Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer

Remziye E Wessel ¹, Nardin Ageeb ², Joseph M Obeid ³, Ileana S Mauldin ⁴, Kate A Goundry ¹, Gabriel F Hanson ¹, Mahdin Hossain ⁵, Chad Lehman ⁵, Ryan D Gentzler ⁶, Nolan A Wages ⁷, Craig L Slingluff Jr ⁴, Timothy N J Bullock ^5,8, Sepideh Dolatshahi ^1,5,✉,0, Michael G Brown ^5,9,10,11,0

Abstract

Background

Major histocompatibility complex class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression.

Methods

We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single-cell neighborhoods from mIF images followed by multivariate discriminant analysis.

Results

Spatial quantitation of tumor cell MHC-I expression revealed intratumoral and intertumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56⁺ cell numbers in patient tumors were positively associated with disease-free survival (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56⁺ and CD8⁺ cells (HR=0.199, p<1×10⁻³). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3⁺CD8⁺ T cells and CD3^–CD56⁺ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell–cell communication, we analyzed spatial single-cell neighborhood profiles to delineate the cellular environments of IFNγ^+/– NK cells and T cells. We discovered that both IFNγ⁺ NK and CD8 T cells were more frequently associated with other IFNγ⁺ lymphocytes in comparison to IFNγ^– NK cells and CD8 T cells (p<1×10^–30). Moreover, IFNγ⁺ lymphocytes were most often found clustered near MHC-I⁺ tumor cells.

Conclusions

Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Coassociation of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent colocalization of IFNγ⁺ NK cells with other IFNγ⁺ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.

NeoBiotechnologies’ products were used in this study:

Keywords: Major histocompatibility complex – MHC, T cell, Natural killer – NK, Tumor infiltrating lymphocyte – TIL, Lung Cancer

Publication History:
J Immunother Cancer. 2024 Sep 18;12(9):e009126. doi: 10.1136/jitc-2024-009126

Footnotes:
Funding: This work was funded by a Cancer Team Science Award from the UVA Comprehensive Cancer Center (TNJB and MGB), and a Collaborative Research Award from the UVA Beirne Carter Center for Immunology Research (TNJB, SD and MGB). The UVA Comprehensive Cancer Center Support Grant (P30CA044579) from the NCI additionally supported work performed by the Biorepository and Tissue Research Facility (BTRF) and Molecular Immunologic and Translational Science (MITS) core facilities. REW and GFH received training support from National Institute of General Medical Sciences of the National Institutes of Health (T32 GM145443). JO received training support from National Cancer Institute T32 CA163177 and UVA, Department of Surgery. MH received training support through the UVA College Science Scholar Summer Research stipend. CL received training support through the UVA School of Medicine Summer Medical Research Internship Program.

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Ethics approval: This study involves human participants and because these are analyses of deidentified pathological specimens, they are not considered human subjects (not a clinical trial). However, we received IRB approval for the two cohorts from the University of Virginia Institutional Review Board (IRB): (1) The tissue microarray (TMA) of cohort 1 was collected and analyzed under IRB-HSR#: 18346 and (2) Cohort 2: IRB-HSR # 13310: Identification of Biomarkers in Diseased Human Specimens. Participants gave informed consent to participate in the study before taking part.

Data availability free text: Demographics data for cohorts 1 and 2 are included in online supplemental spreadsheets 1 and 2, respectively. Algorithms developed for this study (O-PLSDA and cell neighborhood scoring) are available on GitHub (https://github.com/Dolatshahi-Lab). Quantitative IHC and mIF data are available as online supplemental spreadsheets 3-4. IHC and mIF images will be made available by the authors on reasonable request.

Presented at: This work, in part, was presented at the 2023 American Association of Immunologists (AAI) Annual Meeting (Abstract 62.08).