CD8 T cells contribute to lacrimal gland pathology in the nonobese diabetic mouse model of Sjögren syndrome

Abstract

Sjögren syndrome is an autoimmune disease characterized by targeted destruction of the lacrimal and salivary glands resulting in symptoms of severe ocular and oral dryness. Despite its prevalence, the mechanisms driving autoimmune manifestations are unclear. In patients and in the nonobese diabetic (NOD) mouse model of Sjögren syndrome, lymphocytic infiltrates consist of CD4 and CD8 T cells, although the role of CD8 T cells in disease pathogenesis has been largely unexplored. Here, we evaluated the contribution of CD8 T cells to lacrimal and salivary gland autoimmunity. Within the lacrimal and salivary glands of NOD mice, CD8 T cells were proliferating, expressed an activated phenotype, and produced inflammatory cytokines. Transfer of purified CD8 T cells isolated from the cervical lymph nodes (LNs) of NOD mice into NOD-severe combined immunodeficiency recipients resulted in inflammation of the lacrimal glands, but was not sufficient to cause inflammation of the salivary glands. Lacrimal gland-infiltrating CD8 T cells displayed a cytotoxic phenotype, and epithelial cell damage in the lacrimal glands was observed in recipients of CD8 T cells regardless of the presence of CD4 T cells. Collectively, our results demonstrate that CD8 T cells have a pathogenic role in lacrimal gland autoimmunity. The gland-specific pathogenicity of CD8 T cells makes them a valuable resource to further understand the mechanisms that discriminate lacrimal versus salivary gland autoimmunity and for the development of new therapeutics that target the early stages of disease.

Figure 1. Gland-infiltrating CD8 T cells display an activated phenotype

(a) Representative flow cytometry plots of gland-infiltrating T cells from male lacrimal (top) or female salivary (bottom) glands from 12–17 week-old NOD mice. Plots were gated on live, CD3ε⁺ singlets. Numbers represent the frequency of cells in indicated gate. Graphs depict cumulative quantification of data from at least 2 independent experiments from male lacrimal (top) or female salivary (bottom) glands, n = 7 per group. Each data point indicates an individual mouse and lines represent the mean. (b) Flow cytometric analyses of gland-infiltrating CD8 T cells from male lacrimal (left) or female salivary (right) glands from mice in a. Plots were gated on live, CD3ε⁺CD8⁺CD4⁻ singlets. Numbers represent the frequency of cells in each gate. (c) Flow cytometric analyses of gland-infiltrating CD4 T cells from male lacrimal (left) or female salivary (right) glands from mice in a. Plots were gated on live, CD3ε⁺CD8⁻CD4⁺Foxp3⁻ singlets. Numbers represent the frequency of cells in each gate.

Figure 2. Transfer of CD8 T cells causes dacryoadenitis, but not sialadenitis, in NOD-SCID recipients

(a) Representative plots to demonstrate CD8 T cells in pre- and post-sort samples. Bulk cervical LN cells from male NOD mice were FACSorted into purified CD8 cells (CD8 only) or CD8-depleted cells (nonCD8). Plots were gated on singlets. Numbers represent the frequency of cells in indicated gate. (b) Quantification of lacrimal (left) and salivary (right) gland inflammation in male NOD-SCID recipients of male NOD donor cells. Recipients received CD8-depleted cells with CD8 T cells added back (nonCD8+CD8: n = 8) or purified CD8 T cells alone (CD8 only: n = 10). Data are pooled from at least 2 independent experiments. Symbols represent individual mice, lines are medians. Boxed symbols represent diffuse inflammation in which individual foci coalesced and could not be accurately enumerated. P values determined by Mann-Whitney U-test. (c) Quantification of salivary (left) and lacrimal (right) gland inflammation in female NOD-SCID recipients of female NOD donor cells. Recipients received nonCD8+CD8: n = 10 or CD8 only: n = 12. Data are pooled from 2 independent experiments. Symbols represent individual mice, lines are medians. P value determined by Mann-Whitney U-test. (d and e) Representative flow cytometry plots of cervical LN cells (d) or lacrimal gland-infiltrating cells (e) from male recipients represented in b. Plots were gated on live, CD3ε⁺ singlets. Numbers represent the frequency of cells in indicated gate. Graphs depict cumulative quantification of data pooled from 2 independent experiments (nonCD8+CD8: n = 8; CD8 only: n = 10) (d) or 1 experiment (nonCD8+CD8: n = 4; CD8 only: n = 8) (e). Each symbol represents an individual mouse, lines are means. P values determined by unpaired Student’s t-test. (f) Ratio of CD4 to CD8 T cells (log transformed) present in the cervical LN or lacrimal glands of male recipients from d and e. Each symbol represents an individual mouse, lines are means. P values determined by unpaired Student’s t-test. (g) Ratio of CD4 to CD8 T cells (log transformed) present in spontaneous male NOD mouse lacrimal gland infiltrates. Data are representative of 2 independent experiments (n = 7). Each symbol represents an individual mouse, lines are means. P value determined by unpaired Student’s t-test.

