Depletion of Alloreactive T-Cells by Anti-CD137-Saporin Immunotoxin

Mice

Female Balb/c (H-2^d), C57BL/6 (H-2^b), and (C57BL/6 × DBA/2)F1 (BDF1; H-2^b/d) mice, 8—10 weeks of age, were purchased from Orient Bio-Charles River (Seongnam, Korea) and used for experiments. OT-I [T-cell receptor (TCR) specific for ovalbumin (OVA) peptide] transgenic mice (19) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). CD137^-/- mice (28) were crossed with OT-I transgenic mice to generate OT-I-CD137^-/-mice. All mice were maintained under specific pathogen-free conditions in the animal facility of the University of Ulsan and used in accordance with the Experimental Animal Guidelines of the University of Ulsan.

Antibodies

3E1 hybridoma cells producing anti-mouse CD137 mAb (41) were provided by Robert S. Mittler (Emory University, Atlanta, GA, USA). Anti-human CD137 [4B4 (BBK-1) and 4785 (BBK-2)] mAbs were described previously (31). They were purified from ascites, and control rat IgG and purified anti-CD3 mAb (OKT3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC), phycoerythrin (PE), or phycoerythrin—Cy5 (PE—Cy5) mAbs conjugated to the following mouse or human cell surface molecules were purchased from eBioscience (San Diego, CA, USA): CD3, CD4, CD8, CD11, CD25, CD44, CD62L, CD137, FoxP3, NK1.1, and annexin V. The FITC-conjugated anti-mouse early endosome antigen-1 (EEA-1) was purchased from BD Bioscience (San Jose, CA, USA). PE- or FITC-conjugated isotype control antibodies were purchased from eBioscience.

Anti-CD137—Saporin or Doxorubicin Immunotoxin

Purified 3E1 or rat IgG was conjugated with saporin by Advanced Targeting Systems (San Diego, CA, USA) to produce anti-CD137—saporin or rat IgG—saporin complex. Doxorubicin alone or doxorubicin and FITC were also conjugated to 3E1 (Peptron, Daejeon, Korea).

Cell Lines

CTLL-R8, obtained from Herbert F. Oettgen (Memorial Sloan-Kettering Cancer Center, New York, NY, USA), is a mouse cytotoxic T-lymphocyte cell line derived from (Balb/c × C57BL/6)F1 mice that were immunized with Balb/c leukemia RL male 1 (40). Mouse P815 (mastocytoma) and EL-4 (thymoma) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). To generate EL-4 cells that overexpress CD137-enhanced green fluorescence protein (EGFP), a full-length CD137 gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using a mouse 4-1BB specific primer set (sense: 5′-CAA GCT TAT GGG AAA CAA CTG T-3′; antisense: 5′-GGG ATC CAG CTC ATA GCC TCC-3′) and complementary DNA (cDNA) prepared from Balb/c splenocytes and cloned into pEGFPN1 (Clontech, Mountain View, CA, USA) after cleavage with restriction endonucleases HindIII and BamHI. EL-4 cells (5 × 10⁶) were suspended in 0.3 ml of Opti-MEM (Life Technologies, Waltham, MA. USA), transferred to a 4-mm gap cuvette, and then mixed with 10 mg of either pEGFPN1—CD137 or empty EGFPN1 vector. Cells were transfected at 960 microfarads and 250 V using a Gene Pulser Electroporation Apparatus (Bio-Rad, Hercules, CA, USA). To establish CD137-expressing stable cell lines, EGFP-positive cells were sorted by MoFlow XDP cell sorter (Beckman Coulter, Indianapolis, IN, USA) after transfected cells were selected at 300 μg/ml of G418 (Sigma-Aldrich).

Isolation of Splenocytes

Single-cell suspensions in phosphate-buffered saline (PBS) were prepared from the spleens, filtered through a sterile 40-μm nylon mesh (BD Falcon, Bedford, MA, USA), and washed. After the erythrocytes were lysed in hemolysis buffer (144 mM NH₄Cl and 17 mM Tris-HCl; pH 7.2; BioLegend, San Diego, CA, USA), the remaining cells were resuspended in PBS for further processes.

