ARTICLE https://doi.org/10.1038/s41467-021-21445-4 OPEN Acquired cancer cell resistance to T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation Enrique J. Arenas 1,2,8, Alex Martínez-Sabadell 1,8, Irene Rius Ruiz 1,2, Macarena Román Alonso 1, Marta Escorihuela1, Antonio Luque1, Carlos Alberto Fajardo3, Alena Gros3, Christian Klein 4 & Joaquín Arribas 1,2,5,6,7 ✉ 1234567890():,; Immunotherapy has raised high expectations in the treatment of virtually every cancer. Many current efforts are focused on ensuring the efficient delivery of active cytotoxic cells to tumors. It is assumed that, once these active cytotoxic cells are correctly engaged to cancer cells, they will unfailingly eliminate the latter, provided that inhibitory factors are in check. T cell bispecific antibodies (TCBs) and chimeric antigen receptors (CARs) offer an opportunity to test this assumption. Using TCB and CARs directed against HER2, here we show that disruption of interferon-gamma signaling confers resistance to killing by active T lympho- cytes. The kinase JAK2, which transduces the signal initiated by interferon-gamma, is a component repeatedly disrupted in several independently generated resistant models. Our results unveil a seemingly widespread strategy used by cancer cells to resist clearance by redirected lymphocytes. In addition, they open the possibility that long-term inhibition of interferon-gamma signaling may impair the elimination phase of immunoediting and, thus, promote tumor progression. 1 Preclinical Research Program, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona 08035, Spain. 2 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid 28029, Spain. 3 Tumor Immunology & Immunotherapy Group, VHIO, Vall d’Hebron Barcelona Hospital Campus, Barcelona 08035, Spain. 4 Roche Innovation Center Zurich, Roche Pharmaceutical Research and Early Development, Schlieren 8952, Switzerland. 5 Department of Biochemistry and Molecular Biology, Universitat Autónoma de Barcelona (UAB), Bellaterra 08193, Spain. 6 Cancer Research Program, IMIM (Hospital del Mar Medical Research Institute), Barcelona 08003, Spain. 7 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain. 8These authors contributed equally: Enrique J. Arenas, Alex Martínez-Sabadell. ✉email: jarribas@vhio.net NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 T cell-engaging therapies, such as T cell bispecific antibodies As expected, resistance was not due to the downregulation of (TCBs)—which are functionally similar but structurally dif- HER2, as measured by Western blot or flow cytometry (Fig. 1i ferent to bispecific T cell engagers, BiTEs—or chimeric anti- and Supplementary Fig. 1d). Further, the binding of HER2-TCB gen receptors (CARs), are raising extraordinary expectations as future to parental and resistant cells was indistinguishable (Fig. 1j). treatments for virtually all cancers (for recent reviews on the subject, The sensitivity of resistant cells to different chemotherapeutic see refs. 1–11). Encouraging these expectations, TCBs and CARs have agents or to T-DM1, an antibody drug conjugate against HER2, been recently approved to treat some hematologic malignancies12–14. was similar to that of parental BT474 cells (Supplementary In contrast, TCBs and CARs against solid tumors tested to date have Fig. 1e). Thus, BT-R cells are specifically resistant to killing by re- failed to show clinical efficacy. This failure prompted intense research directed lymphocytes. and the subsequent identification of mechanisms of primary We reasoned that resistance could arise by inhibition of T cells and acquired resistance, including: (i) defective tumor infiltration or, alternatively, by intrinsic resistance of target cells. Regarding of redirected lymphocytes, (ii) immunosuppressive tumor environ- the first possibility, analysis of a panel of immune checkpoint ments, (iii) downmodulation of the antigen against which TCB inhibitors and co-stimulators19,20, showed no differences between or CAR are directed and, (iv) upregulation of immune checkpoints parental and resistant cells, except the downmodulation of B7-H4 (see, the aforementioned reviews). Different strategies are being (Fig. 1k); however, since this is an inhibitory molecule21, its implemented to overcome these mechanisms of resistance (reviewed downmodulation is not likely a cause of resistance. An array of 80 in ref. 15). cytokines, including immunosuppressive cytokines such as TGFβ All these mechanisms of resistance impinge on the ability of or IL10, revealed no major differences between cocultures T cells to reach cancer cells and/or on the inhibition of T cells. containing parental or resistant cells. The only factor differentially However, little is known about putative intrinsic mechanisms of secreted in cocultures with resistant cells was TIMP2 (Fig. 1l and resistance of cancer cells. That is, mechanisms deployed by tumor Supplementary Fig. 2a). Quantification of TIMP2 levels confirmed cells to resist killing by fully active and correctly engaged T cells. that it is upregulated in BT-R cells (Supplementary Fig. 2b). To TCBs and CARs targeting the cell surface receptor HER2 are a functionally characterize this observation, we downregulated the useful tool to identify such mechanisms. expression of TIMP2 by means of two specific short hairpin RNAs HER2 is a tyrosine kinase and, when overexpressed, a driver for (shRNAs). The resulting cells, which expressed similar levels of breast and gastric cancers16. The downregulation of over- TIMP2 to those of parental BT474 cells (Supplementary Fig. 2c), expressed HER2 may lead to tumor regression17. Thus, in prin- showed resistance to HER2-TCB similar to BT-R cells (Supple- ciple, HER2-driven cancer cells are not likely to downmodulate mentary Fig. 2d). Thus, we concluded that the overexpression of the expression of HER2 to escape TCBs or CARs, making them a TIMP2 by BT-R cells is unrelated to resistance. suitable experimental system to unveil mechanisms of intrinsic Treatment of cocultures of PBMCs and parental BT474 resistance to these therapies. cells with HER2-TCB augmented the secretion of several Using HER2-driven cell lines and patient derived xenografts cytokines characteristic of lymphocyte activation, such as (PDXs), and a TCB and CAR targeting HER2, here we describe TNF-α, Interferon-gamma (IFN-γ), IL13, or IL5. Supporting experiments that unveil a mechanism of resistance to redirected that lymphocyte activation is unaffected by resistant cells, the T cells. This mechanism should be taken into consideration secretion of theses cytokines was similar in assays including when designing strategies to increase the efficacy of cancer BT474 or BT-R cells (Supplementary Fig. 2e). Analysis of PBMC immunotherapies. proliferation, or of different makers of activity, namely, CD69 and granzyme B, confirmed that activation of lymphocytes was unaffected in assays with resistant cells (Supplementary Fig. 2f, Results g). We concluded that lymphocytes are equally activated by Model of resistance to cell killing by redirected lymphocytes. resistant cells. Thus, we focused on possible mechanisms of The structure of the HER2-TCB used in this study has been pre- intrinsic resistance of target cells. viously described18 (see also Supplementary Fig. 1a). Addition of picomolar concentrations of this HER2-TCB to cocultures of Transcriptomic analysis. Analysis by RNA-seq showed that 97 peripheral blood mononuclear cells (PBMCs) and the HER2- genes were acutely upregulated or downregulated in BT-R cells overexpressing BT474 cells led to the killing of the latter (Supple- compared to parental BT474 cells (≥4-fold; p < 10−5) (Fig. 2a). mentary Fig. 1b). It should be noted that the IC50 of HER2-TCB for Gene set enrichment analysis (GSEA) identified different biolo- a given ratio PBMC:BT474 varied depending on the donor of gical processes that differed between parental and resistant cells PBMCs. Similar differences have been previously observed18, and (Fig. 2b). We focused in the IFN-γ response (Fig. 2c), because it are likely due to alloreactions of different intensities, which parallel has been shown that it affects the antitumor response at multiple different degrees of HLA mismatch, as well as T cell fitness that may levels22. Analysis of the protein encoded by IRF1, a gene speci- depend on the donor. For the 1:1 ratio chosen for subsequent fically regulated by IFN-γ signaling23, confirmed that this experiments, the IC50 of HER2-TCB varied from ~40 to ~220 pM. pathway is impaired in resistant cells (Fig. 2d). To generate a model of intrinsic resistance to TCB-mediated cell killing, we treated PBMC:BT474 