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TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor respo

Issuing time:2021-04-16 11:54

Abstract

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8+ T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8+ T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors.

Introduction

Tissue resident Batf3-dependent type 1 dendritic cells (DCs), named cDC1, excel in activation of CD8+ T cells to exogenously acquired antigens, and are essential to detect tumors and to mediate rejection of nascent lesions1,2,3,4. Moreover, intratumoral cDC1 support the function of T cells during T-cell mediated immune clearance5,6, and their abundance in several human cancers correlates with better prognosis5,7,8. cDC1 accumulation in tumors depends on NK cells and CCL58,9 and cause, in turn, recruitment and expansion of tumor specific T cells in situ7,10. These positive correlations likely reflect residual cDC1 activity, as many reports suggest that DCs are negatively affected by signals present in the tumor immune microenvironment (TME)10,11,12,13,14,15,16,17,18.

Previous work established that cDC1-mediated anti-tumor immunity primarily relies on engulfment of dying tumor cells to acquire cell-associated antigens5,6,16. Still, the receptors that control acquisition of tumor cell-associated antigens by cDC1 in tumor tissues, and whether this process is targeted by the suppressive TME has not been investigated. Putative receptors such as CD36, DEC-205, DNGR1/Clec9A, SCARF1 and AXL control recognition/binding of dying cells and cross-presentation of cell associated antigens by cDC119,20,21,22,23,24, in various contexts. TIM4 (T-cell immunoglobulin mucin-4), that recognizes phosphatidylserine (PtdSer) on apoptotic cells (AC), is predominantly expressed on resident peritoneal macrophages25,26,27 and some subsets of DCs28,29,30,31,32. The expression of these phagocytic receptors on tissue resident cDC1, their contribution to tumor antigen uptake and modulation during tumor growth remains however poorly explored.

In this study, we use a transplantable orthotopic KP model of adenocarcinoma of the lung (derived from Kras/p53 KP mice16,33,34) to study the ability of lung resident cDC1 to engulf cell-associated antigens during tumor development. cDC1 actively engulf and present cell-associated antigens to CD8+ T cells at early stages of tumor development, yet antigen uptake, presentation and T cell activation is inhibited in advanced tumors. Gene expression profiling show high expression of TIM4 in normal lungs cDC1 and its downregulation in cells associated with late tumors. In addition, profiling across lung phagocytes indicates that, unlike what has been described in other tissues, TIM4 is not expressed on lung resident macrophages or macrophages associated to lung tumors. Receptor blockade and cDC1 genetic deletion of TIM4 regulates the capacity of cDC1 to engulf dying cells and tumor cells in the lung, and to induce cancer specific CD8 responses that control tumor growth. In human lung adenocarcinomas a combined cDC1/Timd4 gene signature predicts survival in early stages patients, and responses to PD-1 treatment in a cohort of lung adenocarcinoma patients.

