IMMUNOBIOLOGY
`
`Programmed death 1 signaling on chronic myeloid leukemia–specific T cells
`results in T-cell exhaustion and disease progression
`Sabine Mumprecht,1 Christian Schu¨ rch,1 Juerg Schwaller,2 Max Solenthaler,3 and Adrian F. Ochsenbein1,4
`
`1Tumor Immunology, Department of Clinical Research, University of Berne, Berne; 2Department of Research, Childhood Leukemia, University of Basel, Basel;
`and 3Department of Hematology and Central Hematology Laboratory and 4Institute for Medical Oncology, Inselspital, Berne, Switzerland
`
`Chronic myeloid leukemia (CML) is a ma-
`lignant myeloproliferative disease with a
`characteristic chronic phase (cp) of sev-
`eral years before progression to blast
`crisis (bc). The immune system may con-
`tribute to disease control
`in CML. We
`analyzed leukemia-specific immune re-
`sponses in cpCML and bcCML in a retrovi-
`ral-induced murine CML model.
`In the
`presence of cpCML and bcCML express-
`ing the glycoprotein of lymphocytic cho-
`Introduction
`
`riomeningitis virus as a model leukemia
`antigen,
`leukemia-specific
`cytotoxic
`T lymphocytes (CTLs) became exhausted.
`They maintained only limited cytotoxic
`activity, and did not produce interferon-␥
`or tumor necrosis factor-␣ or expand
`after restimulation. CML-specific CTLs
`were characterized by high expression of
`programmed death 1 (PD-1), whereas CML
`cells expressed PD-ligand 1 (PD-L1).
`Blocking the PD-1/PD-L1 interaction by
`
`generating bcCML in PD-1–deficient mice
`or by repetitive administration of ␣PD-L1
`antibody prolonged survival. In addition,
`we found that PD-1 is up-regulated on
`CD8ⴙ T cells from CML patients. Taken
`together, our results suggest that block-
`ing the PD-1/PD-L1 interaction may re-
`store the function of CML-specific CTLs
`and may represent a novel therapeutic
`approach for CML.
`(Blood. 2009;114:
`1528-1536)
`
`Chronic myeloid leukemia (CML) is a clonal myeloproliferative
`disorder resulting from the neoplastic transformation of a hemato-
`poietic stem cell.1 The disease is bi- or triphasic, comprising a
`chronic, an accelerated, and a terminal blast phase in which the
`patients develop an acute leukemia of either myeloid (AML) or,
`less often, lymphoid (ALL) cell type. More than 90% of all CML
`cases are associated with the presence of the Philadelphia chromo-
`some, which results from a reciprocal
`translocation between
`chromosomes 9 and 22 forming the breakpoint cluster region/
`Abelson protein tyrosine kinase (BCR/ABL) fusion protein, a
`constitutively activated tyrosine kinase.2,3 Depending on the pre-
`cise breakpoints in the BCR gene, different forms of BCR/ABL
`fusion protein with different molecular weights can be generated
`(p190BCR/ABL, p210BCR/ABL, and p230BCR/ABL). CML pa-
`tients predominantly express p210BCR/ABL.1
`Currently, BCR/ABL-selective tyrosine kinase inhibitors are
`the standard treatment for CML. However, resistant clones often
`develop during treatment. At present, the only curative treatment
`for CML is allogeneic hematopoietic stem cell transplantation.4
`Several earlier studies suggested that the immune system plays
`an important role in the control of CML. CML cells are susceptible
`to lysis by CD8⫹ T cells5 and natural killer (NK) cells in vitro.6 For
`unknown reasons, CML is the most graft-versus-leukemia–
`sensitive leukemia.7 In addition, cytotoxic T lymphocytes (CTLs)
`directed against leukemia antigens are found in CML patients
`without hematopoietic stem cell transplantation, including CTLs
`specific for BCR/ABL, overexpressed self-proteins such as protein-
`ase-3, and Wilms tumor 1 protein.5,8 However, the physiologic
`relevance of these leukemia-specific CTL responses in the control
`of CML is unknown. The presence of CTL escape mechanisms
`
`during CML disease progression to blast crisis suggests that CTLs
`are involved in the control of the chronic phase of the disease.
