PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis

Abstract

T cell non-Hodgkin lymphomas are a heterogeneous group of highly aggressive malignancies with poor clinical outcomes. T cell lymphomas originate from peripheral T cells and are frequently characterized by genetic gain-of-function variants in T cell receptor (TCR) signalling molecules. 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.

Extended Data Figure 1. Phenotypic characterization of ITK-SYK-induced lymphomas.

a, Survival curves for the ITK-SYK^CD4-Cre mice and littermate controls (CD4-Cre). The p-value was determined using the two-sided log-rank test, and the median survival was 281 days vs. not reached. b, Histologies of lymph node and liver after H&E staining of tissues from a sick ITK-SYK^CD4-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 represent 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-SYK^CD4-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-SYK^CD4-Cre mouse and clonality of the ITK-SYK^CD4-Cre lymphoma sample. RFU, relative fluorescence unit e, Survival curves for C57BL/6 mice (n=4 recipients for each donor group) which had received polyclonal ITK-SYK^CD4-Cre cells (3x10⁶) from 3-week old ITK-SYK^CD4-Cre mice or clonal ITK-SYK^CD4-Cre cells from diseased ITK-SYK^CD4-Cre mice with lymphoma (n=4 donors for each group). P=two-sided log-rank test f, Histological analysis of lymphoma samples in tamoxifen-induced ITK-SYK^CD4-CreERT2 animals. Lymph node with a loss of histomorphological architecture. Multifocal nodular infiltrations of lymphoblasts into kidney and lung are shown. Scale bars represent 100 µm and 20 µm (inset). g, Flow cytometric analyses of TCRβ-, CD4- and CD44-stained splenic cells in a tamoxifen-induced ITK-SYK^CD4-CreERT2 mouse and wild-type littermate (Control). b, c, d, f, g, Representative data from at least ten analysed mice.

Extended Data Figure 2. Lymphomas with transposon insertions within Pdcd1.

a, Overall survival of the Rosa26^LSL-ITK-SYK;Rosa26^LSL-PB;ATP2;CD4-Cre mice (red) and littermate ITK-SYK^CD4-Cre mice (blue). The Rosa26^LSL-PB;ATP2;CD4-Cre mice (black) did not display any signs of disease. The p-value was determined using two-sided log-rank test, and the median survival was 255 days vs. 289 days. b, Flow cytometric analyses of TCRβ, CD4 and eGFP expression in a typical lymphoma sample from a Rosa26^LSL-ITK-SYK;Rosa26^LSL-PB;ATP2;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 represent 200 µm and 20 µm (inset). d, Pdcd1 mRNA transcript levels in eGFP⁺ FACS-sorted peripheral lymphocytes from Rosa26^LSL-ITK-SYK;Rosa26^LSL-PB;ATP2;CD4-Cre lymphoma samples without any detectable transposon cassettes within the Pdcd1 locus (None, n=4) or with a single insertion (Single, n=4) or multiple Pdcd1-located transposon cassettes (Multiple, n=4). Pdcd1 expression levels were normalized to eGFP transcript levels. P=Tukey’s post hoc test. Shown are the mean±s.e.m and individual data points. e, Twenty unique transposon integration sites mapped to the murine 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 Rosa26^LSL-ITK-SYK;Rosa26^LSL-PB;ATP2;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 murine Engrailed 2 splice acceptor; pA, SV40 polyadenylation signal; MSCV, murine stem cell virus long terminal repeat; LunSD, splice donor from exon 1 of murine Foxf2. b, c, Representative data from at least ten analysed mice. f, The splice junctions were amplified from the cDNA of at least eight lymphomas.

Extended Data Figure 3. PDCD1 alterations in human lymphomas.

a, 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 (top). The grey horizontal bar indicates the genomic region that was affected by a PDCD1 copy number gain in T-NHL patient ATL074 (middle). Histograms showing the number of human genome-aligned RNAseq 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 (bottom). b, The dashed box indicates the genomic region on human chromosome 2 that is shown in detail in the lower part of the panel (top). CNAs in PDCD1 that were detected in CTCL patients are shown (bottom). 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 kbp. 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. CNA, copy number aberration; kbp, kilo base pairs; Mbp, million base pairs

Extended Data Figure 4. Oncogenic T cell signalling induces a PD-1 inhibitory loop.

