T cell non-Hodgkin lymphomas are a heterogeneous group of highly aggressive malignancies with poor clinical outcomes1. T cell lymphomas originate from peripheral T cells and are frequently characterized by genetic gain-of-function variants in T cell receptor (TCR) signalling molecules1,2,3,4. Although these oncogenic alterations are thought to drive TCR pathways to induce chronic proliferation and cell survival programmes, it remains unclear whether T cells contain tumour suppressors that can counteract these events. Here we show that the acute enforcement of oncogenic TCR signalling in lymphocytes in a mouse model of human T cell lymphoma drives the strong expansion of these cells in vivo. However, this response is short-lived and robustly counteracted by cell-intrinsic mechanisms. A subsequent genome-wide in vivo screen using T cell-specific transposon mutagenesis identified PDCD1, which encodes the inhibitory receptor programmed death-1 (PD-1), as a master gene that suppresses oncogenic T cell signalling. Mono- and bi-allelic deletions of PDCD1 are also recurrently observed in human T cell lymphomas with frequencies that can exceed 30%, indicating high clinical relevance. Mechanistically, the activity of PD-1 enhances levels of the tumour suppressor PTEN and attenuates signalling by the kinases AKT and PKC in pre-malignant cells. By contrast, a homo- or heterozygous deletion of PD-1 allows unrestricted T cell growth after an oncogenic insult and leads to the rapid development of highly aggressive lymphomas in vivo that are readily transplantable to recipients. Thus, the inhibitory PD-1 receptor is a potent haploinsufficient tumour suppressor in T cell lymphomas that is frequently altered in human disease. These findings extend the known physiological functions of PD-1 beyond the prevention of immunopathology after antigen-induced T cell activation, and have implications for T cell lymphoma therapies and for current strategies that target PD-1 in the broader context of immuno-oncology.
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We thank N. Prause and K. Burmeister for providing technical assistance and S. Ogawa, K. Kataoka, R. P. Lifton and J. Choi for providing access to NGS data from patients with T cell lymphoma. This study used data generated by the Department of Pathology and Tumor Biology of Kyoto University. This work was supported by research grants from the DFG (SFB 1054/B01 and RU 695/6-1) and ERC (FP7, grant agreement no. 322865) awarded to J.R.
The authors declare no competing financial interests.
Reviewer Information Nature thanks V. Boussiotis and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, Survival curves for the ITK-SYKCD4-cre mice and littermate controls (CD4-Cre). P value was determined using the two-sided log-rank test, and the median survival was 281 days versus not reached. b, Histological analysis of lymph node and liver after H&E staining of tissues from a sick ITK-SYKCD4-cre mouse on a C57BL/6 genetic background. The architecture of the lymph node is disrupted by a diffuse infiltration of lymphoblastoid cells. Multinodal perivascular infiltrations of lymphoblasts are also detectable in the liver. Scale bars, 200 μm and 20 μm (insets). c, Flow cytometric analyses of forward scatter area (FSC-A), which was used as a parameter to detect cell size, and TCRβ, CD4 and eGFP expression in lymphoma tissues from a sick ITK-SYKCD4-cre mouse. Peripheral lymphocytes from a CD4-Cre littermate mouse served as the control. d, GeneScan analysis of TCR gene rearrangements. Fragment size distributions of fluorochrome-labelled PCR products of the Vβ1-20;Jβ2 junction demonstrating polyclonal repertoires of a healthy CD4-Cre mouse (control), a 3-week-old ITK-SYKCD4-cremouse and clonality of the ITK-SYKCD4-crelymphoma sample. RFU, relative fluorescence units. e, Survival curves for C57BL/6 mice (n = 4 recipients for each donor group) that had received polyclonal ITK-SYKCD4-crecells (3 × 106) from 3-week-old ITK-SYKCD4-cremice or clonal ITK-SYKCD4-cre cells from diseased ITK-SYKCD4-cre mice with lymphoma (n = 4 donors for each group). P value determined by two-sided log-rank test. f, Histological analysis of lymphoma samples in tamoxifen-induced ITK-SYKCD4-creERT2 animals. Lymph node with a loss of histomorphological architecture. Multifocal nodular infiltrations of lymphoblasts into kidney and lung are shown. Scale bars, 100 μm and 20 μm (inset). g, Flow cytometric analyses of TCRβ- and CD4-stained splenic cells in a tamoxifen-induced ITK-SYKCD4-creERT2 mouse and wild-type littermate control. h, CD44-stained lymphocytes extracted from the liver from the ITK-SYKCD4-creERT2 mouse in g. Data in b–d and f–h are representative from at least 10 analysed mice.
