Figure 4: Evidence of enhanced T cell functionality in the CD8+ PD-1^HIperipheral compartment.

A) Clinical details of 4 patients analyzed prospectively for changes in peripheral blood T cell functionality. NST indicates no special type. B) Polyfunctionality of PD-1^HICD4+ and PD-1^HICD8+ T cells isolated from PBMCs in 4 patients prior and after NAC (>1000 individual cells/sample/timepoint) was determined by Isoplexis single-cell cytokine profiling. Polyfunctionality is defined as the percentage of cells capable of producing ≥ 2 cytokines following CD3/CD28 stimulation. The percentage of cells in each sample capable of secreting 2, 3, 4, or 5+ cytokines are depicted in stacked bars. Characteristics of each of the 4 patients are shown above the bars. Patients with TNBC (Pt. 1 and Pt. 4) had greater increases in polyfunctionality in the CD8+ compartment with NAC. C) Heatmap representation of log cytokine signal intensity of each cell in each patient sample, pre and post NAC. Each row represents one PD-1^HiCD8+ T cell. White indicates no cytokine secreted. D) TCRβ chain repertoire analysis in CD8+ peripheral blood T cells. Upper plots indicate the number of individual T cells sequenced plotted by sample on the left Y axis; number of clonotypes (unique CDR3 amino acid sequences) plotted by sample on the right Y axis. In the lower graph, each sample is divided into the number of clonotypes comprising expanded (hyper-expanded, large, medium, small, and rare) compositions of the detected repertoire (categories divided by orders of magnitude of fraction of total repertoire). E) The fraction of repertoire clonotypes identified in PD-1^HI versus PD-1^NEG CD8+ T cells (before or after NAC) classified as ‘hyperexpanded’ or ‘large’ (comprising >0.1% of repertoire). P value represents a 2-sample 2-tailed t-test.