Results

cDC1 fail to uptake cell associated antigens in late lung tumors tissue

To explore uptake of cell associated antigens by lung resident cDC1 during tumor development, we established orthotopic tumors by intravenous inoculation (i.v.) of KrasG12D/+;p53−/− primary tumor cells (KP)33,34. We defined two time points for analysis, early tumors (KP(e), days 10–14) when the tumor area occupies between 5–10% of the lung parenchyma and a late time point, when nodules occupy more than 30% of the total lung area (KP (l), days 28–35) (Supplementary Fig. 1a). Mice injected with PBS were used as control (normal lungs, nLung). Established tumors showed an increased frequency of neutrophils, unchanged frequencies of SiglecF+ CD11c+ alveolar macrophages (AM) and Ly6C monocytes and decreased frequencies of T cells (Supplementary Fig. 1b). The DCs compartment (Supplementary Fig. S1c–e) in late tumors was characterized by an increment of mo-DCs and cDC2 over cDC1, in line with previous data16,18. Expression of cDC1 lineage specific and maturation markers was not affected in tumor-associated cDC1 (Supplementary Fig. 1f). To examine the ability of cDC1 to engulf dying cells, we first administered labeled apoptotic thymocytes intratracheally (i.t.) to control or tumor bearing animals. Quantification of fluorescence associated with phagocytes 2 h after inoculation showed a high uptake by AM and a lower uptake by cDC1 and cDC2 (Figs. 1a and S2a). Uptake by cDC2 and AM remained unchanged in early and late tumors as compared to controls, whereas uptake by cDC1 was significantly inhibited in late tumors (Fig. 1a). To directly examine the uptake of cellular tumor fragments in tissues, we generated a KP-BFP reporter line. At early time points after tumor inoculation we observed a higher BFP fluorescence in lung cDC1 than in cDC2 and AM, likely due to faster degradation of endocytic cargo in the latter subsets (Supplementary Fig. 2b)5,35. In more advanced tumors, BFP associated with cDC1 was significantly decreased whereas it remained unchanged in cDC2 and AM, suggesting a selective reduction in the capacity to engulf tumor cells by cDC1 (Fig. 1b and Supplementary Fig. 2b). To visualize the uptake at the singe cell level we next isolated cDC1 from control, early, and late KP tumor-bearing mice and incubated them ex-vivo with labeled apoptotic thymocytes. cDC1 from normal lung and early tumors captured and engulfed dying cells efficiently. In contrast, cDC1 isolated from late tumors were impaired in binding to and phagocytosis of dying cells (Fig. 1c, d and Supplementary Fig. 2c, d). We concluded that the TME of KP tumors inhibits the intrinsic capacity of cDC1 to engulf dying cells. cDC1 preferentially cross-present tumor associated antigens acquired via phagocytosis to activate tumor specific CD8+ T cells5,6,24. To understand the impact of defective uptake on the capacity to cross-present antigens, we generated a KP-OVA line expressing low levels of the model antigen OVA (OVA, clone G11, Supplementary Fig. 3A). KP-OVA tumors grew less than the parental KP tumors and showed smaller nodules at late time points (Supplementary Fig. 3b). Depletion of CD8+ T cells during the first 2 weeks upon tumor challenge led to a dramatic increase in tumor burden indicating that tumor containment was mediated by CD8+ T cells (Supplementary Fig. 3c). Staining with the 25D-1.16 antibody to detect cross-presented peptide:MHC OVA on tissue cDC1 showed high antigen density in early KP-OVA tumor bearing lungs and low signal in late tumor bearing lungs (Fig. 1e and Supplementary Fig. 3d). To further probe changes in the capacity to cross-present tumor associated antigens we isolated cDC1, cDC2, and AM from mice bearing early or late KP-OVA tumors. In early tumors, cDC1 were the only subset capable of inducing efficient activation of OVA specific CD8, in line with previous reports (Fig. 1f and Supplementary Fig. 3e)3,5,6,36. This property was significantly inhibited in cDC1 from late tumors indicating that cross-presentation of antigens picked-up in tumor tissue is lost in advanced tumors (Fig. 1f). To confirm suppression of cross-presentation of cell-associated antigens, we delivered OVA-loaded apoptotic thymocytes in the lung. CD8 T cells proliferated in control and early tumor bearing animals but not in in late tumor bearing animals (Fig. 1g). Spontaneous IL-12 production in vivo was slightly decreased yet cells responded efficiently to ex-vivo restimulation suggesting that cytokine signals by DC1 are not deeply compromised (Supplementary Fig. 3f, g). In addition, peptide pulsed cDC1 induced robust activation of OVA specific CD8+ T cells, indicating normal interaction with T cells and no impairment in co-stimulatory signals (Supplementary Fig. 3i). Cross-presentation of soluble protein antigens delivered ex-vivo was diminished in late tumor cDC1, in line with previous reports13(Supplementary Fig. 3j). Thus, by dissecting individual steps of DC1 activity, we evidenced a primary defect in antigen capture by tumor tissues resident cDC1.

Fig. 1: Uptake of cell associated tumor antigens by cDC1 is inhibited in advanced lung tumors.

a Control mice (nLung), or mice carrying early (e) or late (l) KP tumors were challenged i.t. with CTV labeled apoptotic thymocytes. Two hours later total lung cell suspensions were analyzed by flow-cytometry to assess the fraction of phagocytes associated to fluorescence. Representative flow cytometry plots of cDC1 (gated on CD45+/Cd11c+/SiglechF/MHC-IIhigh/CD11b) and quantification of uptake, expressed as percentage of CTV+ cDC1. Data were examined over two independent experiments: nLung n = 8, KP(e) n = 5, KP(l) n = 5, one-way ANOVA followed by Tukey’s post-test. bMice were challenged with KP-BFP cells. Lungs were harvested at early or late time points after challenge to prepare total lung cell suspensions. Representative flow cytometry plots displaying the percentage of BFP+ cDC1 and the corresponding quantification. Data are pooled from two experiments with n = 8 mice per group, two-tailed unpaired t-test. c, d Sorted-cDC1 cells from nLung, early or late whole tumor tissues were incubated for 30’ (binding, c) or 2 h (uptake, d) with CFSE labeled apoptotic thymocytes. c Representative micrographs showing cDC1 binding to thymocytes. The percentage of cDC1 in contact with at least one thymocyte was quantified on 30 individual cells acquired on separated fields in two independent experiments. d Representative micrographs showing thymocytes uptake. The percentage of cDC1 that has engulfed at least one cellular fragment is plotted as uptake. Data are representative of 30 cells/condition in two independent experiments. In c and d one-way ANOVA followed by Tukey’s post-test. e Tumors were induced by i.v. injection of KP-OVA expressing cells. Cross-presentation by lung cDC1 was detected by flow cytometry on whole lung tissues by labeling with peptide:MHC OVA specific antibody (25D1.16) (gated on CD45+/Cd11c+/Siglech-F/MHC-IIhigh/CD11b). Representative histograms and quantification of the percentage of positive cells based on the gate depicted on histograms. Data are from two independent experiments: KP(e) n = 4, KP(l) n = 3 (cDC1 were pooled from two mice for analysis), two-tailed unpaired t-test. f cDC1 were sorted from whole lung cell suspension of early and late KP-OVA bearing tumors and mixed with CFSE-labeled OVA specific T cells (OT-I). Representative CFSE peaks and quantification of proliferation (cells undergoing at least two cycles of proliferation over total T cells). Each point corresponds to cDC1 isolated from KP(e) n = 3 or KP(l) n = 4 mice in three independent experiments, two-tailed unpaired t-test. g CSFE labeled OT-I were transferred into nLung, early, and late tumor bearing animals. OVA-loaded apoptotic thymocytes were administered i.t. and T cell proliferation in the mediastinal lymph node (MLN) was evaluated 2 days later. Data are from two independent experiments, nLung n = 9/7, KP(e) n = 4 or KP(l) n = 7, one-way ANOVA followed by Tukey’s post-test. All data are expressed as mean ± SEM. Source data are provided as a Source Data file.