a, Overall survival of the Rosa26LSL-ITK-SYK;Rosa26LSL-PB;ATP2-H32;CD4-cre mice (red) and littermate control ITK-SYKCD4-cre mice (blue). The Rosa26LSL-PB;ATP2-H32;CD4-cre mice (black) did not display any signs of disease. P value was determined using two-sided log-rank test, and the median survival was 255 days versus 289 days. b, Flow cytometric analyses of TCRβ, CD4 and eGFP expression in a typical lymphoma sample from a Rosa26LSL-ITK-SYK;Rosa26LSL-PB;ATP2-H32;CD4-cre mouse. c, Histological analysis of the mouse shown in b with multinodal perivascular infiltrations of lymphoblasts into the liver (left) and lung (right). Scale bars, 200 μm and 20 μm (inset). d, Pdcd1 mRNA transcript levels in eGFP+ FACS-sorted peripheral lymphocytes from Rosa26LSL-ITK-SYK;Rosa26LSL-PB;ATP2-H32;CD4-cre lymphoma samples without any detectable transposon cassettes within the Pdcd1 locus (none, n = 4) or with a single insertion (n = 4) or multiple Pdcd1-located transposon cassettes (n = 4). Pdcd1 expression levels were normalized to eGFP transcript levels. P value determined by Tukey’s post hoc test. Data are mean ± s.e.m., and individual data points are shown. e, Twenty unique transposon integration sites mapped to the mouse Pdcd1 locus. The vertical black bars indicate the genomic positions of the insertion. The red arrows indicate the orientations of the transposon cassettes. f, Nucleotide sequences of the Pdcd1-piggyBac mRNA splice junctions as determined by Sanger sequencing of amplified cDNA from Rosa26LSL-ITK-SYK;Rosa26LSL-PB;ATP2-H32;CD4-cre lymphoma samples. The cDNA was generated from sorted eGFP+ lymphoma cells in which at least one Pdcd1 transposon cassette could be detected. Note that the 5′ piggyBac transposon-specific inverted terminal repeat nucleotide sequence (PB5) can function as a cryptic splice acceptor and a splice donor site. Hence, PB5 may act as a gene trapping sequence by itself14. En2SA, exon 2 located mouse En2 splice acceptor; pA, SV40 polyadenylation signal; MSCV, murine stem cell virus long terminal repeat; LunSD, splice donor from exon 1 of mouse Foxf2. Data in b and c are representative from at least 10 analysed mice. The splice junctions in f were amplified from the cDNA of at least eight lymphomas.
a, Top, the dashed box indicates the genomic region q37.3 on human chromosome 2 that is shown in more detail in the middle of the panel. Middle, the grey horizontal bar indicates the genomic region that was affected by a PDCD1 copy number gain in a patient (ATL074) with T cell NHL2. Bottom, histograms showing the number of human genome-aligned RNA-seq reads from peripheral blood mononuclear cells (PBMCs) of two healthy donors and lymphoma cells of patient ATL074 at the PDCD1 locus and a non-coding region near the PDCD1 3′ UTR. b, Top, the dashed box indicates the genomic region on human chromosome 2 that is shown in detail in the bottom of the panel. Bottom, CNAs in PDCD1 that were detected in patients with cutaneous T cell lymphoma (CTCL)6 are shown. The vertical dashed lines indicate the genomic position of the PDCD1 locus. Black dots represent the logarithmic tumour/normal copy number ratio; each dot represents 1 kb. The grey lines indicate logarithmic value of the median tumour/normal copy number ratio. The arrow indicates a bi-allelic PDCD1 loss in sample CTCL08.
a, b, Jurkat T cells (a) or primary human CD4+ T cells (b) were infected with retroviruses carrying ITK–SYK or kinase-defective ITK–SYK (ITK–SYKKD) together with GFP or GFP alone (control). PD-1 surface expression was determined by flow cytometry. c, PD-1 expression after acute ITK–SYK signalling in primary T cells from tamoxifen-treated ITK-SYKCD4-creERT2 mice was assessed by a flow cytometric analysis. ITK-SYKCD4-creERT2 mice received 1 mg tamoxifen, and the cells were collected 48 h later, cultured in vitro and analysed at the indicated time points. The data in a and b are from three experiments with similar results. Results in c are representative from three analysed mice.
