and non-lymphatic organs, and of CD4⁺CD8α⁺ dp thymocytes are shown. Two independent experiments with n = 4–10 dogs in total were performed. The numbers in the flow cytometry plots indicate percentages. Appropriate gating was confirmed by internal negative (CD8α⁺ single-positive (sp) T cells) and positive (CD4⁺ sp T cells) controls (B). Presented is the FoxP3 expression of CD4⁺ sp and CD8α⁺ sp T cells in canine lung being representative for all analyzed organs. (C) Proportions of FoxP3 expression of CD4⁺ sp (green hexagons) and CD4⁺CD8α⁺ dp (red triangles) T cells were analyzed in indicated organs (tLN: tracheobronchial lymph node, mLN: mesenteric lymph node, PP: Peyer’s patches, IEL: intraepithelial lymphocytes of the small intestine). Each symbol represents one individual dog, the horizontal bars indicate median values. The Mann-Whitney U test was performed to check for statistical significance between CD4⁺ sp and CD4⁺CD8α⁺ dp T cells (two-tailed; * p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 6. CD4⁺CD8α⁺ double-positive (dp) T cells of the small intestinal epithelium express the cytotoxic molecule granzyme B.

(A) Representative results of granzyme B expression in CD4⁺CD8α⁺ dp T cells of small intestinal intraepithelial lymphocytes (IEL) are shown in comparison to CD4⁺ single-positive (sp) and CD8α⁺ sp T cells. The numbers in the flow cytometry plots indicate percentages. (B) Quantification of the frequency of granzyme B⁺ cells among CD4⁺ sp (green hexagons), CD8α⁺ sp (blue diamonds), and CD4⁺CD8α⁺ dp (red triangles) T cells in mesenteric lymph node (mLN), Peyer’s patches (PP) and IEL is depicted. Each symbol represents one individual dog, the horizontal bars indicate mean values. Statistical analysis was performed by One-way analysis of variance (ANOVA) with Bonferroni post hoc test (*** p < 0.001).