Background: Immune checkpoint inhibitors (ICI) have shown durable benefit in a subset of non-small cell lung cancer (NSCLC) patients. The composition of the tumour microenvironment (TME) is becomingly increasingly recognised as an important factor to predict response to therapy. Methods: Here, we applied digital spatial profiling of the tumour and stromal compartments from a 2^nd line NSCLC ICI-treated cohort (n = 41 patient) and standard of care (SOC), platinum treated NSCLC cohort (n = 47), to identify tissue-based signatures of response to therapy. Results: We demonstrate by mIHC that the interaction of CD68⁺ macrophages with PD1⁺, FoxP3⁺ cells is significantly enriched in ICI refractory tumours (p = 0.012). Patients sensitive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p = 0.028) within the tumour compartments, which corresponded with the increased expression of IL2 mRNA (p = 0.001) within their stroma. Immuno-inhibitory markers CTLA-4 (p = 0.021) and IDO-1 (p = 0.023) were supressed in ICI-responsive patients. Tumour CD44 (p = 0.02) was depleted in the response group and corresponded inversely with significantly higher stromal expression of one of its ligands, SPP1 (osteopontin, p = 0.008). Analysis of dysregulated transcripts indicated the potential inhibition of stromal interferon-gamma (IFNγ) activity, estrogen-receptor and Wnt-1 signalling activity within the tumour cells of ICI responsive patients. Cox survival analysis indicated tumour CD44 expression was associated with poorer prognosis (HR = 1.61, p = 0.01), consistent with its depletion in ICI sensitive patients. Similarly, stromal CTLA-4 (HR = 1.78, p = 0.003) and MDSC/M2 macrophage marker ARG1 (HR = 2.37, p = 0.01) were associated with poorer outcome while BAD (HR = 0.5, p = 0.01) appeared protective. The SOC cohort paralleled similar roles for immune checkpoints and pro-apoptotic markers, with LAG3 (HR = 3.81, p = 0.04) indicating poorer outcome, and BIM (HR = 0.16, p = 0.014) with improved outcome. Interestingly, stromal mRNA for E-selectin (HR = 652, p = 0.001), CCL17 (HR = 70, p = 0.006) and MTOR (HR = 1065, p = 0.008) were highly associated with poorer outcome in ICI treated patients, indicating pro-tumourigenic features in the tumour microenvironment that may facilitate ICI resistance. Conclusions: Through multi-modal approaches, we have dissected the characteristics of NSCLC treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy.