Physiological and Functional Effects of Dominant Active TCRα Expression in Transgenic Mice

Abstract

A T cell receptor (TCR) consists of α- and β-chains. Accumulating evidence suggests that some TCRs possess chain centricity, i.e., either of the hemi-chains can dominate in antigen recognition and dictate the TCR's specificity. The introduction of TCRα/β into naive lymphocytes generates antigen-specific T cells that are ready to perform their functions. Transgenesis of the dominant active TCRα creates transgenic animals with improved anti-tumor immune control, and adoptive immunotherapy with TCRα-transduced T cells provides resistance to infections. However, the potential detrimental effects of the dominant hemi-chain TCR's expression in transgenic animals have not been well investigated. Here, we analyzed, in detail, the functional status of the immune system of recently generated 1D1a transgenic mice expressing the dominant active TCRα specific to the H2-K^b molecule. In their age dynamics, neither autoimmunity due to the random pairing of transgenic TCRα with endogenous TCRβ variants nor significant disturbances in systemic homeostasis were detected in these mice. Although the specific immune response was considerably enhanced in 1D1a mice, responses to third-party alloantigens were not compromised, indicating that the expression of dominant active TCRα did not limit immune reactivity in transgenic mice. Our data suggest that TCRα transgene expression could delay thymic involution and maintain TCRβ repertoire diversity in old transgenic mice. The detected changes in the systemic homeostasis in 1D1a transgenic mice, which are minor and primarily transient, may indicate variations in the ontogeny of wild-type and transgenic mouse lines.

Keywords: TCR repertoire; aging; allogeneic immune response; chain-centric TCR; dominant active TCRα; proteomics; thymic involution; transgenesis; transgenic mice.

Figure 1

The figure was changed accordingly. Effects of the dominant active TCRα expression on the thymus homeostasis of 1D1a transgenic mice. Wild-type B10.D2(R101) (WT) and transgenic 1D1a (TG) mice at the ages of 3, 6, and 12 months (3 Mo, 6 Mo, and 12 Mo, respectively) were used to analyze the absolute thymocyte count (×10⁶ per thymus) (A) and the proportion (%) of main thymocyte subpopulations as determined by the expression of CD4 and CD8 markers by flow cytometry (B). The absolute counts (×10⁶) of double-negative (CD4−CD8−, DN), double-positive (CD4+CD8+, DP), and single-positive CD4+ and CD8+ thymocytes (C) in the thymus of 3 Mo (left panel), 6 Mo (middle panel), and 12 Mo (right panel) WT and TG mice were then calculated. Data are presented as mean ± SEM (n = 6–10). * p < 0.05; ** p < 0.01 (unpaired Student t-test).

Figure 2

Effects of the dominant active TCRα expression on the spleen homeostasis of 1D1a transgenic mice. The spleens of wild-type B10.D2(R101) (WT) and transgenic 1D1a (TG) mice at the ages of 3, 6, and 12 months (3 Mo, 6 Mo, and 12 Mo, respectively) were isolated and analyzed. (A) The total leukocyte count (×10⁶ per spleen). (B–D) Flow cytometry analyses of spleen T cell populations in WT and TG mice. (B) The proportion (%) of T cells (CD3+). (C) The proportion (%) of CD4+, CD8+, and double-negative CD4−CD8− T cells. (D) The proportion (%) of CD8+ T cells with the phenotype of naïve cells (CD62L+CD44−), central memory cells (CD62L+CD44+), and effectors (CD62L−CD44+). Data are presented as mean ± SEM (n = 5–10). * p < 0.05; ** p < 0.01 (unpaired Student t-test).

Figure 3

Changes in the TCRβ repertoire of peripheral T cells in 1D1a transgenic mice with the age-dependent dynamics. Leukocytes were isolated from the blood of wild-type B10.D2(R101) (WT) and transgenic 1D1a (TG) mice at the ages of 3, 6, and 12 months (3 Mo, 6 Mo, and 12 Mo, respectively). The proportion (%) of CD3+ cells expressing different TCRVβ families was evaluated by flow cytometry in the pool of peripheral leukocytes of WT (A) and TG (B) mice. Data are presented as mean ± SEM (n = 5–6). * p < 0.05; ** p < 0.01 (Kruskal–Wallis test).

