A MLR-Based Approach to Analyze Regulators of T Lymphocyte Activation In Vivo

Abstract

Depending on the context, robust and durable T lymphocyte activation is either desirable, as in the case of anti-tumor responses, or unwanted, in cases of autoimmunity when chronic stimulation leads to self-tissue damage. Therefore, reliable in vivo models are of great importance to identify and validate regulatory pathways of T lymphocyte activation. Here, we describe an in vivo mixed-lymphocyte-reaction (MLR) approach, which is based on the so-called parent-into-F1 (P → F1) mouse model in combination with the congenic marker CD45.1/2 and cell proliferation dye-labeling. This setup allows us to track adoptively transferred allogenic CD4⁺ and CD8⁺ T lymphocytes and analyze their phenotype as well as the proliferation by flow cytometry in the blood and spleen. We could show hypo-reactive responses of T lymphocytes isolated from knockout mice with a known defect in T lymphocyte activation. Thus, this MLR-based in vivo model provides the opportunity to analyze positive regulators of T cell responses under physiological conditions of polyclonal T lymphocyte activation in vivo.

Keywords: GVHD model; MLR; T cell activation; allogenic response; in vivo model.

Figure 1

The P → F1 approach to analyze T cell activation in vivo in spleen and blood. (A) Experimental setup: 8 × 10⁶ wild type; eFluor670 proliferation dye-labeled CD3⁺ T cells were adoptively transferred i.v. to either syngenic or allogenic recipients. Transferred T cells were analyzed from spleen and blood of recipient mice by flow cytometry at the indicated time points. (B) Spleen weight and splenocyte counts at day 3 and 8 after ACT. (C) The frequency of transferred T cells (CD45.2⁺) in the spleens of allogenic or syngenic recipients (both CD45.1⁺/2⁺) was determined by flow cytometry using CD45.1 and CD45.2 specific antibodies. (D) T cell proliferation was analyzed based on dilution of the proliferation dye eFluor670 with each cell division. Proliferation of transferred CD4⁺ and CD8⁺ T cells is depicted as percent of divided cells. (E) The frequency of naive and effector/memory subsets among transferred CD4⁺ and CD8⁺ T cells was determined by CD44 and CD62L staining. Data are represented as individual values plus mean. Representative FACS dot plots or histograms of individual mice are shown. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; unpaired t-test.

Figure 2

Kinetic of allogenic T cell activation in blood sample. Transferred T cells were analyzed from the blood at day 1, 3, 6 and 8 after transfer for (A) their frequency among lymphocytes, (B) proliferation of CD4⁺ and CD8⁺ T cells as well as (C) naive and effector/memory status—analogous to what is shown in Figure 1. Data are represented as individual values plus mean. Representative FACS histograms of individual mice are shown.

Figure 3

Using PKCθ^-/- T cells in the modified P → F1 approach as an example of a hypo-reactive T cell response. (A) Spleen weight and splenocyte counts were determined at day 8 after ACT of 8 × 10⁶ wild type or PKCθ-deficient CD3⁺ T cells to allogenic recipients. (B,C) Flow cytometric analysis of the frequency (B) or proliferation (C) of either transferred wild type or PKCθ^-/- T cells in the spleens of allogenic recipients at day 3 and 8 after ACT. (D) Naive and effector/memory subsets of transferred CD4⁺ and CD8⁺ T cells, either wild type or PKCθ^-/-, were analyzed at day 8 in the spleens of allogenic recipients. Data are represented as individual values plus mean. Representative FACS dot plots or histograms of individual mice are shown. ns = not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001; (A,D) unpaired t-test; (B,C) one-way ANOVA following Bonferroni’s post hoc test.

Figure 4

Impaired allogen-induced activation of PKCθ-deficient T-cells can be detected in blood. (A) Proliferation and (B) CD4⁺ and CD8⁺ T cell subsets (naive and effector/memory) were analyzed at the same time points by flow cytometry from blood. Data are represented as individual values plus mean. Data are represented as individual values plus mean. Representative FACS histograms of individual mice are shown. ns = not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001; one-way ANOVA following Bonferroni’s post hoc test.

Figure 5

Titration of transferred T cell numbers. (A) Representative pictures of spleens on day 8 after transfer of 1, 2, 4 or 8 million wild type T cells are depicted. (B) Frequency of the transferred T cells among splenocytes and from blood at day 8 after ACT was determined by flow cytometry. (C) Proliferation and (D) T cell activation status (naive and effector/memory) of transferred wild type CD4⁺ and CD8⁺ T cells was analyzed at day 8 by CD62L and CD44 staining and flow cytometry. Data are represented as individual values plus mean. Representative FACS dot plots of individual mice are shown. ns = not significant, * p < 0.05, **** p < 0.0001; one-way ANOVA following Bonferroni’s post hoc test.