cocultures with HER2-TCB, allowed the cells to recover, and repeated the treatment with a IFN-γ signaling is required for efficient killing by redirected fresh batch of PBMCs and HER2-TCB (Fig. 1a). At every round, lymphocytes. To determine whether the downmodulation of we controlled that the alloreaction of PBMCs on target cells was IFN-γ signaling was related to resistance, first we used a blocking low enough to allow the specific activity of the HER2-TCB. After antibody. The anti-IFN-γ efficiently impaired killing of parental 6 months, the resulting cells, BT-R, showed an IC50 for the BT474 cells mediated by the HER2-TCB, in 2D or 3D cultures HER2-TCB approximately tenfold higher than that of parental (Fig. 3a). Similarly, blocking IFN-γ prevented killing of target cells (156 pM vs. >1 nM) (Fig. 1b). Resistance was also observed cells by HER2-CAR T cells (Fig. 3b). in three dimensional cultures (Supplementary Fig. 1c) and in vivo IFN-γ acts on cytotoxic lymphocytes as well as on tumor cells. (Fig. 1c, d). BT-R cells were also resistant to a trastuzumab-based Maximal cytotoxic activity of T cells requires IFN-γ. On the other second generation HER2-CAR (described in Supplementary hand, IFN-γ impairs the proliferation of tumor cells by inhibiting Fig. 1a), in vitro and in vivo (Fig. 1e–h). their progression through the cell cycle and promoting their 2 NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 ARTICLE Fig. 1 Generation and characterization of resistant cells. a Schematic showing the assay of HER2-TCB in cocultures. b Cocultures of PBMCs with parental BT474 or resistant BT-R cells were treated with different concentrations of HER2-TCB (PBMC:target cell ratio 1:1) for 72 h. Then, viable cells were quantified by flow cytometry using EpCAM as a marker. c Schematic showing in vivo treatment with HER2-TCB. Totally, 107 BT474 or BT-R cells were injected orthotopically into NSG mice. When tumors reached ~200 mm3 (dark background), 107 PBMCs were injected i.p. Then animals were treated i.v. with 0.125 mg/kg HER2-TCB as indicated (red arrows). d Tumor volumes are represented as averages ± SD (standard deviation) (n = 4 per arm). e Schematic showing the HER2-CAR assay. f Parental BT474 cells or resistant BT-R cells were cocultured with different ratios of CAR T cells for 48 h. Cell numbers were calculated and expressed as in (b). g Schematic showing in vivo treatment with HER2-CAR T cells. Mice were injected with BT474 or BT-R cells as in (c). When tumors reached ~200 mm3, 3 × 106 HER2-CAR T-positive cells were injected i.p. h Tumor volumes are represented as averages ± SD (BT474, n = 6; BT-R, n = 8). i Levels of HER2, normalized to BT474 cells, were determined by Western Blot. j Cells were stained with HER2-TCB and analyzed by flow cytometry. Results were normalized to BT474 cells. k Cells were stained with antibodies against the indicated factors. Results are presented as the MFI of staining in BT474 and BT-R cells. l, Levels of 80 cytokines and growth factors were measured using a commercial array in the media conditioned by cocultures of BT474 cells and PBMCs or BT-R cells and PBMCs treated with HER2-TCB. Results are presented as the fold change of BT-R relative to BT474 cells. b **p = 0.005, *p = 0.02, **p = 0.006. f ***p < 0.001. k *p = 0.04, two-tailed t test. d, h **p < 0.01, ***p < 0.001, two-way analysis of variance (ANOVA) and Bonferroni correction. Data are presented as mean ± SD of four (b, f, j, k), or three (i) independent experiments. Source data are provided as a Source Data file. apoptotic death (reviewed in ref. 22). Thus, the blocking antibody Fig. 3a, b), and prevented the killing induced by IFN-γ (Fig. 3f). could prevent the killing of target cells by impairing the activation Irrespectively of the method, diminishing the expression of IFNGR1 of cytotoxic lymphocytes, blocking the action of IFN-γ on target resulted in resistance to HER2-TCB in coculture assays (Fig. 3g). cells, or a combination of both effects. Consistent results were obtained when we examined the sensitivity To directly test the effect on target cells, we treated BT474 cells to HER2-CAR T cells (Fig. 3h). To validate the results obtained with IFN-γ. As shown in Fig. 3c, IFN-γ induced the death of BT474 in vitro, we showed that the knock-out of IFNGR1 caused resistance cells by inducing apoptosis, as shown in the increase of annexin V+ to the HER2-TCB also in vivo (Fig. 3i). The knockdowns of JAK1 cells (Fig. 3d). In contrast, resistant cells were unaffected by the or STAT1 from parental BT474 cells also resulted in resistance same treatment. This result shows that IFN-γ is likely part of the to the HER2-TCB (Supplementary Fig. 3c, d). Thus, disrupting mechanism used by cytotoxic cells to kill target cells, and indicates IFN-γ signaling by different means induces resistance to redirected that BT-R cells became resistant by impairing IFN-γ signaling. lymphocytes. To confirm this hypothesis, we impaired IFN-γ signaling The effects of the IFN-γ-blocking antibodies or of the knock- in target cells, by knocking-out the IFNGR1 gene through down of IFNGR1 were not a particularity of BT474 cells, similar CRISPR–Cas9 technology, or by downmodulating the expression effects were observed when assaying different HER2-positive of IFNGR1 with shRNAs from parental BT474 cells (Fig. 3e). As cultures from breast cancer PDXs and an additional cell line expected, both methods impaired IFN-γ signaling (Supplementary (Supplementary Fig. 4a–i). Furthermore, impairment of IFN-γ NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 Fig. 2 Transcriptomic analysis. a Heatmap showing the 97 most differentially expressed genes in BT-R cells compared to parental BT474 cells (≥4-fold; p < 10−5). p Value was obtained from the DESeq2 analysis of the RNA-seq. Genes with FDR < 5% and |shrunken fold change| > 1.5 were considered significant. b Pathways showing positive and negative enrichment in BT-R compared with BT474 cells as determined by GSEA. Only statistical gene sets are shown (NOM p value < 0.05). c IFN-γ response GSEA signature in resistant cells, compared to parental BT474. p Value corresponds to the NOM p value obtained by GSEA in the HALLMARK database. d Levels of IRF1 upon treatment with IFN-γ in BT474 and BT-R cells were determined by Western blot analysis. Results of three independent quantifications, normalized to treated BT474 cells, are presented as mean ± SD. ***p < 0.001, two-tailed t test. Source data are provided as a Source Data file. signaling by knocking down IFNGR1, led to resistance to HER2- the dimerization and transport to the nucleus of the former, TCB in cell lines from HER2-positive ovarian and lung cancers where dimeric pSTAT1 regulates the expression of different (Supplementary Fig. 4j, k). genes, including IRF122 (Fig. 4a). Analysis of these components in our coculture assays revealed little or no difference in the Components of the IFN-γ signaling pathway in resistant cells. secretion of IFN-γ or in the expression of IFNGR1 and 2, JAK1 The intracellular signaling pathway activated by IFN-γ is or STAT1 by resistant cells (Fig. 4b–e). In contrast, we observed relatively simple. Dimeric IFN-γ triggers the formation of a a marked reduction of JAK2 expression (Fig. 4f), indicating that complex of four receptor molecules (two IFNGR1 and two downmodulation of JAK2 could result in the impairment of IFNGR2), four JAK kinases (two JAK1s and two JAK2s) and IFN-γ signaling. Consistently with this hypothesis, pSTAT1 two molecules of the STAT1 transcription factor. The sub- levels were significantly decreased in resistant cells when trea- sequent phosphorylation of the STAT1s by the JAKs leads to ted with IFN-γ (Fig. 4g). 