a, b, Flow cytometric analyses of TCRβ, CD4, PD-1 and eGFP expression in single-cell suspensions from the spleen (a) or kidney, liver and lung (b) from an ITK-SYKCD4-creERT2;Pdcd1−/− mouse at 1 week after tamoxifen injection. Flow cytometric analysis of FSC-A was used as a parameter to detect cell size. c, Lymph node and lung histology by H&E staining of tissues from a diseased ITK-SYKCD4-creERT2;Pdcd1−/− mouse 1 week after tamoxifen induction. The lymph node architecture was disrupted by a diffuse neoplastic lymphoid infiltration. In the lung, neoplastic lymphoid cells were located in intra- and perivascular regions with a multifocal nodal infiltration pattern. Scale bars, 100 μm and 20 μm (inset). d, Histology by H&E staining of lymph node and liver tissues of an NSG recipient mouse 13 days after the transplantation of 5 × 107 lymphomatous ITK-SYKCD4-creERT2;Pdcd1−/− splenocytes. Abnormal infiltration of lymphoblastoid cells is visible. Scale bars, 100 μm and 20 μm (insets). e–g, Flow cytometric analysis of FSC-A and TCRβ, CD4 and eGPF expression in lymphoma cells in the spleen (e, f), kidney, liver and lung (g) from the same mouse as in d. Data in a–c are representative from three independent experiments, each with three biological replicates. Results in d–g are representative from one of six NSG recipient mice from two independent experiments.
a, Representation of the experimental strategy for inducing ITK–SYK expression via TAT-Cre in PD-1-competent (Rosa26LSL-ITK-SYK) or PD-1-deficient (Rosa26LSL-ITK-SYK;Pdcd1−/−) cells for subsequent transfer into wild-type C57BL/6 recipient mice. b, Survival curves of the C57BL/6 recipients (n = 6 recipients per genotype) transplanted with 5 × 105 TAT-Cre-treated Rosa26LSL-ITK-SYK or Rosa26LSL-ITK-SYK;Pdcd1−/− CD4+ T cells (n = 6 donor mice per genotype). P value determined by two-sided log-rank test. c, Flow cytometric analysis of ITK–SYK-expressing eGFP+ peripheral blood lymphocytes from C57BL/6 mice that received TAT-Cre-treated PD-1-competent (Rosa26LSL-ITK-SYK) or PD-1-deficient (Rosa26LSL-ITK-SYK;Pdcd1−/−) cells on days 7 and 21 after transplantation. The genotypes of the donor mice are indicated. d, Histology and immunohistochemistry of the indicated organs from a sick primary recipient mouse after the transfer of TAT-Cre-treated Rosa26LSL-ITK-SYK;Pdcd1−/− T cells showing the expansion of a malignant T cell population. Scale bars, 100 μm (top) and 20 μm (bottom). e, f, Flow cytometric analysis of CD4-stained lymphocytes derived from the spleen (e) or indicated organs (f) of the mouse shown in d. FSC-A was used as a parameter to detect cell size of splenic Rosa26LSL-ITK-SYK;Pdcd1−/− T cells compared to the TAT-Cre-treated C57BL/6 CD4+ T cells (control). g, Survival of secondary C57BL/6 recipient mice (n = 6) that received 1 × 105 TAT-Cre-induced Rosa26LSL-ITK-SYK;Pdcd1−/− lymphoma T cells from diseased primary C57BL/6 recipients (n = 6 biological replicates). h, H&E-stained histological sections from lymph node and liver tissues from a diseased C57BL/6 mouse that had received 1 × 105 TAT-Cre-treated Rosa26LSL-ITK-SYK;Pdcd1−/− T cells as the secondary recipient from a sick C57BL/6 primary recipient. Scale bars, 100 μm and 20 μm (insets). i, j, Flow cytometric analysis as in e and f but for the secondary recipient from h. Characteristic flow cytometric profiles in c were measured in two independent experiments. Results in d–f and h–j are representative results from one out of six analysed mice.
a, Flow cytometric analysis of FSC-A, which was used as a parameter to detect cell size, and TCRβ, PD-1 and eGPF expression in splenic lymphoma cells from a diseased ITK-SYKCD4-creERT2;Pdcd1+/− mouse at 2 weeks after tamoxifen injection (0.25 mg). CD4+ T cells from an untreated wild-type C57BL/6 mouse served as control. b, c, Flow cytometric analysis of CD4, eGFP and PD-1 expression in single-cell suspensions of the indicated dissociated organs from the same mouse as in a. d, Histology analysis after H&E staining and immunohistochemical staining with anti-CD3 or anti-Ki-67 antibody of the liver tissue from the same animal as in a–c. Abnormal infiltration of lymphoblastoid cells is visible. Scale bars, 100 μm and 20 μm (insets). e, Flow cytometric analyses of PD-1 receptor expression on ITK–SYK-expressing eGFP+ T cells from ITK-SYKCD4-creERT2, ITK-SYKCD4-creERT2;Pdcd1+/− and ITK-SYKCD4-creERT2;Pdcd1−/− mice are shown. f, Flow cytometric analysis of PD-1 expression on ITK–SYK-expressing eGFP+ T cells. Cells isolated from the kidney, liver and lung of diseased ITK-SYKCD4-creERT2;Pdcd1+/− or ITK-SYKCD4-creERT2;Pdcd1−/− mice from the experiment presented in Fig. 4c. Data are mean ± s.d., and individual data points are shown. ND, not detectable. g, Fifty million splenic cells from diseased ITK-SYKCD4-creERT2;Pdcd1+/− mice (n = 3 donors) were intravenously transferred to NSG mice (n = 3 