Immunology and Inflammation

Thymic self-recognition-mediated TCR signal strength modulates antigen- specific CD8⁺ T cell pathogenicity in non-obese diabetic mice

Chia-Lo Ho
Li-Tzu Yeh
Yu-Wen Liu
Jia-Ling Dong
Huey-Kang Sytwu author has email address

Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan
National Institute of Infectious Disease and Vaccinology, National Health Research Institutes, Miaoli, Taiwan
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

https://doi.org/10.7554/eLife.99897.2

Open access
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Figures and data

Unique phenotypic traits of naïve NOD CD8⁺ T cells are stratified by CD5 expression.
In (A) to (G), flow cytometry analysis of total naïve CD8⁺ T cells (CD44^loCD62L^hi; grey), and the upper 5% (CD5^hiCD8⁺; red) and lower 5% (CD5^loCD8⁺; blue) based on CD5 expression, from the spleen and PLNs of normoglycemic female NOD mice aged 6 to 8 weeks. (A) Representative flow cytometry histograms and the corresponding (B) geometric mean fluorescence intensity (gMFI) of CD5, p-CD3ζ and p-Erk expression shown in the indicated samples. Additionally, the percentages of (C) transcription factors T-bet and Eomes, along with (D) cytokines Granzyme B, TNF-α, IFN-γ and IL-2 in CD8⁺ T cells are also presented in the indicated samples. The gMFI for effector/memory T cell-related markers includes (E) CXCR3, CD122, CD127 and (F) CD69, PD-1, KLRG-1, CD44 and CD25. (G) Representative flow cytometry plots (left) and gMFI (right) of positive IGRP-tetramer staining in CD8⁺ T cells compared among naive total CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ populations. Data represent mean ± SD. The data presented in (A) to (F) represent two experiments. The sample size was n = 5-10 (B), n = 6-10 (C), n = 7-10 (D), n = 5-10 (E) n = 6-10 (F) and n = 3 (G) per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA (B–D, F, G) and unpaired, two-tailed t test (E).

The gene expression profile of naïve CD5^hiCD8⁺ T cells reveals a poised phenotype for effector T cells in autoimmune diseases.
(A) RNA-Seq performed on the sorted 10% upper and lower CD5-expressing of naïve CD8⁺ T cells (CD5^hiCD8⁺ and CD5^loCD8⁺) from 6-8-week-old normoglycemia female NOD mice to understand the gene expression profile of CD5^hiCD8⁺ T cells and comparing that to the CD5^lo counterpart to gain insights into their potential role in autoimmune diseases. (B) The volcano plot displaying the transcripts that are upregulated and downregulated by RNA sequencing of naïve CD5^hiCD8⁺ T cells compared to CD5^loCD8⁺ T cells. The plot shows the log10 fold change of each transcript and the significance of the change, measured by the negative log10 of the adjusted P-value (adj.p). Genes with higher fold changes and lower adj.p values (red plots) indicate a significant change in expression between the two groups. (C) The gene set enrichment score plot for gene ontology terms (T cell activation, external side of plasma membrane, positive regulation of immune system process, and regulation of catalytic activity). Higher scores indicate a higher degree of enrichment of these processes in naïve CD5^hiCD8⁺ T cells compared to the CD5^lo counterpart.(D) The dot plots of the significant KEGG gene set enrichment in the top 10 enriched pathways, reinforcing the presence of a poised phenotype for effector T cells in autoimmune diseases in naïve CD5^hiCD8⁺ T cells. The data presented in (A) to (D) represent two experiments. In (A) to (D), n=10 mice per CD5^hiCD8⁺ or CD5^loCD8⁺ group, with pooled spleen and PLN cells for sorting obtained from a total of 20 normoglycemia female NOD mice aged 6 to 8 weeks old. Significance is determined using a false discovery rate (FDR) adjusted P-value < 0.05 and validated using multiple testing correction method Benjamini-Hochberg correction.