4 NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 ARTICLE Fig. 3 IFN-γ response is required for efficient killing by redirected lymphocytes. a Left, cocultures of PBMCs with BT474 or BT-R cells were treated with different concentrations of HER2-TCB in presence of an IgG control or an IFN-γ blocking antibody for 72 h. Then, viable cells were quantified by flow cytometry using EpCAM as a marker. Right, BT474 cells were grown in 3D and treated with HER2-TCB in presence of an IgG control (−) or an IFN-γ blocking antibody (+) for 72 h. Viable BT474 cells were quantified by flow cytometry using EpCAM as a marker. Results were normalized to untreated cells. b Parental BT474 cells were cocultured with different ratios of CAR T cells for 48 h in the presence of an IgG control or an IFN-γ blocking antibody. Then, cell numbers were calculated and expressed as in (a). c Parental BT474 or resistant BT-R cells were treated with different concentrations of IFN-γ for 5 days. Cell numbers were estimated with the crystal violet staining assay. d Parental BT474 or resistant BT-R cells were treated with 1 μg/ml of IFN-γ and the percentages of apoptotic cells were determined by means of Annexin V+ cells measured by flow cytometry. e Cells were stained with anti-IFNGR1 or isotype antibody and analyzed by flow cytometry. IFR1 stands for IFNGR1. f Sensitivity of the indicated cells to IFN-γ was analyzed as in (c). g The indicated cells were treated with different concentrations of HER2-TCB and analyzed as in (a). h The indicated cell lines were cocultured with CAR Ts and analyzed as in (b). i Totally, 6.5 × 107 BT474 cells or the same cells knock-out for IFNGR1 were injected orthotopically into NSG mice. Mice were treated as described in Fig. 1c. Tumor volumes are represented as averages ± SD (empty, n = 8; IFR1 KO, n = 4). a Left, **p = 0.001, *p = 0.05; right, *p = 0.01. b ***p < 0.001, **p = 0.002. c ***p < 0.001. d **p = 0.007. f Left, **p = 0.002, ***p < 0.001, **p = 0.003, **p = 0.0013; right, ***p < 0.001, **p = 0.002 (shIFNGR1 #31); **p = 0.009, **p = 0.006, ***p < 0.001, **p = 0.003 (shIFNGR1 #92); *p = 0.02, **p = 0.004, ***p < 0.001 (shIFNGR1 #97). g Left, ***p < 0.001; right, ***p < 0.001, **p = 0.004, *p = 0.02 (shIFNGR1 #31); *p = 0.046, *p = 0.047, *p = 0.03 (shIFNGR1 #92); *p = 0.05, *p = 0.02, *p = 0.02 (shIFNGR1 #97). h Left, ***p < 0.001; right *p = 0.02, ***p < 0.001 (shIFNGR1 #31); ***p < 0.001, *p = 0.02 (shIFNGR1 #92); ***p < 0.001, **p = 0.005, **p = 0.005 (shIFNGR1 #97). Two-tailed t test. i **p < 0.01, ***p < 0.001, two-way ANOVA and Bonferroni correction. Data are presented as mean ± SD of three (a–c, f, g right, h), four (d), or six (g left) independent experiments. Source data are provided as a Source Data file. Resistant cells cultured for up to three months in the absence of JAK2 expression. Among the different acetylation marks, any selective pressure showed resistance similar to that of BT-R H3K27Ac is considered one of the best markers of active cells (Supplementary Fig. 5a) and similar levels of JAK2 promoters and enhancers and it is tightly associated to gene (Supplementary Fig. 5b). Therefore, resistance is stable and does expression24. Thus, we concluded that JAK2 is likely down- not require selective pressure. We hypothesized that this stable regulated epigenetically in BT-R cells. downmodulation of JAK2 and, thus, resistance could be maintained epigenetically. To test this hypothesis, we treated BT-R cells with different well-characterized drugs that interfere The downmodulation of JAK2 causes resistance to redirected with epigenetic modifications, including the DNA demethylating lymphocytes. To establish the functional relevance of the agent 5-AZA, the histone demethylating agent DZNEP, and the downmodulation observed, we transduced resistant cells with a pan-HDAC inhibitor Trichostatin A (TSA). While demethylating vector encoding JAK2 (Fig. 5a). As expected, overexpression of agents did not affect JAK2 transcription, the HDAC inhibitor JAK2 restored IFN-γ signaling (Supplementary Fig. 5c). Of note, upregulated JAK2 expression in BT-R cells (Fig. 4h), indicating overexpression of JAK2 resulted in re-sensitization to killing by that de-acetylation of histone H3 may contribute to the silencing T cells redirected via TCB or CAR and in the re-sensitization to of JAK2. Direct analysis of H3K27Me3 and H3K27Ac marks the death induced by IFN-γ (Fig. 5b–d). Confirming the relevance showed reduced levels of the latter in the JAK2 promoter of BT-R of these in vitro results, overexpression of JAK2 restored sensi- cells (Fig. 4i), confirming that histone acetylation may regulate tivity to the TCB in vivo (Fig. 5e). NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 Fig. 4 Components of the IFN-γ signaling pathway in resistant cells. a Schematic showing the IFN-γ signaling pathway. b The levels of IFN-γ in the media conditioned by cocultures with BT474 or resistant BT-R in the presence of HER2-TCB was determined by ELISA. c The levels of Interferon-gamma receptors 1 and 2, normalized to BT474, were determined by quantitative real-time PCR (left) or flow cytometry with specific antibodies (right). d–f Levels of JAK1, STAT1, and JAK2 (mRNA and protein), normalized to BT474, as determined by quantitative real-time PCR (left) or Western blot (right). g Levels of phosphoSTAT1 in parental and resistant cells were determined by Western Blot. Results were normalized to treated BT474 cells. h BT-R cells were treated with indicated compounds for 48 h. Then, the levels of the mRNA encoding JAK2 were determined by RT-PCR and normalized to the levels in cells treated with vehicle. i Levels of H3K27Me3 and H3K27Ac histone marks in the promoter of JAK2 in BT474 and BT-R cells as measured by ChIP followed by quantitative real-time PCR. Results were normalized to the levels of an IgG control antibody in each sample, and then normalized to the levels in BT474 cells. ***p < 0.001, two-tailed t test. Data are presented as mean ± SD of two (b, i left), three (c, d, h, i right) or four (e–g) independent experiments. Source data are provided as a Source Data file. 6 NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 ARTICLE Fig. 5 The downmodulation of JAK2 causes resistance to redirected lymphocytes. a Levels of JAK2 in BT-R cells or the same cells stably transfected with a vector encoding JAK2 were determined by Western blot. Blot shown is representative of four independent experiments. b Cocultures of PBMCs with the indicated cells were treated with different concentrations of HER2-TCB for 72 h. Then, viable target cells were quantified by flow cytometry using EpCAM as a marker. c The indicated cells were treated with different ratios of CAR T cells. Cell numbers were calculated and expressed as in (b). d The indicated cells were treated with different concentrations of IFN-γ for 5 days. Cell numbers were estimated with the crystal violet staining assay. e Totally, 6.5 × 106 BT-R cells or the same cells expressing JAK2 were injected orthotopically into NSG mice. When tumors reached ~200 mm3 (dark background), 107 PBMCs were injected i.p. Then animals were treated i.v. with 0.125 mg/kg HER2-TCB as indicated (red arrows). Tumor volumes are represented as averages ± SD (n = 7 per arm). f Levels of JAK2 in BT474 cells stably expressing a non-targeting shRNA (shc) or an shRNA targeting JAK2 (shJK2) were determined by Western blot. Results were normalized to BT474 shc. g–i The indicated cells were analyzed as in (b–d), respectively. j Totally, 6.5 × 106 BT474 cells stably expressing a non-targeting shRNA (shc) or an shRNA targeting JAK2 (shJAK2) were injected into NSG mice and treated as in (e). Tumor volumes are represented as averages ± SD (shc, n = 10; shJAK2, n = 5). b **p = 0.005, **p = 0.003, **p = 0.002, **p = 0.002. c **p = 0.002, ***p < 0.001. d ***p < 0.001. g *p = 0.05, *p = 0.05, *p = 0.02. h, i ***p < 0.001, two-tailed t test. e, j *p < 0.05, ***p < 0.001, two-way ANOVA and Bonferroni correction. Data are presented as mean ± SD of three (b–d, h, i) or four (f, g) independent experiments. Source data are provided as a Source Data file. Conversely, knock-down of JAK2 from parental cells impaired Fig. 6f). After two rounds of treatment, the tumors were no IFN-γ signaling (Fig. 5f and Supplementary Fig. 5d), induced longer sensitive to the HER2-TCB (Supplementary Fig. 6g). resistance to redirected lymphocytes and to cell death induced by Again, the resistant PDX showed unaltered levels of HER2 but IFN-γ in vitro (Fig. 5g–i), and resistance to the HER2-TCB strongly decreased levels of JAK2 (Supplementary Fig. 6h, i). in vivo (Fig. 5j). Thus, resistance to HER2-TCB in different contexts results in the Conceivably, impairment of any of the components that same defect: downmodulation of JAK2 and impaired IFN-γ transduce its signal results in defective response to IFN-γ. To signaling in tumor cells. determine if different components can be downmodulated in different resistant models, we generated an independent resistant Models of resistance to IFN-γ. Treatment of BT474 cells with BT474 model following an in vivo approach. We recovered cells increasing concentrations of IFN-γ starting at the IC50 during from the residual tumor that remained after TCB-treatment, re- 4 months resulted in resistant cells (designed BT-RG), with an IC50 grafted them into mice and repeated the process (Supplementary comparable to that of cells resistant to the HER2-TCB (BT-R) Fig. 6a). After the second round of selection, we obtained cells (>1000 ng/ml) (Fig. 6a). As expected, IFN-γ signaling was down- resistant