Accumulation of tissue-resident natural killer cells, innate lymphoid cells, and CD8+ T cells towards the center of human lung tumors

ABSTRACT Lung cancer is a leading cause of cancer-related death worldwide. Despite recent advances in tissue immunology, little is known about the spatial distribution of tissue-resident lymphocyte subsets in lung tumors. Using high-parameter flow cytometry, we identified an accumulation of tissue-resident lymphocytes including tissue-resident NK (trNK) cells and CD8+ tissue-resident memory T (TRM) cells toward the center of human non-small cell lung carcinomas (NSCLC). Chemokine receptor expression patterns indicated different modes of tumor-infiltration and/or residency between trNK cells and CD8+ TRM cells. In contrast to CD8+ TRM cells, trNK cells and ILCs generally expressed low levels of immune checkpoint receptors independent of location in the tumor. Additionally, granzyme expression in trNK cells and CD8+ TRM cells was highest in the tumor center, and intratumoral CD49a+CD16− NK cells were functional and responded stronger to target cell stimulation than their CD49a− counterparts, indicating functional relevance of trNK cells in lung tumors. In summary, the present spatial mapping of lymphocyte subsets in human NSCLC provides novel insights into the composition and functionality of tissue-resident immune cells, suggesting a role for trNK cells and CD8+ TRM cells in lung tumors and their potential relevance for future therapeutic approaches.


Introduction
Lung cancer is one of the most common causes of cancerrelated death. Current immunotherapeutic approaches mainly focus on circulating T cells which infiltrate the tumor. However, little is known about the distribution of tissueresident lymphocyte subsets such as tissue-resident NK (trNK) cells, innate lymphoid cells (ILCs), and tissue-resident memory T (T RM ) cells within lung tumors.
TrNK and T RM cells, defined by co-expression of CD69, CD49a, and CD103, are readily identified in human lung 1,2 . ILCs which express high levels of CD69 are considered tissueresident irrespective of CD49a or CD103 expression [2][3][4] . TrNK cells are characterized by a CD16 − phenotype but differ markedly from conventional circulating CD16 − NK cells both at the protein and transcriptome levels 2 . Frequencies of CD16 − NK cells are increased in lung tumors [5][6][7] , suggesting that tumorinfiltrating NK cells are, at least in part, comprised of trNK cells. Additionally, a fraction of NK cells in human lung tumors have been reported to express immune checkpoint receptors, including PD-1, CTLA-4, and TIM-3 8,9 , potentially inhibiting cytotoxic function of intratumoral NK cells.
In addition to NK cells and T RM cells, ILC1, ILC2, and ILC3 have been identified in human lung tumors in several studies 10,11 . While ILC2s are reduced in frequency in lung tumor tissue compared to tumor-free lung tissue, NKp44 + ILC3s have been suggested to aid the formation of intratumoral lymphoid structures, indicating a relevance for ILC subsets within lung tumors 10 .
Compared to trNK cells and ILCs, CD8 + T RM cells have been studied in more detail in lung cancer. CD8 + T RM cells are more frequent in lung tumors compared to peritumoral lung tissue 12 , and their frequencies are associated with greater overall survival 13,14 . Intratumoral CD8 + T RM cells express inhibitory receptors, including CTLA-4, TIM-3, TIGIT, and CD39, which are largely confined to CD8 + T RM cells [12][13][14] . Co-expression of CD39 and CD103 identifies tumor-reactive CD8 + T cells, strongly indicating a functional relevance for CD8 + T RM cells in the control of lung tumors 14 . To date, it remains unknown whether tissue-resident lymphocytes other than CD8 + T RM cells accumulate in human lung tumors and whether these cells share common traits with respect to expression of inhibitory receptors, chemokine receptors, and effector functions.
Here, we dissected the landscape of tissue-resident lymphocytes in patients with lung cancer in order to map common and distinct features of tissue-resident lymphocytes in human lung tumors. We show that trNK cells and CD8 + T RM cells accumulated toward the center of non-small cell lung carcinomas (NSCLC), associated with slightly overlapping yet distinct chemokine receptor expression patterns on trNK cells and CD8 + T RM cells. In line with low expression of immune checkpoint receptors, trNK cells from the tumor center were responsive to ex vivo target cell stimulation.

Lung tissue collection
Thirty-seven patients undergoing lobectomy for suspected lung cancer were included, with patient-matched tumor tissues (peritumoral, margin, center) from 17 patients, nonmatched tumor-distal tissue from 19 patients, and matched tumor-distal tissue from one patient. All tumor tissues were derived from NSCLC, while two additional tumor-distal tissues were derived from carcinoid tumors. Definition of the distinct tumor areas was controlled by pathologists. Patients with records of preoperative chemotherapy and/or radiotherapy, strong immunosuppressive medication and/or hematological malignancy were excluded. Clinical and demographic details of the patients are summarized in Table 1. The Regional Ethical Review Board in Stockholm approved the study (permit 2018/1819-31/1), and all donors gave informed written consent prior to sample collection.