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^KD together with GFP or GFP alone (control; GFP). 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-SYK^CD4-CreERT2 mice was assessed by a flow cytometric analysis. ITK-SYK^CD4-CreERT2 mice received 1 mg tamoxifen, and the cells were harvested 48 hours later, cultured in vitro and analysed at the indicated time points. a, b The data from three experiments with similar results. c Representative results from three analysed mice.

Extended Data Figure 5. Phenotypic characterization of PD-1 deficient lymphomas.

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-SYK^CD4-CreERT2;PD-1^-/- mouse at one week after tamoxifen (TAM) injection. Flow cytometric analysis of forward scatter area (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-SYK^CD4-CreERT2;PD-1^-/- mouse one 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 represent 100 µm and 20 µm (inset). Scale bars represent 100 µm and 20 µm (insets). d, Histology by H&E staining of lymph node and liver tissues of an NSG recipient mouse 13 days after the transplantation of 5x10⁷ lymphomatous ITK-SYK^CD4-CreERT2;PD-1^-/- splenocytes. Abnormal infiltration of lymphoblastoid cells is visible. Scale bars represent 100 µm and 20 µm (insets). e, f, 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. a, b, c Representative data from three independent experiments, each with three biological replicates. d, e, f, g Representative results from one of six NSG recipient mice from two independent experiments.

Extended Data Figure 6. Serial transplantation of PD-1-deficient lymphomas.

a, Representation of the experimental strategy for inducing ITK-SYK expression via TAT-CRE in PD-1-competent (Rosa26^LSL-ITK-SYK) or PD-1-deficient (Rosa26^LSL-ITK-SYK;PD-1^-/-) 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 5x10⁵ TAT-CRE-treated Rosa26^LSL-ITK-SYK or Rosa26^LSL-ITK-SYK;PD-1^-/-CD4⁺ T cells (n=6 donor mice per genotype). P=two-sided log-rank test c, Flow cytometric analysis of ITK-SYK/eGFP expression in peripheral blood lymphocytes from C57BL/6 mice that received TAT-CRE-treated PD-1-competent (Rosa26^LSL-ITK-SYK) or PD-1-deficient (Rosa26^LSL-ITK-SYK;PD-1^-/-) 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 Rosa26^LSL-ITK-SYK;PD-1^-/- T cells showing the expansion of a malignant T-lymphocyte population. Scale bars represent 100 µm (top row) and 20 µm (bottom row). 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, forward scatter area, which was used as a parameter to detect cell size of splenic Rosa26^LSL-ITK-SYK;PD-1^-/- 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 1x10⁵ TAT-CRE-induced Rosa26^LSL-ITK-SYK;PD-1^-/- lymphoma T-cells from diseased primary C57BL/6 recipients (n=6 biological replicates). h, H&E-stained histologic sections from lymph node and liver tissues from a diseased C57BL/6 mouse that had received 1x10⁵ TAT-CRE-treated Rosa26^LSL-ITK-SYK;PD-1^-/- T cells as the secondary recipient from a sick C57BL/6 primary recipient. Scale bars represent 100 µm and 20 µm (insets). i, j, Flow cytometric analysis as in e, f but for the secondary recipient from h. c, Characteristic flow cytometric profiles measured in two independent experiments. d, e, f, h, i, j, Representative results from one out of six analysed mice.