`Documented escape mechanisms include the expression of Fas
`ligand during blast crisis,9 the deletion of high-avidity CTLs
`specific for a leukemia-associated self-antigen,10 and the develop-
`ment of functional blocks in the caspase activation pathway in
`AML cells.11
`Coinhibitory molecules are essential for the maintenance of
`T-cell homeostasis, self-tolerance, and tolerance to chronic infec-
`tions. Programmed death 1 (PD-1) is a member of the immunoglobu-
`lin (Ig) superfamily, which is inducibly expressed on T cells,
`B cells, and activated monocytes.12 To mediate inhibitory signals
`through PD-1, the binding of either of the 2 ligands to the receptor
`is necessary. PD-ligand 1 (PD-L1, B7-H1) is expressed constitu-
`tively on resting T cells, B cells, dendritic cells (DCs), macro-
`phages, mesenchymal stem cells, some parenchymal cells, and
`cultured bone marrow-derived mast cells. PD-L1 expression is
`further up-regulated after activation.13 PD-ligand 2 (PD-L2, B7-
`DC) is only inducibly expressed on DCs, macrophages, and
`cultured bone marrow-derived mast cells.13 Recently, PD-1 signal-
`ing has been identified as an important mechanism of antigen-
`specific T-cell dysfunction in chronic infections such as lympho-
`cytic choriomeningitis virus (LCMV) infection in mice14 or HIV15
`and hepatitis C virus (HCV)16 infections in humans. In addition, it
`has been shown that PD-1 leads to functional impairment of
`tumor-infiltrating T cells in solid tumors.17 In contrast, mechanisms
`determining the function of leukemia-specific CTLs have not yet
`been analyzed in detail.
`In the present study, we analyzed the immunosurveillance of
`chronic phase CML (cpCML) and blast crisis CML (bcCML) in a
`
`Submitted September 16, 2008; accepted April 25, 2009. Prepublished online
`as Blood First Edition paper, May 6, 2009; DOI 10.1182/blood-2008-09-
`179697.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
`
`An Inside Blood analysis of this article appears at the front of this issue.
`
`© 2009 by The American Society of Hematology
`
`1528
`
`BLOOD, 20 AUGUST 2009 䡠 VOLUME 114, NUMBER 8
`
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`BLOOD, 20 AUGUST 2009 䡠 VOLUME 114, NUMBER 8
`
`EXHAUSTION OF CML-SPECIFIC T CELLS BY PD-1 IMMUNOBIOLOGY
`
`1529
`
`murine retroviral bone marrow transduction and transplantation
`model.18 We found that leukemia-specific CTLs were functionally
`exhausted and phenotypically characterized by high expression of
`PD-1, in both cpCML and bcCML. Blocking PD-1 signal transduc-
`tion by using PD-1–deficient recipient mice or by administration of
`␣PD-L1 antibody reversed CML-specific T-cell tolerance, and the
`time to disease progression was increased.
`
`Methods
`
`Mice
`
`C57BL/6 mice were purchased from Harlan. The p14 T-cell receptor (TCR)
`transgenic mice19 and H8 transgenic mice20 were obtained from the Institute
`for Laboratory Animals. CD45.1⫹ mice were obtained from C. Mueller
`(University of Berne). PD-1–deficient mice21 were provided by T. Honjo
`(Kyoto University). Animal experiments were performed with sex- and
`age-matched mice and approved by the Experimental Animal Committee of
`the Canton of Berne and performed according to Swiss laws for animal
`protection.
`
`Viruses, peptide, and retroviral vectors
`
`LCMV, strain WE, and Docile were provided by R. M. Zinkernagel
`(University Hospital, Zurich) and propagated, as described.22,23 The LCMV-
`GP, amino acids 33-41 (glycoprotein [gp]33, KAVYNFATM), was pur-
`chased from NeoMPS SA. The retroviral vectors pMSCV-p210 BCR/ABL-
`IRES-GFP, pMSCV-p210 BCR/ABL-pgk-neo, and pMSCV-NUP98/
`HOXA9-IRES-GFP (MSCV, mouse stem cell virus; IRES, internal ribosomal
`entry site; GFP, green fluorescent protein; neo, neomycin) were a gift from
`D. G. Gilliland (Havard Medical School), and the packaging vector pIK6
`was purchased from Cell Genesys.24-26
`
`51Cr-release assay
`
`Spleens were isolated and analyzed after 5 days of in vitro restimulation in a
`standard 51Cr-release assay, as described.27 The cytotoxicity assay of ex
`vivo isolated p14 T cells was performed as follows: 6 ⫻ 105 CD45.1⫹ cells,
`purified by magnetic cell sorting (MACS; Miltenyi Biotec), were either
`used directly or restimulated for 5 days with 3 ⫻ 105 irradiated gp33-
`pulsed naive C57BL/6 splenocytes in the presence of 50 U/mL murine
`interleukin (IL)-2.
`
`Cells and retroviral particle production
`
`Retroviral particles were generated by transient cotransfection of 293-
`T cells with the respective MSCV vector and pIK6, using Superfect
`transfection reagent (Qiagen), according to the manufacturer’s protocol.
