T cells drive negative feedback mechanisms in cancer associated fibroblasts, promoting expression of co-inhibitory ligands, CD73 and IL-27 in non-small cell lung cancer

ABSTRACT The success of immune checkpoint therapy shows tumor-reactive T cells can eliminate cancer cells but are restrained by immunosuppression within the tumor micro-environment (TME). Cancer associated fibroblasts (CAFs) are the dominant stromal cell in the TME and co-localize with T cells in non-small cell lung cancer. We demonstrate the bidirectional nature of CAF/T cell interactions; T cells promote expression of co-inhibitory ligands, MHC molecules and CD73 on CAFs, increasing their production of IL-6 and eliciting production of IL-27. In turn CAFs upregulate co-inhibitory receptors on T cells including the ectonucleotidase CD39 promoting development of an exhausted but highly cytotoxic phenotype. Our results highlight the bidirectional interaction between T cells and CAFs in promoting components of the immunosuppressive CD39, CD73 adenosine pathway and demonstrate IL-27 production can be induced in CAF by activated T cells.


Introduction
Stromal cells influence all phases of the immune response. From directing migration of naïve T cells in the lymph nodes and facilitating their proliferation to limiting clonal expansion and effector function, the appropriate activity of stromal cells optimizes immune responses. 1 Cancer-associated fibroblasts (CAFs) however present an impediment to effective antitumor immunity, 2 promoting tumor growth and immunosuppression. [3][4][5] In non-small cell lung cancer (NSCLC) the majority of tumor-associated T cells are found within the stroma, amidst a dense network of CAFs, suggesting CAF/T cell interactions are likely to influence T cell function in situ. In the Lewis lung carcinoma model deletion of fibroblast activation protein (FAP) + expressing CAFs slows tumor growth in an IFN-γ/TNF-α dependant fashion 6 showing CAFs restrain anti-tumor immunity. Under inflammatory conditions fibroblasts can be induced to express MHC-II 7 and uptake and process antigen 3 increasing their potential for interactions with T cells. Expression of CD73 on CAFs limits T cell expansion via production of immunosuppressive adenosine. 8 Thus CAFmediated suppression of T cell function in the tumor microenvironment (TME) represents a critical late-stage barrier to effective anti-tumor immunity.
Tumor infiltrating T cells (TILs) in NSCLC display characteristics of T cell exhaustion including a hierarchical loss of effector function, expression of multiple co-inhibitory receptors and enhanced susceptibility to apoptosis. 9 The most profound exhaustion is seen in tumor reactive T cells which, after multiple rounds of stimulation, are unable to effectively eliminate the target antigen. 10,11 During chronic viral infection exhausted T cells express CD39 (NTPDase1) 12 and single-cell RNA-sequencing identified an enrichment of exhausted CD39 expressing T cells in NSCLC tumor versus normal nontransformed lung tissue. 13 Enrichment of CD39 + T cells is a common feature of solid organ malignancies including head and neck cancer, renal cell carcinoma and gastric adenocarcinoma. 14 CD39 expression requires TCR stimulation 15 and identifies T cells which have recently encountered antigen allowing differentiation of tumorreactive CD39 + T cells from CD39 − bystander T cells. 10 Consequently, there is particular interest in CD39 expression in TILs where expression allows enrichment for tumor-reactive T cells without knowledge of their fine specificity. 16 CD39 is an ectonucleotidase which converts ATP/ADP to adenosine monophosphate decreasing the availability of proinflammatory ATP and promoting production of immunosuppressive adenosine. 17 Loss of CD39 augments CD8 + T cell responses, 18 inhibits its enzymatic activity increasing cytokine production 15 and enhances anti-tumor responses. 19 Although the suppressive effects of CD39 are best described in Tregs 17 they have also been reported for CD39 + CD8 + T cells 20,21 raising the possibility that CD39 + cytotoxic T cells (CTLs) may directly contribute to immunosuppression in the TME. The critical factors and cellular interactions driving CD39 expression in TILs are incompletely defined but modulating CD39 expression and/or function may provide an opportunity to slow development of the most profound T cell exhaustion and strengthen anti-tumor immunity.