to HER2-TCB, which were named BT-vR (Supplemen- modulated in these resistant cells (Supplementary Fig. 5e). In vitro tary Fig. 6b). The levels of cell surface HER2 in resistant cells were assays showed that IFN-γ resistant cells were also resistant to the unaltered (Supplementary Fig. 6c), and we observed a down- HER2-TCB and to HER2-CAR T cells (Fig. 6b, c). Further, IFN-γ modulation of JAK2 similar to that in the in vitro selected BT-R resistant cells were also resistant to the HER2-TCB in vivo (Fig. 6d). cells (Supplementary Fig. 6d). Lymphocyte infiltration, stained Thus, cells selected because of their resistance to IFN-γ showed with anti-CD3 antibodies, was similar in tumors generated by similar characteristics to those selected for resistance to TCBs, parental and resistant cells (Supplementary Fig. 6e), showing that further supporting the relevance of the IFN-γ pathway in resistance deficient infiltration of lymphocytes was not the cause of to redirected lymphocytes. resistance. Finally, in contrast with parental cells, IRF1 was Importantly, as was the case with HER2-TCB resistant cells, undetectable in resistant cells (Supplementary Fig. 6e), confirm- the levels of JAK2 were significantly reduced in BT-RG cells ing defective IFN-γ signaling in resistant cells. (Fig. 6e). Next, we performed gain-of-function experiments; that To select resistant cells from another source and in different is, analysis of IFN-γ signaling (Supplementary Fig. 5f) and the conditions, we implanted a HER2-positive PDXs in mice sensitivity to IFN-γ, HER2-TCB and HER2-CAR of BT-RG cells humanized with CD34+ hematopoietic stem cells as previously transfected with JAK2 (Fig. 6f–h). The results confirmed the described18. Once the tumors reached ~200 mm3, mice were causal role of JAK2 downmodulation on resistance to killing by treated with the HER2-TCB, the tumors regressed, were allowed redirected lymphocytes, independently of the method to generate to regrow and the treatment was repeated (Supplementary cells resistant to IFN-γ signaling. NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 Similarly to BT-R cells, treatment of BT-RG cells with drugs levels of an antigen, or if it also applies to T cells engaged through that interfere with epigenetic modifications showed an effect of TCR/MHC-antigen synapses. the pan-HDAC inhibitor Trichostatin A (TSA) on the expression In this regard, loss-of-function mutations in JAK1 or JAK2 of JAK2 (Fig. 6i). In addition, BT-RG showed reduced levels of have been shown to arise in tumors that progressed to treatment H3K27Ac in the promoter of JAK2 (Fig. 6j). Thus, we concluded with immune checkpoint inhibitors, showing that IFN-γ pathway that JAK2 is also downregulated epigenetically in BT-RG cells. may also be critical for the killing of target cells by the TCR/ Chronic treatment with IFN-γ is a convenient way to obtain MHC–antigen complex. In addition, recently the down- models of resistance. Thus, to confirm our conclusions, we treated modulation of genes upregulated by IFN-γ has been correlated cultures from the two HER2-postive PDXs with IFN-γ to obtain with resistance to immune checkpoint blockade33,34, further resistant cells. In both cases, resistance to IFN-γ was acquired supporting the relevance of IFN-γ signaling on the sensitivity of through downmodulation of JAK2. Consistent with the results target cells to killing by cytotoxic lymphocytes. obtained with BT474 cells, IFN-γ resistance induced resistance to The results presented here, have practical implications, particu- killing by TCB; further, JAK2 overexpression rescued completely larly when considering the widespread use of JAK2 inhibitors in the the phenotype (Supplementary Fig. 7). We concluded that breast clinic34. Our results imply that the systemic use of these inhibitors, cancer cells acquire resistance to IFN-γ and, thus, resistance to currently approved to treat myelofibrosis and hydroxyurea resistant redirected lymphocytes, by downregulating JAK2 (Fig. 6k). or intolerant polycythemia vera to alleviate the exacerbated acti- vation of the immune system, may impact on cancer immunoe- diting and, in some contexts, favor tumor progression. Discussion Redirection of lymphocytes, via TCBs or CARs, is already Methods approved to treat some hematological malignancies. This success Study design. This study was designed to identify the mechanisms that trigger the contrasts with the failures in the treatment of solid tumors. In resistance against HER2-TCB and -CAR. In order to generate acquired resistance order to overcome this lack of efficacy, many efforts have focused models and to identify the mechanism described in this work, in vitro and in vivo on counteracting the inhibitory effect that the tumor micro- functional assays were performed, using both tumor cell lines and PDXs. Experiments consisted of combining tumor cells with human lymphocytes and TCBs or CARs environment exerts on lymphocytes. Only recently, intrinsic targeting HER2. For in vivo models, two sources of human immune cells were used: mechanisms or resistance, such as defective death receptor sig- PBMCs and CD34+ hematopoietic stem cells. All human samples were obtained with naling causing resistance to CARs directed against CD19, have informed consent and following institutional guidelines under protocols approved by been described25. The simple approach used in this study allowed the Institutional Review Boards (IRBs) at Vall d’Hebron Hospital. Animal work was performed according to protocols approved by the Ethical Committee for the Use of to unveil additional mechanisms deployed by cancer cells to resist Experimental Animals at the Vall d’Hebron Institute of Oncology. For in vivo killing by a fully active lymphocyte with unrestricted access to its experiments, two to five mice were used per group, and they were randomized by target cell. tumor size. Mice that died before the end of the experiments for reasons unrelated to The impairment of the IFN-γ pathway does not have dis- treatment or that did not have detectable percentages of human immune cells were excluded. Because of ethical reasons, we ended the experiments before the full cernible effects on cell proliferation, as readily shown by the development of graft-versus-host disease or when tumor volume surpassed 1500 knockdown or knockout of IFNGR1, JAK1, STAT1, or JAK2, but mm3. Experiments were performed in a blinded fashion. severely affects the sensitivity of cells to the killing by redirected lymphocytes. Thus, alterations conducting to impairment of IFN- Cell lines and primary cultures. BT474 (#HTB-20), SKBR3 (#HTB-30), HEK293T γ signaling in tumor cells are likely to arise in patients under the (#CRL-11268), SKOV3 (#HTB-77), and H1781 (#CRL-5894) were obtained from selective pressure imposed by TCBs or CARs. ATCC (Manassas, VA, USA). GP2-293 cells (#631458) were obtained from Activated T cells engage target cells via a cytolytic synapse Clontech. PDX433 (ER+/PR−/HER23+) was extracted by core needle biopsy formed between the TCR and the MHC-antigen complex. These (CNB) from a breast cancer patient’s metastasis in the liver. PDX667 (HER2+) was extirpated by CNB from a breast cancer patient’s metastasis in the skin. PDX118 synapses cause the death of the target cells via perforin- and (ER+/PR−/HER23+) comes from a skin metastasis collected by CNB. All these granzyme-induced apoptosis. In addition, FAS-L produced by PDXs have been established at VHIO following institutional guidelines. The IRBs activated lymphocytes causes death of cells expressing the FAS at Vall d’Hebron Hospital provided approval for this study in accordance with the receptor26,27. It is generally assumed that this system suffices to Declaration of Helsinki. Written informed consent was obtained from all patients who provided tissue samples. kill target cells and that TCBs and CARs trigger the same Cell lines were cultured under standard conditions in complete medium mechanism. The results presented here show a previously (DMEM F-12 medium (#21331, Gibco) supplemented with 10% fetal bovine serum unnoticed role of the IFN-γ pathway in the killing of HER2- (FBS) (#10270, Gibco), 1% L-glutamine (#X0550, Biowest), and 1% positive cancer cells by lymphocytes, at least those redirected via penicillin–streptomycin (#P4333, Sigma-Aldrich)). Cells were genetically modified to acquire resistance to certain antibiotics or to downmodulate or overexpress TCBs or CARs targeting HER2. different genes. Cells were routinely tested for the absence of mycoplasma Compared with TCR/MHC-antigen synapses, the contacts contamination using the MycoAlertTM Mycoplasma detection kit (#LT07-318, mediated by TCBs or CARs have some particularities (reviewed Lonza). Cell lines were not authenticated in-house. in ref. 4). First, contacts formed via TCR or CARs function independently of MHC28. On the other hand, the affinities of PBMC isolation. PBMCs were isolated from fresh buffy coats obtained from TCRs for TCR/MHC-antigen complexes are in the range of healthy donors through the Blood and Issue Bank of Catalonia (BST). Blood was 1–100 µM, whereas the affinities of TCBs and CARs are typically diluted 1:3 with 1× phosphate-buffered saline (PBS) and transferred to a 50 ml below 100–10 nM29,30. Finally, few complexes of a given antigen falcon tube with Ficoll-Paque PLUS (#70-1440-02, GE Healthcare) at a 1:3 ratio, following the manufacturer’s instructions. After obtaining the buffy coat, red blood with its cognate MHC are expressed per target cells31; in contrast, cells (RBC) were lysed with 1× RBC lysis buffer (#00-4333-57, Invitrogen) for 4 targets of TCBs and CARs may number in the 1000–10,000 s, or min. Obtained PBMCs were counted and frozen with Cryostor CS10 (#07959, in the 100,000 s in the case of HER2-amplified tumors32. In fact, Stemcell Technologies) at −80 °C for in vitro and in vivo experiments. differences between the synapses established by TCR/MHC- antigen and CARs have been directly shown33. Probably because Generation of resistant cells in vitro. To generate the BT-R model, BT474 cells of these differences, while the activation of T cells by TCR/ stably expressing hygromycin resistance were treated with a 3:1 ratio PBMC:Tumor MHC–antigen synapses requires additional second signals, TCBs and a concentration of 67.5 pM HER2-TCB in PBMC media (RPMI 1640 (#61870, Gibco), 10% heat-inactivated FBS, 1% L-glutamine, 1% HEPES (#H0887, Sigma- and CARs suffice to activate lymphocytes. Future work should Aldrich), 1% MEM nonessential amino acids solution (#11140, Gibco), and 1% clarify if the dependence of IFN-γ signaling is a particularity of T penicillin–streptomycin). After 72 h, media was removed and replaced with com- cell redirected via TCBs or CARs against cells expressing high plete medium containing 100 μg/ml hygromycin (#10687010, Gibco) during 7 days 8 NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 ARTICLE Fig. 6 Model of resistance to IFN-γ. a Parental BT474 cells or resistant BT-R or BT-RG cells were treated with different concentrations of IFN-γ for 5 days. Cell numbers were estimated with the crystal violet staining assay. b Cocultures of PBMCs with the indicated cells were treated with different concentrations of HER2-TCB for 72 h. Then, viable target cells were quantified by flow cytometry using EpCAM as a marker. c The same cells as in (a) were treated with different ratios of CAR T:Tumor cell. Cell numbers were obtained by means of EpCAM counts by flow cytometry. d Totally, 107 BT474 or resistant BT-RG cells were injected orthotopically into NSG mice. When tumors reached ~300 mm3 (dark background), 107 PBMCs were injected i.p. Then animals were treated i.v. with 0.125 mg/kg HER2-TCB as indicated (red arrows). Tumor volumes are represented as averages ± SD (BT474, n = 8; BT-RG, n = 6). e The levels of JAK2 (mRNA and protein) as determined by quantitative real-time PCR (left) or Western blot (right). Results were normalized to BT474 cells. f–h The indicated cells were analyzed as in (a–c), respectively. i BT-RG cells were treated with indicated compounds for 48 h. Then, the levels of the mRNA encoding JAK2 were determined by RT-PCR and normalized to the levels in cells treated with vehicle. j Levels of H3K27Me3 and H3K27Ac histone marks in the promoter of JAK2 in BT474 and BT-RG cells as measured by ChIP followed by quantitative real-time PCR. Results were normalized to the levels of an IgG control antibody in each sample, and then normalized to the levels in BT474 cells. k Schematic drawing summarizing our findings. a **p = 0.002, ***p < 0.001. b **p = 0.009, **p = 0.006, *p = 0.013, **p = 0.002. c **p = 0.008, ***p < 0.001. e ***p < 0.001. f *p = 0.02, **p = 0.004, **p = 0.002, ***p < 0.001, **p = 0.002. g **p = 0.005, ***p < 0.001. h **p = 0.0015, ***p < 0.001. i, j ***p < 0.001. Two-tailed t test. d *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA and Bonferroni correction. Data are presented as mean ± SD of three (a–c, e–i), two (j left) or four (j right) independent experiments. Source data are provided as a Source Data file. to specifically kill remaining PBMCs. The process was repeated several times. 2.5 mM EDTA, 1% bovine serum albumin (BSA) (#A9647, Sigma-Aldrich), 5% Resistant population was obtained after 6 months. horse serum (#26050, Gibco)) in polypropylene V-bottom 96-well plates Interferon-gamma resistant models were established by culturing cells in (#651201, Greiner Bio-One). Twenty minutes later, samples were centrifuged presence of increasing IFN-γ (#300-02, Peprotech) concentrations, starting at 100 and cells were stained with the epithelial cell marker anti-human EpCAM ng/ml and reaching 1 μg/ml. In the three models (BT474-RG, PDX667-RG, and (#324212, BioLegend) at 1:300 concentration in FACS buffer in ice for 30 min. PDX433-RG) resistance was obtained after 4 months of treatment. After a wash with 1× PBS, samples were resuspended in the viability marker Zombie Aqua at 1:1000 (#423101, BioLegend) in 1x PBS and acquired on LSR Fortessa, using BD FACSDiva software (BD Biosciences). Number of T cell cytotoxicity assays. All target cells were seeded in 96-well flat bottom plates alive cells was analyzed with FlowJo software (BD Life Sciences) by means of (0.01 × 106 cells/well) (#353075, Corning Life Sciences). Effector PBMCs were EpCAM counts. Gating strategy for tumor EpCAM+ counts is shown in added to each well at the indicated ratio in PBMC medium. Different concentra- Supplementary Fig. 8. tions of HER2-TCB were added to the wells. The plates were incubated for 72 h. When treated with CAR Ts, target cells were seeded as described above. After At the endpoint cells were harvested with trypsin–EDTA (#25300096, Gibco) 24 h, effector CAR Ts targeting HER2 were added to each well at the indicated and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS 1×, NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 ratios in PBMC medium. The plates were incubated for 48 h. Endpoint was assayed Paclitaxel Hospira, Pfizer), doxorubicin (#23214-92-8, Farmiblastina, Pfizer), or T- as previously explained. DM1 (#1018448-65-1, Kadcyla, Roche). Treatment lasted for 3 days in the case of the For the IFN-γ blocking experiments, tumor cells were seeded as explained chemotherapeutic agents paclitaxel and doxorubicin, and 6 days when treated with T- before, and in addition to the PBMCs and HER2-TCB/CAR Ts, either 20 μg/ml of DM1. Cell death was assayed by crystal violet staining of alive cells and read at the anti-human IFN-γ (#506512, Biolegend) or mouse IgG control (#400123, Infinite M200 Pro Multimode Microplate Reader (TECAN). Biolegend) was added in the coculture. Experiment lasted 72 or 48 h respectively and endpoint was assayed as previously explained. Western blot. For Western blot, protein extracts were isolated by lysing the cells in homemade lysis buffer (130 mM NaCl, 0.01% NP-40, 1% glycerol, 2 mM EDTA pH 3D organoid assay. Tumor cells were seeded in 48-well plates (103 cells/well) in a 8 and 20 mM Tris-HCl pH 7.4), supplemented with phosphatase inhibitors 5 μM β- drop of 20 μl of matrigel (#356235, Corning). Each drop was dispensed in the center glycerolphosphate, 5 μM sodium fluoride, 1 μM sodium orthovanadate and cOm- of the well and incubated for 15 min at RT. After matrigel was solidified, 250 μl of pleteTM, EDTA-free protease inhibitor cocktail (#COEDTAF-RO, Sigma-Aldrich, 1 3D breast tumor organoid media35, were added to each well. Media was replaced tablet per 10 ml lysis buffer). Protein extracts were sonicated for 10 s at 4.5 V to twice a week and 3D formation was assessed after 15 days. Organoids per well were break the cell apart. Tubes were centrifuged 19,000 ×g 10 min and supernatant was counted and assumed that each of them consisted of approximately 50 cells. collected. Organoid media was removed and 3D structures were cocultured with PBMCs at a Protein lysates were resolved by sodium dodecyl sulfate polyacrylamide gel 2:1 ratio in PBMC media and treated with HER2-TCB at 1 nM for 72 h. electrophoresis and then transferred to a 0.45 μm nitrocellulose membrane For the IFN-γ blockade experiment, same assay was followed but adding either (#10600002, GE Healthcare Biosciences). Totally, 20–30 μg of protein lysate was 20 μg/ml of anti-human IFN-γ or mouse IgG control to