`After 48 hours, virus-containing supernatant was harvested. For determina-
`tion of retroviral titers, BA/F3 cells were infected with different amounts of
`retroviral supernatant using polybrene transfection reagent (10 ␮g/mL;
`Sigma-Aldrich). After 48 hours, retroviral
`titers were determined by
`enumerating GFP-positive cells by flow cytometry. Alternatively, to assess
`virus titers of the vector containing a neocassette, BA/F3 cells were infected
`and then cultivated for 5 days in the presence or absence of 0.8 mg/mL
`G-418 sulfate (Gibco). Surviving cells were assessed with trypan blue
`staining.
`
`retroviral particles with polybrene (6.7 ␮g/mL) and 0.01 M HEPES (N-2-
`hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid) through spin infection
`(90 minutes at 1258g, 30°C). A total of 105 transduced bone marrow cells
`was injected intravenously into previously irradiated (4.5, 6.5, or 9.5 Gy)
`syngeneic recipient mice (transduction efficiency: 24.0% ⫾ 9.8%).
`
`Antibodies and flow cytometry
`
`␣CD8-allophycocyanin (APC), ␣CD4-biotin, ␣B220-biotin, ␣I-Ab-major histo-
`compatibility complex (MHC) class II-biotin, ␣CD45.1-phycoerythrin (PE) and
`-APC, ␣CD11c-biotin, ␣ interferon (IFN)-␥-PE, ␣ tumor necrosis factor (TNF)-
`␣-PE, streptavidin-PE and APC, ␣NK1.1-biotin, ␣PD-1-biotin, ␣PD-L1-biotin,
`␣PD-L2-biotin, rat IgG2a isotype-biotin, and human ␣PD-1-PE were obtained
`from eBioscience. ␣GR-1-PE, ␣CD8-peridinin chlorophyll protein (PerCP)-
`Cy5.5, ␣CD4-PerCP-Cy5.5, ␣PD-1-PerCP-Cy5.5, ␣V␣2-biotin and PE, ␣Sca-1-
`APC, ␣MAC-1-PE-Cy7, ␣C-kit-PE-Cy7, mouse IgG1-PE isotype control,
`human ␣CD8-fluorescein isothiocyanate, and human ␣CD45-PerCP-Cy5.5 were
`obtained from BD Pharmingen. MHC class I (H-2Db) tetramer-PE complexed
`with gp33 or nuclear protein (np)396 was obtained from ProImmune and used
`according to the manufacturer’s protocol. Intracellular staining was performed, as
`described.28 Relative fluorescence intensities were measured on a BD LSRII flow
`cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar). The
`frequency of IFN-␥– and TNF-␣–producing CD8⫹ T cells was calculated as
`gp33-pulsed minus nonpulsed samples.
`
`Adoptive transfer experiments and reisolation of transferred
`CD8ⴙ T cells
`
`P14 ⫻ CD45.1 T cells were isolated and purified by MACS for CD8⫹V␣2⫹
`T cells. A total of 2.5 to 4 ⫻ 106 CD8⫹Va2⫹CD45.1⫹ cells was injected
`intravenously into CML mice, naive C57BL/6 control mice, C57BL/6 mice
`that were infected with 104 plaque-forming units (pfu) of LCMV-WE, and
`C57BL/6 mice chronically infected with 107 pfu of LCMV Docile (all
`recipient mice were CD45.1⫺). CML disease progress was monitored by
`fluorescence-activated cell sorter (FACS) analysis of blood, and on the days
`indicated, CD45.1⫹ T cells from CML mice, naive C57BL/6 mice, and
`C57BL/6 mice infected with LCMV were isolated by MACS.
`
`Proliferation assays
`
`For ex vivo proliferation of transferred p14 T cells, 2 ⫻ 105 CD45.1⫹ cells
`were restimulated for 4 days with 2 ⫻ 105 irradiated gp33-pulsed or
`nonpulsed naive C57BL/6 splenocytes. For in vitro proliferation, naive p14
`CD8⫹ T cells and p14 ⫻ PD-1⫺/⫺ CD8⫹ T cells (2 ⫻ 105) were restimu-
`lated with irradiated (50 Gy) FACS-sorted H8-cpCML cells (BCR/ABL-
`GFP⫹), FACS-sorted H8-bcCML cells (NUP98/HOXA9-GFP⫹), H8 spleno-
`cytes, or C57BL/6 splenocytes (0.4 ⫻ 105). 3H-thymidine incorporation
`assay was performed, as described.22 The proliferation index was calculated
`as the ratio between gp33-pulsed and nonpulsed samples (ex vivo prolifera-
`tion assay) and as the ratio between H8-CML cells or H8 splenocytes and
`C57BL/6 splenocytes used as stimulators (in vitro proliferation assay).
`
`Cytometric bead array assay
`
`A total of 1.5 ⫻ 105 CD45.1⫹ cells was restimulated for 18 hours with
`3 ⫻ 105 irradiated gp33-pulsed or nonpulsed naive C57BL/6 splenocytes.
`Cytokine production in supernatant was analyzed with a mouse T helper
`cell 1/2 cytokine cytometric bead array assay according to the manufactur-
`er’s protocol (BD Biosciences).