Processing of tissue specimens
Lung tissue was processed as previously described 15 . Briefly, macroscopically tumor-free human lung tissue, taken either as distal as possible from the tumor site (defined as 'distal'), or from a site in close proximity (within 2.5 cm) to the tumor (defined as 'peritumoral'), together with a part taken from the tumor margin and tumor center, were transferred into ice-cold PBS or Krebs-Henseleit buffer 15 and stored at 4°C for maximal 18 h until processing. Tissue was digested using collagenase II (Sigma-Aldrich) and DNase (Roche) for 30 min at 37°C. After digestion, RPMI supplemented with 10% FCS (Thermo Scientific), 1 mM L-glutamine (Invitrogen), 100 U/ml penicillin, and 50 μg/ml streptomycin (Invitrogen) (R10 medium) was added, and the cell suspension was passed through a 40-μm cell strainer and washed twice in R10 medium. Mononuclear cells from lung cell suspensions were isolated by density gradient centrifugation (Lymphoprep, Axis Shield).

Flow cytometry
Cells were stained with antibodies against extracellular and intracellular proteins (Supplementary Table S1). Secondary stainings were performed using streptavidin BB630 (BD Biosciences), or streptavidin BV650 (Biolegend) and Live/ Dead Aqua (Molecular probes, Life Technologies). After surface staining, cells were fixed and permeabilized using a FoxP3/Transcription Factor staining kit (eBioscience). Samples were analyzed on a BD Symphony equipped with five lasers (BD Biosciences), and data were analyzed using FlowJo 10.7.1 (BD Biosciences).

Degranulation assay
NK cell degranulation was analyzed as previously described 15 . In brief, fresh lung mononuclear cells were resuspended in R10 medium and rested for 15-18 h at 37°C. Cells were cocultured with or without K562 cells for 6 h with anti-human CD107a (BV421, H4A3; BD Biosciences). Brefeldin A and monensin (GolgiPlug and GolgiStop, BD Biosciences) were added for the last 5 h of incubation.

Statistics
Statistics were analyzed in GraphPad Prism 9. Wilcoxon matched-pairs signed rank test or Mann-Whitney test were used for matched or unmatched pairs of data, respectively, unless otherwise stated.

Tissue-resident NK cells and CD8 + T RM cells are enriched in human NSCLC tumors
First, we analyzed the landscape of CD16 − NK cells, ILCs, and T cell subsets in tumor-free (distal), peritumoral, tumor margin, and tumor center tissues of human lung tumors (Figure 1a; gating strategies in Figure S1A-D). Frequencies of CD16 − NK cells of total NK cells as well as of ILCs (defined as  CD3 − CD127 + CD161 + cells) of total CD3 − lymphocytes were higher toward the tumor center compared to peritumoral or distal tissue (Figure 1b, c). In contrast, frequencies of CD8 + and CD4 + T cells of total T cells were similar between the different tissue areas (Figure 1b, c, Figure S1B).
Expression of CD69, a marker for tissue-resident lymphocytes, was more frequently expressed toward the tumor center on CD16 − NK cells and CD8 + T cells and in a similar but non-significant trend on CD4 + T cells, while nearly all ILCs expressed CD69 at all locations studied (Figure 1d, Figure  S1E, F). Next, we analyzed the distribution of cell subsets with additional features of tissue-residency, such as the expression of CD49a and CD103. Both CD16 − NK cells and CD8 + T cells expressed CD49a and CD103 more frequently in the tumor center as compared to the tumor margin, peritumoral, and distal sites (Figure 1e, f). In contrast, CD4 + T cells expressed similar levels of CD49a and CD103 throughout all areas ( Figure S1E, F). On ILCs, CD49a expression was generally low; however, CD103 expression was elevated in the tumor center as compared to tumor-free distal tissue (Figure 1e, f). In line with these results, tissue-resident lymphocytes were slightly more frequent in the tumor center (Figure 1g). Among the tissueresident lymphocytes, CD8 + T RM cells comprised the predominant tissue-resident population in the tumor and peritumoral areas, while CD4 + T RM cells dominated in tumordistal tissue (Figure 1g, Figure S1G), indicating proportional changes of the lymphocyte composition in tumor-free tissue in close proximity to the tumor. The stratification of data based on the NSCLC-tumor histotype, i.e. adenocarcinoma (ADC) or squamous cell carcinoma (SCC), revealed comparable patterns of trNK cell and CD8 + T RM cell accumulation toward the tumor center, independent of tumor type specificity or cancer stage (Figure 1h, Figure S1H-J). Together, our results suggest an accumulation of tissue-resident lymphocytes, mainly constituted by CD8 + T RM cells, in the center of human NSCLC tumors.