Lung cDC1 fail to activate CD8+ tumor responses in late tumors

To examine whether reduced antigen presentation by cDC1 along with tumor progression correlates to impairment of CD8+ T cell responses in vivo, we tracked endogenous CD8+ T cell over time in the blood of animals challenged with KP-OVA. Circulating IFN-γ+ CD8+ T cells were observed by day 14 after challenge, peaked at day 21 and declined to almost baseline by day 28 (Fig. 2a, b). The same trend was observed in lung tissue CD8+ T cells when comparing early and late time points (Fig. 2c). Immunohistochemistry showed high density of CD8+ T cells in early nodules as opposed to few CD8+ T cells confined at the tumor border in late tumors (Fig. 2d). Because loss of T cell responses may have resulted from selection of OVA negative tumor cells in late tumors33, we verified OVA expression by flow cytometry and immunohistochemistry. The percentage of OVA expressing CD45 cells was similar in early and late tumors (Supplementary Fig. 4a). The intensity of OVA staining was similar in small nodules in early tumors and large nodules in late tumors (Supplementary Fig. 4b), indicating that the antigen is still expressed in advanced lesions. To distinguish cell intrinsic exhaustion of CD8+ T cells from defective antigen presentation, we analyzed proliferation of naïve CFSE labeled CD8+ OVA specific T cells transferred into early and late tumor bearing-lungs. Proliferation was induced efficiently in early tumors but not in late tumors, suggesting lack of priming by APC in situ (Fig. 2e, f). In addition, proliferation was reduced in Batf3−/− mice3, indicating that cDC1 are required for full T cell activation in this model (Fig.2g, h). Together these data indicate that inefficient priming of tumor specific CD8+ T cells in established tumors coincides with loss of antigen presentation by tissue resident cDC1.

Fig. 2: CD8+ T cell responses and cross-presentation of tumor antigens by cDC1 are inhibited in late tumors.

a, b Representative plots and quantification of the kinetic of IFN-γ production by endogenous blood CD8+ T cells in KP-OVA challenged mice. Data are from two independent experiments at day 7–28 or 3 independent experiments at day 14–21. In b Kruskal–Wallis followed by Dunn’s post-test day 7 n = 6, day 14 n = 15, day 21 n = 6, day 28 n = 4 mice. c Percentage of IFN-γ producing endogenous CD8+ T cells in lungs bearing early or late KP-OVA tumors. KP(e) n = 7, KP(l) n = 8 mice in two independent experiments, two-tailed unpaired t-test. d CD8+ T cells localization in early and late KP-OVA tumors. Scale bar 200 µm. Quantification of the density of CD8+ T cells as a function of the nodule area was calculated automatically on three consecutive sections/sample. Data are from KP(e) n = 11, KP(l) n = 7, two-tailed unpaired t-test. e, f Proliferation profile of OT-I in the MLN (e) or lung (f) of mice bearing early or late KP-OVA tumors was measured 2 days after transfer. Proliferation index (cells that underwent at least two cycles of proliferation) and absolute numbers of proliferated cells are depicted. In e % of proliferation KP(e) n = 12, KP(l) n = 7; absolute numbers KP(e) n = 6, KP(l) n = 4. In f %proliferation KP(e) n = 12, KP(l) n = 7; absolute numbers KP(e) n = 7, KP(l) n = 5. Data are from three independent experiments, two-tailed unpaired t-test. g, h KP-OVA tumors were induced in WT and Batf3−/− mice. g Proliferation and IFN-γ production of adoptively transferred OVA specific CD8 T cells and IFN-γ production by endogenous CD8+ T cells in the lung. %proliferation WT n = 3, Batf3−/− n = 3; IFNγ+OTI (%) and IFNγ+CD45.2+CD8(%) WT n = 4, Batf3−/− n = 4; two-tailed unpaired t-test. h IFN-γ production by OT-I in MLNs. Data represent one of two independent experiments (n = 4 mice/group), two-tailed unpaired t-test. All data are expressed as mean ± SEM. Source data are provided as a Source Data file.