`
`CML model
`
`PD-L1 blockade
`
`Bone marrow donor mice were pretreated with 150 mg/kg 5-fluorouracil
`intraperitoneally (Sigma-Aldrich). After 6 days, the bone marrow was
`flushed out from femurs and tibias. Erythrocytes were removed and the
`bone marrow cells were incubated in transplant media (RPMI/10% fetal
`calf serum with recombinant murine IL-3 [6 ng/mL; BD Biosciences],
`recombinant murine stem cell factor [10 ng/mL; Biocoba], and recombinant
`human IL-6 [10 ng/mL; BD Biosciences]) for 24 hours. A total of 4 ⫻ 106
`cells was transfected twice on 2 consecutive days with the respective
`
`bcCML mice were treated intraperitoneally every third day with 200 ␮g of
`␣PD-L1 monoclonal antibody (clone 2G9; BioXCell) or IgG from rat
`serum (I8015; Sigma-Aldrich) starting on the day of transplantation.
`
`CD8 depletion
`
`bcCML mice were treated intraperitoneally on day 0 and day 2, and from
`then on weekly with 100 ␮g of␣CD8 monoclonal antibody (YTS 169.4).
`
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`1530
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`MUMPRECHT et al
`
`BLOOD, 20 AUGUST 2009 䡠 VOLUME 114, NUMBER 8
`
`Figure 1. Model of CML-like disease in mice. (A) Schema of
`retroviral bone marrow transduction and transplantation to irradi-
`ated recipient mice. (B) Donor bone marrow (CD45.2⫹) was
`transferred into CD45.1⫹ recipient mice (irradiated with 4.5 Gy).
`Twenty days later, PBMCs were stained for GR-1, CD8, CD4,
`B220, and NK1.1, and analyzed by flow cytometry. Representa-
`tive dot plots of 1 of 6 mice are shown. (C) Survival of cpCML (—,
`n ⫽ 12) and bcCML (哹, n ⫽ 18) mice is shown in Kaplan-Meier
`plots. Statistical comparison was performed with the log-rank test.
`(D) Phenotypical characterization of GFP⫹ leukemic cells in
`cpCML and bcCML mice. Cells were gated on granulocytes in
`control C57BL/6 mice and on transduced granulocyte populations
`in cpCML and bcCML mice. One representative staining of 5 is
`shown.
`
`The treatment depletes CD8⫹ T cells to below the detection limit of flow
`cytometry analysis (data not shown).
`
`CML patients
`
`Human blood sample collections were approved by the ethical committee of
`the Canton of Berne, Switzerland, and patients gave their written informed
`consent in accordance with the Declaration of Helsinki. The average age of
`healthy donors was 40.6 ⫾ 13.8 years (3 [female] and 5 [male]) and of
`cpCML patients 46.0 ⫾ 12.2 years (3 [female] and 5 [male]). All cpCML
`patients expressed p210BCR/ABL.
`
`Statistical analysis
`
`Data are presented as the mean plus or minus SEM. The significance of the
`differences in Kaplan-Meier survival curves was determined using the
`log-rank test (2-tailed). The significance between groups of human samples
`was determined by using unpaired Student
`test (2-tailed) and the
`t
`correlation with the Spearman correlation (2-tailed, ␣ ⫽.05).
`
`Results
`
`CML-like disease in immunocompetent mice
`
`It has been shown previously that BCR/ABL expression in bone
`marrow cells leads to cpCML in mice.29 Coexpression of NUP98/
`HOXA9 leads to accumulation of immature myeloid cells and
`progression to blast crisis.18 Therefore, we transduced bone mar-
`row from C57BL/6 mice with retroviral particles expressing
`p210BCR/ABL alone, or cotransduced bone marrow cells with
`p210BCR/ABL and NUP98/HOXA9 retroviral particles. Trans-
`duced bone marrow cells were adoptively transferred into irradi-
`ated syngeneic recipient mice (Figure 1A).
`In previous studies, recipient mice were lethally irradiated.2,29,30
`This completely prevented a leukemia-specific immune response
`by the host and made it impossible to study the immunosurveil-
`
`lance of CML. In this study, we titrated the irradiation dose and the
`number of retroviral particles NUP98/HOXA9 and BCR/ABL
`needed to generate cpCML and bcCML originating from donor
`bone marrow cells in a host with a largely intact immune system.
`To address whether immune cells for the control of the leukemia
`originate from recipient or donor bone marrow, transduced bone
`marrow cells were transferred into CD45.1⫹ recipient mice. CD8⫹
`and CD4⫹ T cells, B cells, and NK cells were found to originate
`from donor bone marrow (CD45.1⫺) when recipient mice were
`irradiated with 9.5 Gy (data not shown). In contrast, when recipient
`mice were irradiated with 4.5 Gy (Figure 1B) or 6.5 Gy (data not
`shown), immune cells were found to originate from the recipient
`mouse, whereas the leukemia cells (GR-1⫹) originated from the
`cotransduced donor bone marrow.