A full understanding of factors in the TME promoting expression of multiple co-inhibitory receptors and their ligands, immunosuppressive pathways and pro and antiinflammatory cytokines is necessary to direct the rational development of therapies. Many immunosuppressive elements of the TME, including upregulation of PD-L1, production of indolamine-2,3-dioxygenase (IDO) and elevated numbers of Tregs, are induced as a consequence of the anti-tumor immune response itself. 22 In order to investigate how activated T cells influence the phenotype of CAFs and vice versa we measured phenotypic and functional changes in both populations during co-culture in primary NSCLC samples. Activated T cells increase expression of PD-1 ligands, MHC-II, CD73 and production of immunomodulatory cytokines (including IL-6 and IL-27) by CAFs, which in turn promoted expression of multiple co-inhibitory receptors including CD39, PD-1, Tim3 and production of IL-10 by T cells. The bidirectional interactions of CAF and T cells in the tumor stroma could act to promote immunosuppression and T cell exhaustion and provide a target to prevent loss of function in tumor-reactive T cells.

Exhausted CD39 + T cells co-localize with FAP hi CAFs in NSCLC
In human NSCLC we found a locally elevated frequency of CD39 + T cells in the tumor versus paired non-cancerous lung (NCL) tissue (Figure 1a), consistent with published reports. 11,16 This remains true in both CD4 + and CD8 + compartments ( Figure 1(b,c)). Co-expression of CD39 and CD103 is common in CD8 + T cells but in stark contrast the majority of CD39 + CD4 + T cells are CD103 − (Figure 1a). Treg cells are enriched in the TME where they express high levels of CD39 (a feature of highly activated Tregs 23 with enhanced stability of suppressive function at inflammatory sites 24 ) and lack of expression of CD103 (Figure 1a Sup Figure 1a-c). Therefore, distinct patterns of CD39 and CD103 expression describe tumor-reactive CTLs (CD8 + /CD39 + /CD103 + ) and tumor infiltrating Tregs (CD4 + /CD39 + /CD103 − ). Combining data on CD3 + T cells from multiple paired tumor and non-cancerous adjacent lung tissues illustrates significant enrichment of both of these populations in cancerous tissue (Figure 1d). Among CD8 + CTLs, the CD39 + express the highest levels of multiple co-inhibitory molecules including PD-1, Tim3 and Lag3 (Figure 1d) as well as the hierarchical loss of effector function associated with the development of exhaustion. Reduced capacity for TNF-α production was most pronounced in CD39 hi CD103 + CD8 + T cells while these cells retained the highest capacity for granzyme B production and showed greater evidence of proliferation ex-vivo by Ki67 staining (Sup SFigure 2a), in line with the findings of Canale et al. 11 in human breast cancer. CD39 + CD4 + T cells showed reduced TNF-αα production and lacked capacity for IFN-γ production as would be expected of Tregs. Notably we found a relative lack of CD4 + CD103 + T cells in tumor versus non-cancerous adjacent lung tissue (Figure 1d/1 F) and that the small population of CD103 + cells remaining had the highest capacity for IFN-γ production (Supp Figure 2) illustrating a relative lack of CD4 + T cells with maximum effector function in the TME.
CAFs in NSCLC can be differentiated from normal fibroblasts in non-cancerous adjacent lung tissue by elevated expression of fibroblast activation protein (FAP) (figure 1f, gating strategy Sup Figure 3). High levels of FAP expression in the stromal compartment of the tumor is associated with T cell retention in the stroma (Sup SFigure 4) where CD39 + CD8 + T cells are found in close association with FAP + CAFs ( Figure 1(g, h)/Sup SFigure 4).

