To examine alterations in the Treg and Tconv repertoires at higher resolution, we sacrificed 1MOG244.2 mice prior to immunization or 23-25 days after EAE induction. Spleen and, for mice with EAE, CNS cells were collected and pooled from 5-6 identically treated mice per group. Pooling was performed due to low post-isolation T-cell yields from the CNS of retrogenic mice. The cells were sorted based on CD4 and the presence or absence of GFP-Foxp3 expression. RT-PCR was performed to analyze the paired β chains in each organ and cell type. V8.2-C or V8.2-J2.7 primers were used to amplify TCR cDNA. We established a robust system to PCR amplify TCRβ cDNA without contamination, and the same experiment was repeated twice. Five PCR reactions were performed on each cDNA to avoid PCR bias. The 400 bp DNA segments were TA cloned into pCR2.1 vector, 50-100 clones were picked and sequenced the with T7 primer. Finally, we submitted the sequences in bulk to the IMGT (immunogenetics information system; http://imgt.cines.fr/) web server for identifying the TCR gene use. Totally ~4000 sequences were collected. The identical experiment was performed twice.
We initially surveyed V8.2-C sequences to determine the distribution of J among V8.2+ TCR. In total, 1829 sequences were assessed. Twelve different J
regions were found in these TCR sequences. J used by Treg and Tconv cells showed no significant difference in either spleen or CNS (Figure 4-1C and D). This hinted that Treg cells have a diverse V8.2+ TCR repertoire as Tconv cells, and this diversity was not
altered by autoimmunity circumstances. Proportions of J used by either the Foxp3+ or
Foxp3- populations showed no significant differences between the CNS and periphery in mice with EAE (p > 0.05, Figure 4-1A and B). Comparison of pre- and post-immune splenocytes also failed to identify changes in J use among Foxp3+ T-cells and showed a
significant difference in Foxp3- cells only for a single J, J2.4 (6.2 ± 0.5% pre versus
14.1 ± 0.5% post; p = 0.012). This indicates preserved diversity in the V8.2+ population
with EAE induction, especially when comparing the CNS-infiltrating and splenic T-cell populations of diseased mice.
We next surveyed the more circumscribed V8.2-J2.7 repertoire. V8.2was selected due to the predominate expression of V8.2 among MOG-specific T-cells in H- 2b mice (Mendel Kerlero de Rosbo and Ben-Nun, 1996) and J2.7 due to its use in the original V8.2+J2.7+ TCR chain partner for the 1MOG244.2 TCR (Alli et al., 2008).
Despite the theoretical potential for formation of >106 unique TCR sequences with this V-J constraint, we therefore knew that V8.2+J2.7+ TCR had the potential to pair
with the 1MOG244.2 to form MOG-specific TCR. An estimated 95% confidence interval for the upper limit of the maximum frequency (φmax) of unobserved TCR CDR3
sequences for individual cell types/samples in different experiments ranged from 1.4 to 3.1% for different samples and cell types (Table 4-1). The φmax confidence interval (CI)
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Figure 4-1. J used in Tconv and Treg cells. CD4+ T-cells from retrogenic mice,
preimmune or EAE induction, were flow cytometrically sorted into CD4+Foxp3- and CD4+Foxp3+ populations. After a few steps of RT-PCR, TA clone and sequencing, CDR3b regions were identified through IMGT. Comparisons were made between Tconv (A) and Treg (B) cells from unimmunized and immunized splenocytes and CNS T-cells from mice with EAE. Comparisons were also made between Tconv and Treg cells in immunized CNS T-cells (C) and in immunized splenocytes (D). * P < 0.05.
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Table 4-1. Isolation of TCR V sequences from retrogenic mice.
Experiment Disease scores T-cell type Sequence
Isolated
sequences V-J max 95% CI V-C V-J
1 (Pre-
immune) NA Spleen Foxp3- Total 137 155
Unique 131 55 2.25% Spleen Foxp3+ Total 152 183
Unique 150 64 1.91% 2 (Pre-
immune) NA Spleen Foxp3- Total 102 161
Unique 97 54 2.17% Spleen Foxp3+ Total 128 123
Unique 124 62 2.85%
3 (EAE, Day 23)
1,2,3,3,
4 Spleen Foxp3- Total 159 167
Unique 97 52 2.09% Spleen Foxp3+ Total 131 141
Unique 57 46 2.48% CNS Foxp3- Total 207 256 Unique 90 42 1.36% CNS Foxp3+ Total 147 226 Unique 88 59 1.54% 4 (EAE, Day
25) 1,1,1,2,2,2 Spleen Foxp3- Total 160 208
Unique 71 48 1.68% Spleen Foxp3+ Total 144 114
Unique 82 49 3.08%
CNS Foxp3- Total 207 211
Unique 82 45 1.65% CNS Foxp3+ Total 155 207
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95% certainty of positive detection. For example, there were a total of 155 amino sequences in splenocyte Treg of pre-immune sample in experiment 1, if the frequency of particular CDR3 sequence in the population is above 2.25%, the chance for it to be detected is 95%. This indicates that the collected sequences were adequate to survey common sequences in each cellular cohort.
Among the V8.2-J2.7 sequences obtained, 452 unique amino acid sequences were identified. Interestingly, 54 (~12%) of sequences were shared, or public sequences, among the 4 independent experiments. Public sequences were over-represented among more commonly identified sequences, with 74% of public sequences present within the 100 most frequent sequences. An increased probability of identifying sequences in multiple independent samples when their sequence frequency is high is expected, and implies that the observed proportion of public sequences underestimates total
representation due to non-identification of low frequency public sequences. 16/54 public sequences were found in TCR exclusively used by Treg, 14/54 in Tconv TCR, and 24/54 in sequences identified both in Treg and Tconv. The CDR3 amino acid sequences of public sequences were diverse and no sequence patterns were identified.
Abundance coverage estimator analysis (ACE) of pooled sequences, an indicator of total sequence diversity, indicated a limited diversity consistent with findings in other systems, with values of 271 and 337 for pre-immune, and 191 and 171 for post-immune V8.2+, J2.7+ Tconv and Treg splenocytes respectively. This restricted diversity and
the high proportion of public sequences indicates that the retrogenic repertoire is heavily sculpted and that few of the potential sequences that may form from V-D-J
rearrangement are realized in the retrogenic mice. Interestingly, the original chain partner for the 1MOG244.2 TCR (ASGDAGTGYEQY) was among the public
sequences identified in the CNS Foxp3- populations of both EAE induction experiments, though not in pre-immune analyses. This suggests that receptors relevant to EAE in C57BL/6 mice, which served as the source of the 1MOG244.2 hybridoma, play a role in the 1MOG244.2 retrogenic mice.
Among public TCR, 20 CDR3 sequences were identified as common to both preimmune T-cells and T-cells from mice with EAE. TCR sequences appearing
predominantly in Foxp3+ or Foxp3- populations preimmune showed the same orientation, Foxp3+ or Foxp3-, in mice with EAE for 19 of the 20 receptors. This implies that TCR sequence rather than disease status is the primary force governing T-cell Foxp3
expression.