Gene expression profiling shows loss of TIM4 in lung tumor associated cDC1

To explore the molecular basis of loss of antigen acquisition in late tumor, we compared the transcriptional profile of cDC1 isolated from control lung (nLung) or late KP tumor-bearing lungs. Factors defining the cDC1 lineage were similarly expressed in control and tumor associated KP cDC1, indicating that cDC1 maintained their identity (Fig. 3a). Gene expression analysis returned more than 40 differentially expressed transcripts (false discovery rate ≤5% and absolute fold change ≥2; Supplementary Fig. 5a and Supplementary Table 1). Transcripts enriched in tumor-associated cDC1 belonged preferentially to the interferon alpha pathway, intracellular trafficking and antigen processing (Supplementary Fig. 5b–d). We focused on phagocytic receptors to find a mechanism for loss of antigen uptake. As shown in Fig. 3b, c; Timd4, encoding for the PtdSer receptor TIM425, was highly expressed in nLung cDC1 and strongly downregulated in late tumors. Other receptors implicated in binding and engulfment of apoptotic cells (Clec9a, Cd36, Axl, Lrp11, Ly75, Stab1, MerTK, Marco,Tyro3, Scarf1) showed no significant modulation, with the exception of Cd300lf, a second receptor for PtdSer known to inhibit efferocytosis in DCs37, and Cd209a(DC-SIGN), which was slightly down-modulated. Since selective high expression of TIM4 by lung cDC1 had never been reported we validated our data by analysis of publicly available datasets. Tabula Muris single cells dataset from normal lung tissues (Nature. https://doi.org/10.1038/s41586-018-0590-4) confirmed co-expression of Timd4 with markers of cDC1 in a cluster of myeloid cells (Fig. 3d). Importantly, analysis of a recently generated scRNA dataset from control and KP tumor murine lungs16 confirmed selective expression of TIM4 in the cDC1 subset and a significant decrease in tumors condition (Fig. 3e). Flow cytometry confirmed high expression of TIM4 protein in lung cDC1 and lack of expression in the other lung phagocytic subsets (Supplementary Fig. 6a, b). Further profiling across multiple mouse tissues38 showed the expected high expression in tissue resident macrophages, in a consistent fraction of Langerhans cells in the skin and in a fraction of dermal cDC132. Slight TIM4 expression was present on liver cDC1, on a small fraction of migratory cDC1/cDC2 in mediastinal LNs and in splenic and bone marrow cDC1/cDC2, confirming the highest expression on lung cDC1 in naive mice (Supplementary Fig. 6c, d).

Fig. 3: Selective cDC1 expression of the PtdSer receptor TIM4 in lung phagocytes and downregulation in tumors.

a cDC1 were isolated from nLung or late KP tumors to analyze transcriptional profiles. The graph shows expression of cDC1 and cDC2 lineage specific genes in nLung-sorted and tumor-sorted cDC1. Data are expressed as mean ± SD with nLung n = 4, KP(l) n = 3. b Volcano plot showing the fold change of genes encoding scavenger receptors (significantly modulated genes are depicted in red and blue, using a q-value < 5%, a false discovery rate (FDR) ≤ 5% and a fold change ≥2 or ≤2), using the Significance Analysis of Microarray (SAM) algorithm. c The dot-plot shows the relative abundance of genes in b using a color and size scale of linear expression values. d T-distributed stochastic neighbor embedding (tSNE) plots of single cells expression profiles in mouse lungs (Tabula Muris). Colors represent clusters of different subsets based on global similarity of gene expression. The cluster corresponding to DC-macrophages was further analyzed to assess co-expression of cDC1 markers and Timd4 transcripts, as depicted in the boxes. e scRNA seq of naive and KP lung tumors from16 were analyzed to visualize Timd4-expressing cells in three subpopulation of lung DCs. Violin plots show expression of Timd4 in cDC1, cDC2, and mregDCs. Statistical analysis was performed using Seurat R package, taking in consideration both number of expressing cells and levels of expression. f Representative histograms and quantification of the percentage of cells expressing TIM4 in nLung, early and late lung tumors (the gate for identification of positive and negative cells is depicted in the histogram). Data are from nLung n = 3, KP(e) n = 6, KP(l) n = 9 mice. Significance was determined by one-way ANOVA followed by Tukey’s post-test; data are expressed as mean ± SEM. g Representative histograms of TIM4 expression on total CD45 and CD45+ cells and all lung phagocytic subsets in nLung and late KP lungs. Alveolar macrophages (AM), interstitial macrophages (IM), tumor associated macrophages (TAM), monocytes (Mono). Each peak is overlaid on the corresponding FMO controls (black line). Source data are provided as a Source Data file.