T cell activation promotes expression of co-inhibitory ligands, MHC molecules and CD73 on CAF
Co-culture of five independently generated NSCLC CAF lines with PBMC revealed a distinct pattern of responsiveness to activated T cells. T cell activation resulted in increased expression of MHC-I, induction of MHC-II expression, elevated expression of the co-inhibitory ligands PD-L1 and PD-L2 and increased expression of the ectonucleotidase CD73 (Figure 2a, b). Anti-CD3/CD28 stimulated PBMC produced around 4322 pg/ml IFN-γ and 75 pg/ml TNF-αα and changes in coinhibitory molecule expression could be reversed by neutralization of IFN-γ and TNF-αα (Figure 2a, b). Increased coinhibitory ligand expression could also be induced by culture of CAFs in the presence of conditioned media from activated T cells (( Figure 2c) which contained 3224 pg /ml IFN-γ and 547 pg/ml TNF-α). The effect of IFN-γ was dominant in modulating MHC-I, PD-L1/2 and CD73 expression and was solely responsible for induction of MHC-II expression ( Figure  2c). However, clear synergistic effects of co-exposure to IFN-γ and TNF-αα resulted in the highest levels of MHC-1, PD-L1 and PD-L2 ( Figure 2d). Thus, activated T cells induce phenotypic changes in CAFs which may both increase the capacity of CAFs for antigen presentation and suppress further T cell activation via PD-1 interactions with PD-L1/PD-L2 or adenosine production by CD73.
CAFs promote expression of multiple co-inhibitory receptors including CD39 and increase production of IFNγ and IL-10 in activated T cells Activation in the presence of CAFs induced phenotypic changes in both CD8 + (Figure 3a,b) and CD4 + (Figure 3c, d) T cells with upregulation of co-inhibitory receptors PD-1, Tim3 and LAG-3 as well as CD39 (Figure 3a,b) resulting in a phenotype resembling that of tumor infiltrating T cells (Figure 1a). Significant upregulation of CD39, PD-1, Tim3 and LAG-3 was seen in multiple experiments but CD103 levels were not enhanced by CAFs (Figure 3a-d). Pre-exposure of CAF to IFN-γ and TNF-α (to increase PD-L1 and PD-L2 expression) prior to co-culture with PBMCs did not further increase the degree of CAF-induced co-inhibitory molecule expression suggesting increased PD-1-signaling does not account for the increased co-inhibitory receptor expression (Sup SFigure 4). Co-culture also increased production of IFN-γ and IL-10 ( Figure 3e, f). CAF did not increase the frequency of IFN-γ producing cells but rather increased the amount of cytokine produced by both CD8 + and CD4 + T cells (Figure 3e and Sup SFig 6). CAF significantly increased both the amount of IL-10 produced (Figure 3e) and the frequency of IL-10 producers among CD8 + but not CD4 + T cells (Sup SFigure 5). CAFs themselves did not produce either IFN-γ or IL-10 ( Figure 3e, f). The cytokine enhancing function of CAF is consistently seen as five independently generated NSCLC CAF lines showed capacity to upregulate IFN-γ ( Figure 3g) and IL-10 ( Figure 3h) production when in co-culture with activated T cells.

