The T Cell Receptor Repertoire in Neuropsychiatric Systemic Lupus Erythematosus

Abstract

Objective: In systemic lupus erythematosus (SLE), widespread T cell infiltration into target organs contributes to inflammation and organ damage. Autoreactive T cells become aberrantly activated in this disease due to dysfunctional T cell receptor signaling that lowers the activation threshold. Characterizing the T cell repertoire can provide further insight into the specific homing and proliferation of these T cells into lupus target organs. In the spontaneous lupus model, MRL/lpr, the TCR repertoire has not been fully elucidated, especially for T cells infiltrating the brain. Our aim was to investigate and compare the TCR repertoire between MRL/lpr mice and its congenic controls, MRL/MpJ, and within MRL/lpr tissues. Methods: Spleen, salivary gland, and brain choroid plexus were isolated from female MRL/lpr mice and MRL/MpJ mice. The TCRβ CDR3 region was analyzed by multiplex PCRs and sequencing. Results: Significant differences were seen not only between the MRL/lpr and MRL/MpJ spleens, but also between MRL/lpr tissues. The TCR repertoire in MRL/lpr choroid plexus tissues had significantly increased clonality and sequence homology compared to MRL/lpr spleen and salivary gland. The consensus sequence, CASSQDWGGYEQYFF, was identified in the MRL/lpr choroid plexus repertoire. Conclusions: The TCR repertoire in lupus prone mice is not uniform between target organs, and suggests that T cells are specifically recruited into the choroid plexus of MRL/lpr mice. Further studies are needed to determine the antigen specificities for these infiltrating T cells in target organs of lupus mice, and their possible contribution to the pathogenesis of neuropsychiatric disease and other lupus manifestations.

Keywords: MRL/lpr; T cell receptor; choroid plexus; neuropsychiatric lupus; salivary gland; systemic lupus erythematosus.

Figure 1

Comparison of the TCR repertoire between MRL/lpr and MRL/MpJ spleens. The TCR repertoires of MRL/lpr (n = 9) and MRL/MpJ (n = 5) spleens were analyzed. (A) Sample clonality. (B) Number of unique productive TCR clones. (C) Frequency of the top productive TCR clones. (D) Selective increased TCRB V gene usage in MRL/lpr spleen repertoires compared to MRL/MpJ. (E) Specific decreased TCRB V gene usage in MRL/lpr spleen repertoires compared to MRL/MpJ. Each dot indicates one mouse and bar graphs represent mean ± SD. P-values were determined with an unpaired T-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

T cell infiltration and subsets in MRL/lpr tissues. (A) Immunofluorescent staining for CD4 (green) and CD8 (red) T cell infiltration in choroid plexus (top) and salivary gland (bottom), magnification = 20X. (B) Percentage of CD4+ T cells (left), CD8+ T cells (center), and double negative T cells (right) in MRL/lpr tissues by flow cytometry; CP, choroid plexus; Gland, salivary gland; LN, lymph node. Values represent percentages of parent CD3+ T cells. (C) Sample clonality of MRL/lpr spleen (n = 9), salivary gland (n = 4), and choroid plexus tissues (n = 9). (D) Normalized number of unique productive clones in MRL/lpr tissue repertoires calculated by dividing the total number of unique productive clones to the amount of DNA analyzed. (E) Sum of the top 10 productive rearrangement frequencies. P-values were determined with one-way ANOVAs, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 3

The choroid plexus exhibits distinct TCR profiles compared to other MRL/lpr tissues. (A) Frequency of CDR3 lengths compared to Gaussian distributions in MRL/lpr choroid plexus (left), salivary gland (middle), and spleen (right) TCR repertoires. (B) Selective increased TCRB V gene usage in MRL/lpr choroid plexus tissue. (C) Specific decreased TCRB V gene usage in MRL/lpr choroid plexus tissue. (D) Frequency of TCRB D (top) and J gene (bottom) usage in MRL/lpr tissues. P-values determined by one-way ANOVAs, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4

TCR sequence homology in the MRL/lpr choroid plexus. (A) Overlapping TCR sequences in ≥ 6 MRL/lpr choroid plexus tissues compared to MRL/lpr splenic and salivary gland tissues and MRL/MpJ splenic tissues. Underlined residues indicate matching amino acid sequences to identified consensus sequence, CASSQDWGGYEQYFF. (B) Similarity scores, measuring the amount of overlap between two TCR repertoires, generated within each MRL/lpr tissue. (C) Visual representation of epitope cluster analysis (IEDB.org) of identified TCR sequences with 80% overlapping. Red circle indicates sequences similar to the consensus sequence identified. LPR CP = MRL/lpr CP tissues, LPR SG = MRL/lpr salivary gland tissues, LPR SP = MRL/lpr spleen tissues, MpJ SP = MRL/MpJ spleen tissues. P-values calculated with one-way ANOVA, *p < 0.05, **p < 0.01.