To verify the kinetic of TIM4 downregulation in tumors we analyzed early stages that correspond to functional cDC1. We observed a slight, not significant, trend of reduction (Fig. 3f), providing a causal link between receptor levels and engulfment capacity (Fig. 1a, b). Interestingly, orthotopic KP injection in one lobe was sufficient to induce a slight reduction in the contra-lateral lobe (Supplementary Fig. 7a). Expression was not detected in any of the other lung phagocytes in late tumors, including TAMs, nor in the stroma or tumor cells (CD45 negative fraction) (Fig. 3g), in contrast to previous data in breast tumors and melanoma29,31. A slight reduction in TIM4 was observed also in early autochthonous KP tumors (Supplementary Fig. 7b), and in cDC1 associated to Lewis lung carcinoma tumors (Supplementary Fig. 7c).

TIM4 on lung cDC1 control uptake of cell-associated antigens

TIM4 plays a non-redundant role for tethering and engulfment of apoptotic cells in peritoneal macrophages27,39. Whether TIM4 may function as a receptor for dead-cell uptake in the DCs compartment or in cDC1, is poorly documented28. To analyze the role of TIM4 during engulfment of BFP-KP cells, we first verified exposure of phosphatidylserine by tumor cells growing in lung tissues (Supplementary Fig. 7d). We next administered TIM4 blocking antibodies or isotype controls during the initial phases of tumor engraftment. Receptor blockade was effective and did not alter maturation or absolute numbers of phagocytes excluding bystander effects (Supplementary Fig. 7e). TIM4 blockade caused a significant reduction in the fraction of cDC1 engulfing BFP tumor cells, without affecting the uptake by the other subsets (Fig. 4a and Supplementary Fig. 7f). We further tested the impact of TIM4 blockade on phagocytosis of dying thymocytes. TIM4 blocking antibodies in nLung selectively reduced the uptake by cDC1 almost to the level found in KP tumors, and had no additive effect when administered to tumor lungs (Fig. 4b) (Supplementary Fig. 7g). Uptake was reduced as well in cDC1 isolated from wt or Timd4−/− mice and incubated with apoptotic thymocytes labeled with pH Rodo, a probe that becomes fluorescent only under acidic conditions39,40 (Fig. 4c). Blockade or genetic deletion of TIM4 impacted similarly on the subsequent step of antigen presentation. Blocking antibodies decreased antigen presentation of KP-OVA antigens (peptide:MHC OVA complexes labeling) by tissue cDC1 (Fig. 4d), and proliferation of adoptively transferred CD8 T cells into mice receiving OVA loaded apoptotic thymocytes (Fig. 4e). Finally, cDC1 isolated from early KP-OVA lungs of animals treated with TIM4 blocking Abs failed to induce proliferation of OVA specific T cells ex-vivo (Fig. 4f). The same reduction was observed when using Tim4−/− animals as recipient (Fig. 4g). We concluded that TIM4 expression in lung cDC1 is required to capture and present cell-associated antigens in normal lungs and early tumor tissues.

Fig. 4: TIM4 controls capture and cross-presentation of cell-associated antigens in lung resident cDC1.

a Mice were challenged with KP-BFP and treated with blocking antibodies to TIM4 or isotype control as depicted. The uptake by lung cDC1 was quantified 5 days after tumor challenge. Dot plot and quantification of one representative of two experiment with n = 3 mice/group are shown. bControl mice or late KP tumor bearing mice were pre-treated with isotype control or αTIM4 antibodies and challenged with CTV-labeled apoptotic thymocytes. The percentage of lung phagocytes associated to fluorescence was evaluated by flow-cytometry 2 h after challenge. Representative plots and quantification of two independent experiments are shown, Isotype n = 7, Isotype KP(l) = 9, αTIM4 n = 8, αTIM4-KP(l) n = 3. c Uptake of pH rodo loaded thymocytes by lung cDC1 in WT and Timd4−/− mice. Representative histograms and quantifications (percentage and MFI of pH rodo+ cDC1) for one of three independent experiments with n = 3 mice/group are shown. dCross-presentation by lung cDC1 (labeling with 25D1.16 antibody specific for MHC class-I:OVA peptide complex) in day 7 KP-OVA bearing animals treated with TIM4 blockade. Representative histograms showing MFI values and quantification of five animals/group in two independent experiments. e In vivo proliferation of adoptively transferred OT-I induced by i.t. injection of OVA-loaded apoptotic thymocytes, in the presence of TIM4 blocking antibodies or isotype control as depicted in the scheme. Graphs show frequencies and absolute numbers of proliferated CD8+ T cells in the MLN (at least one cycle of proliferation). Data are representative of two independent experiments. % proliferation Isotype n = 8, αTIM4 n = 7; Absolute numbers five mice/group. f Ex-vivo OT-I proliferation induced by cell-sorted cDC1 from day 7 KP-OVA lung tumors under TIM4 blockade or isotype treatment. Representative dilution profile and quantification from three independent experiments (each point corresponds to the pool cDC1 from three mice), Isotype n = 9, αTIM4 n = 7. g As in f, cDC1 were isolated from day 7 KP-OVA lung tumors induced in WT or Tim4d−/−animals (n = 5 mice in two independent experiments). All data are depicted as mean ± SEM. Significance was determined by one-way ANOVA followed by Tukey’s post-test for b, or two-tailed unpaired t-test. Source data are provided as a Source Data file.