`Transduction of bone marrow with BCR/ABL retroviral par-
`ticles alone (105 U) or cotransduction with NUP98/HOXA9 (106 U)
`regularly induced cpCML or bcCML, respectively. In contrast,
`transduction with NUP98/HOXA9 retroviral particles alone did not
`induce leukemia up to 3 months after transplantation. bcCML
`rapidly progressed and animals died within 3 weeks, whereas
`cpCML mice lived up to 6 weeks (Figure 1C). Because the
`retroviral vectors BCR/ABL in cpCML and NUP98/HOXA9 in
`bcCML coexpressed the fluorescent dye GFP, the development of
`CML-like disease was followed with flow cytometry of peripheral
`blood (Figure 1D). The majority of granulocytes were GFP
`positive, indicating that these cells expressed BCR/ABL in cpCML
`or NUP98/HOXA9 in bcCML. In addition, the granulocyte popula-
`tion in the blood of cpCML and bcCML mice was increased to
`9 ⫻ 107 granulocytes/mL blood compared with the C57BL/6
`control (⬍ 2 ⫻ 106 granulocytes/mL blood; data not shown).
`Immature myeloid blast cells (MAC-1⫹, GR-1⫹, c-kit⫹) were
`detectable in bcCML. In contrast, all transduced GFP⫹ leukemic
`cells in cpCML phenotypically resembled mature granulocytes
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`BLOOD, 20 AUGUST 2009 䡠 VOLUME 114, NUMBER 8
`
`EXHAUSTION OF CML-SPECIFIC T CELLS BY PD-1 IMMUNOBIOLOGY
`
`1531
`
`(MAC-1⫹, GR-1⫹, c-kit⫺) comparable with untransduced granulo-
`cytes in naive C57BL/6 mice (Figure 1D). Therefore, by reducing
`the dose of irradiation given to recipient mice (4.5 or 6.5 Gy) and
`by administering defined amounts of retroviral particles, it was
`possible to reproducibly induce cpCML and bcCML in recipient
`mice with a largely intact immune system.
`
`Leukemia-specific CTLs are not detectable in CML disease
`progression
`
`To analyze antigen-specific immune responses in vivo, bone
`marrow from H8 transgenic mice was transduced with the retrovi-
`ral particles. In this experimental setup, all leukemia cells ex-
`pressed the immunodominant CTL epitope gp33 of LCMV on
`MHC class I molecules as a model leukemia antigen (H8-CML
`mice). The leukemia-specific CTL response was analyzed in
`H8-CML mice at different time points after bone marrow transplan-
`tation. Transduction of bone marrow with 0.01 ⫻ 106 U of
`BCR/ABL or cotransduction with 0.01 ⫻ 106 U of BCR/ABL and
`0.1 ⫻ 106 U of NUP98/HOXA9 retroviral particles induced a
`transient increase of GFP-positive granulocytes in recipient mice,
`but by day 30 all leukemia cells were eliminated (data not shown).
`Tetramer staining revealed that recipient mice that eliminated CML
`developed a LCMV-gp33–specific CTL response (Figure 2A). In
`contrast, if bone marrow cells were transduced with 0.1 ⫻ 106 U of
`BCR/ABL alone or together with 106 U of NUP98/HOX-A9
`retroviral particles, H8-cpCML and H8-bcCML persisted and
`progressed to the death of the animals (disease kinetics comparable
`with Figure 1C). In H8-cpCML and H8-bcCML mice, LCMV-gp33–
`specific CTLs were not detectable by tetramer staining (Figure 2A)
`or by intracellular staining for IFN-␥ or TNF-␣ after in vitro
`restimulation with gp33 (Figure 2B). In addition, CTLs isolated
`from cpCML and bcCML mice were not able to lyse gp33
`peptide-pulsed target cells ex vivo after 5 days of in vitro
`restimulation (Figure 2C). Naive C57BL/6 mice and LCMV-
`immune mice that have been infected 8 weeks previously with
`200 pfu of LCMV-WE were used as controls (Figure 2A-C).
`To test whether gp33-specific CTLs can be activated in mice
`with CML-like disease, H8-cpCML, H8-bcCML mice, and control
`C57BL/6 mice were infected with 200 pfu of LCMV. Eight days
`later, the frequencies of CML-specific CTLs (gp33) and of CTLs
`specific for an unrelated viral epitope (np396) were analyzed by
`tetramer staining. In H8-cpCML and H8-bcCML mice, np396-
`specific CTLs, but not gp33-specific CTLs, were detectable (Figure
`2D). In contrast, naive C57BL/6 mice infected with LCMV
`mounted gp33- and np396-specific CTL responses. These results
`demonstrate that (a) leukemia cells were able to induce a specific
`CTL response, and (b) in the presence of CML-like disease with
`high leukocytes counts, leukemia antigen-specific CTLs were
`not present.
