Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion

Abstract

Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFNγ-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFNγ pathway, allowing us to distinguish it as a novel mechanism of immune resistance.We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.

Upregulated CD58 is associated with clinicopathological characteristics and poor prognosis of patients with pancreatic ductal adenocarcinoma

“…One of the challenges in cancer immunotherapy is the resistance of immune checkpoint blockade (ICB) in the tumor microenvironment. Recently, Frangieh et al [ 50 ] found that CD58 expression was diminished in melanoma tissues from ICB-resistant patients. In cells surviving T/NK co-culture, CD58 level was reduced, which favored resistance to T/NK-cell-mediated killing.…”

CD58 Immunobiology at a Glance

“…Although immune checkpoint blockade (ICB) therapy has exhibited unprecedented clinical efficacy in tumor treatment ( 213 , 214 ), ICB still lacks efficacy in the majority of cancer patients ( 215 ). A recent study reported that the surface expression of CD58 was strongly reduced in tumor cells of melanoma patients with ICB resistance compared with that of untreated patients ( 216 ). CD58 loss induced immune evasion in different co-culture models with CTLs, and the PD-L1 expression was elevated in CD58-knockout melanoma cells ( 216 ).…”

“…A recent study reported that the surface expression of CD58 was strongly reduced in tumor cells of melanoma patients with ICB resistance compared with that of untreated patients ( 216 ). CD58 loss induced immune evasion in different co-culture models with CTLs, and the PD-L1 expression was elevated in CD58-knockout melanoma cells ( 216 ). These data illustrated that the loss of CD58 facilitated immune evasion possibly via different mechanisms, including deficiency of T cell costimulation, reduction of T cell adhesion, and even synergy of the corepressor PD-L1.…”

“…Thus, elevating CD58 expression is likely to contribute to the alleviation of ICB resistance in melanoma patients. In particular, the expression of CD58 was independent of the IFN-γ pathway, and the loss of CD58 led to immune escape without affecting MHC expression ( 216 ), indicating that it differs from the known mechanisms of ICB resistance.…”

Current and Prospective Methods for Assessing Anti-Tumor Immunity in Colorectal Cancer

“…Thus, in the study profiling myeloid cells in glioblastoma, CITE-seq revealed new cell markers for subsets of TAMs and dendritic cells (DCs) that were not identified in scRNA-seq analysis [ 108 ]. Novel extensions of CITE-seq, such as ECCITE-seq, that integrates pooled CRISPR screens into CITE-seq measurements and Perturb-CITE-seq, which combines pooled genetic perturbation screens with CITE-seq, were used to investigate molecular mechanisms underlying cancer immunotherapy resistance [ 109 , 110 , 111 ]. Although, to the best of our knowledge, the methods described above have not been being applied in CRC yet, CITE-seq or its modifications can expand our knowledge of intratumor heterogeneity and immune evasion in this deadly disease.…”