TIM4 controls initiation of anti-tumor CD8 immunity

To directly assess the impact of TIM4-mediated engulfment on anti-tumor CD8 responses we administered TIM4 blocking antibodies during tumor challenge. TIM4 blockade prevented induction of OVA specific endogenous effector CD8+ T cells in the peripheral blood of KP challenged mice (Fig. 5a), and reduced the activation of adoptively transferred OVA specific T cells (Supplementary Fig. 7h). To further investigate the impact of TIM4 during T cell priming we inoculated KP-OVA tumors into wild-type or Timd4−/− animals. Numbers of IFN-γ+ CD8+ T cells at day 7 after challenge were significantly decreased in Timd4−/− mice as compared to WT controls (Fig. 5b). Moreover, absolute numbers of total CD8+ T cells and OVA pentamer CD8+ were diminished in the lung of Timd4−/− mice (Fig. 5c, d). To overcome potential bystander effects of systemic TIM4 blockade and pinpoint precisely on cDC1, we generated mixed bone marrow chimeras using mixtures of wild-type:Batf3/− or Timd4−/−Batf3−/− bone marrow (Supplementary Fig. 8a–c). IFN-γ production and accumulation of CD8+ T cells was decreased in animals carrying a specific deletion of TIM4 in the cDC1 compartment (Fig. 5e, f). Most importantly, containment of tumor growth was reduced in Timd4−/−:Batf3−/− chimeras as shown by more abundant lesions and larger nodules than in animals reconstituted with wild-type:Batf3−/− bone marrows (Fig. 5g). Thus, selective ablation of TIM4 in cDC1 reduces protective anti-tumor responses. We finally tested whether TIM4 contributes to rejection induced by immunogenic chemotherapy, which favor APC activation41,42. We used a combination of oxaliplatin and cyclophosphamide (oxa/cycl), previously shown to induce CD8+ T cell responses and rejection in this same KP model34. Animals were challenged with KP tumors and treated with oxa/cycl at day 7 and 14 after challenge (Fig. 6a). The drug increased CD8/CD4 ratio both in LNs and lung tissues and significantly reduced tumor burden with respect to untreated animals (Fig. 6b–d). The efficacy of oxa/cycl treatment was abrogated in Batf3−/− mice (Fig. 6e), indicating that responses induced by immunogenic therapy require cDC1 in this model. Of note, addition of TIM4 blocking antibodies one day before oxa/cycl administration resulted in reduced efficacy of immunogenic therapy (Fig. 6b–d), denoting that TIM4 plays a role during capture of drug-induced apoptotic cells to induce anti-tumor responses.

Fig. 5: TIM4 promotes anti-tumor CD8+ T cell responses and tumor control.

a Effect of TIM4 blockade on activation of endogenous CD8+ T cells in the blood of KP-OVA challenged mice. The percentage of IFN-γ+ CD8 and OVA specific-IFN-γ+ CD8 (Vα2/Vβ5+) as a function of total CD8 and Vα2/Vβ5+ T cells, respectively, at various time points after challenge. Isotype n = 5, αTIM4 n = 4, data represent one of two independent experiments. bd Activation of anti-tumor T cell responses in WT and Timd4−/ animals 7 days after challenge with KP-OVA. bRepresentative plots and frequencies of IFN-γ+ endogenous CD8+ T cells in MLN. c Absolute numbers of CD8+ T cells and d OVA-specific CD8+ T cells (pentamers+) accumulated at tumor site. Data are representative of two experiments (n = 5 mice/group). eg Lethally irradiated CD45.1 WT mice were injected with a 1:1 mixture of CD45.2 bone marrows of the indicated genotypes to generate bone marrow chimeras. Ten weeks later mice were inoculated with KP-OVA tumors. eRepresentative plots and frequencies of IFN-γ+ CD8+ T cells in MLN 10 days after tumor challenge (n= 6 mice/group). f Absolute numbers and quantification of CD8+ T cells accumulated at tumor site with Batf3−/−;WT n = 6 and Batf3−/−;Timd4−/− n = 5 mice. g Tumor growth in bone marrow chimeras was assessed 28 days after tumor challenge. Representative sections and quantification of tumor nodules calculated automatically on four consecutive sections of three lobes/animal and expressed as a function of the total lobe area. Each value in the plot represents the average of the three lobes and four sections for each mouse. All data are mean ± SEM. Significance was determined by two-way ANOVA followed by Sidak’s post-test for a, two-tailed unpaired t-test for bf, and two-tailed Mann–Whitney test for g. Source data are provided as a Source Data file.

