Bi-Allelic TCRα or β Recombination Enhances T Cell Development but Is Dispensable for Antigen Responses and Experimental Autoimmune Encephalomyelitis

Abstract

Dual TCRα-expressing T cells outnumber dual TCRβ-expressing cells by ~10:1. As a result, efforts to understand how dual TCR T cells impact immunity have focused on dual TCRα expression; dual TCRβ expression remains understudied. We recently demonstrated, however, that dual TCRβ expression accelerated disease in a TCR transgenic model of autoimmune arthritis through enhanced positive selection efficiency, indicating that dual TCRβ expression, though rare, can impact thymic selection. Here we generated mice hemizygous for TCRα, TCRβ, or both on the C57BL/6 background to investigate the impact bi-allelic TCR chain recombination has on T cell development, repertoire diversity, and autoimmunity. Lack of bi-allelic TCRα or TCRβ recombination reduced αβ thymocyte development efficiency, and the absence of bi-allelic TCRβ recombination promoted γδ T cell development. However, we observed no differences in the numbers of naïve and expanded antigen-specific T cells between TCRα+/-β+/- and wildtype mice, and TCR repertoire analysis revealed only subtle differences in Vβ gene usage. Finally, the absence of dual TCR T cells did not impact induced experimental autoimmune encephalomyelitis pathogenesis. Thus, despite more stringent allelic exclusion of TCRβ relative to TCRα, bi-allelic TCRβ expression can measurably impact thymocyte development and is necessary for maintaining normal αβ/γδ T cell proportions.

Fig 1. Characterization of single TCR T cell mice.

A) Splenocytes were collected from WT and single TCR T cell C57BL/6 mice and analyzed by flow cytometry for co-expression of Vα2 with Vα3.2 or Vα8.3 (top panels) or co-expression of Vβ6 with any of a panel of fourteen other Vβ proteins (bottom panels) in CD3⁺ CD4⁺ T cells. Representative flow plots from three independent experiments show the presence of dual TCRα and β populations in WT (boxed populations in left panels) that are absent in single TCR T cell mice (right panels). B) Flow cytometric analysis of splenocytes from WT and single TCR T cell mice reveals equivalent CD3 expression among CD4⁺ and CD8⁺ T cells. C) Developmental T cell stages (left) and peripheral T cell subsets (right) from WT and single TCR T cell mice were analyzed and enumerated by flow cytometry (n = 6). D) The number of γδ T cells in the lymph nodes and spleen of adult WT or single TCR T cell mice as determined by flow cytometry (B220^-, CD11b^-, CD11c^-, CD3⁺ and TCRγδ⁺). Results shown are mean +SEM (n = 3 for TCRα^+/- and TCRα^+/- TCRβ^+/-, n = 2 for TCRβ^+/-, and n = 6 for WT). Student’s t-test was used to determine p values; ***p<0.001. Flow cytometry plots are in Log10 fluorescence scale.

Fig 2. The peripheral TCR repertoire remains diverse despite the absence of bi-allelic TCR rearrangements.

TCRβ chain sequencing was performed on DNA purified from T cell-enriched splenocytes from WT and single TCR T cell mice. A) The number of unique productive TCRβ sequences in WT and single TCR T cell mice are similar (n = 3/genotype). Individual Trbv usage in B) productively and C) non-productively rearranged WT and single TCR T cell mice is displayed as a percentage of total unique sequences ranging from ~11,000 to ~35,000 sequences per mouse (n = 3/genotype). Results shown are mean +SEM. Two way ANOVA with Bonferroni posttest with a 95% confidence interval was used to determine p value. ** and *** indicate p<0.01 and p<0.001 respectively.

Fig 3. Bi-allelic TCRα and β chain recombination affect thymic selection efficiency.

T cell-depleted bone marrow was collected from WT C57BL/6 (CD45.1 and CD90.2) mice and combined with that of either TCRα^+/-, TCRβ^+/-, or TCRα^+/- TCRβ^+/- (all CD45.2 and CD90.2) mice in equal proportions and injected into lethally-irradiated C57BL/6 CD90.1 mice. Thymus, spleen, and lymph nodes were collected 10 weeks following transplantation. The ratios of WT:TCRα^+/-, WT:TCRβ^+/-, and WT:TCRα^+/- TCRβ^+/- (CD45.1:CD45.2) cells were determined by flow cytometric analysis of A) thymocytes at the indicated developmental stages and B) peripheral lymphocyte subsets. All ratios are adjusted to the peripheral B cell CD45.1:CD45.2 ratio in each mouse, as an internal control. The data plotted include the mean and SEM (n = 7–8 mice/group). Student’s t-test was used to determine p value, relative to the WT:WT control. *, **, and *** indicate p<0.05, p<0.01, and p<0.001 respectively. Student’s t-test was used to determine p values of CD45.1:CD45.2 ratios pre/post α and β (DP_pre/DP_post and DN1/DP_pre respectively). ††† indicates p<0.001 for pre/post selection comparisons.

Fig 4. Bi-allelic TCR rearrangements do not impact the foreign antigen TCR repertoire.

Splenocytes and lymph node cells were collected from naïve, 2W1S-, B8R-, or gp33-peptide immunized WT and single TCR T cell mice, followed by magnetic tetramer enrichment of antigen-specific populations. Antigen-specific T cells were then stained and analyzed by flow cytometry. A, C, E) Representative plots of naïve (left) and immunized (right) WT and single TCR T cell mice showing 2W1S:A^b-, B8R:K^b-, or gp33:D^b-specific populations respectively. B, D, F) Numbers antigen-specific T cells in naïve and antigen immunized WT and single TCR T cell mice. Each symbol represents one mouse. No statistically significant differences between the two genotypes were present at either of the time points (Student’s t-test). Flow plots are in Log10 fluorescence scale.

Fig 5. Bi-allelic TCR rearrangements do not alter self antigen-specific T cell repertoire.

Splenocytes and lymph nodes were collected from naïve and MOG_35-55- or PLP_178-191-immunized WT and single TCR T cell mice. Cells were enriched for antigen-specific populations using tetramers and magnetic cell sorting. Antigen specific T cells were then stained and analyzed by flow cytometry. A) Representative flow cytometry plots and B) numbers of MOG_38-49:A^b- specific T cells in naïve (left) and immunized (right) WT and single TCR T cell mice. C) Representative flow cytometry plots and D) number of PLP_178-191:A^b-specific populations in naïve (left) and immunized (right) WT and single TCR T cell mice. C) Dual tetramer staining was used to identify the PLP_178-191:A^b-specific cells in naïve WT and single TCR T cell mice due to their rarity. In B and D, each symbol represents one mouse. No statistically significant differences between the two genotypes were present at either of the time points (Student’s t-test). Flow plots are in Log10 fluorescence scale.

Fig 6. Dual TCR T cells are not required for initiation of EAE.

EAE was induced in WT and single TCR T cell mice by immunization with 200 μg MOG_35-55 or 50 μg PLP_178-191 plus adjuvant. EAE severity scores were determined daily for up to 21 days post injection. A and B) Average EAE scores of MOG_35-55- and PLP_178-191- induced EAE in WT and single TCR T cell mice over time. Data points are depicted as the mean +/- SEM. One-way ANOVA was used to determine p values (n = 12-15/group). C and D) Percentage of WT or single TCR T cell mice with an EAE score equal to or greater than 1 in MOG_35-55- and PLP_178-191- induced EAE. The p values for Kaplan-Meier curves were calculated using log rank test with Prism software (GraphPad); no significant statistical difference was observed.