Figure 3. Lacrimal gland inflammation occurs in the absence of CD8 T cells

(a) Quantification of lacrimal gland inflammation in male NOD-SCID recipients of sorted male NOD donor cervical LN cells. Recipients received CD8-depleted cells with CD8 T cells added back (nonCD8+CD8; n = 13) or CD8-depleted cells alone (nonCD8; n = 19). Data are pooled from 3 independent experiments. Symbols represent individual mice, lines are medians. P value determined by Mann-Whitney U-test. (b) Representative flow cytometry plots of cells isolated from the cervical LN from recipients in a. Plots were gated on live, CD3ε⁺ singlets. Numbers represent the frequency of cells in indicated gates. Graphs depict cumulative quantification of data pooled from 3 independent experiments. Symbols represent individual recipients, lines are means. P values determined by unpaired Student’s t-test. (c) Representative flow cytometry plots of cells isolated from the lacrimal glands from recipients in a. Plots were gated on live, CD3ε⁺ singlets. Numbers represent the frequency of cells in indicated gates. Graphs depict cumulative quantification of data from 1 experiment (nonCD8+CD8: n = 4; 8 only: n = 9). Symbols represent individual recipients, lines are means. P values determined by unpaired Student’s t-test.

Figure 4. CD8 and CD4 T cells each contribute to the early stages of lacrimal gland autoimmunity

Quantification of inflammation in male lacrimal glands from 11–14 week old WT (n = 28), CD8^null (n = 14), or CD4^null (n = 15) NOD mice or 20–24 week old WT (n = 7), CD8^null (n= 20), or CD4^null (n = 10) NOD mice. Symbols represent individual mice, lines represent medians. Significant differences between mouse groups and age groups were identified by two-way ANOVA on focus score ranks (P < 0.0001 and P < 0.01, respectively) with no significant interaction between the two (P = 0.109). Tukey’s HSD post-hoc test P values are shown. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Figure 5. CD8 T cells cause dacryoadenitis in the absence of CD4 T cells

(a) Representative flow cytometry plots to demonstrate CD8 T cells in pre- and post-sort samples. Bulk cervical LN cells from male NOD mice were FACSorted based on expression of CD8α. Plots were gated on singlets. Numbers represent the frequency of cells in indicated gate. (b) Cumulative quantification of CD4 T cells in the peripheral blood of male NOD-SCID recipient mice that received purified CD8 T cells from male NOD donors and were treated weekly with anti-CD4 antibody (n = 12) or isotype control antibody (n = 10). Data are representative of 2 independent experiments. Symbols represent individual mice, lines represent means. P values determined by unpaired Student’s t-test. (c) Quantification of lacrimal gland inflammation in male NOD-SCID recipients in b. Symbols represent individual mice, lines represent medians. P value determined by Mann-Whitney U-test. (d and e) Representative flow cytometry plots of cells isolated from the cervical LN (d) or lacrimal glands (e) of male NOD-SCID recipient mice treated as in b. Plots were gated on live, CD3ε⁺ singlets. Graphs depict cumulative quantification of CD8 or CD4 T cells pooled from 2 independent experiments. Symbols represent individual mice, lines represent means. P values determined by unpaired Student’s t-test. (f) Ratio of CD4 to CD8 T cells (log transformed) present in the cervical LN or lacrimal glands of male recipients from b. Each symbol represents an individual mouse, lines are means. Data are representative of 2 independent experiments. P values determined by unpaired Student’s t-test.

Figure 6. CD8 T cells mediate destruction of lacrimal gland epithelial cells

(a and b) Activated caspase-3 immunostaining. Representative microscopic fields of lacrimal glands with focus of inflammation (a, asterisks) or lacking inflammation (b). Arrows and inset represent activated caspase-3⁺ cells located within the inflammatory focus (a). Arrowheads represent activated caspase-3⁺ cells located outside of inflammatory cell focus (a) or in area of normal tissue devoid of inflammatory foci (b). (c) Quantification of the number of activated caspase-3⁺ cells in lacrimal gland sections from NOD mice that spontaneously developed lacrimal gland inflammation (n = 5), or NOD-SCID mice that received CD8 only cells (n = 9) or nonCD8+CD8 cells (n = 4) based on location: within inflammatory foci (in), outside of inflammatory foci (out), or in areas of normal gland tissue (normal). Activated caspase-3⁺ cells were determined by averaging the values from 2 images per gland. Symbols represent individual mice, lines are means. P value determined by two-way ANOVA demonstrates a significant difference between locations (i.e., for each group “in” is greater than “out” or “normal”) but no significant difference between groups (P = 0.68) and no significant interaction between group and location (P = 0.34). (d) Correlation between the total number of activated caspase-3⁺ cells per mm² and the number of foci per mm² in whole lacrimal gland sections from recipients of CD8 only cells (n = 4), recipients of nonCD8+CD8 cells (n = 4), or male NOD mice with spontaneous disease (n = 5). Symbols represent individual mice. P and r values were determined by Spearman correlation. (e) Correlation between the number of activated caspase-3⁺ cells within foci and percentage of CD4 T cells in the lacrimal glands of mice that received CD8 only T cells. Symbols represent individual mice. P and r values were determined by Pearson correlation. (f) Flow cytometric analyses of CD8 T cells isolated from the cervical LN or lacrimal glands of 12–16-week old male NOD mice. Cells were gated on live, CD3ε⁺CD19⁻CD8⁺ singlets prior to assessing surface expression of CD107a or staining for intracellular granzyme B. Numbers represent the frequency of cells in each gate. Graphs depict cumulative quantification of data from 2 independent experiments (n = 6 per group). P values determined by unpaired Student’s t-test.