Fig. 6: TIM4 is required for tumor rejection induced by immunogenic chemotherapy.

a Scheme of experimental design. Mice were challenged with KP cells and left untreated or treated with oxa/cycl and anti-TIM4 or isotype as depicted in the scheme. b, c Analysis of the CD8/CD4 T cell ratio in the MLNs (b), Untreated n = 10, Oxa/Cycl+Isotype n = 9, Oxa/Cycl+αTIM4 n = 9; and in the lungs (c), Untreated n = 10, Oxa/Cycl+Isotype n = 10, Oxa/Cycl+αTIM4 n = 9; of mice 2 weeks after the last treatment. Data are from two independent experiments. d Effect of TIM4 blockade on the efficacy of the immunogenic chemotherapy. Tumor burden was assessed at day 28 by calculating the area covered by tumor nodules on four consecutive sections of three lobes/animal and expressed as a function of the total lobe area. Results are from untreated n = 13, Isotype+Oxa/Cycl n = 14 and Oxa/Cycl+αTIM4 n = 14 in two independent experiments. e WT and Batf3−/− mice were challenged with KP cells and treated with oxa/cycl or left untreated. Treatment efficacy is expressed as the percentage of reduction in tumor area over not treated control calculated in 14 WT and 4 Batf3−/− mice. All data are expressed as mean ± SEM. Significance was determined by one-way ANOVA followed by Tukey’s post-test for bd and two-tailed Mann–Whitney test in e. Source data are provided as a Source Data file.

TIM4 expression in lung adenocarcinomas correlates with signatures of protective immune cells and predicts responses to PD-1 checkpoint blockade

To investigate the relevance of our observations in human cancers, we explored TIM4 expression in the Cancer Genoma Atlas (TCGA) dataset. We found a correlation between Timd4 transcripts and a gene signature specific for human cDC1 and CD8 effector T cells in early stage patients (Fig. 7a) (Supplementary Table 2 and refs. 7,8). To further explore the potential prognostic impact of TIM4, we analyzed Kaplan–Meier survival curves in the TCGA dataset of lung adenocarcinoma. Interestingly, early-stage patients expressing a high cDC1 signature complemented by Timd4 showed a small yet significant advantage in survival (Fig. 7b). Based on these observations, we reasoned that TIM4 may be important to determine responsiveness to therapies that promote CD8 responses. To this goal, we analyzed a recent dataset comparing the transcriptional profiles before treatment of responders and non-responders to anti PD-1 immunotherapy (GSE126044). Remarkably, Timd4 expression was significantly higher in patients who responded to therapy than in non-responders (Fig. 7c), suggesting that TIM4 confers an advantage under the selective pressure of immunotherapy. A cDC1 signature and a CD8 effector signature had a similar predictive power of positive responses to therapy (Fig. 7d), indicating that Timd4 could be an additional biomarker to identify tumors with intact antigen presentation.

Fig. 7: Timd4 expression correlations and predictive value in human lung adenocarcinoma.

a Correlation between Timd4 transcripts and gene signatures for cDC1 and cytotoxic T cells (gene signatures described in Supplementary Table 2) within lung adenocarcinoma cases (LUAD) in TCGA. Pearson correlation coefficient in the cor.test function of R stats package with two.sided as alternative hypothesis. b Kaplan–Meier survival curves showing probability of survival for stage-I LUAD cases from the TCGA dataset (n = 219). The two groups with either high or low expression of cDC1 or cDC1 + Timd4, were identified based on the classification rule described in the ref. 60 and detailed in methods. Survival curves were compared using the log-rank (Mantel–Cox) test. c, dPatients from cohort GSE126044 that responded or not to PD-1 therapy were classified based on baseline pre-treatment expression of Timd4 (c), cDC1 and CD8 cytotoxic T cells signatures (d). Pvalues are calculated using the two-tailed Mann–Whitney test; responder n = 5, Non-responder n = 11. Source data are provided as a Source Data file.