Purification of CD4⁺ or CD8⁺ T-Cells

Mouse or human CD4⁺ and CD8⁺ T-cells were isolated from mouse splenocytes or human peripheral blood mononuclear cells (PBMCs) by immunomagnetic bead selection, according to the manufacturer's protocols (Miltenyi Biotech, Auburn, CA, USA). In brief, a single-cell suspension in magnetic-activated cell sorter (MACS) buffer was incubated with anti-CD4 or anti-CD8 mAb microbeads for 15 min and transferred onto the column attached to the cell separator. Cells bound to microbeads were eluted with MACS buffer from the column detached from the cell separator. Human PBMCs were taken at the University of Ulsan Clinic from healthy volunteers (37- and 41-year-old males), after obtaining written informed consent. This study was reviewed and approved by the Institutional Review Board of the Ulsan University.

Internalization of Anti-CD137 mAb

CTLL-R8 cells and CD137—EGFP-transfected EL-4 cells were incubated with PE-conjugated anti-CD137 mAb or isotype control antibody (1 μg/ml) for 30 min on ice. Cells were then washed three times with PBS to remove unbound anti-CD137 mAb and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (WelGENE, Daegu, Korea) containing 10% FBS (WelGENE) and antibiotics (Gibco, Carlsbad, CA, USA) for 30, 60, 120, or 240 min. Cells were harvested at the indicated times and fixed for 15 min with 4% paraformaldehyde (Sigma-Aldrich), washed three times with PBS, and permeabilized in 0.25% Triton X-100 (Sigma-Aldrich). Cells were then stained with FITC-conjugated anti-EEA-1 mAb or isotype control antibody (1 μg/ml) for 1 h and attached to a poly-L-lysine (Sigma-Aldrich)-coated slide after three washes with PBS. Slides mounted with Flouromount G (Southern Biotech, Birmingham, AL, USA) were analyzed for the subcellular localization of CD137 and EEA-1 using Olympus FV500 confocal microscopy (Olympus, Center Valley, PA, USA). In some experiments, mouse or human CD4⁺ and CD8⁺ T-cells were activated by anti-mouse or human CD3 mAb, respectively, for 24 h and used to examine the internalization of anti-mouse or human CD137 mAb. CD137 expression on activated T-cells was confirmed by staining them with anti-CD137 mAb, as described below.

Flow Cytometry

Cells were preincubated in a blocking buffer [PBS containing 2.4G2 mAb (eBioscience), 0.2% bovine serum albumin (BSA; Sigma-Aldrich), and 0.1% sodium azide (Sigma-Aldrich)], and stained with relevant mAbs at 4°C for 30 min. Cells were then washed two times with cold FACS buffer (PBS containing 0.2% BSA and 0.1% sodium azide) and analyzed by flow cytometry using a FACS CANTO II with Diva software (BD Biosciences). To detect apoptosis, cells were stained with anti-annexin V mAb (1 μg/ml) according to the manufacturer's protocol. For intracellular staining of anti-interferon-γ (IFN-γ, cells were restimulated with 200 ng/ml phobol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) plus 500 ng/ml ionomycin (Calbiochem, Billerica, MA, USA) or 1 μg/ml OVA_257—264 peptide (Peptron) for 5 h. One hour later, GolgiStop (BD Pharmingen, San Jose, CA, USA) was added to inhibit the export of cytokines. Cells were then stained with anti-CD8 mAb for 15—30 min at 4°C. After fixation and permeabilization with IC Fixation/Permeabilization Buffer (eBioscience), cells were stained with IFN-γ mAb (1 μg/ml; eBioscience) according to the manufacturer's instructions.

Viability Test and Thymidine Incorporation Assay

Splenocytes from wild-type (WT), CD137^-/-, OT-I, or OT-I-CD137^-/- mice were stimulated with anti-CD3 mAb (0.5 μg/ml) or OVA_257—264 peptide (1 μg/ml) for 24 h. After confirmation of CD137 expression on CD4⁺ and CD8⁺ T-cells, cells (2 × 10⁶ cells/well) were aliquoted into a 24-well culture plate (BD Falcon) and then treated with various concentrations of anti-CD137—saporin immunotoxin or control immunotoxin (Advanced Targeting Systems). Cells were further cultured at 37°C for 48 h and stained with anti-annexin V and anti-CD4 or anti-CD8 mAb (1 μg/ml for each). Apoptotic cells were analyzed by FACS Canto II. For the thymidine incorporation assay, splenocytes were stimulated as described above, and cells were distributed to a 96-well cell culture plate (BD Falcon) at 2 × 10⁵ cells/well. