Background: PD-1 axis blockade induces lasting clinical responses in ~20% of patients with advanced NSCLC. However, most patients eventually develop resistance. Acquired resistance is poorly understood, but may be mediated by alternative immune suppressive pathways. Methods: Using multiplex immunofluorescence we simultaneously measured levels of DAPI, CD3 (D7AE6), PD-1 (EH33), TIM-3 (D5D5R) and LAG-3 (17B4) in 11 whole tissue sections obtained from patients with NSCLC before PD-1 axis blockade and after acquired resistance (8 cases with progression on-treatment and 3 with progression off-therapy). Markers were measured in the whole tumor area or in CD3+ T-cells using fluorescence co-localization. The association between markers and changes upon acquired resistance were studied. Results: Expression of PD-1, TIM-3 and LAG-3 was seen in all cases with membranous staining pattern and signal predominantly located in CD3+ T-cells. Levels of TIM-3 and LAG-3 in T-cells were significantly correlated (Spearman’s R = 0.65, P = 0.001), but were not associated with PD-1 (R = -0.03, P = 0.86 for TIM-3 and PD-1; and R = 0.24, P = 0.28 for LAG-3 and PD-1). Compared to pre-treatment samples, 6 cases (55%) showed significantly higher levels of PD-1 or LAG-3 on acquired resistance and 5 cases (45%) had higher TIM-3. Of these, 4 cases had higher levels of the 3 markers and were on-therapy at progression. Lower levels of PD-1, TIM-3, and LAG-3 were found on acquired resistance in 5 (45%), 6 (55%), and 4 (36%) cases, respectively. Four of these cases showed lower levels of all inhibitory receptors, 3 of which were off-therapy at progression. Only one case had no change in LAG-3 levels. Conclusions: PD-1, TIM-3 and LAG-3 were expressed in the majority of NSCLCs with signal predominantly located in T-lymphocytes. Among acquired resistance cases, higher levels of PD-1, TIM-3 and LAG-3 were associated with progression on-treatment. Lower levels of the markers were associated with progression off-therapy. Although multiple mechanisms may exist, up-regulation of alternative immune inhibitory receptors such as TIM-3 and LAG-3 could mediate acquired resistance to PD-1 axis blockers in a proportion of NSCLCs.