Background: In mice, cryo combined with cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade increases antigen-specific TILs, generating a synergistic rejection of secondary tumors. In humans, breast cancer antigens are shared across pts in low frequency and are incompletely characterized. Thus, to evaluate for antigen-specific TILs in pts receiving cryo and/or the anti-CTLA-4 antibody, ipi, we employed deep sequencing of TCR CDR3 region DNA, which provides a quantitative measurement of the frequency of individual T cell clones, each putatively reactive to a unique antigen. Methods: 19 pts were treated with cryo (7 pts), single-dose ipi at 10mg/kg (6 pts), or cryo+ipi (6 pts). Core biopsy (Bx) +/- cryo was performed 7-10 days prior to standard-of-care mastectomy and ipi was given 1-5 days before core Bx. From available specimens, DNA was sequenced using the immunoSEQ™ assay. Utilizing CDR3 sequence copy number, the frequency of each unique T cell clone was determined. Nonparametric analyses were conducted on derivative metrics including T cell %, clonal overlap, and clonality. Results: In core Bx’s, T cell % (median 6.8%, 0.6-30.7%) and clonality (median 0.13, 0.09-0.4) varied across pts, with a higher T cell % in poorly differentiated lesions (p=.03). Cryo reduced T cell counts but induced a polyclonal infiltrate (p=.02) with low clonal overlap, indicating influx of new clones. For the 3 ipi alone pts for whom Bx and mastectomy tissue were available, clonal repertoire appeared closer to baseline, as measured by clonality and overlap. Combination cryo+ipi frequently expanded the proportion of the most dominant (top 5) clones (3/6 cases), an effect which was rarely observed with monotherapy (1/8 cases). Conclusions: Cryo-associated polyclonality and influx of new clones may be related to cryo-mediated cell death, antigen release, and T cell engagement. A cryo+ipi mediated surge of the most dominant clones may reflect synergistic activation of a clonal subset. These findings call for further characterization of therapy-induced dominant clones, and support repertoire analyses in breast cancer. Clinical trial information: NCT01502592.