Background: Immune checkpoint blockade with PD-1 inhibitor pembrolizumab was effective in RS but not CLL in a phase 2 study (NCT02332980). Analyzing the immune signatures including PD-L1 expression and T cell diversity is important for understanding the differential responses. Methods: 15 CLL and 14 RS patients in NCT02332980 were included. Expression of PD-L1 in lymph node (LN) was analyzed by immunohistochemistry staining. Peripheral blood (PB) and LN T cell diversity was analyzed using the ImmunoSEQ platform (Adaptive Biotechnology), which quantifies the clonailty of T cells by deep sequencing of the CDR3 region of T cell receptor (TCR). Data analysis (Student’s t-test and Mann-Whiney U test) was done by GraphPad Prism (v7). Results: PD-L1 expression was significantly lower in CLL (n = 6) vs RS (n = 12) patients (mean 5.5% vs 22.9%, P= 0.003). A control CLL cohort (n = 11, Mayo CLL tissue registry) also had lower PD-L1 expression (7.7%, P= 0.002 vs RS). RS Patients progressed on ibrutinib or chemotherapy had similar PD-L1 expression (25.3% vs 19.6%, P= 0.416). PB TCR clonality at trial baseline was significantly lower in RS (n = 13) vs CLL (n = 13) patients (median 0.098 vs 0.342, P= 0.026). Patients progressed on ibrutinib or chemotherapy had similar TCR clonality (CLL: 0.34 vs 0.24, P= 0.524; RS: 0.11 vs 0.09, P= 0.534). 5 RS patients achieved an objective response (1 CR, 2 PR, 1 CMR and 1 PMR), 4 of which were after progression on ibrutinib. For CLL patients, only 1 nodal reduction was seen. 4 RS patients had prior CLL samples available for TCR analysis. 2 patients had a notable decrease of TCR clonality (0.21-0.04 and 0.17-0.03) from CLL (PB) to RS state (LN), and achieved CR and PR, respectively. 2 other patients with stable clonality (0.04-0.05 and 0.03-0.08) had SD and PMR, respectively. PB TCR clonality did not change significantly with pembrolizumab (CLL [n = 11]: P= 0.775; RS [n = 11]: P= 0.830; paired t-test). Conclusions: RS patients had higher expression of PD-L1 and lower TCR clonality (more diverse T cells) compared with CLL patients. The distinct immune signatures may explain their differential responses to PD-1 blockade. The dynamic changes of TCR clonality during Richter’s transformation may predict a response to PD-1 blockade.