Naïve CD5^hiCD8⁺ and CD5^loCD8⁺ T cells exhibit distinct expression in effector/memory T cell gene-expression clusters.

Naïve CD5^hiCD8⁺ and CD5^loCD8⁺ T cells exhibit distinct expression in effector/memory T cell gene-expression clusters.

CD5 expression distinguishes CD8⁺ T cell subsets with differential activation and proliferation.
(A-K) Naïve CD8⁺ T cells were isolated from the spleen of 6–8-week-old normoglycemic female NOD mice and stimulated with the indicated conditions. (A) Diagram depicting experimental setup using varying concentrations of anti-CD3 (0–10 μg/ml) and anti-CD28 (2 μg/ml; 0 μg/ml for control) for 2 days. (B, G upper/left panels) Representative flow cytometry plots, with statistical summaries shown in (B-H). Comparison of CD5 (B), fold change of p-CD3ζ (C), p-Erk (D), CD69 (E), CD44 (F), proliferation levels (CTV dilution %) (G) and fold change of CD127 expression (H) between the upper and lower 5% of CD5-expressing CD8⁺ T cells (CD5^hiCD8⁺ and CD5^loCD8⁺, respectively). Fold change values in (C-F, H) were normalized to each group’s baseline at 0 μg/ml anti-CD3 stimulation, where total CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ were each set to 1. (I) Histogram comparing CD69 expression between the upper and lower 10% of CD5-expressing CD8⁺ T cells (CD5^hiCD8⁺ and CD5^loCD8⁺) after stimulation with anti-CD3/CD28 dynabeads or PMA plus Ionomycin for specified durations. (J) Comparison of Ki-67 expression and IFN-γ production between CD5^hiCD8⁺ and CD5^loCD8⁺ T cells.,(K) Proliferation of CD5^hiCD8⁺ and CD5^loCD8⁺ T cells assessed by [methyl-3H] thymidine incorporation (cpm; counts per minute) after stimulation with the indicated amounts of anti-CD3/CD28 dynabeads or PMA plus Ionomycin for 2 days. (L) Comparison of CD5 gMFI in CD8⁺CD69⁺ (upper gate of flow dot plot) to that in CD8⁺CD69^- (lower gate of flow dot plot) population by histogram (CD8⁺CD69⁺ in solid red line; CD8⁺CD69^- in grey), across spleen and PLNs from normoglycemic female NOD mice aged 6 to 8 weeks. The statistical results of the percentages of activated (CD8⁺CD69⁺) population (left) and the ratio of activated to non-activated CD5 gMFI (right) are shown. Data represent mean ± SD. The data presented in (B) to (H) represent two experiments. The sample size was n = 10 (B-H), n = 3 (J), n = 3-7 (K), and n = 4 (L) per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by two-way ANOVA (B)-(K) and unpaired, two-tailed t test (L).