CXCR3 + CXCR6 + trNK cells and CD8 + T RM cells accumulate within the tumor center
The increase in trNK cells, ILCs, and CD8 + T RM cells toward the tumor center suggested a selective recruitment or retention of these cells at this location. To identify whether tissueresident lymphocyte subsets share common tumorinfiltration capacities, we analyzed the expression of chemokine receptors relevant for mediating recruitment of leukocytes to the tumor microenvironment [16][17][18] . Expression of CXCR3, CXCR6, CCR2, and CCR5 differed between trNK cells, ILCs, CD8 + T RM cells, and CD4 + T RM cells (Figure 2a, Figure S2A), as well as between locations within the tumor and tumor-distal lung tissue (Figure 2b). CXCR3 expression was high on trNK cells and CD8 + T RM cells (Figure 2b, c) and did not differ between ADC and SCC ( Figure S2B, C). Notably, the frequency of CXCR6 + trNK cells and CD8 + T RM cells increased toward the tumor center (Figure 2b, c). However, this observation was specific to SCC and not observed in ADC ( Figure S2B, C). Conversely, the proportion of trNK cells and CD8 + T RM cells expressing CCR2 decreased (Figure 2b, c). This reduction was particularly noticeable on trNK cells derived from SCC, potentially attributable to their overall low expression. Nonetheless, it was evident in both cancer subtypes for CD8 + T RM cells ( Figure S2B, C). CCR5 expression on trNK cells and CD8 + T RM cells was similar at all locations (Figure 2b, c). Except for CCR2, chemokine receptor expression was low on ILCs, with no or only minor changes throughout all areas (Figure 2b, Figure S2B, C). Co-expression patterns of chemokine receptors were considerably more diverse on CD8 + T RM cells than on trNK cells (Figure 2c). Strong increases toward the tumor center were observed for CXCR3 + CXCR6 + cells (population 13) and CCR5 + CXCR3 + CXCR6 + CD8 + TRM cells (population 9), concurrent with a decrease in CCR5 single-positive (population 12) as well as CCR2 + CCR5 + CXCR6 + (population 3) CD8 + T RM cells (Figure 2c). A large proportion of CXCR3 + CXCR6 + CD8 + T RM cells but not trNK cells also coexpressed CCR5 (population 9), indicating a potential role for CCR5 specifically on CD8 + T RM cells. In contrast, CD8 + T RM cells expressing only CXCR3 (population 14) and coexpressing CXCR3, CCR2, and CCR5 (population 2) were largely confined to the distal non-tumor area (Figure 2c).
Altogether, although chemokine receptor co-expression patterns differed between trNK cells and CD8 + T RM cells, increased CXCR6 expression on both cell types toward the tumor center indicate that CXCR6 constitutes a key-receptor for mediating the accumulation of trNK cells and CD8 + T RM cells in lung tumors.

Immune checkpoint receptor expression increases on tumor-infiltrating CD8 + T RM cells but not trNK cells or ILCs
Despite the capacity to infiltrate tumors, intratumoral NK cells and T cells have been suggested to be largely functionally inert 19,20 , in part resulting from upregulation of immune checkpoint receptors/exhaustion markers such as PD-1 21,22 , TIM-3 23,24 , TIGIT 25 , and CD39 26,27 . Since little is known about the expression of immune checkpoint receptors on tissue-resident innate lymphocytes, we assessed their expression on trNK cells, ILCs, CD8 + T RM , and CD4 + T RM cells in the distinct tumor locations (Figure 3a, b and Figure S3A). In most donors, immune checkpoint receptors were expressed by few trNK cells and ILCs at all locations, whereas CD8 + T RM cells displayed high immune checkpoint receptor expression in the tumor tissues (Figure 3a, b). Expression of TIGIT and TIM-3 on trNK cells, and all immune checkpoint receptors analyzed on CD8 + T RM and CD4 + T RM cells increased toward the tumor center (Figure 3b, Figure S3A). Although TIGIT was expressed by a higher frequency of trNK cells in the tumor center compared to peritumoral and distal tissue, expression remained considerably lower than that of CD8 + T RM cells at all locations (Figure 3b). In contrast to other immune checkpoint receptors, TIGIT was not expressed on ILCs (Figure 3b), which, however, more frequently expressed CD39 toward the tumor center (Figure 3b). Immune checkpoint receptor expression on trNK cells, ILCs, and CD8 + T RM cells did not differ significantly between ADC and SCC ( Figure S3B).
Together, our data indicate that CD8 + T RM cells in lung tumors would be substantially more affected by common immune checkpoint therapies than trNK cells and ILCs.

