T cell populations in the pancreatic lymph node naturally and consistently expand and contract in NOD mice as disease progresses

Abstract

Nonobese diabetic (NOD) mice develop spontaneous autoimmune Type 1 diabetes (T1D) that results from the destruction of insulin secreting β cells by diabetogenic T cells. The activation of autoreactive T cells occurs in the pancreatic lymph nodes (PLN) from where effector T cells migrate to the pancreas. This study was designed to explore whether T cell populations in the NOD PLN expand in a predictable and reproducible way during disease progression. Complementary determining region (CDR) 3 length spectratype analysis of 19 TCR Vβ families was used to identify the relative frequency of T populations in PLN of 4 and 10 week old NOD mice and mice at T1D onset. Significant and highly reproducible changes in specific T cell populations were detected in 14 of Vβ families tested at all stages of disease. However, of these, the CDR3 spectratype of only four Vβ families was significantly more perturbed at T1D onset than in 10 week old mice. Intriguingly, when diabetes was induced in 10 week old mice with cyclophosphamide (CYP) the same four Vβ families, Vβ5.1, Vβ9, Vβ10, and Vβ15, were again significantly more perturbed than in the untreated non-diabetic age matched mice. Taken together the data show that while T cell responses in PLN of NOD mice are heterogeneous, they are ordered and consistent throughout disease development. The finding that within this heterogeneous response four Vβ families are significantly more perturbed in diabetic mice, whether spontaneous or induced, strongly suggests their selection as part of the disease process.

Figure 1. Identification of expanded T cell populations in the TCR Vβ 12 family in PLN

In this example, the relative areas of individual CDR3 peaks were calculated as a percentage of the total area under all peaks for Vβ12. The mean and SD of each relative peak area for Vβ12 was calculated for thymus (n=5) and the mean ± 3 SD was plotted for each peak (bars). The relative peak area for T cell populations with different CDR3 lengths in individual 4 week old NOD mice (n=11) are plotted as x in the graph. The peaks in PLN that are significantly different from the equivalent peak in thymus are indicated with asterisk above the respective bar: ***, p<0.0001; **, p<0.001 or *, p<0.01 by ANOVA. The x-axis shows the CDR3 length in amino acid and the y-axis shows the relative peak area (%).

Figure 2. The majority of 4 week old NOD mice display the same altered TCR Vβ repertoire in pancreatic lymph nodes

The Vβ CDR3 spectratype of 19 Vβ families was compared between thymus (n=5) and PLN (n=11) of 4 week old NOD mice. The bars represent the mean ± 3 SD of relative peak area for each CDR3 length in each Vβ family for thymus. The relative peak area for each CDR3 length in each Vβ family for PLN in individual mice is indicated by an x on the same plot. Peaks in PLN that are significantly different from the equivalent peak in thymus in at least 80% of mice tested are indicated with asterisk above the respective bar: ***, p<0.0001; **, p<0.001 or *, p<0.01 by ANOVA. The x-axis shows the CDR3 length in amino acid and the y-axis shows the relative peak area (%).

Figure 3. The TCR repertoire is more diverse at 10 weeks of age than at 4 weeks of age

The global perturbation index was calculated for each mouse at 4 weeks of age (n=11), 10 weeks of age (n=20) and at diabetes onset (n=14). Each dot represents an individual mouse. The mean and SD is shown for each group. Statistical differences between groups are calculated using unpaired t test and are shown as *** p<0.0001 or ** p<0.001.

Figure 4. The reduction in global perturbation index between 4 and 10 weeks of age is not reflected by a reduction in total perturbation index of individual Vβ families

The total perturbation index (%) for each Vβ family in each mouse was calculated for 4 (n=11) and 10 week old mice (n=20), and mice at diabetes onset (n=14). The mean and SD is shown for each Vβ family, and each dot represents an individual mouse. Vβ4 is significantly less perturbed at 10 weeks than at 4 weeks of age (A), while the total perturbation index of Vβ8.2 and Vβ8.3 increased during disease progression (B). Other Vβ families are either more perturbed at T1D onset (C) or do not change during disease progression (D). Significant changes between time points was calculated using unpaired t test and are shown as *** p<0.0001; ** p<0.001 or * p<0.01.

Figure 5. Cyclophosphamide (CYP) accelerates changes that naturally occur between 10 weeks of age and TID onset

Eight week old female NOD mice were treated with (n=10) or without (n=20) CYP and monitored for diabetes onset. One week after the last CYP injection when all treated mice were diabetic, PLN were isolated and the CDR3 spectratype for all 19 TCR Vβ families determined for individual mice. (A) Total perturbation index for Vβ families that expand naturally and after CYP treatment between 10 weeks of age and TID onset. (B) Total perturbation index for Vβ families that expand only after CYP treatment. The total perturbation index for each Vβ family was calculated for each mouse. The mean and SD is shown for each Vβ family, and each dot represents an individual mouse. (C) Global perturbation index for treated and untreated mice. The global perturbation index was calculated for each mouse and the mean ± SD is shown. Statistical analysis was performed using unpaired t test and statistical differences between the corresponding groups is shown as *** p<0.0001; ** p<0.001 or * p<0.01.