Figure 4

Analysis of the TCRα repertoire of peripheral T cells in 1D1a transgenic mice. Leukocytes were isolated from the blood of wild-type B10.D2(R101) (WT) and transgenic 1D1a (TG) mice at the age of 3 months. (A) The proportion (%) of T cells expressing different TCRVα families was evaluated by flow cytometry in the pool of peripheral CD3+ leukocytes. (B) The proportions (%) of CD4+ and CD8+ cells were assessed in the pool of peripheral CD3+ leukocytes of WT and TG mice. (C) The proportion (%) of T cells with endogenous TCRVα families was analyzed individually for CD4+ and CD8+ subsets of peripheral CD3+ cells of WT and TG mice. Data are presented as mean ± SEM (n = 4).

Figure 5

Effects of the dominant active TCRα expression on the development of an allogeneic immune response in 1D1a transgenic mice. Splenocytes from wild-type B10.D2(R101) (WT) and transgenic 1D1a (TG) mice were isolated and cultured in the mixed lymphocyte reaction with mitomycin C-treated splenocytes of C57BL6 (BL/6) or FVB mice for 72 h. The level of responder cells proliferation was measured by ³H-thymidine incorporation. Indices of the antigen-induced immune response were calculated as described in the Section 4 for 3-month-old (A), 6-month-old (B), and 12-month-old (C) WT and TG mice. (D) The mean indices of the immune response to the specific (BL/6) and third-party (FVB) alloantigens in WT and TG mice aged 3–12 months (3 Mo, 6 Mo, and 12 Mo). In aged (12 Mo) WT and TG mice, two distinct but equal groups of animals were detected: those with unchanged or decreased antigenic responses. Thus, for 12 Mo mice, the mean response indices were calculated individually for the indicated groups of animals. Data are presented as mean ± SEM (n = 3–12). * p < 0.05; ** p < 0.01 (Kruskal–Wallis test and Mann–Whitney U-test).

Figure 6

Effects of the dominant active TCRα expression on the development of the humoral immune response and the delayed-type hypersensitivity reaction in transgenic 1D1a mice. Wild-type B10.D2(R101) (WT) and transgenic 1D1a (TG) female mice at the ages of 3, 6, and 12 months (3 Mo, 6 Mo, and 12 Mo, respectively) were immunized with sheep red blood cells (SRBCs). (A) The number of plaque-forming cells (×10³) in the spleens of mice was counted on day 5 post-immunization. (B) Immunoglobulin levels (μg/mL) in the blood serum of non-immunized (intact) 3- and 6-month-old WT and TG female mice were measured by ELISA. (C) The level of the delayed-type hypersensitivity (DTH) reaction (%) in mice was assessed 24 h after injection of the SRBCs resolving dose. To evaluate cytokine production, WT and TG female mice (n = 3) were immunized with SRBCs, and three days later, their splenocytes were isolated and cultured for 24 h with 3 μg/mL of concanavalin A (+ConA) or without stimulation (-ConA). Levels of IFN-γ (D) and IL-4 (E) production in cell cultures were measured by ELISA. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01 (unpaired Student t-test and Mann–Whitney U-test).

Figure 7

Proteomics analysis of the blood plasma proteins of transgenic 1D1a mice. The blood plasma of female and male wild-type B10.D2(R101) (WT) and transgenic 1D1a (TG) mice at the ages of 3, 6, and 12 months was subjected to chromatography-mass spectrometry analysis. Each sex and age group contained three animals. Comparative bioinformatics analyses were performed pair-wise for WT and TG of each sex and age group using the Perseus platform by MaxQuant. Data on significant differences in the plasma proteome between each sex and age group of WT and TG mice are presented as the fold change of the protein level in the plasma of TG mice relative to the respective level in the plasma of WT mice. (A) Proteome changes in the blood plasma of TG females at the age of 3 months. (B) Proteome changes in the blood plasma of TG females at the age of 12 months. (C) Proteome changes in the blood plasma of TG males at the age of 3 months.