Engineering hematopoietic grafts: Purified allogeneic hematopoietic stem cells plus expanded CD8+ NK-T cells in the treatment of lymphoma
2001; Elsevier BV; Volume: 7; Issue: 10 Linguagem: Inglês
10.1016/s1083-8791(01)70014-6
ISSN1523-6536
AutoresMichael R. Verneris, Maki Ito, Jeanette Baker, Arash Arshi, Robert S. Negrin, Judith A. Shizuru,
Tópico(s)Immunotherapy and Immune Responses
ResumoExpanded CD8 + NK-T Cells Mediate GVT With Minimal GVHD 533 B B & M Twho underwent transplantations for a variety of malignancies [12][13][14].Purified HSCs are immunologically naive and therefore will not cause GVHD.However, as we recently showed in a mouse model, allogeneic HSCs are devoid of measurable GVT activity [15].Thus, we [15][16][17][18] and others [5][6][7]19,20] have pursued clinically feasible methodologies to obtain cell populations that mediate GVT activity without GVHD.Many of these approaches require large quantities of cells and extensive technical manipulations to generate tumor specific populations, thereby limiting their general applicability to patients.We previously described, for both mice [21] and humans [16,22], a population of ex vivo expanded lymphoid cells that demonstrate in vitro cytotoxicity against a variety of syngeneic and allogeneic tumors, without having had prior exposure.These cells are derived from peripheral blood lymphocytes (PBLs) that are cultured with timed addition of growth factors and cross-linking anti-CD3 MoAbs.After approximately 3 weeks in culture, a phenotypically homogeneous population (>90%) expands that expresses CD3, CD8, and the T-cell receptor (TCR) α/β, and 20% to 60% of cells express natural killer (NK)-cell markers, resulting in a >100-to 1000-fold expansion of this otherwise rare population of cells [21].Here, we report on the in vivo activity of expanded CD8 + NK-T cells in a murine model of persistent lymphoma following transplantation of purified allogeneic HSCs.Expanded CD8 + NK-T cells demonstrate significant GVT activity with minimal to no GVHD, even when transplanted across major histocompatibility complex (MHC) barriers.The expanded cells can be detected in the peripheral circulation for weeks and require an intact perforin-dependent cytolytic pathway to confer protection from lymphoma growth.Our studies show that expanded CD8 + NK-T cells are a phenotypically unique population that appears to have innate tumor immunity but are not pan-reactive to alloantigens. MATERIALS AND METHODS MiceRecipient mice were 7-to 10-week-old BALB/c mice (H-2 d , Thy-1.2).Thy-1.1 congenic C57BL/Ka.Thy-1.1 mice (H-2 b , Thy-1.1, CD45.2) were used as allogeneic HSC and CD8 + NK-T cell donors.In selected studies, C57BL/Ka.Thy-1.2mice congenic at the Thy-1.2 and CD45 loci (H-2 b , Thy-1.2, CD45.1) were used as CD8 + NK-T cell donors.Target cells for the fluorescence-activated cell sorting (FACS)-based killing assay were obtained from C57BL/Ka.Thy-1.1 or BALB/c mice.Mice were bred at Stanford University (Department of Comparative Medicine).In selected studies, the following immune-defective mice were used as donors for expanded CD8 + NK-T cells: perforin-knockout (C57BL/6-Pfp tmlSdz ) and Fas ligand (FasL)-mutant mice (B6Smn: Fas gld ) (Jackson Laboratories, Bar Harbor, ME).All mice were maintained in specific pathogen-free conditions in temperature-controlled and light-cycled rooms. Expansion of CD8 + NK-T CellsSingle-cell suspensions were prepared from donor splenocytes.Cells were cultured in RPMI 1640 media supple-mented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 50 mmol/L 2-mercaptoethanol.Murine interferon-γ (IFNγ) (Genetics Institute, Cambridge, MA), 1000 U/mL, was added on day 1.After 24 hours, the cells were transferred to flasks coated with 50 ng/mL of the anti-CD3 MoAb (145-2C11; Pharmingen, San Diego, CA) and 300 IU/mL of human recombinant interleukin-2 (rIL-2) (Chiron, Emeryville, CA).The cultured cells were supported with fresh media and rhIL-2 every 2 to 3 days. Morphology AnalysisExpanded CD8 + NK-T cells (10 6 cells) were suspended in 100 µL phosphate buffered saline (PBS) and cytospun at 800 rpm onto bovine serum albumin-coated glass slides.The cytospun cells were stained with Wright-Giemsa stain. Phenotype AnalysisFACS analysis of either splenocytes or expanded CD8 + NK-T cells was performed.For analysis of unactivated splenocytes, 6-to 10-week-old C57BL/Ka.Thy-1.1 mice were killed and their splenocytes isolated.Red blood cells were removed using osmotic lysis (ACK), and the remaining cells were washed with FACS staining buffer consisting of PBS supplemented with 2% FCS.In experiments using expanded CD8 + NK-T cells, cells were washed with FACS buffer (as above).To prevent nonspecific antibody binding, cells were first treated for 15 minutes with Fc receptor blocking antibodies (α-CD16/CD32, Pharmingen).The cells were then stained for 15 to 30 minutes with either fluorescein isothiocyanate (FITC)-or phycoerythrin (PE)conjugated MoAbs directed against CD3 (145-2C11), CD8 (Ly 3.2, 53-5.8),TCRγ/δ (GL3), NK1.1 (Ly 55, PK136), TCRα/β (Η57-597), Ly49A (A1), Ly49C/I (5E6), Ly49D (4E5), Ly49F (HBF-719), Ly49G2 (4D11), Ly49I (YLI-90), and NKG2A/C/E (20d5) or appropriate isotype controls (all from Pharmingen).In select experiments (multicolor FACS analysis), CD3-allophycocyanin (145-2C11; Pharmingen), CD8-Texas red (Ly 3.2, 53-5.8)(gift from Irving Weissman, Stanford University, Stanford CA), CD94-biotin (18d3) and second-step strepavidin-PE (Pharmingen), and neutra-Avidin-cascade blue (Molecular Probes, Eugine, OR) were used.Excess antibody was washed off and cells were resuspended in FACS staining buffer.Propidium iodine (PI; Sigma, St. Louis, MO) was added at 1 mg/mL to allow exclusion of dead cells.Cells were analyzed using either a single-laser FACScan or a triple-laser FACS instrument (with Moflops electronics) that was modified and made available through the FACS shared-user group at Stanford University.Data analysis was performed using Flowjo (Tree Star Inc, San Carlos, CA), version 3.2. HSC PurificationPurified HSCs were obtained by modification of the methods described by Spangrude et al. [10].MoAbs were the kind gift of Dr. I. Weissman (Stanford University, Stanford, CA), with the exceptions of anti-CD3 MoAb (145-2C11) and anti-CD8 MoAb (53-6.7)which were obtained from Pharmingen.Bone marrow (BM) was obtained from donor mice by flushing tibiae and femurs with Hanks Balanced Salt Solution (HBSS; Applied Scientific, San Francisco,
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