the coculture with PBMCs. loaded per experiment. Membranes were incubated with 5% BSA or 5% non-fat At the endpoint, organoids were disaggregated by adding 500 μl of trypsin for milk in TBS-T (1× tris-buffered saline with 0.1% tween 20 (#P7949, Sigma- 30 min at 37 °C. Then, cells were collected and incubated for 30 min on ice to Aldrich)). After blocking, membranes were incubated overnight with primary liquify matrigel. Fully disaggregated organoids were washed and stained as antibodies. previously explained. Number of alive cells was analyzed with FlowJo software by After washing, membranes were incubated with horseradish peroxidase- means of EpCAM counts. Gating strategy for tumor EpCAM+ counts is shown in conjugates antibodies (GE Healthcare) for 1 h. Membranes were developed with Supplementary Fig. 8. Immobilon Western Chemiluminescent HRP Substrate (#WBKLS0500, Millipore) and protein bands were visualized in AmershamTM Imager 600 (GE Life Sciences). Flow cytometry. Cells were harvested with StemPro Accutase (#A1110501, Gibco) Antibodies used were: HER2 (#AM134, Biogenex), JAK1 (#3344, Cell Signaling and resuspended in FACS buffer. Twenty minute later, samples were centrifuged Technology (CST)), JAK2 (#3230, CST), pSTAT1 (#9167, CST), STAT1 (#9172, and cells were incubated for 30 min with the specified antibody. After a wash and CST), IRF1 (#sc-497, Santa Cruz Biotechnology (SC)), and GAPDH (#ab128915, Zombie Aqua staining, samples were acquired on LSR Fortessa. The following Abcam). All antibodies were used at 1:1000 concentration in 5% BSA except antibodies were used: hPDL1 (#329736), hCD80 (#305221), hCD86 (#305425), GAPDH (1:5000). Quantification of protein levels was done with ImageJ (National hGalectin-9 (#348905), hB7-H3 (#351003), hB7-H4 (#358103), hHVEM Institutes of Health). Quantifications are the result of ≥3 independent biological (#318805), hICOS-L (#309403), h41BB-L (#311503), hOX40-L (#316307), replicates. All original Western blots are provided in the Source data file. hIFNGR1 (#308606), hIFNGR2 (#308504), all from Biolegend at 1:100 dilution. As an isotype control, PE mouse IgG Isotype Ctrl (#400114, Biolegend) was used RNA isolation and qRT-PCR. Total RNA was isolated from adherent cells by using at 1:100. RNeasy Mini Kit (#74106, Qiagen) according to the manufacturer’s protocol. RNA In the case of HER2 staining, cells were incubated in FACS buffer for 30 min was eluted in RNase-free water and quantified using NanoDropTM 2000 spectro- with Trastuzumab (#180288-69-1, Herceptin, Roche) at 2.5 μg/ml. After two photometer (Thermo Fisher Scientific). washes with PBS, a secondary conjugated antibody Anti-human Alexa-488 (#A- cDNA was prepared from 1 μg template RNA using the high capacity cDNA 11013, Invitrogen) was incubated with the cells at a concentration of 1:500 for 30 reverse transcription Kit (#4368813, Applied Biosystems) according to the min. Cells were then washed with 1× PBS and resuspended in Zombie Aqua manufacturer’s protocol. viability marker and acquired on LSR Fortessa. HER2-TCB binding assay was Real-time quantification of transcript abundance was determined by qRT-PCR performed by incubation of cells with 10 nM HER2-TCB for 30 min, followed by using the TaqMan Gene Expression probes (Applied Biosystems) and TaqMan the procedure described above. Universal Master Mix II (#4440039, Applied Biosystems), in 384-well plates in PD-L2 staining consisted of a primary and secondary antibodies incubation. 7900HT Fast Real-Time PCR System (Applied Biosystems), following the The incubation with the primary antibody, anti-PD-L2 (#130098525, Miltenyi manufacturer’s protocol. Biotec), lasted 30 min at 1:11 concentration, and after a wash with 1× PBS, samples The following TaqMan probes were used: TIMP2 (Hs00234278_m1), IFNGR1 were incubated with the secondary antibody anti-biotin (#130113292, Miltenyi (Hs00988304_m1), IFNGR2 (Hs00194264_m1), JAK1 (Hs01026983_m1), JAK2 Biotec) at 1:50 for 30 min. After a wash and Zombie Aqua staining, samples were (Hs01078136_m1), STAT1 (Hs01013996_m1), GAPDH (Hs02758991_g1). Data acquired on LSR Fortessa using BD FACSDiva software. was analyzed with sodium dodecyl sulfate (SDS) software, RQ Manager, and The activation marker CD69 (#310914, Biolegend) in CD8+ cells (#344712, DataAssist software (Applied Biosystems), using the 2−ΔCT method. GAPDH was Biolegend) was used in order to assess T-cell activation after 72 h of coculture with used as an endogenous control. tumoral cells and 67.5 pM HER2-TCB (both at 1:300 concentration). Ki-67 intracellular staining was performed in PBMCs after 72 h of coculture with BT474 cells using different concentrations of HER2-TCB. First, cells were Drugs targeting epigenetic modifications. Cells were treated with 5 μM of dif- incubated with zombie aqua for 25 min at 1:500 concentration at room ferent drugs for 48 h: 5-Azacytidine (#A2385, Sigma-Aldrich), 3-Deazaneplanocin temperature (RT). Then, cells were washed and stained with a CD8 antibody as A (DZNEP, #HY-10442, MedChemExpress) or Trichostatin A (TSA, #HY-15144, previously explained. After a wash with 1× PBS, cells were fixed with 200 μl of fresh MedChemExpress). Then, RNA was isolated and subjected for JAK2 expression by prepared (1 part of concentrate with 3 parts of diluent) fixation/permeabilization RT-qPCR. working solution (#00-5523-00, Invitrogen) for 25 min on ice in dark. Supernatant was removed after a centrifugation and cells were permeabilized with 1× permeabilization buffer for 25 min on ice in dark. After supernatant was Chromatin immunoprecipitation (ChIP). Indicated cells were grown to 70% removed, cells were stained with anti-Ki-67 antibody (#563756, BD Biosciences) in confluence, collected, and subsequently cross-linked with 1% formaldehyde shak- 1× permeabilization buffer at 1:300 concentration for 1 h at RT, washed with 1× ing at 37 °C temperature for 10 min. Reaction was quenched by incubating the PBS and acquired on LSR Fortessa. Flow cytometry data was analyzed with FlowJo samples with 125 mM Glycine (#BP381, Fisher Scientific) for 5 min. Cells were software (BD Life Sciences). Gating strategy for T-cell proliferation, activation pellet at 5 × 106 cells/vial and stored at −80 °C. marker analysis, and for median fluorescence intensity (MFI) measures is shown in Cell pellets were resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, Supplementary Fig. 8. 50 mM Tris pH 8) with 1:200 Protease Inhibitor Cocktail Set III (#535140, Merck Millipore) for 30 min on ice. Samples were then sonicated to generate fragments of IFN-γ cytotoxicity assays. Tumor cells were seeded in flat bottom 96-well plates DNA between 100 and 600 bp with the Bioruptor (Diagenode). After 20 min on ice, (0.01 × 106 cells/well). After 24 h cells were treated with different concentrations of samples were centrifuged at 19,000 × g and supernatant was diluted 1/10 with IFN-γ. Treatment lasted for 5 days and cell death was assayed by crystal violet Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH staining of alive cells. Cells were fixed for 30 min with 10% glutaraldehyde, washed, 8, 167 mM NaCl), in order to decrease concentration of SDS. Samples were and stained for other 30 min with 0.1% crystal violet (#548-62-9, Sigma-Aldrich). incubated with 10 μl of Dynabeads protein A (#10002D, Invitrogen) and 1 μg of After three washes with water, plates were let dry overnight. For the readout, 100 μl irrelevant antibody Rabbit IgG (#I8140, Sigma-Aldrich) per IP in that sample, as a of 10% acetic acid were added to each well and absorbance was read at 560 nm preclearing. Incubations lasted for 3 h rotating at 4 °C. Magnets were used to using an Infinite M200 Pro Multimode Microplate Reader (TECAN). discard the beads, and the samples were separated per IP, saving 10% for the input. Totally, 3 μg of corresponding antibody was added at each tube and samples were incubated overnight rotating end over end at 4 °C. The antibodies used were: Antitumor drugs cytotoxic assays. Tumor cells were seeded as described above. Rabbit IgG, anti-H3K27Me3 (#07-449, Merck Millipore) and anti-H3K27Ac After 24 h cells were treated with different concentrations of paclitaxel (#33069-62-4, (#ab4729, Abcam). 