Fig 3. Frequency of the characteristic HEL-specific CD4+ T cell clone in antigen- naive and immunized mice.

(A) 4 week old BALB/c mice were injected with PBS or HEL emulsified in IFA and four day later removed by surgical excision. The mice were then sacrificed at 12 weeks and for focused Vβ8.2Jβ1.5 TCR next generation repertoire analysis was conducted on splenic T cells. Significance calculated by Student’s t-test. Graphs of copy number vs. distinct nucleotide CDR3 sequence are shown. Red arrow indicates location of HEL-specific Vβ8.2Jβ1.5 CDR3 sequence(CDR3=CASGTGNNQAPL). Results are representative of at lest three independent experiments. (B) Spleen cells from unimmunized 12 week old BALB/c mice were harvested and stained with antibodies specific to CD4, CD25, CD44, CD127, and CD62L. Lymphocytes were then FACS sorted to isolate pure populations of memory (CD4+, CD25med/low, CD44 high, CD62L−/+) T cells. Isolated memory CD4+ T cells were cocultured for 96 hr with in vitro generated BMDCs in presence or absence of HEL. RNA was extracted for focused Vβ8.2Jβ1.5 TCR next generation repertoire analysis. We were unable to generate focused Vβ8.2Jβ1 TCR libraries from BMDCs cultured alone in presence or absence of HEL. These confirms absence of T cell in our BMDCs preparations. Resulting reads were then analyzed using an in-house developed software. Graphs of copy number vs. distinct nucleotide CDR3 sequence are shown. Red arrow indicates location of HEL-specific Vβ8.2Jβ1.5 CDR3 sequence(CDR3=CASGTGNNQAPL).