Activated T cells elevate production of IL-6 and induce production of IL-27 in CAFs
Having shown CAFs actively promote the development of key phenotypic characteristics of exhausted tumor infiltrating lymphocytes and production of the immunosuppressive cytokine Figure 1. CAF co-localize with C39 + T cells in the tumor microenvironment. a) Representative staining of CD8 + (upper panels) and CD4 + (lower panels) T cells from paired non-cancerous lung (NCL) or tumor for CD39 and CD103. b) Proportion of CD8 + T cells co-expressing CD39 and CD103 in paired NCL/tumor samples (** P = .005). c) Proportion of CD103-CD39 + cells within CD4 + T cells from paired NCL and tumor samples (n = 9) (*** P = .0007). d) tSNE plots showing concatenated data files gated on CD45 + CD3 + T cells from four paired NCL and tumor samples, gates indicate the location of CD8 + CD39 + cells, CD4 + CD39 + cells and CD4 + CD103 + cells. The distribution of cells derived from non-cancerous lung (NCL) and tumor samples is shown and heat maps illustrate the relative expression of CD103, CD39, PD-1, Tim3 and LAG3. e) The frequency of CD4 + cells expressing CD103 in paired NCL and tumor samples (n = 11) (** P = .0075). f). FAP expression on CD45-EpCAM − CD31 − CD90 + stromal cells derived from NCL or paired tumor samples (n = 12) (*** P = .0005). g). Representative Immunohistochemistry illustrating localistaion of FAP, CD8, CD39 and CD103 expressing cells in a section of NSCLC, scale bar represents 100 µm. h) Distribution of CD39 + and CD103 + CD8 + T cells illustrates enrichment of CD39 + cells within the stroma (*** P = .0008) and CD103 + cells within NSCLC tumors (*** P = .006) (n = 6). Two tailed paired T tests were used for all statistical analyses. IL-10 we sought to identify factors produced by CAFs which might drive these changes. Induction of CD39 requires TCR stimulation (in vitro and in vivo 11,15,25 ) and IL-6, 11 IL-27 26 and TGF-β 16,27 have been reported to increase CD39 expression. The ability of CAF to produce IL-6 is well documented and can have pro-tumor effects. 28 Activated T cells significantly increased IL-6 production by CAFs and this was blocked by neutralizing TNF-α and IFN-γ ( Figure 4a). T cell-conditioned media (containing 3224 pg /ml IFN-γ and 547 pg/ml TNF-α) also increased IL-6 production by CAFs and this was inhibited by neutralizing TNF-α and to a lesser extent IFN-γ ( Figure 4b). Confirmatory experiments with recombinant cytokines showed TNF-α is most potent in driving IL-6 by NSCLC CAFs (Figure 4c). IL-27 favors development of Th1 responses during T cell priming but also exerts potent effects on activated T cells including promoting production of IL-10 29,30 and inducing expression of multiple co-inhibitory receptors. 31 While none of the nine NSCLC derived CAF lines tested produced IL-27 spontaneously we were surprised to discover that activated T cells (which produced around 4322 pg/ml IFN-γ and 75 pg/ml TNF-α) induce production of IL-27 by fibroblasts (Figure 4d). IL-27 was induced in CAFs by the synergistic action of recombinant TNF-α and IFN-γ confirming the capacity of CAF to produce IL-27 in the absence of other cell types ( Figure 4e). Thus IFN-γ and TNF-α act in concert to upregulate components of the antigen presentation pathway, maximize expression of PD-1 ligands, CD73 and production of IL-6 and IL-27 by CAF.
Our results describe a scenario wherein IFN-γ and TNF-αβ produced by activated T cells promote upregulation of MHC molecules, co-inhibitory ligands and the immunosuppressive ectonucleotidase CD73 on the surface of CAFs as well as elevating their production of IL-6 and inducing production of IL-27. In turn CAFs induce characteristics of T cell exhaustion including upregulation of multiple co-inhibitory molecules such as PD-1, Tim3 and LAG-3 and the TGF-β mediated upregulation of CD39 replicating the phenotypic characteristics of the most highly activated tumor reactive cells in CAF-rich stroma of NSCLC tumors.

Discussion
Activated fibroblasts have the potential to uptake, process and present antigen to T cells 32 and the consequences of CAF/T cell interactions in the tumor microenvironment can dictate the efficacy of anti-tumor immune responses. 3,6,8,33,34 Although low co-stimulatory molecule expression means fibroblasts are generally inefficient compared to professional antigen presenting cells they can activate memory T cells 35 and in the right cytokine environment can initiate immune responses. 32 Single cell RNA sequencing has recently identified MHC-II expressing CAF with the capacity to present to CD4 + T cells in pancreatic ductal adenocarcinoma highlighting the potential for CAF to interact with T cells in the TME. 36 The majority of tumor infiltrating T cells are previously activated, displaying features of a tissue resident memory (TRM) phenotype 37 and co-expressing multiple co-inhibitory molecules. 9,10,13 Though these cells show signs of exhaustion they are not entirely dysfunctional 38,39 but rather exquisitely sensitive to environmental conditions. The success of immune checkpoint therapy shows that reducing the negative signals received by tumor reactive T cells can be sufficient to unleash effective antitumor immunity. While targeting individual signaling pathways such as PD-1/PD-L1 or CTLA-4 can be effective it is the overall balance of positive and negative signals which determines outcome and justifies the many combined approaches to immunotherapy currently under study.