Extended Data Figure 7. Characterization of Pdcd1- heterozygous lymphomas.

a, Flow cytometric analysis of forward scatter area (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-SYK^CD4-CreERT2;PD-1^+/- mouse at two weeks after tamoxifen injection (0.25 mg). CD4⁺ T cells from an untreated wild-type C57BL/6 mouse (Control) 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 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, b, c. Abnormal infiltration of lymphoblastoid cells is visible. Scale bars represent 100 µm and 20 µm (insets). e, Flow cytometric analyses of PD-1 receptor expression on ITK-SYK⁺/eGFP⁺ T cells from ITK-SYK^CD4-CreERT2, ITK-SYK^CD4-CreERT2;PD-1^+/- and ITK-SYK^CD4-CreERT2;PD-1^-/- mice are shown. f, Flow cytometric analysis of PD-1 expression on ITK-SYK⁺/eGFP⁺ T lymphocytes. Cells isolated from the kidney, liver and lung of diseased ITK-SYK^CD4-CreERT2;PD-1^+/- (labelled with PD-1^+/-) or ITK-SYK^CD4-CreERT2;PD-1^-/- (labelled with PD-1^-/-) mice from the experiment presented in Fig. 4c. Shown are the mean±s.d. and individual data points; n.d., not detectable. g, Fifty million splenic cells from diseased ITK-SYK^CD4-CreERT2;PD-1^+/- 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. † indicates 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 5x10⁷ splenic cells from a TAM-induced ITK-SYK^CD4-CreERT2;PD-1^+/- 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 represent 100 µm and 20 µm (insets). a, b, c, d, Representative data from three independent experiments, each with at least three biological replicates. e, Histograms representing one out of three analysed mice per genotype. f, PD-1 expression on eGFP⁺ T cells isolated from the lung, kidney, and liver was measured in three biological replicates per genotype. g, The data from a single experiment that was independently repeated once with similar results. h, i, Representative results from one of six analysed mice from two independent experiments. j, The results are characteristic for three mice that were analysed in two independent experiments.

Extended Data Figure 8. Anti-PD-L1 triggers lethal ITK-SYK⁺ lymphoproliferation.

a, b, c, Analyses of the antibody-treated mice from the experiment shown in Fig. 4d. Flow cytometric data of forward scatter area (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 represent 100 µm and 20 µm (inset). d, Fifty million splenic cells from anti-PD-L1-treated, sick ITK-SYK^CD4-CreERT2 mice (n=3 biological replicates) were intravenously transferred to NSG recipient mice (n=3). 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-SYK^CD4-CreERT2 mouse from the experiment presented in Fig. 4f. The mouse received an anti-PD-1 antibody treatment that started ten days after tamoxifen administration. a, b, c, Representative data from one out of nine analysed mice in four independent experiments. d, The data from a single experiment that was repeated once with similar results. e, Histologies are characteristic for four mice that were analysed in two independent experiments.

Extended Data Figure 9. PD-1 regulates PI3K signalling.

a, Human T-NHL HuT 78 cells were infected with retroviruses carrying wild-type PD-1 (PD-1) or a signalling-incompetent variant of PD-1 (PD-1 YFYF; ITIM/ITSM motifs mutated in the PD-1 cytoplasmic tail) together with GFP or GFP alone (control; GFP). 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- (PD-1) or GFP only-transduced (GFP) HuT 78 cells that had been co-cultured with PD-L1-expressing human dendritic cells for 24 hours. c, Intracellular flow cytometric analyses from a similar experiment as in b but with wild-type-PD-1 (PD-1) and Y223F/Y248F-mutated-PD-1 (PD-1 YFYF) transduced HuT 78 cells. d, TAT-CRE-induced Rosa26^LSL-ITK-SYK;PD-1^-/- 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 PI3Kδ 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) or vehicle into mice. ITK-SYK^CD4-CreERT2;PD-1^-/- T cells were induced in vivo with tamoxifen (0.25 mg). On day five after induction, 5x10⁴ eGFP⁺ T cells were transplanted into NSG recipient mice. Five days later, the mice received a single gavage of idelalisib (10 mg/kg) or vehicle control. Four hours later, spleen-derived single-cell suspensions were analysed by flow cytometry via Phosflow. a, Results from two independent experiments with comparable outcomes. b, c Experiments were performed three times, each with similar results. d, Experiment was performed with four biological replicates; one representative replicate is shown. e, Experiment was performed twice with similar results.