CD5^hiCD8⁺ T cells-transferred group exhibits severe insulitis and disease phenotype.
(A) Diabetes incidence (left) in NOD Rag1^-/- mice transferred with naïve CD5^hiCD8⁺, CD5^loCD8⁺, or CD8⁺ T cells, combined with CD4⁺ T cells, respectively. The islet histological analyses and insulitis scoring (right) for the mice after a 4-week transfer in the respective transferred groups showed a higher diabetes frequency in the CD5^hiCD8⁺ group compared to the CD5^loCD8⁺ group. The cells were sorted from the 10% upper and lower of CD5 expression of naïve CD8⁺ T cells (CD5^hiCD8⁺ and CD5^loCD8⁺ T cells) from the spleen and PLNs 6-8-week-old normoglycemic female NOD mice and adoptively transferred to 6-8- week-old female NOD Rag1^-/- recipients via i.p. injection. Urine glucose concentrations of the four groups of mice were monitored weekly for diabetes incidence. Diabetes was defined as glycosuria > 500 mg/dl in two consecutive tests. Insulitis scoring criteria can be found in the supplementary materials and methods. (B) Percentages and cell numbers of CD8⁺ T cells in spleen and PLNs, analyzed from CD5^hiCD8⁺ and CD5^loCD8⁺ cells-transferred group, respectively, after 4-week adoptive transfer. (C) Percentages of TNF-α, IFN-γ and IL-2 production in CD5^hiCD8⁺ and CD5^loCD8⁺ group, respectively, from spleen or PLNs, analyzed by flow cytometry, with representative flow dot plots (left) and statistical results (right). (D) Percentages of T-bet, Eomes and Granzyme B-expressing in CD5^hiCD8⁺ and CD5^loCD8⁺ group, respectively, from spleen or PLNs, analyzed by flow cytometry, with representative flow dot plots (left) and statistical results (right). (E) Comparison of differential gMFI expression of CD5, CD25, CD44, CD69, CD122, CD127, KLRG1 and PD-1 between CD5^hiCD8⁺ and CD5^loCD8⁺ group from spleen or PLNs. (F) Comparison of percentages of central memory T cells (defined by CD44^hiCD62L^hi) (upper) and positive IGRP-tetramer staining (lower) between CD5^hiCD8⁺ and CD5^loCD8⁺ groups from PLNs, with representative flow dot plots (left) and statistical results (right). Representative flow cytometry plots include FMO negative control for accurate IGRP-tetramer gating. Data represent mean ± SD. Sample size was n = 5-11 (A), and n = 3 (B-F) per group. *P < 0.05, **P < 0.01, ***P < 0.001, by log-rank test (A), by two-way ANOVA (B) and (E) and unpaired, two-tailed t test (C), (D) and (F).

The high CD5-linked TCR basal signals positively correlate with thymic selection in autoimmune-poised NOD8.3 mice.
(A) CD5, p-CD3ζ, p-Erk and IL-2 expression levels compared between the lower 5% (hollow blue) and upper 5% (hollow red) CD5 expression in thymocytes of normoglycemia female NOD mice aged 6 to 8 weeks old across DN, DP and CD8SP stages. (B-H) Comparison of various marker expression levels between thymocytes of normoglycemia female NOD (solid blue) and NOD8.3 (solid red) mice aged 6 to 8 weeks old across different selection stages. (B) CD5 gMFI in NOD and NOD8.3 thymocytes across DN, DP and CD8SP stages. (C) Percentages of p-CD3ζ (left) and the corresponding gMFI of p-CD3 (right) compared between NOD and NOD8.3 thymocytes in the indicated stages DN, DP and CD8SP. (D) Percentages of DN, DP and CD8SP composition compared between NOD and NOD8.3 thymocytes. (E) Percentages of indicated DN stages (DN1, DN2, DN3 and DN4) in the DN subset compared between NOD and NOD8.3 mice. DN stages are defined as follows: DN1 (CD44⁺CD25^-), DN2 (CD44⁺CD25⁺), DN3 (CD44^-CD25⁺) and DN4 (CD44^-CD25^-), as depicted in the gating shown in Supplementary Figure 3A, middle panels. (F) CD5 gMFI (upper) and corresponding percentages (lower) in the pre-positive selection (TCRβ^-CD69^-) and post-positive selection (TCRβ⁺CD69⁺) populations in DP thymocytes compared between NOD and NOD8.3 mice. The presence of higher TCR basal signals (indicated by higher CD5 gMFI) in NOD8.3 in the pre-positive selection stage leads to an increased percentage of TCRβ⁺CD69⁺ cells in the post-selection stage compared to NOD mice, as shown in Supplementary Figure 3F. (G) gMFI of CD122, CD127, CD44 and CD25 assessed in DP thymocytes compared between NOD and NOD8.3 mice. (H) Percentages of virtual memory (V_memory) cells in CD8SP thymocytes compared between NOD and NOD8.3 mice. Data represent mean ± SD. The data presented in (B) to (H) represent two experiments. The sample size was n = 10 (A), n = 10-11 (B), n = 3-6 (C), n = 9-11 (D), n = 7-11 (E), n = 8-11 (F) n = 10-11 (G) and n = 8-9 (H) per group. *P < 0.05, **P < 0.01, ****P < 0.0001, by unpaired, two-tailed t test (A- H).

The protective effect of transgenic Pep on diabetogenesis is negated in dLPC/NOD8.3 mice.
(A) Spontaneous diabetes incidence (left) and insulitis scoring (right) of female non-Tg NOD, dLPC/NOD, NOD8.3 and dLPC/NOD8.3 mice. Insulitis scoring was performed on the islets from the pancreata of 6-8-week-old mice before the onset of diabetes in each group. Insulitis scoring criteria can be found in the supplementary materials and methods. Urine glucose concentrations of mice were monitored weekly for spontaneous diabetes incidence. Diabetes was defined as glycosuria > 500 mg/dl at two consecutive tests. (B-G) Characterization of various aspects of TCR signal strength and effector/memory T cell phenotypes among female normoglycemia non-Tg NOD (blue), dLPC/NOD (black), NOD8.3 (red) and dLPC/NOD8.3 (grey) mice at the age of 6 to 8 weeks old. (B) CD5 gMFI of CD8⁺ T cells in the spleen (left) and PLNs (right). (C) Percentage of p-CD3ζ⁺CD8⁺ T cells in the spleen (left) and PLNs (right). (D) Percentage of p-Erk⁺CD8⁺ T cells in the spleen (left) and PLNs (right). (E) gMFI of effector T cell markers CD69 and KLRG1 in CD8⁺ T cells from the spleen (upper) and PLNs (lower). (F) gMFI of memory T cell markers CD25, CD122 and CD44 in CD8⁺ T cells from the spleen (upper) and PLNs (lower). (G) Percentage of IL-2⁺-, TNF-α⁺-, and IFN-γ⁺CD8⁺ T cells in PLNs. Data in (B) to (G) represent mean ± SD. The data presented in (B) to (G) represent two experiments. The sample size was n = 7-10 (A), n = 4-11 (B), n = 3-7 (C), n = 3-7 (D), n = 5-11 (E), n = 5-11 (F) and n = 3-9 (G) per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by log-rank test (A), by one-way ANOVA (B-F) and unpaired, two-tailed t test (G).

Examination of the CD5^hi population reveals TCR repertoires linked to autoimmune disorders.
(A) Comparison of TCR repertoire differences between CD5^hiCD8⁺ and CD5^loCD8⁺ T cell populations conducted using bulk RNA sequencing, followed by TCR repertoire analysis using the MiXCR pipeline. Naïve CD5^hiCD8⁺ and CD5^loCD8⁺ cells were sorted from pooled spleens and PLNs of 6–8-week-old normoglycemic female NOD mice, following a similar procedure as outlined in Figure 2A, with 1.5 x 10⁶ cells per group. The TCR repertoire of naïve CD5^hiCD8⁺ T cells exhibited shorter CDR3 lengths, higher clone overlap values within group comparisons, pathogenic-related CDR3 motifs and greater hydrophobicity and diversity in the TRA chain compared to CD8⁺CD5^lo cells. (B-E) Comparison of the TRAV gene segment usage (B), TRAJ gene segment usage (C), TRBV gene segment usage (D) and TRBJ gene segment usage (E) within TRA variable and TRB variable genes between CD5^hiCD8⁺ (red) and CD5^loCD8⁺ (blue) cells. (F-J) Comparison of CDR3α (left) or CDR3β (right) length in amino acids (F), CDR3 clonal overlap by overlapped count matrix (G), top 20 CDR3 motifs (H), average hydrophobicity score (I) and diversity (J) of CDR3α (left) and CDR3β (right) between CD5^hiCD8⁺ and CD5^loCD8⁺ T cells. In (G), the number in each cell indicates the count of shared clonotypes between sample pairs; the detailed method for constructing the Overlapped Count Matrix is provided in the Supplementary methods section. (H) Occurrence frequencies of the top 20 CDR3 motifs from mapped- CDR3α (left) and CDR3β (right) sequencings in CD5^hiCD8⁺ and CD5^loCD8⁺ cells, with the high-occurrence rate of the CDR3α motif “SGGSNYKLTF” (underlined in red) specific to T1D-related IGRP peptide in the CD8⁺CD5^hi population. (I) Average hydrophobicity score defined by summing up the hydrophobicity score in each amino acid in the CDR3 motif and dividing by the CDR3 length, then averaging by total counts in each group. Diversity definition in (J) is shown in Materials and Methods section Clonotype repertoire metrics formulas. In (B-E), each point represents 10 mice, with two points for each group, totaling n = 20 mice (F-J) for all CD5^hi or CD5^lo samples. *P < 0.05, **P < 0.01, ***P < 0.001, by two-way ANOVA (B-E), by Welch two sample t-test (F), and unpaired, two-tailed t test (I) and (J).