The beneficial effects of targeting CAFs, in experimental models, rely on the presence of an adaptive immune response 6,40 and increase the effectiveness of checkpoint inhibitors indicating CAFs suppress T cell responses and facilitate suppression via PD-1 in vivo. 33 In vitro studies show CAFs can limit T cell proliferation, 41 alter patterns of cytokine production, [42][43][44] promote apoptosis 3 and upregulate coinhibitory receptor expression. 44 We have extended these findings to show human NSCLC-derived CAFs also induced expression of CD39 on activated T cells via TGF-β. There is increasing interest in CD39 + T cells in the TME due to the demonstration that CD39 expression identifies tumor reactive CTLs. 10,16 The frequency of CD39 + cells in lung cancer also correlates with the mutation status of the epidermal growth factor receptors on tumor cells suggesting high levels of neoantigen presentation are reflected in a higher frequency of exhausted responder T cells. 10 Re-invigorating exhausted tumor reactive T cells is the goal of immunotherapy and CD39 + T cells display an exhausted phenotype expressing the highest levels of multiple co-inhibitory receptors including PD-1/Tim3 and LAG-3. 11,16 Cells expressing multiple coinhibitory receptors are the most profoundly functionally impaired and likely to require inhibition of multiple immune checkpoints to regain effective function. 45 PD-1 hi tumor infiltrating T cells typically express multiple co-inhibitory receptors and their frequency is predictive of both responsiveness to PD-1 blocked and survival in NSCLC patients 38 suggesting reinvigoration is possible and that the presence of PD-1 hi /CD39 + T cells in the TME indicates the potential exists for an effective anti-tumor immune response. Our results suggest that T cell activation in the TME modulates CAF function igniting a negative feedback mechanism which raises PD-1 expression along with CD49b, Tim3, LAG-3 and CD39 on activated T cells whilst simultaneously elevating PD-L1/PD-L2 and CD73 expression on CAFs themselves.
IFN-γ displays a dual role in tumor immunology, it promotes immunity via activation of cytotoxic T cells and upregulating expression of MHC-I molecules on tumor cells, but also promotes suppression by inducing expression of PD-1 ligands (Reviewed) . [46] Similarly, while IFN-γ upregulates expression of MHC-I and induces expression of MHC-II in fibroblasts 7 it promotes expression of PD-L1 and PD-L2 42 and CD73. PD-1/PD-L1 interactions control the magnitude of immune responses and contribute to peripheral tolerance and dynamic regulation of PDL-1 expression allows efficient local regulation. The highest levels of PD-L1 expression are found in the most immunologically active tumors demonstrating links between the vigor of the anti-tumor response and the suppressive nature of the TME. 47 While PD-L1 can be induced in many cell types expression of PD-L2 is mostly restricted to antigen presenting cells. However, CAFs express higher levels of PD-L2 than normal fibroblasts in lung, colon, pancreatic and breast cancer 3 and PD-L2 mediated signaling has been implicated in CAF mediated immunosuppression. 3 In breast cancer distinct CAF subtypes have been described and the dominant FAPhi subset of immunosuppressive CAF express the highest levels of PD-L2 and CD73. 4 IFN-γ and TNF-α show synergistic activity in regulating many aspects of immunity including production of iNOS 48 IL-6, 49 IL-8 and CXCL-10. 50 IFN-γ and TNF-αα also promote the highest level of costimulatory molecule expression in fibroblasts. 