Figure 1. Counter-regulation of oncogenic T cell signalling in vivo.

a, Frequencies of ITK-SYK-expressing eGFP⁺ lymphocytes in the blood of the ITK-SYK^CD4-CreERT2 mice following single injections of tamoxifen (n=3 mice per dose). r=Pearson’s correlation coefficient b, Gene set enrichment analyses of wild-type naïve CD4⁺ T cells (CD4 naïve) and ITK-SYK^CD4-CreERT2 T cells at 4 (ITK-SYK 4d) and 7 days (ITK-SYK 7d) after tamoxifen injection. FDR=colour intensity of the circle. NES=circle diameter. Blue/red indicates the group in which a signature was positively enriched; n=3 biological replicates for each group. NES, normalised enrichment score; FDR, false discovery rate c, Cohorts shown in a were followed over one year (n=3 biological replicates per dose). The data in the dashed box correspond to the data in a. † indicates animals that had to be euthanized because of lymphoma. P=Fisher’s exact test. The test was performed on pooled data from two independent experiments. Representative data from one experiment are shown.

Figure 2. Identification of PDCD1 alterations in T cell lymphoma.

a, Schematic representation of the transposon screen to discover T cell lymphoma genes. Transposons from the ATP2 transgene were mobilized by piggyBac in ITK-SYK-expressing T cells. The tumour cells were then FACS-sorted based on eGFP expression, and the transposon insertion sites were identified by next-generation sequencing and bioinformatics analysis. b, Transposon insertion densities within pooled lymphomas (n=30 mice) for the murine chromosome 1, total: 2732 insertions and Pdcd1 locus: 23 insertions. The p-value was calculated with a one-sided Poisson distribution based test, which assumes transposon insertions occurring at a constant rate. c, Flow cytometric analysis of PD-1 expression on lymphoma cells from diseased Rosa26^LSL-ITK-SYK;Rosa26^LSL-PB;ATP2;CD4-Cre mice. Lymphoma sample without any transposon cassette within the Pdcd1 gene (No insertion), with a single transposon cassette located in the Pdcd1 locus (Single insertion) or with multiple transposon cassettes within Pdcd1 (Multiple insertions). d, The dashed box indicates the genomic region q37.3 on human chromosome 2 that is shown in detail in the lower part of the panel (top). Mono- or bi-allelic deletions of the PDCD1 gene detected in human T cell lymphoma patients are shown (bottom). The horizontal bars indicate the regions that were affected by the CNAs. The colour of the bar indicates the type of CNA. CNA, copy number aberration; Mbp, million base pairs c, Representative results from at least 15 mice. d, Results of an analysis that included all available WGS data within one published dataset.

Figure 3. Oncogenic T cell signalling induces a PD-1 inhibitory loop.

a, PD-1 expression on the peripheral blood lymphocytes from an ITK-SYK^CD4-CreERT2 mouse as measured by flow cytometry 96 hours after tamoxifen injection. b, Normalized transcript read counts of reads that were mapped to the Pdcd1 locus and detected in the RNAseq dataset from naïve CD4⁺ T cells (Naive) or ITK-SYK-expressing eGFP⁺CD4⁺ T cells from ITK-SYK^CD4-CreERT2 mice 4 days after the tamoxifen injection (ITK-SYK). Shown are the mean±s.e.m and individual data points. FPKM, fragments per kilobase of exon per million fragments mapped; n.d., not detectable indicates FPKM<0.1; n=3 mice per group. c, Gene Set Variation Analysis (GSVA) of fifty primary human T-NHL RNAseq samples is shown. The analysis was performed based on the geneset GO_antigen_receptor_mediated_signaling_pathway. d, PDCD1 FPKM distributions from the samples in a are shown. The T-NHL patients (n=50) were classified according to their enrichment score. P=one-sided unequal variances Student’s t-test. The FPKM distributions are shown as box plots. The 0.25-, 0.5- and 0.75-quantile is depicted by the box, minimum and maximum by the whiskers. e, PDCD1 FPKM distributions from the positive samples in d are shown. The lymphomas were classified according to their PDCD1 copy number status. All T-NHLs with positive enrichment score were included (n=28). P=one-sided unequal variances Student’s t-test. The FPKM distributions are shown as box plots. The 0.25-, 0.5- and 0.75-quantile is depicted by the box, minimum and maximum by the whiskers; n.d., not detectable indicates FPKM<0.1. f, Intracellular flow cytometric analyses of PTEN, p-AKT and p-PKCθ levels in primary ITK-SYK-expressing T cells stimulated with PD-L1 or Fc-fragment-coated microspheres. a, Representative results from three analysed mice. f, Representative data from three independent experiments with two biological replicates per experiment.