Thymocyte self-reactivity impacts the effector functions and memory cell formation of auto-reactive T cells in the T1D model NOD mice.

CD5^hiCD8⁺ T cells display enhanced TCR signaling, effector profiles and autoreactive potential in NOD mice.
Representative FLOW analysis plots show a comparison of positive expression percentages in (A) proximal TCR signaling molecules p-CD3ζ and p-Erk, (B) transcription factors T-bet and Eomes, and (C) cytokines Granzyme B, TNF-α, IFN-γ and IL-2 within total naïve CD8⁺ (grey), CD5^hiCD8⁺ (red), CD5^loCD8⁺ (blue) cells, and fluorescence minus one (FMO) controls (light grey) from spleen (left) and PLNs (right). (D) Corresponding percentages of IGRP-tetramer positive CD8⁺ T cells among total naïve CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ populations from spleen and PLNs. (E) Representative flow cytometry plots (left) and corresponding gMFI and proportions (right) of Ins B15-23-pentamer positive CD8⁺ T cells compared among total naïve CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ populations from spleen and PLNs. In (D) and (E) all panels, total naïve CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ are colored grey, red and blue, respectively. Data represent mean ± SD. Sample size was n = 3–5 per group. *P < 0.05, **P < 0.01, by one-way ANOVA.

CD5^hiCD8⁺ T cells display enhanced TCR signaling, effector profiles and autoreactive potential in NOD mice.
Representative FLOW analysis plots show a comparison of positive expression percentages in (A) proximal TCR signaling molecules p-CD3ζ and p-Erk, (B) transcription factors T-bet and Eomes, and (C) cytokines Granzyme B, TNF-α, IFN-γ and IL-2 within total naïve CD8⁺ (grey), CD5^hiCD8⁺ (red), CD5^loCD8⁺ (blue) cells, and fluorescence minus one (FMO) controls (light grey) from spleen (left) and PLNs (right). (D) Corresponding percentages of IGRP-tetramer positive CD8⁺ T cells among total naïve CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ populations from spleen and PLNs. (E) Representative flow cytometry plots (left) and corresponding gMFI and proportions (right) of Ins B15-23-pentamer positive CD8⁺ T cells compared among total naïve CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ populations from spleen and PLNs. In (D) and (E) all panels, total naïve CD8⁺ T cells, CD5^hiCD8⁺ and CD5^loCD8⁺ are colored grey, red and blue, respectively. Data represent mean ± SD. Sample size was n = 3–5 per group. *P < 0.05, **P < 0.01, by one-way ANOVA.