`
`CML-specific CTLs have residual effector functions
`
`To study CML-specific CTL responses in more detail, purified p14
`TCR transgenic CD8⫹ T cells (CD45.1⫹CD8⫹V␣2⫹) specific for
`LCMV-gp33 were adoptively transferred to H8-bcCML mice and
`to naive and LCMV-infected control mice. After transfer to
`H8-bcCML mice, p14 CD8⫹ T cells rapidly expanded in blood
`(Figure 3A) and spleen (Figure 3B). The specific CTLs reached a
`frequency of 25% of total
`lymphocytes 6 days after transfer.
`Thereafter, their number rapidly declined, reaching a stable fre-
`quency of approximately 2.8% (⫾ 1.9%), which is slightly above
`the frequency of transferred p14 CTLs in naive C57BL/6 control
`
`mice. In addition, the leukemia-specific TCR chain V␣2 was
`down-regulated on CML-specific CTLs in blood (mean V␣2⫹ p14
`42.1% ⫾ 25.8%;
`CD8⫹ T cells: CML,
`naive C57BL/6,
`92.3% ⫾ 3.5%; LCMV-infected, 79.2% ⫾ 16.8%; Figure 3C) and
`spleen (data not shown), indicating TCR ligation and CTL activa-
`tion. P14 CD8⫹ T cells transferred to C57BL/6 control mice did not
`proliferate or down-regulate the specific TCR, whereas p14 CD8⫹
`T cells transferred to LCMV-infected mice rapidly expanded. Due
`to the large number of transferred specific CTLs, LCMV-WE was
`rapidly eliminated and V␣2 expression was already reconstituted in
`most animals at day 3 after transfer (Figure 3A-C). The down-
`regulation of the TCR V␣2 did not correlate with the percentage of
`leukemic GFP⫹ granulocytes when analyzed in blood (Spearman
`correlation, P ⫽ .49, r ⫽ ⫺0.17; Figure 3D). We concluded that
`naive specific CTLs in H8-bcCML mice were initially activated
`after TCR ligation and expanded. However, the majority of these
`CML-specific CTLs were deleted, and only a small fraction
`persisted long-term.
`Memory CD8⫹ T cells in chronic infections with persisting
`antigen are either physically deleted or functional impaired, a
`process termed exhaustion.31 To analyze the degree of functional
`impairment of specific CD8⫹ T cells in the presence of H8-bcCML,
`adoptively transferred p14 CD8⫹ T cells were isolated from
`spleens of H8-bcCML mice and of naive and LCMV-infected
`control mice 6 days after transfer. P14 CTLs isolated from H8-
`bcCML mice produced hardly any proinflammatory cytokines such
`as IFN-␥, TNF-␣, or IL-2 after in vitro restimulation (Figure 4A).
`In contrast, p14 CTLs isolated from LCMV-infected mice pro-
`duced high amounts of IFN-␥ and TNF-␣ (Figure 4A). In addition,
`p14 CTLs isolated from H8-bcCML mice did not lyse peptide-
`pulsed target cells directly ex vivo (data not shown), and had only
`limited lytic capacity after 5 days of in vitro restimulation (Figure
`4B). Therefore, the cytokine profile and lytic function of LCMV-
`specific CD8⫹ T cells define effector CTLs, whereas the character-
`istic of CML-specific CD8⫹ T cells is consistent with partial
`exhaustion.
`To analyze whether the lytic function of the transferred CTLs is
`sufficient to influence the leukemia load, we followed the fre-
`quency of GFP⫹ granulocytes before and 6 days after the transfer
`of p14 CD8⫹ T cells. In most animals, the frequency of GFP⫹
`granulocytes was at least transiently reduced (Figure 4C). This
`indicates that the transferred naive p14 CD8⫹ T cells acquired
`effector function in vivo sufficient
`to transiently reduce the
`leukemia load.
`To assess the proliferative capacity of the isolated p14 CD8⫹
`T cells, 3H-thymidine incorporation was measured after in vitro
`restimulation. P14 CTLs from C57BL/6 control mice with a naive
`phenotype proliferated vigorously in contrast to effector p14 CTLs
`isolated from acute LCMV-infected mice (Figure 4A-B,D). P14
`CTLs isolated from H8-bcCML mice neither expanded efficiently
`in vitro (Figure 4D) nor exhibited effector functions (Figure 4A-B).
`Thus, CML-specific CTLs are partially exhausted with limited
`residual effector functions.