recipients). The fold changes of ITK–SYK-expressing eGFP+ lymphocytes in the peripheral blood of the recipients are indicated. Dagger symbols indicate animals that had to be euthanized because of lymphomas. h, i, Flow cytometric analysis of the FSC-A, TCRβ, PD-1, CD4 and eGFP expression in spleen cell suspension (h) or lymphoma cells isolated from the lung, kidney and liver (i) of an NSG mouse that received 5 × 107 splenic cells from a tamoxifen-induced ITK-SYKCD4-creERT2;Pdcd1+/− mouse. CD4+ T cells from an untreated wild-type C57BL/6 mouse served as control. j, Spleen and liver histology after H&E staining of organ sections of the NSG recipient mouse presented in h, i. Infiltration of a lymphoblastoid cell population in the spleen resulting in a complete loss of the normal architecture. Mixed periportal and intrasinusoidal infiltrations of neutrophilic granulocytes, lymphocytes and lymphoblasts into the liver with multifocal periportal hepatocellular necroses. Scale bars, 100 μm and 20 μm (insets). Data in a–d are representative from three independent experiments, each with at least three biological replicates. Histograms in e represent one out of three analysed mice per genotype. Data in f are from three biological replicates per genotype. Data in g are from a single experiment that was independently repeated once with similar results. Results in h and i are representative from one out of six analysed mice from two independent experiments. The results in j are characteristic for three mice that were analysed in two independent experiments.
a–c, Analyses of the antibody-treated mice from the experiment shown in Fig. 4d. Flow cytometric data of FSC-A, which was used as a parameter to detect cell size, and TCRβ, CD4, PD-1 and eGPF expression in lymphomatous cells isolated from the spleen (a), kidney, liver and lung (b). c, Histology and immunohistochemistry of liver sections from the same mouse demonstrating the expansion of blastoid T cells. Scale bars, 100 μm and 20 μm (inset). d, Fifty million splenic cells from anti-PD-L1-treated, sick ITK-SYKCD4-creERT2 mice (n = 3 biological replicates) were intravenously transferred to NSG recipient mice (n = 3 recipients). The fold change of eGFP+ lymphocytes in the peripheral blood of recipients over time is shown. e, Liver and lymph node histology after H&E staining of tissue sections from a diseased ITK-SYKCD4-creERT2 mouse from the experiment presented in Fig. 4f. The mouse received an anti-PD-1 antibody treatment that started 10 days after tamoxifen administration. Data in a–c are representative from one out of nine analysed mice in four independent experiments. The data in d are from a single experiment that was repeated once with similar results. Histology analyses in e are characteristic for four mice that were analysed in two independent experiments.
a, Human T cell NHL HuT 78 cells were infected with retroviruses carrying wild-type PD-1 or a signalling-incompetent variant of PD-1 (Y223F/Y248F (YFYF); ITIM/ITSM motifs mutated in the PD-1 cytoplasmic tail) together with GFP or GFP alone (control). PD-1 surface expression was determined by flow cytometry. b, Intracellular flow cytometric analyses of PTEN, p-AKT and p-PKCθ levels in wild-type PD-1-tranduced or GFP-only-transduced HuT 78 cells that had been co-cultured with PD-L1-expressing human dendritic cells for 24 h. c, Intracellular flow cytometric analyses from a similar experiment as in b but with HuT 78 cells transduced with wild-type PD-1 or mutated PD-1 (YFYF). d, TAT-Cre-induced Rosa26LSL-ITK-SYK;Pdcd1−/− cells isolated from diseased C57BL/6 recipient mice (see Extended Data Fig. 6b) were cultured in vitro in the presence of the indicated concentration of the PI3K inhibitor idelalisib or DMSO. Cell viability was determined over time. e, Ex vivo phosphorylation status of the AKT kinase after the oral administration of idelalisib (10 mg kg−1) or vehicle into mice. ITK-SYKCD4-creERT2;Pdcd1−/− T cells were induced in vivo with tamoxifen (0.25 mg). On day 5 after induction, 5 × 104 eGFP+ T cells were transplanted into NSG recipient mice. Five days later, the mice received a single gavage of idelalisib (10 mg kg−1) or vehicle control. Four hours later, spleen-derived single-cell suspensions were analysed by flow cytometry via Phosflow. Results in a are from two independent experiments with comparable outcomes. Experiments in b and c were performed three times, each with similar results. The experiment in d was performed with four biological replicates; one representative replicate is shown. The experiment in e was performed twice with similar results.
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Wartewig, T., Kurgyis, Z., Keppler, S. et al. PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis. Nature 552, 121–125 (2017). https://doi.org/10.1038/nature24649
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