Background: The phase II SWOG S1801 study showed an improved event-free survival with anti-PD-1 (PD1) neoadjuvant immunotherapy (neoIT) vs adjuvant PD1. One hypothesis explaining this benefit is the presence of tumor-draining lymph nodes (tdLN; defined as the nearest node to the tumor without direct involvement) as a potential reserve of stem-like (TCF7+) T cells, crucial to a good response to IT. We sought to analyze the immune infiltrate of the tumor-involved LN (ie TME) and tdLN from patients (pts) achieving major pathological response (MPR: complete [pCR] or near-complete [near-pCR] pathological response) vs non-MPR (partial [pPR] or no [pNR] pathological response). Methods: Pts with stage III melanoma treated with 6 weeks of PD1-based neoIT (PD1 + Lenvatinib) were included (NeoPele clinical trial; NCT04207086). Multiplex fluorescent immunohistochemistry of the TME before and after neoIT, and of the tdLN after neoIT was analyzed (T cell panel: CD3, TCF7, CD103, FoxP3, CD39 and Sox10; and B cell panel: CD20, CD21, CXCR5, TCF7, CD3 and SOX10). Lymphoid aggregates, characterized by clusters of CD21+ and CXCR5+ immune cells, were quantified. Results: Of the 20 pts, 11 (55%) had MPR (8 [40%] with pCR and 3 [15%] with near-pCR) and 9 (45%) had a non-MPR (4 [20%] with pPR and 5 [25%] with pNR). At baseline, MPR pts had a higher % of T cells (11% vs 3%, p = 0.0127) and follicular B helper T cells (CXCR5+; 29% vs 9%, p = 0.0293) than non-MPR pts in the TME. NeoIT led to an increase in the % of T cells, mainly in pts with MPR (median +30%; p = 0.0156) vs non-MPR (median +7%; p = 0.0312) pts, including an increase in the % of tumor-reactive (CD39+) T cells (p = 0.0195), but a decrease in % of tissue-resident stem-like (CD103+ TCF7+) T cells (p = 0.0195). Within the B cell compartment, there was an increase in the % of CD21+ B cells and mature (CD21+ CXCR5+) B cells in MPR vs non-MPR pts. At week 6, MPR pts maintained a higher % of T cells (36% vs 11%, p = 0.0172), follicular B helper T cells (20% vs 9%, p = 0.0133), and mature B cells (10% vs 2%, p = 0.0021) in the TME compared with non-MPR. NeoIT led to a significant increase in the number of lymphoid aggregates in the TME from MPR pts (median +42; p = 0,0156), but no difference in non-MPR pts. No differences were observed in the tdLN based on pathological response. Conclusions: MPR pts had a higher density of T cells in the TME at baseline, and a more differentiated and activated immune profile after neoIT compared with non-MPR pts. MPR pts had a significant increase in the number of lymphoid aggregates at week 6 compared to non-MPR pts; however, the role of these lymphoid aggregates, including the follicular B helper T cell subset and mature B cells, promoting a good pathological response to neoIT is yet to be clarified. Clinical trial information: NCT04207086.