51 In addition, we found they have synergy in eliciting the highest observed level of PD-L1 /PD-L2 and CD73 expression in NSCLC CAFs as well as increasing production of IL-6 and initiating production of IL-27. Besides IFN-γ and TNF-α IL-6 and IL-27 can also promote PD-L1 expression 52 and their production may further amplify upregulations induced by IFN-γ and TNF-α. The finding that IFN-γ and TNF-α induce IL-27 production by CAFs identifies a novel immunoregulatory function in CAF ignited by activated T cells. IL-27 is a heterodimeric cytokine of the IL-6/IL-12 cytokine families composed of the Epstein-Barr virus (EBV) induced gene 3 (EBI3) and the IL-27p28 subunits and plays important roles in the initiation and regulation of immune responses (reviewed) . [53] IL-27 has potent anti-tumor effects both directly, 54 via inhibiting angiogenesis 55 and in promoting granzyme B expression and expansion of effective anti-tumor T cells. [56][57][58] However IL-27 can also restrain immune responses, it promotes CD39 expression in tumor infiltrating Tregs, 26 drives expansion of IL-10 producing Tr1 cells 30,59 and increases expression of multiple co-inhibitory receptors 31 as well as the co-inhibitory ligands PD-L1 and PD-L2. 60 IL-27 promotes expression of the co-inhibitory receptor Tim3 and production of IL-10 61 and in the absence of IL-27 R-signaling tumor infiltrating T cells retain functionality and more effectively prevent tumor growth, confirming its relevance to T cell responses in the TME. While we were unable to demonstrate a role for IL-27 in CAF-mediated induction of CD39, Tim3 or IL-10 in T cells it is possible that IL-27 signaling was not entirely blocked by our neutralizing antibody to IL-27. Huang et al 62 recently described of a role for IL-27 in maintaining the capacity of CD8 + T cells to proliferate and avoid apoptosis during chronic inflammation by promoting IRF1 expression. This highlights the potential of IL-27 to support the maintenance of a pool of T cells ready to respond to checkpoint therapy and raises the possibility that activated stromal cells could positively impact T cell survival via IL-27 production.
Fibroblasts express both chains of the IL-27-receptor, WSX-1 and Gp130, and respond to IL-27 with STAT-1 activation and increased expression of IL-6, CXCL10 and ICAM-1. 63,64 While several studies have shown responsiveness of fibroblasts to IL-27, evidence of IL-27 production by fibroblasts has only been reported in response to the synthetic TLR3 ligand poly I:C. 65 Collectively these studies indicate fibroblast responses to IL-27 have the potential to impact both tumor cell growth and T cell responses. Whether IL-27 production by CAF occurs in the TME and exerts autocrine effects on fibroblasts and/or paracrine effects on T cells in the TME will be the source of future investigation.
CD39 expression can shape immunity in the long term, interfering with the formation T follicular helper cells 66 and long lived memory T cells. 67 Expression of CD39 on CD4 + T cells in aged individuals correlates with increased susceptibility to apoptosis which can be reversed by CD39 inhibition illustrating a role for CD39 in regulating T cell longevity. 67 Increased susceptibility to apoptosis is also a feature of tumor infiltrating T cells and limits effective anti-tumor immunity 47 and increasing CD39 expression may represent a novel means by which CAF favor T cell apoptosis in the TME. CD39 expression also limits CD8 + T cell responses in atherosclerotic lesions, 15 during chronic viral 12 and bacterial infection 18 providing a common suppressive pathway in chronic inflammation. Our results suggest stromal cells, by promoting CD39 expression in T cells, can contribute to the generation of a locally suppressive microenviroment. During chronic inflammation this may represent a means of retaining responsiveness to the initiating antigen while preventing activation or limiting expansion of newly recruited T cells, potentially responsive to autoantigens released during inflammation, and thus preventing epitope spreading. While this would be desirable in true autoimmunity it would limit responsiveness to neoantigens and hinder effective immunity in cancer.