Discussion

Cells of the myeloid lineage have been delineated with increasing precisions in the TME revealing their complexity, plasticity, and key roles in promoting or restricting tumor growth. Among these, cDC1 have emerged as invariably linked to positive outcomes across multiple preclinical models and human tumors5,6,7,8,16. A key function enabling cDC1 anti tumoral properties is the efficient capture and internalization of tumor cell fragments for cross-presentation to CD8+ T cells5,6,19,20,21,36,43,44. Here, we show that this exquisite cDC1 property is targeted by the microenvironment of lung adenocarcinoma. Past investigations to explain suppression of DCs activity in the TME have mostly focused on post internalization events like processing of soluble antigen, cellular metabolism, reduced maturation10,11,12,14,45. In contrast, the mechanism underlying acquisition of tumor antigens in tissues, and its modulation during tumor progression have been poorly explored. By comparing cDC1 resident in healthy lung tissues to those exposed to early or late lung tumors, we have been able to trace the evolution of antigen capture as a function of tumor progression. We uncover that cDC1 are key to sample tumor antigens and initiate tumor specific CD8 responses in early lesions. Instead, at later tumor stages, antigen acquisition become inefficient, coinciding with loss of antigen presentation and fading of T cell responses. Thus, a major hurdle to sustain tumor specific CD8 responses is suppression of antigen acquisition by the most efficient cross-presenting subset in tissues.

By analyzing expression of scavenger receptors, we identified the loss of the PtdSer receptor TIM4 as one major mechanism underlying cDC1 deactivation in lung murine tumors. Additional cell intrinsic changes, such as defective cross-presentation and impaired cytokine production, likely contribute to overall cDC1 suppression. However, by impinging on the initial step of antigen acquisition, we propose that loss of TIM4 may dominate over downstream events. The uptake of dead cells has been attributed to selective expression of receptors, that are capable to bind to and promote engulfment of dying cells in cDC119,20,21,22,23,24. These studies were mostly performed using lymphoid resident cDC1 and model antigens, whereas the receptors involved in acquisition of tumor antigens by cDC1 resident in different tumor tissue have been less investigated. TIM4 binding to PtdSer in macrophages is key to engulf and clear dying cells and its genetic deletion promotes development of autoimmunity40,46,47,48. The pattern of expression and specific function of TIM4 in the DCs compartment has remained controversial, likely because of a lack of proper definition of DCs subset in past analysis26,28,29,49,50,51. To fill this gap, we performed an extensive characterization of DCs subsets across mouse tissues using recently established gating strategies38. Besides the well-established expression in most tissue resident macrophages, we found a selective expression of TIM4 in mouse lung cDC1 that was higher than in any other tissues DCs. Curiously, alveolar lung macrophages are among the few resident macrophages that do not express TIM4. It will be extremely intriguing to identify the tissue factors that control transcriptional and epigenetic regulation of TIM4 expression52,53 in steady state myeloid cells. A second important issue relates to the function of TIM4 in tumors and the mechanism behind receptor downregulation. TIM4 expression was reported to be upregulated in macrophages and DCs infiltrating melanoma and mammary mouse tumors, and to be associated with suppression of immune responses in colorectal mouse models29,31,54. In contrast, mouse lung tumors do not induce recruitment of myeloid cells expressing TIM4 but rather suppress its expression on cDC1, ablating T cell priming. These observations suggest a highly specific tissue and context dependent regulation, that would be important to investigate in more tumor types and human tumors. A recent report showed decrease of TIM4 and consequent loss of clearance in aging macrophages caused by p38 activation55, suggesting a possible mechanism of receptor loss in cDC1.

In this study, we used blocking antibodies and genetic deletion to demonstrate the key role of TIM4 in engulfment of dying cells and tumor cells and cross-presentation of cell associated antigens by lung cDC1. Moreover, TIM4 blockade inhibited responses to a combination of immunogenic drugs previously shown to induce T cell responses in this same tumor model34,56, indicating its role in promoting processing of released antigens. Together these evidences underscore the role of TIM4 in cDC1 as a PtdSer receptor implicated in capture of tumor antigens in cDC1, which contributes to surveillance of nascent tumors and initiation of tumor protective responses in mouse lung tumors. These findings somehow conflict with a previous study showing that TIM4 induces degradation of tumor antigens by autophagy, thereby inhibiting immunity29. This discrepancy likely arises from the different antigen processing machinery in bone-fide cDC1 as opposed to macrophages or CD11c/MHC-IIhigh cells and the different tumor tissue analyzed.

Gene expression from the Cancer Genoma Atlas (TCGA) showed a correlation between Timd4 transcripts and a gene signature for human cDC18, and effector CD8 T cells and better survival for patients expressing high levels of cDC1 and Timd4. In addition, a small trial showed a better response to checkpoint blockade in patients, suggesting an association between TIM4 expression and protective immune responses in human lung tumors. Further analysis of scRNA-seq data, flow cytometry and immunohistochemistry are needed to establish the precise pattern of expression across human tissues and its significance for the human disease.

In conclusion, this study identifies TIM4 as a lung cDC1 phagocytic receptor implicated in immune surveillance of early murine tumors, that is targeted at later stages of tumor progression impairing antitumor responses. Initial evidences support the potential of TIM4 as a biomarker for stratification of human lung tumors.

Article classification: Biological abstract
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