10 NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 ARTICLE Samples were incubated with 50 μl of prewashed dynabeads and incubated 3 h reaching 0.25 mg/kg. After two passages, resistant tumors (termed as 118-vR) were rotating at 4 °C. Dynabeads were then washed 3 times with low salt and 3 times obtained. with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8, and 150 or 500 mM NaCl respectively) and 2 times with LiCl buffer (250 mM LiCl, Immunohistochemistry. The following primary monoclonal antibodies were used: 1% NP-40, 1% NaDOC, 1 mM EDTA, 10 mM Tris pH 8). Samples were then anti-IRF1 (#HPA063131, Atlas Antibodies) and anti-CD3 (#790-4341, Ventana incubated with 48 μl of elution buffer (0.4% SDS, 5 mM EDTA, 10 mM Tris pH 8, Medical Systems (Ventana)). For immunohistochemistry, fixed tissue samples 300 mM NaCl) supplemented with 2 μl proteinase K (#RPTOTKSOL, Roche). embedded in paraffin were sectioned at 4 μm thickness. Sections were heated at Then, samples were incubated shaking 1 h at 55 °C and subsequently overnight at 60 °C, deparaffinized with xylene and hydrated with two steps of incubation with 65 °C. Input samples were treated the same way. DNA was purified from the eluted different dilutions of ethanol. samples with the MinElute PCR Purification Kit (#28006, Qiagen) following the When stained with anti-IRF1, antigen retrieval was performed by boiling the manufacturer’s instructions. samples for 20 min in citrate buffer pH 6 (#S2369, Agilent). Endogenous Finally, qPCR was performed with the ChIP samples using SYBR green reagent peroxidase was blocked by incubating the samples with 3% peroxide hydrogen (#733-1390, Quantabio). A 179-bp segment of the JAK2 promoter was amplified (#108597, Merck Millipore) diluted in absolute methanol for 20 min. Slides were with the following primers: 5′-GGATGTGAGTGGGAGCTGAG-3′ (sense) and also blocked with 3% BSA in 1× PBS for 10 min. Samples were then incubated 5′-GAGATAACACCCACCGGCTA-3′ (antisense). Data shown is the result of overnight with the primary antibody anti-IRF1 diluted 1:650 in EnVision FLEX normalizing the specific signal of each antibody (normalized to the IgG control Antibody Diluent (#K8006, Agilent). Next, the slides were incubated with EnVision signal) of BT-R or BT-RG to the parental BT474 cells. System-HRP labeled polymer anti-rabbit secondary antibody (#K4003, Agilent). Samples were then stained with DAB substrate chromogen (#K3468, Agilent) for Humanized xenograft models. In the PBMCs humanized xenograft models, NSG 1–4 min and counterstained with harris hematoxylin (#H3404, Vector mice were injected orthotopically in two flanks with 6.5 × 106 or 107 tumor cells in Laboratories) for 2 min, followed by dehydration with ethanol and xylene, and 100 μl of 1:1 PBS:matrigel. Once tumor size reached a specified volume, animals finally mounted in DPX. were intraperitoneally injected with 107 PBMCs obtained from healthy donors. Immunohistochemical staining of CD3 was performed using a Discovery After 24 h, animals started treatment and were treated biweekly with HER2-TCB ULTRA autostainer (Ventana). Heat-induced antigen retrieval was executed using (0.125 mg/kg) or vehicle intravenously. Cell Conditioning 1 (#950-124 Ventana) for 40 min at 95 °C. Endogenous In the HER2-CAR in vivo experiment, NSG mice were injected orthotopically peroxidase block was performed with the CM inhibitor from the ChromoMap with 107 tumor cells. Once tumors were around 200-300 mm3, animals were DAB kit (#760-159, Ventana) for 8 min. Then, the anti-CD3 primary antibody, intraperitoneally treated once with 3 × 106 HER2-CAR T-positive cells. ready to use, was applied 32 min at 36 °C. Next, samples were incubated for 8 min To obtain immunodeficient mice with a reconstituted human immune system, with detection kit UltraMap anti-Rabbit HRP (#760-4315, Ventana). Reactions CD34+ cells were purified from human cord blood obtained through the Blood and were detected using the ChromoMap DAB Kit. Finally, the slides were Tissue Bank of Catalonia. Blood was diluted 1:2 with 1× PBS + 2 mM EDTA and counterstained with Haematoxylin II (#790-2208, Ventana) 8 min and Bluing transferred to a 50 ml falcon tube with 15 ml of Ficoll–Paque PREMIUM (#70- Reagent (#760-2037, Ventana) 4 min, followed by dehydration with ethanol and 1440-02, GE Healthcare), following the manufacturer’s manual. After obtaining the xylene, and mounted in DPX. mononuclear cells, resting RBC were lysed with 1× RBC lysis buffer for 4 min. Slides were scanned in the NanoZoomer 2.0-HT slide scanner (Hamamatsu CD34+ cells were purified by negative selection by incubating the mononuclear Photonics) and visualized in the NDP.view2 software (Hamamatsu Photonics). cells with EasySep Human Progenitor Cell Enrichment Cocktail with Platelet Depletion (#19356, StemCell Technologies), following manufacturer’s protocol. Cytokine array. Detection of 80 cytokines (ENA-78, G-CSF, GM-CSF, Gro a/b/g, Purity of the remaining cell mix was checked with anti-human CD34 (#60013, CXCL1, CCL1, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, StemCell) and anti-human CD45 (#304008, Biolegend) staining at 1:300 IL-12, IL-13, IL-15, IFN-gamma, MCP-1, MCP-2, MCP-3, M-CSF, CCL22, MIG, concentration in FACS buffer. Samples were acquired in LSR Fortessa and MIP-1 beta, MIP-1 delta, RANTES, SCF, SDF-1 alpha, CCL17, TGF beta, TNF percentage of CD34 and CD45 cells were analyzed in FlowJo. Obtained cells were alpha, TNF beta, EGF, IGF-1, Angiogenin, OSM, TPO, VEGF-A, PDGF-BB, Leptin, frozen with Cryostor CS10 at −80 °C. BDNF, CXCL13, CCL23, CCL11, CCL24, CCL26, FGF-4, FGF-6, FGF-7, FGF-9, CD34+ cells were injected intravenously in 5-week-old female NSG mice FLT-3 ligand, Fractalkine, GCP-2, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, previously treated with busulfan (15 mg/kg) to remove the hematopoietic system of IGFBP-4, IL-16, CXCL10, LIF, LIGHT, MCP-4, MIF, MIP-3 alpha, NAP-2, NT-3, the mice. After 4–5 months, levels of humanization were checked by flow NT-4, OPN, OPG, PARC, PLGF, TGF beta 2, TGF beta 3, TIMP-1, and TIMP-2) cytometry and mice with more than 30% of human CD45+ cells in blood were used was conducted in culture supernatants of untreated and treated BT474 and BT-R for in vivo experiments. Totally, 107 tumor cells were orthotopically implanted per cells. Cells were seeded at 1 × 106 cells per 6 mL of PBMC medium alone or in flank, and once these reached ~200 mm3, animals were randomized and treated coculture with a ratio 3:1 of PBMCs and 67.5 pM of HER2-TCB. After 48 h of biweekly with HER2-TCB (0.25 mg/kg) or vehicle (intravenously). coculture, supernatant was harvested and frozen. In order to detect differences in At the end of the experiments, tumors were analyzed. Tumors were cut into parental and resistant cells cytokine secretomes, a human cytokine array (#AAH- small pieces and divided into samples for IHC, protein, flow cytometry, or CYT-5, RayBiotech) was used with these supernatants following the manufacturer’s reinjection. Samples for IHC were fixed and embedded in paraffin. Samples for recommendations. Quantification of cytokine levels was done with ImageJ following western blot were incubated with lysis buffer, supplemented with phosphatase and manufacturer’s instructions. protease inhibitors, in BashingBead lysis tubes (#S6003, Zymo Research) and homogenized in Precellys Evolution Homogenizer (Bertin Technologies). Samples for flow cytometry and reinjection were digested in 300 U/ml ELISA. In order to assay TIMP2 release, a TIMP2 ELISA (#DY971, R&DSystems) collagenase IA (#C2674, Sigmsa-Aldrich) and 100 U/ml hialuronidase IS (#H3506, was used with the same supernatants as specified above, following the manu- Sigma-Aldrich) in DMEM F-12 medium. After 1 h of incubation at 37 °C with facturer’s manual. shaking at 10 × g, the mixture was filtered through 100 μm strainers. RBC were In the case of IFN-γ ELISA, supernatants from treated BT474 and BT-R were lysed with 1× RBC for 5 min RT. After a wash with 1× PBS, samples were counted obtained at the conditions described above, with different timepoints (24, 48, and and either reinjected or stained for HER2 and EpCAM as previously explained and 72 h). The culture supernatants were used to perform a human interferon-gamma acquired on LSR Fortessa. Data were analyzed with FlowJo software (BD Life ELISA (#31673539, Immunotools), following manufacturer’s instructions. Sciences). Gating strategy for MFI measures is shown in Supplementary Fig. 8. All mice in this study were kept within Home Office limits of 22 °C ± 2 °C, Granzyme B activity. Target cells (BT474 or BT-R) were seeded, 0.25 × 106 in 60 55–65% humidity and run on a 12 h light/dark cycle that runs from 8 a.m. to 8 p.m. mm plates (#430166, Corning), cocultured with PBMCs at a PBMC:Target cell ratio of 3:1 and treated with HER2-TCB at a concentration of 67.5 pM for 72 h. After 72 h, tumor cells and PBMCs were harvested and lysed with 100 μl of lysis Generation of in vivo resistant cells. In the generation of BT474 resistant to buffer. Lysed cells were centrifuged at 21,000×g