Gene expression patterns and enrichment are comparatively analyzed between naïve CD5^hi and CD5^loCD8⁺ T cells.
(A) Gene-concept network of CD5^hi versus CD5^lo DEGs across key immune process GO terms. This interaction picture displays a gene- concept network highlighting genes associated with the top three GO terms: “external side of plasma membrane” (GO:0009897), “T cell activation” (GO:0042110), and “cytokine production involved in immune response” (GO:0002367) in CD5^hi versus CD5^lo DEGs. Circle size represents the gene counts, while the color spectrum, ranging from green to red, indicates the fold change in gene expression, spanning from less than 0 to a 6-fold change. (B) Comparison of gene expression patterns of CD5^hiCD8⁺ (x-axis) versus CD5^loCD8⁺ (y-axis) T cells, using core gene signatures from clusters I to X (top panel). The two lower panels represent a similar comparison to the top panel but specifically focusing on the core gene signature differences in (left) clusters I, II, III, IX, and X, and (right) clusters IV to VIII between CD5^hi and CD5^lo T cells (lower panel); colors in plots (genes) match colors of clusters (key); red diagonal lines indicate a difference in expression of twofold; blue diagonal line indicates x = y. *P < 0.001 and **P < 0.00001 (t-test). Data are representative of two independent experiments. (C) The gene set enrichment score plot for biological process (BP) of GO terms (lymphocyte activation, cell activation, leukocyte activation, regulation of leukocyte activation, and immune system process). Higher scores indicate a higher degree of enrichment of these processes in naïve CD5^hiCD8⁺ than in CD5^loCD8⁺ cells.

Thymocyte development and respective selection profiles are compared between NOD and NOD8.3 mice.
(A) Representative FLOW dot plots showing thymocytes (CD4 and CD8), DN thymocytes (CD25 and CD44), pre-positive selection (TCRβ^-CD69^-), and post-positive selection (TCRβ⁺CD69⁺) stages in DP thymocytes from NOD and NOD 8.3 mice. (B) Representative gMFI histograms of CD5, p- CD3ζ, p-Erk and IL-2 levels (left) and the IL-2 gating representative plot (right) compared between the lower 5% and upper 5% CD5 expression in thymocytes of normoglycemia female NOD mice aged 6 to 8 weeks old across DN, DP and CD8SP stages. (C) Cell numbers of thymocytes from NOD and NOD8.3 mice at the DN, DP and CD8SP stages. (D) Representative CD5 and p-CD3ζ gMFI histograms in thymocytes across DN, DP and CD8SP stages from NOD and NOD8.3 mice. (E) Cell numbers of thymocytes at the indicated DN stages (DN1, DN2, DN3 and DN4) from NOD and NOD8.3 mice. DN stages are defined as presented in Supplementary Figure 3A, middle panels. (F) CD5 gMFI distribution at the pre-positive selection (upper; TCRβ^-CD69^-) or post-positive selection (lower; TCRβ⁺CD69⁺) stage in DP thymocytes from NOD and NOD8.3 mice. (G) Representative FLOW dot plots for virtual memory (Vmemory) as shown in Figure 5H in NOD or NOD8.3 mice. In all panels from (A) to (G), NOD CD5^lo cells or NOD mice are represented in blue, and NOD CD5^hi cells or NOD8.3 mice are represented in red. Data represent mean ± SD. The data presented in (C) and (E) represent two experiments. The sample size was n = 10-11 (C) and n = 7-11 (E) per group. *P < 0.05, **P < 0.01, ****P < 0.0001, by unpaired, two-tailed t test (C) and (E).

Thymocyte-stage self-reactivity correlates peripheral diabetogenic T cell proliferation profiles in NOD, BDC2.5 and NOD8.3 mice.
(A) CD5 gMFI comparison in the DN, DP and CD8SP stages of thymocytes (upper panel) among NOD (grey), BDC2.5 (blue) and NOD8.3 (red) mice. The received TCR signal (dictated by CD5 level) during thymocyte selection dictates the peripheral cell proliferation level, shown in (B and C). (B) Thymidine analog Edu incorporation in CD8⁺ T cells from spleen (upper) or PLNs (lower) compared among NOD (grey), BDC2.5 (blue) and NOD8.3 (red) mice. Representative FLOW cytometry dot plots (left) and corresponding statistical results (right) are presented. (C) T cell proliferation level in BDC2.5 (left panel) and NOD8.3 (right panel) compared to NOD mice. Naïve T cells were isolated from 6-8-week-old normoglycemia female NOD, BDC2.5 and NOD8.3 mice and stimulated with the indicated amounts of anti-CD3 and anti-CD28 (1 μg/ml). Cell proliferation was measured by the incorporation of [methyl-3H] thymidine. Data represent mean ± SD. The data presented in (B) represent two experiments. The sample size was n = 5 (B) and n = 2-3 (C) per group. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA (B) and unpaired, two-tailed t test (C).