Functionally CD39 can act in concert with CD73 to promote immunosuppression by converting ATP to immunosuppressive adenosine. 17 Adenosine contributes to immunosuppression within the TME 68 and the CD39 antagonist ARL-67156 enhances the function of intratumoral T cells. 69 Deletion of the A2A adenosine receptor increases the efficiency of anti-tumor CAR T cells. 70 Mixed bone marrow chimera experiments show that CD73 expression in CAFs limits T cell response and prevents tumor rejection. 8 Consequently there is increasing interest in targeting these ectoenzymes as inhibitory checkpoints in cancer. While Tregs express both ectonucleotidases 17 tumor infiltrating CTLs in NSCLC do not express CD73 11 and while some studies report CD39 + CTLs can suppress the proliferation of CD39-responder cells 21,25 others found they lacked suppressive potential. 11 Notably CD39 and CD73 can synergistically promote immunosuppression even when expressed on different cell types as has been demonstrated in the case of suppressive Foxp3-Tr1 cells. 71 High level expression of CD73 is a feature of the most immunosuppressive CAF subset in breast cancer 4 and colorectal cancer 8 and we found activated T cells further increased CD73 expression in NSCLC CAFs.
The ability of cancer associated fibroblasts to modify their phenotype in response to anti-tumor immunity presents a dynamic barrier to effective immunotherapy, calibrating the suppressive capacity of CAFs to the magnitude of the immune response. In addition to the classically described regulation via PD-1 we have shown that CAF/T cell interactions upregulate the components of adenosine mediated immunosuppression increasing expression of CD73 on CAF and CD39 of T cells as well as driving IL-27-production. The burgeoning interest in combined targeting of PD-1-signaling and adenosine production should factor in the potential role of CAFs in amplifying these pathways on the frontline of anti-tumor immunity.

Ethics Statement
Healthy volunteer blood was obtained following informed consent and the study was approved by Lothian Regional Ethics Committee (REC) (REC No: 20-HV-069) prior to enrollment in the studies.

Samples and NSCLC tissue digestion
Cancer/lung tissue and blood samples were obtained following approval by NHS Lothian REC and facilitated by NHS Lothian SAHSC Bioresource (REC No: 15/ES/0094). All participants provided written informed consent NSCLC tissues and adjacent non-cancerous lung samples were collected from patients undergoing surgical resection with curative intent. Tumors >30 mm in diameter had areas from within macroscopic tumor and distal non-cancerous lung dissected by the attending pathologist. Fresh samples were processed immediately or stored in media overnight, then minced as finely as possible with scissors in bijoux prior to incubation with 1 mg/ml Collagenase IV (Merck), 1 mg/ ml DNase 1 (Merck), 50 U/ ml hyaluronidase (Stemcell Technologies) in DMEM (Life technologies) for 1 hr at 37°C with agitation. After digestion, samples were passed through 100 µM filters and then 70 µM filters to remove debris and centrifuged at 350 x g for 5 mins at room temperature. Supernatant was removed prior to red cell lysis (Merck) and counting. Typically single cell suspensions were surface stained and analyzed directly by flow cytometry. Cells for cryopreservation were stored in recovery media® (Thermo Fisher) and frozen at −80 o C in overnight before transfer to liquid nitrogen storage.
PBMC from NSCLC patients were isolated from EDTA anti-coagulated whole blood samples collected 1 day prior to surgery and isolated using lymphoprep (Stemcell technologies). PBMC from healthy donors were obtained from consented adults in accordance with local regulations. When not used immediately PBMC were cryopreserved in liquid nitrogen.

Generation of T cell conditioned media
Sections of tumor samples around 1 cm 3 were embedded in low melting point agarose and 400 µM slices cut using a Compresstome® VF-300-0Z vibrating microtome (precisionary). Individual slices were cultured in 24 well plates with media containing 3000 IU IL-2 (Gibco) until cells grew out to cover the bottom of the well (around 1 week). Cells were maintained in IL-2 containing media at a density of 0.8-2 x 10 6 /ml. T cells (>98% CD3+) were plated in 12 well plates at 5 x 10 5 /ml and stimulated with anti-CD3/anti-CD28 (both 1 µg/ml Bio-Xcell/Biolegend) for 48 hrs. After 48 hours cells were harvested centrifuged at 350 x g for 5 minutes and supernatants were drawn off, filtered through 0.22 um syringe filters and stored at −20 until use. Conditioned media were added to CAF cultures at a 1:1 vol:vol ratio (unsupplemented CAF cultures had half the media replaced at the same time to control for dilution effects).