Figure 4. PD-1 is a haploinsufficient tumour suppressor in vivo.

a, ITK-SYK^CD4-CreERT2 and ITK-SYK^CD4-CreERT2;PD-1^-/- mice (n=3 mice per genotype) received single tamoxifen injections (0.25 mg). The percentages of ITK-SYK-expressing eGFP⁺ blood lymphocytes were determined using flow cytometry. b, Fifty million splenic cells from diseased ITK-SYK^CD4-CreERT2;PD-1^-/- mice (n=3 donors) were intravenously transferred to NSG mice (n=3 recipients). The percentage of eGFP⁺ lymphocytes in the recipient’s blood is indicated. NSG, NOD SCID Il2rg^−/− c, Survival of ITK-SYK^CD4-CreERT2, ITK-SYK^CD4-CreERT2;PD-1^+/- and ITK-SYK^CD4-CreERT2;PD-1^-/- mice (n=6, n=12 and n=14). All mice received a single tamoxifen dose on day zero. P=two-sided log-rank test d, The percentages of ITK-SYK-expressing eGFP⁺ peripheral blood lymphocytes of checkpoint inhibitor-treated ITK-SYK^CD4-CreERT2 mice were determined by FACS. ITK-SYK^CD4-CreERT2 mice received single tamoxifen injections. Beginning twenty-four hours later, the mice received 200 µg of anti-PD-L1 or control antibody (n=3 mice per condition) every second day. e, Anti-CD3 antibody-stained liver section from a diseased anti-PD-L1-treated ITK-SYK^CD4-CreERT2 mouse from the experiment shown in d, Scale bars represent 100 µm and 20 µm (inset). f, Survival of ITK-SYK^CD4-CreERT2 mice that received checkpoint inhibitor treatment starting ten days after tamoxifen administration. ITK-SYK^CD4-CreERT2 mice received single injections of tamoxifen on day zero (0.25 mg). Starting ten days after injection, mice were administered 200 µg of anti-PD-L1 or anti-PD-1 antibody or control antibody (n=4 mice per condition) every third day. P=two-sided log-rank test g, Phosflow analyses of PTEN, p-AKT and p-PKCθ levels in ITK-SYK-expressing T cells isolated from the C57BL/6 recipients presented in Extended Data Figure 6b. The genotype of the transplanted cells is indicated. h, Survival of the PI3Kδ inhibitor- or vehicle-treated recipient mice. Tamoxifen-induced ITK-SYK^CD4-CreERT2;PD-1^-/- T cells (n=5 biological replicates, 5x10⁴ cells per transplant) were transplanted into NSG mice (n=5 recipients per group). P=two-sided log-rank test. † indicates animals that were euthanized because of lymphomas. a, Representative data from three independent experiments, each with three biological replicates. b, The data from a single experiment that was independently repeated once with comparable results. c, Pooled data from two independent experiments. d, Representative data from four independent experiments, each with three biological replicates per antibody. e, Representative histology from one out of nine analysed mice. f, Data from a single experiment, which was repeated once with similar results. g, Representative results from two out of eight analysed mice in two independent experiments. h, Pooled survival data from two experiments.