Immunomodulatory effects of transgenic Pep on effector T cell functions are analyzed.
(A) Cell proliferation levels of naïve CD44^loCD62L^hiCD8⁺ T cells (left) isolated from 8-10-week-old normoglycemia female NOD or NOD8.3 upon stimulation with anti- CD3/CD28 dynabeads (middle) or PMA/Ionomycin (right) treatment, measured by the incorporation of [methyl-3H] thymidine (cpm; counts per minute). (B) Cell proliferation levels of CD8⁺ T cells isolated from 8-10-week-old normoglycemia female NOD, dLPC/NOD and dLPE/NOD mice upon anti-CD3/CD28 stimulation. Purified CD8⁺ T cells were stimulated with the indicated amounts of anti-CD3 (0.625, 1.25 and 2.5 μg/ml, respectively) combined with anti-CD28 (1 μg/ml). Cell proliferation was measured by the incorporation of [methyl-3H] thymidine (shown by simulation index). (C) Proliferation levels of splenocytes isolated from 8-10-week-old normoglycemia female NOD8.3 and dLPE/NOD8.3 mice upon stimulation with the indicated amounts of IGRP_206-214 peptides or ConA. Cell proliferation was measured by the incorporation of [methyl-3H] thymidine (cpm; counts per minute). (D) Percentages of effector/memory CD44^hi-, CD69⁺-, and CXCR3⁺CD8⁺ T cells in PLNs of NOD, dLPC/NOD and dLPE/NOD mice. (E) Percentages of TNF-α⁺-, IFN-γ⁺-, and TNF-α⁺IFN-γ⁺CD8⁺ T cells in 8-10-week-old normoglycemia female NOD, dLPC/NOD and dLPE/NOD mice. Isolated cells were stimulated with anti-CD3/CD28 (1 μg/ml) for 48 hours. After stimulation, the percentages of cytokine-producing CD8⁺ T cells were measured by flow cytometry analysis. Notably, in (B), (D) and (E), a stepwise increase in transgenic Pep expression from nontransgenic NOD to dLPC/NOD and dLPE/NOD mice within the litters born at the same time was confirmed in our previous study (27). Data represent mean ± SD. The data presented in (A) to (E) represent two experiments. The sample size was n = 5-7 (A), n = 5-7 (B), n = 6 (C), n = 5-7 (D) and n = 5-7 (E) per group. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired, two-tailed t test (A) and (C) and one-way ANOVA (B, D and E).

The pairing of VJ gene segments in the TCR repertoire is compared between CD5^hiCD8⁺ and CD5^loCD8⁺ cells.
(A) TRA variable (TRAV) and (B) TRB variable (TRBV) genes in CD5^hi (red dot) and CD5^lo (blue dot) groups were examined. Notably, if the same count of a specific VJ gene segment usage occurred in CD5^hi and CD5^lo cells, the outcome of overlapping dot color would be purple. n = 20 mice for each CD5^hi and CD5^lo group.

The genotyping results of NOD, dLPC/NOD, NOD8.3 and dLPC/NOD8.3 mice using the indicated F’ and R’ primers are shown.
(A) The Ptpn22 F’ and R’ primers used for genotyping were designed in our previous study to evaluate the existence of the transgene Ptpn22 (Ptpn22: 853 bp) (27). The primer sequence for TCRβ8.3 can be referred to (Verdaguer et al., 1997) (TCRβ8.3: 260 bp). (B-D) Genotyping results for transgenic Ptpn22 and TCRβ8.3 expression in pups #35 to #53 from male NOD8.3 crossed with female dLPC/NOD mice are presented.

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