Different activation profiles of tumor-infiltrating tissue-resident lymphocytes
Low expression of immune checkpoint receptors on trNK cells led us to assess functional properties in comparison to CD8 + T RM cells (Figure 4). Immune checkpoint receptor-expressing trNK cells and CD8 + T RM cells expressed more Ki67 compared to checkpoint receptor-negative cells (Figure 4a). Furthermore, the tumor center was enriched with trNK cells and CD8 + T RM cells expressing granzymes (Figure 4b, c). While granzyme A expression did not differ between ADC and SCC, granzyme B expression was significantly higher on CD8 + T RM cells in SCC in the tumor center compared to the corresponding region in ADC ( Figure S4A, B). However, both trNK cells and CD8 + T RM cells lacked perforin expression (Figure 4b, c), which was consistent for both ADC and SCC ( Figure S4A, B), despite it being readily detectable in CD16 + NK cells in all tumor areas ( Figure S4C, D). As expected, ILCs and CD4 + T RM cells did not express any or very low levels of cytotoxic effector molecules (Figure S4A, B). In contrast to trNK cells and CD8 + T RM cells, their non-tissue-resident counterparts (defined as CD49a − CD103 − CD16 − NK cells and CD49a − CD103 − CD8 + T cells, respectively) expressed granzyme A and B less frequently in the tumor center ( Figure S4C, D). These expression patterns suggest different cytotoxic capacities of tissue-resident and non-tissueresident lymphocyte subsets toward the center of human lung tumors. Indeed, the analysis of target cell responsiveness revealed that CD49a + CD16 − NK cells from the tumor center degranulated stronger than CD49a − CD16 − NK cells (Figure 4d, e), suggesting the functional competence of trNK cells in the tumor center.
Together, these results reveal different effector profiles of tissue-resident lymphocyte subsets depending on their location within lung tumors. While conventional intratumoral NK cell and T cell subsets are considered hypofunctional 19,20 , our results suggest that trNK cells are tumor target cell-responsive.

Discussion
Here, we studied the distribution and phenotypic traits of tissue-resident lymphocyte subsets across human lung tumors and in tumor-free lung tissue. Human trNK cells have been described in tumor-free lung 2 , however, their distribution in lung tumors is largely unexplored. Our study revealed an accumulation of trNK cells, ILCs, and CD8 + T RM cells toward the center of human lung tumors, which may, in particular for trNK cells and CD8 + T RM cells, be linked to chemokine receptors such as CXCR3 and CXCR6, which are relevant for NK cell and T cell infiltration into tumors [28][29][30] . Both CXCR3 and CXCR6 were expressed at highest frequencies on tumor center trNK cells and CD8 + T RM cells, respectively. Additionally, our data suggest that the CCL5/CCR5 axis might play a role in tumor-infiltration of CD8 + T RM cells, in line with a strong correlation between the expression of CCL5 and CD8 + T cell infiltration across different types of cancer 31 . In addition to varying infiltration capacities, the observed phenotype could have been induced by the tumor-microenvironment or in response to other infiltrating leukocytes. For example, circulating NK cells cocultured with a head and neck SCC cell line and IL-15 upregulated CD49a and CD103, indicating promotion of a tissue-residency phenotype via the tumor microenvironment 32 . Therefore, other mechanisms leading to the accumulation of specific cell subsets such as apoptosis, metabolic differences, and lineage-dependent development must be considered.
Despite functional exhaustion, upregulation of immune checkpoint receptors on CD8 + T RM cells and trNK cells, albeit at low frequencies, indicates an immune-activating environment 33 . Indeed, tumor-infiltrating CD103 + T RM cells are more functional and cytotoxic as compared to their CD103 − counterparts (reviewed in 34 ). Furthermore, the expression of both granzyme A and granzyme B was highest in trNK cells and CD8 + T RM cells in the tumor center. Notably, granzyme B expression was significantly higher on CD8 + T RM cells in the tumor center in SCC compared to ADC.
Intratumoral CD49a + NK cells were readily responsive to tumor target cells, in contrast to previous reports 35 . However, effector cell function in the tumor might be compromised due to lack of perforin expression in these cell subsets. It remains to be determined whether trNK cells can rapidly upregulate perforin upon stimulation as shown for CD8 + T RM cells in influenza infection 1 . Together with a low immune checkpoint receptor expression, this would make trNK cells a relevant alternative for future therapeutic applications in solid tumors. Manipulating the interaction of intratumoral chemokines with their cognate receptors on trNK cells and CD8 + T RM cells might represent another suitable immunotherapeutic approach to modulate cell subset infiltration and retention (reviewed in 36 ). Additionally, trNK and/or CD8 + T RM cells could be expanded and primed ex vivo and harnessed for targeted lysis of the tumor cells. Future analyses will determine whether trNK cells and CD8 + T RM cells can be directly or indirectly harnessed for optimization and development of novel treatment strategies for patients with solid tumors.