for 10 min at 4 °C to pellet cell HER2-TCB, the humanized PBMCs xenograft model was used. NSG mice were nuclei and other cell debris. Supernatants were harvested and assayed for pro- injected orthotopically with 107 tumor cells. Once tumor size reached 300 mm3, tease activity. Reaction was performed in a non-treated 96-well-plate (#442404, animals were intraperitoneally injected with 107 PBMCs obtained from healthy Thermo Fisher Scientific). Each well contained 25 μl of lysis supernatant, donors. After 24 h, animals started the treatment and were treated biweekly with an granzyme B substrate Ac-IEPD-pNA (#368057, Sigma-Aldrich) at a final con- increasing concentration of HER2-TCB or vehicle intravenously. HER2-TCB centration of 300 μM and reaction buffer (0.1 M HEPES pH 7.0, 0.3 M NaCl, treatments started from 0.0325 mg/kg, and concentration was gradually increased 1 mM EDTA) in a total volume of 250 μl/well. Mixtures were incubated at 37 °C until reaching 0.25 mg/kg. After two passages, resistant tumors (termed as BT-vR) overnight and color reaction generated by the cleavage of the pNA substrate was were obtained. measured at a wavelength of 405 nm with the Infinite M200 PRO (Tecan) plate In the case of the generation of PDX118 resistant model to HER2-TCB, CD34+ reader36. humanized xenograft model was used. Humanized mice containing >30% hCD45+ in peripheral blood were orthotopically implanted with 107 tumor cells. Once tumors reached 300 mm3, animals started biweekly treatment with an increasing Annexin V assay. Cells were treated with 1 μg/ml of IFN-γ for 5 days. In order to concentration of HER2-TCB or vehicle intravenously. HER2-TCB treatments assay the number of apoptotic cells, cells were harvested, washed, and stained with started from 0.0325 mg/kg, and concentration was gradually increased until APC-Annexin V (1:20 from stock, #550475, BD Pharmigen) for 15 min RT. After a NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21445-4 wash with 1× PBS, samples were resuspended in 1:500 Propidium Iodine (PI) HER2-CAR T production. To produce CAR Ts against HER2, a vector plasmid (#81845, Sigma-Aldrich) viability marker and acquired on LSR Fortessa. Data was coding for HER2-CAR was synthesized and cloned into pMSGV-1 retroviral vector analyzed with FlowJo software (BD Life Sciences). Annexin V+ cells were con- (Genscript, Netherlands). Then, stocks of HER2-CAR (pMSGV1-HER2-VL-VH- sidered as apoptotic cells. Gating strategy is shown in Supplementary Fig. 8. H8) retrovirus were produced as follows. Firstly, culture plates were coated with Poly-D-Lysine 0.001% w/v in 1× PBS for 1 h at RT, to increase cell attachment. After 1 h, PBS was removed and GP2-293 cells seeded. The day after, cells were Viral tumor cells infections. For lentivirus production, HEK293T cells were first transfected with 0.3 μg/ml of envelope plasmid RD-114 (a gift from Alena Gros’ incubated for 2 h with 25 μM chloroquine (#C6628, Sigma-Aldrich) to increase Lab, VHIO) and 0.7 μg/ml of transfer plasmid (HER2), with 4.6 μl/ml of Lipo- transfection rate. Cells were then transfected with 1 μg/ml of pMD2.G (#12259, fectamine 2000 transfection reagent (#11668, Invitrogen), in DMEM F-12 without Addgene) envelope expressing plasmid, 1.2 μg/ml of psPAX2 (#12260, Addgene) supplements. After 8 h, media was changed with complete medium. Three days lentiviral packaging vector, and 1.2 μg/ml of the specific lentiviral vector, using 10 later, cell supernatant containing retrovirus particles was collected and filtered with μg/ml of polyethylenimine (PEI) (#24765, Polysciences) as transfection agent. 0.45 μm polyvinylidene fluoride (PVDF) filters. Twenty-four hour after transfection, growth medium was replaced with complete PBMCs were stimulated with 50 ng/ml of α-CD3 (OKT3) (#16-0037-85, medium. After 48 h, viral particles-containing supernatant was harvested and fil- Thermo-Fisher) and 300 IU/ml IL-2 (#703892-4, Novartis) in PBMC media for 48 tered with 0.45 μm PVDF filters (#SLHV033RS, Millipore). h before transduction. The day before transduction, 6-well plates were coated with For infections, target cells were seeded in 6-well plates (0.5 × 106 cells/well). 2 ml of 10 μg/ml retronectin (#T100A, Takara) in 1× PBS overnight at 4 °C. The After 24 h, being the confluence around 75%, tumor cells were incubated with the day of transduction, cell supernatant containing retroviral particles was centrifuged viral supernatants and 8 μg/ml polybrene (#H9268, Sigma-Aldrich), and in the retronectin-precoated 6-well plates for 2 h at 2000 × g at 32 °C. Next, viral centrifuged 45 min at 1000 × g. After 24 h, medium was replaced with complete supernatant was removed and stimulated PBMCs were added on top in PBMC medium. Twenty-four hour later, infected cells were selected with either 100 μg/ml media with 300 IU/ml IL-2 at a concentration of 2 × 106 PBMCs in 4 ml/ hygromicin in BT474 cells, 20 μg/ml blasticidin (#ant-bl, Invivogen) in the case of transduction well. Plates were centrifuged for 10 min at 500 × g at 32 °C. After 48 h, Lenti-Cas9-2A-Blast (#73310, Addgene) BT474 infected cells, or 1 μg/ml CAR Ts were transferred to cell culture flasks (#156367 and #156499, puromycin (#P8833, Sigma-Aldrich) in the rest of infections. Selection was ThermoFisher Scientific) to continue its expansion. Five days after transduction, subsequently maintained for one week. CAR expression levels were checked. A minimum of 20% CAR positive cells was In order to generate resistance to HER2-TCB, BT474 were infected with used for experiments. After 13 days of expansion, CAR Ts were frozen down in pBABE-hygro (#1765, Addgene), conferring them resistance to hygromicin. Cryostor CS10 at −80 °C and later used for coculture and in vivo experiments. For silencing, the plasmids were obtained from the lentiviral MISSION shRNA Library: TIMP2 (Clones TRCN0000052433, TRCN0000052434), IFNGR1 Statistics. For animal experiments, two-way analysis of variance (ANOVA) with (TRCN0000300831, TRCN0000058792, TRCN0000304197), JAK2 Bonferroni correction posttest was used using Graphpad. In the rest of the cases, (TRCN0000003180), JAK1 (TRCN0000121215, TRCN0000121275), and unpaired parametric t test was used using Excel. Data were considered significative STAT1 silencing (TRCN0000280021, TRCN0000280024), all from Sigma-Aldrich. when p < 0.05. As a control, tumor cells were infected with scramble shRNA (#1864, Addgene). To overexpress JAK2 in BT-R cells, JAK2 (V617F)-pcw107v5 was used (#64610, Addgene), and empty vector pcw107 (#62511, Addgene) was used as control. Reporting summary. Further information on research design is available in the Nature To generate the BT474 KO IFNGR1 cell line, cells were infected with Lenti- Research Reporting Summary linked to this article. Cas9-2A-Blast (#73310, Addgene). After selected with blasticidin, cells were infected with either a CRISPR gRNA targeting IFNGR1 (#HS5000021477, Sigma) or the Data availability LV04 control universal gRNA vector (#CRISPR18, Sigma). These gRNAs confer The RNAseq data in this study have been deposited in Sequence Read Archive (SRA) puromycin resistance and BFP expression, and cells were selected with 1 μg/ml database and are accessible through the SRA Bioproject accession code PRJNA674313 puromycin. To obtain KO IFNGR1 cells, these were stained with hIFNGR1 as (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA674313). Source data are explained before, and BFPhigh/IFNGR1 negative expressing cells were sorted in available as a Source Data file. The remaining data are available within the Article, FACSAria I Digital Cell Sorter (BD Biosciences), obtaining a pool of cells. Supplementary Information or available from the authors upon request. Validation of KO IFNGR1 cells was done by IFNGR1 staining. 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JAK2 inhibitors for myeloproliferative neoplasms: article’s Creative Commons license and your intended use is not permitted by statutory what is next? Blood 130, 115–125 (2017). regulation or exceeds the permitted use, you will need to obtain permission directly from 35. Sachs, N. et al. A living biobank of breast cancer organoids captures disease the copyright holder. To view a copy of this license, visit http://creativecommons.org/ heterogeneity. Cell 172, 373–382 (2018). e10. licenses/by/4.0/. 36. Yang, J., Pemberton, A., Morrison, W. I., Connelley, T. & Granzyme, B. Is an essential mediator in CD8+ T Cell Killing of Theileria parva-infected cells. Infect. Immun. 87, 397 (2019). © The Author(s) 2021 NATURE COMMUNICATIONS | (2021)12:1237 | https://doi.org/10.1038/s41467-021-21445-4 | www.nature.com/naturecommunications 13