`
`Myeloid leukemia cells suppress T-cell proliferation through
`PD-1/PD-L1 interaction
`
`CTLA-4 and PD-1 are 2 well-known coinhibitory molecules that
`are of importance in the maintenance of peripheral tolerance.32
`Therefore, we analyzed their expression on total and on CML-
`specific CD8⫹ T cells. In H8-bcCML mice, PD-1 was highly
`expressed on the transferred CML-specific p14 CTLs in the blood
`(data not shown) and spleen (Figure 5A). In contrast, PD-1 was not
`
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`1532
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`MUMPRECHT et al
`
`BLOOD, 20 AUGUST 2009 䡠 VOLUME 114, NUMBER 8
`
`expressed on transferred p14 CD8⫹ T cells in C57BL/6 control
`mice, and was only expressed at low levels on p14 CTLs in acutely
`LCMV-WE–infected mice. However, as shown previously,14 PD-1
`expression was high on p14 CTLs in mice chronically infected with
`LCMV Docile. The expression of PD-1 on transferred p14 CTLs in
`H8-bcCML mice was substantially higher than in mice with
`persistent LCMV infection (Figure 5A). Surprisingly, a large
`fraction of total CD8⫹ T cells expressed PD-1 (93.5% ⫾ 1.8% in
`
`Figure 3. Adoptive transfer of leukemia-specific TCR transgenic CD8ⴙ T cells to
`H8-bcCML mice. Purified p14 CD8⫹ T cells (CD45.1⫹CD8⫹V␣2⫹) were adoptively
`transferred to H8-bcCML mice (F), to naive C57BL/6 control mice (⽧), and to
`C57BL/6 mice infected with 104 pfu of LCMV (f). (A-B) The expansion of the
`transferred p14 CD8⫹ T cells (CD45.1⫹) was analyzed in the blood (A) and spleen (B)
`by flow cytometry analysis. (C) The expression of the leukemia-specific TCR V␣2 on
`the transferred p14 CD8⫹ T cells (CD45.1⫹) was analyzed in blood 3 days after
`the TCR V␣2 was
`transfer by flow cytometry analysis. (D) The expression of
`compared with the percentage of leukemic granulocytes (GFP⫹) in the blood 3 days
`after transfer. Results are pooled from 5 independent experiments. Statistical
`comparison was made using the Spearman correlation.
`
`H8-CML mice; data not shown). In contrast, CTLA-4 was not
`expressed on transferred CML-specific CTLs in blood (data not
`shown) and spleen (Figure 5B) in H8-bcCML mice.
`The observation of high rates of PD-1 expression on total CD8⫹
`T cells prompted us to analyze the expression of PD-1 on non–
`leukemia-specific CTLs. Therefore, cpCML and bcCML from
`C57BL/6 bone marrow donors were induced in p14 recipient mice.
`In this experimental setup, 60% of CD8⫹ T cells express the TCR
`V␣2 and are non–leukemia specific. PD-1 was expressed on 3.4%
`(⫾ 2.3%) of p14 CD8⫹ T cells in cpCML and on 7.9% (⫾ 3.2%) in
`bcCML (Figure 5C). Therefore, PD-1 expression in cpCML and in
`bcCML is not restricted to leukemia-specific CTLs. However, the
`majority of leukemia-specific CTLs expressed PD-1 (Figure 5A).
`We next investigated the expression of both PD-Ls on granulo-
`cytes in various organs, such as bone marrow, spleen, inguinal and
`mesenterial lymph nodes, thymus, and peripheral blood. PD-L1
`was up-regulated on leukemic (GFP⫹) granulocytes in H8-cpCML
`and H8-bcCML in spleen (H8-cpCML, 38%; H8-bcCML, 100%)
`and bone marrow compared with the granulocyte population of
`naive C57BL/6 mice (Figure 5D). In contrast, PD-L2 was not
`expressed on leukemic cells in H8-cpCML and H8-bcCML mice
`(Figure 5D).
`
`Figure 2. Presence of leukemia-specific CTLs in CML. Bone marrow from H8 mice
`was transduced with BCR/ABL-GFP or cotransduced with NUP98/HOXA9-GFP and
`BCR/ABL-neo and injected into 4.5 Gy irradiated C57BL/6 mice. (A-B) The frequency
`of gp33-specific CTLs was analyzed by tetramer staining (A) and intracellular IFN-␥
`and TNF-␣ staining (B) in the blood of mice that eliminated H8-cpCML and
`H8-bcCML, in mice with progressive H8-cpCML and H8-bcCML, in naive C57BL/6,
`and in LCMV-immune mice. (C) Splenocytes of H8-cpCML and H8-bcCML mice were
`isolated 20 days after BMT and analyzed in a standard 51Cr-release assay (gp33-
`pulsed target cells [filled symbols]; nonpulsed target cells [open symbols]). CTL
`activity is given as mean ⫾ SEM of 4 CML mice (F), naive C57BL/6 (⽧), and
`LCMV-immune mice (f). (D) H8-cpCML and H8-bcCML mice and C57BL/6 controls
`were infected with 200 pfu of LCMV intravenously. Eight days after LCMV infection,
`the frequency of gp33-specific and np396-specific CTLs was analyzed in the blood
`and spleen by tetramer staining. One representative experiment of 2 is shown.
