Cyto­tox­ic NK Cells Impede Response to Check­point Immunother­a­py in Melanoma with an Immune-Exclud­ed Phenotype

Immune check­point block­ade (ICB) tar­get­ing PD‑1 has trans­formed melanoma ther­a­py, yet half of patients do not ben­e­fit, often due to unclear resis­tance mech­a­nisms. Immune exclu­sion-the inabil­i­ty of lym­pho­cytes to enter the tumor nest-is a key bar­ri­er, and stud­ies using only base­line or late post-treat­ment sam­ples may miss ear­ly immune dynamics.

We hypoth­e­sized that treat­ment-induced changes occur after a sin­gle ICB cycle. In an inter­ven­tion­al study, we pro­filed melanoma with sin­gle-cell RNA-seq, ana­lyz­ing 46 tumor sam­ples from 23 patients, includ­ing 20 matched pre- and ear­ly on-treat­ment biop­sies. We iden­ti­fied 20 immune cell types and linked their abun­dance to response. Con­sis­tent with pri­or work, respon­ders were enriched for CD8+ T cell sub­sets, par­tic­u­lar­ly CD8+ CXCL13+ T cells, at both time­points. Sur­pris­ing­ly, cyto­tox­ic nat­ur­al killer (NK) cells were sig­nif­i­cant­ly enriched in non-respon­ders ear­ly on treat­ment, val­i­dat­ed in two inde­pen­dent melanoma cohorts and one breast can­cer scR­NA-seq cohort.

Spa­tial tran­scrip­tom­ic and pro­teom­ic analy­ses from the same lesions showed that in non-respon­ders, NK and CD8+ T cells co-accu­mu­lat­ed at the tumor rim but failed to pen­e­trate the core, con­sis­tent with immune exclu­sion; respond­ing tumors showed co-local­iza­tion with­in the parenchy­ma. In an immune-exclud­ed mouse melanoma mod­el, phar­ma­co­log­ic NK cell deple­tion enhanced CD8+ T cell infil­tra­tion into the core and enabled tumor clear­ance with anti-PD‑1. Mech­a­nis­ti­cal­ly, NK cells were recruit­ed to exclud­ed regions via CX3CR1 and con­strained CD8+ T cell infil­tra­tion and func­tion. These find­ings iden­ti­fy an unex­pect­ed NK cell – medi­at­ed resis­tance axis and sug­gest tar­get­ing NK recruit­ment or activ­i­ty to over­come immune exclu­sion and improve ICB responses.