Generation of CAF lines
Single cell suspensions from NSCLC tumors were incubated overnight in DMEM 100 U/ L penicillin/streptomycin, 2 mM L-glutamine and 10% FCS (all Gibco). Non-adherent cells were washed away and remaining cells grown to confluence in media supplemented with 1 X Insulin-Transferrin-Selenium. At passage cells were washed in PBS prior to treatment with 0.5% Trypsin EDTA for three minutes at 37°C to lift cells. At the third passage (when uniform CAF lines were free of non-CD90+ cells) CAF lines were cryopreserved. All CAF lines used were at passage 3-6.

Flow cytometry
Cells were washed in PBS, dead cells stained with Zombie UV (Biolegend) according to the manufacturers instructions. Fc receptors were blocked with Trustain FcX (Biolegend) prior to staining with the indicated monoclonal antibodies in PBS 2% FCS (see details in supplementary methods). After staining cells were fixed in 2% PFA (Biolegend). Intranuclear staining (for Foxp3/Ki67) was performed after permeabilization with Foxp-permeabilization buffer (Invitrogen). Intracellular cytokine staining was performed using BD cytofix/cytoperm buffers according to the manufacturers instructions. Prior to staining for intracellular cytokines cells were incubated for 4hrs with 1X Cell activation cocktail (containing Phorbol-12myristate 13-acetate, ionomycin, brefeldin A and monensin (invitrogen)) to stimulate cytokine production. All FACS data was collected on a 6 laser LSR (BD) and analyzed using Flowjo Software (BD). Populations of interest were downsampled to 4000 events and files were concatenated for tSNE analysis, exvivo tumor versus non-cancerous lung or in vitro treatment groups were identified using the sample ID parameter.

IHC/IFF
Formalin fixed paraffin-embedded slides of NSCLC resections (n = 22; 11 Adenocarcinoma, 8 squamous cell carcinoma), and one each of adenosquamous, large cell neuroendocrine and atypical carcinoid) were deparaffinised, rehydrated and antigen retrieval was undertaken with citrate buffer (Abcam ab64214) for 3 × 5 minutes in a microwave. Slides were processed with a commercial DAB staining kit (R&D Sytems, CTS019). Primary antibodies included FAP or CD3 incubated overnight at 4°C on sequentially cut slides. Secondary antibodies were added at RT for 1h and DAB was developed. Slides were counterstained with hematoxylin and mounted. Slides were scored for stromal FAP intensity or stromal CD3 presence on a 4-point scale; 0-no staining, 1-low staining, 2-moderate staining, 3-high staining.
For IFF, six slides with high FAP expression demonstrated by IHC had antigen retrieval undertaken with 0.125 mM EDTA buffer at 110°C for 30 minutes. Primary antibody concentrations were optimized to avoid cross-binding between the steps. Slides were incubated with Pan-CK at RT for 30 minutes followed by development using an AF488 Tyramide Superboost kit (Thermofisher, B40912). Microwave antigen retrieval with EDTA buffer was carried out before samples were sequentially incubated with primary antibody, species appropriate secondary, Qdot conjugation (followed by FAB blocking antibody where appropriate) and blocked for avidin & biotin (Vector Laboratories, SP-2001) and serum-free protein block (Dako, X0909) prior to next primary antibody. The following primary and Qdot conjugates were used; CD39 and Qdot 605, CD8 and Qdot 585, FAP and Qdot 565, CD103 and Qdot 705. Samples were then incubated with Qred for nuclear staining followed by rehydration and mounting. Slides were imaged on Vectra Polaris Imaging System (Akoya Biosciences). Imaging cubes were analyzed using inform software (Akoya Biosciences, v 2.1.10) with masks created for tumor and stromal areas, and the number of cells per area quantified.