`
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`BLOOD, 20 AUGUST 2009 䡠 VOLUME 114, NUMBER 8
`
`EXHAUSTION OF CML-SPECIFIC T CELLS BY PD-1 IMMUNOBIOLOGY
`
`1533
`
`Figure 4. Functional characterization of transferred TCR transgenic CD8ⴙ
`T cells. P14 CD8⫹ T cells were isolated 5 or 6 days after transfer from the spleens of
`H8-bcCML mice, C57BL/6 controls, and LCMV-infected mice. (A) The production of
`IFN-␥, TNF-␣, and IL-2 by p14 CD8⫹ T cells was determined in the supernatant with a
`cytometric bead array assay. Results are given as mean ⫾ SEM of 3 to 10 samples
`per group, pooled from 4 independent experiments. (B) Splenocytes were analyzed
`in a 51Cr-release assay (gp33-pulsed target cells [filled symbols]; nonpulsed target
`cells [open symbols]). CTL activity is given as mean ⫾ SEM of 4 H8-bcCML mice (F),
`C57BL/6 controls (⽧), and LCMV-infected mice (f). One representative experiment
`of 2 is shown. (C) FACS analysis of blood of H8-bcCML mice before and 6 days after
`adoptive transfer of p14 CD8⫹ T cells and control H8-bcCML mice. One representa-
`tive staining of 5 independent experiments is shown. (D) Isolated p14 CD8⫹ T cells
`were restimulated in vitro. 3H-thymidine incorporation of isolated p14 CD8⫹ T cells is
`shown as proliferation index (mean ⫾ SEM of 4-10 mice per group). Results are
`pooled from 3 independent experiments.
`
`To functionally analyze the role of PD-L1 expressed on
`leukemic cells, H8-cpCML and H8-bcCML cells were used as
`stimulators in a 3H-thymidine incorporation assay with either p14
`or p14 ⫻ PD-1⫺/⫺ CD8⫹ T cells as responders. H8-cpCML and
`H8-bcCML cells stimulated only a limited expansion of p14 CD8⫹
`T cells, whereas p14 CD8⫹ T cells proliferated efficiently when
`stimulated with splenocytes from naive H8 mice. In contrast,
`p14 ⫻ PD-1⫺/⫺ CD8⫹ T cells efficiently expanded when stimu-
`lated with H8-cpCML and H8-bcCML cells (Figure 5E).
`In summary, PD-1 is expressed on CML-specific CTLs and
`PD-L1 on leukemic cells with higher expression during bcCML
`than in cpCML.
`
`Prolonged survival of bcCML mice in the absence of PD-1
`signaling
`
`Our data to date using the LCMV-gp33 as model leukemia antigen
`suggested that PD-1 may inhibit leukemia-specific CTLs and lead
`to disease progression. LCMV-gp33 is a foreign antigen that is
`expressed in the H8 transgenic mice under a relatively strong
`promoter. Therefore, the model leukemia antigen used has many
`similarities to the junction peptides derived of BCR/ABL, which
`are similarly expressed under a strong promoter and are novel
`antigens without preexisting self-tolerance. Nevertheless, the H8-
`CML model might overestimate the contribution of CD8⫹ T cells
`and the role of PD-1 in CML-specific tolerance induction. To test
`the physiologic role of PD-1 in CML control, bcCML was induced
`in PD-1–deficient mice and C57BL/6 control mice using trans-
`
`Figure 5. PD-1 and PD-L expression in H8-cpCML and H8-bcCML mice. (A) PD-1
`expression on purified p14 CD8⫹ T cells (CD45.1⫹CD8⫹Va2⫹) isolated from the
`spleen 5 days after transfer. One representative histogram from 3 to 5 mice per group
`is shown. One representative experiment of 2 is shown. (B) CTLA-4 expression on
`purified p14 CD8⫹ T cells (CD45.1⫹CD8⫹V␣2⫹) isolated from the spleen 7 days after
`transfer. (C) cpCML and bcCML were generated from C57BL/6 donors in p14
`recipient mice. PD-1 expression on total CD8⫹ T cells and on unspecific CD8⫹ T cells
`(V␣2⫹) in the spleen was analyzed 20 days after bone marrow transplantation. One
`representative FACS plot of 3 is shown. (D) PD-L1 and PD-L2 expression in the
`spleen and bone marrow of H8-cpCML, H8-bcCML, and naive C57BL/6 mice. Cells
`were gated on GFP-positive gran