Background: Checkpoint inhibitors, such as the PD-1 antibody nivolumab, have been approved for the treatment of several cancer types, including NSCLC. As only a limited subset of NSCLC patients experiences durable benefit, there is a need for early prediction of clinical response. Here, we aim to identify immune markers in peripheral blood samples of NSCLC patients treated with nivolumab. Methods: In 71 NSCLC patients treated with 2^nd line nivolumab (2-weekly), whole blood samples were collected at 3 time points (pre-treatment; prior to 2^nd and 3^rd administration of nivolumab). Multiplex flow cytometry was applied to enumerate 19 immune cell subsets and determine frequencies of T cell subsets positive for 28 markers (single + combinations), enabling classification of T cells according to activation, maturation, co-signaling, and chemotaxis. Best overall response (BOR) within 90 days was assessed using RECIST (v1.1). Standard statistical tests were applied. Results: Patients showing partial response (PR) displayed increased numbers of CD8 T cells prior to and throughout therapy with a median (range) of 560 cells/μl (170-1900), while patients with progressive disease (PD) showed a median of 220 cells/μl (90-1070) (p = 0.022). PR patients also possessed significantly higher frequencies of CD8T cells positive for the maturation marker CD95 (60% (9-87)) at baseline and throughout therapy when compared to PD patients (45% (5-82)) (p = 0.041). With regard to co-signaling, PD patients displayed a significantly lower median frequency of CD8 T cells positive for CD279 (PD-1) and CD366 (TIM3) at baseline (4.8% (1.2-17.6)) and, unlike PR patients, a drop in CD4 T cells positive for multiple co-inhibitory receptors from 24% (8-36) to 17% (8-36) following therapy. In addition, PD patients showed a higher frequency of CD4 T cells positive for co-stimulatory CD28 and CD278 (ICOS) (35% (12-48)). Conclusions: We demonstrate that total numbers of CD8 T cells as well as T cells subsets in peripheral blood, in particular those expressing co-signaling receptors, associate with response to nivolumab in NSCLC patients. When confirmed, these findings will facilitate clinical decision making.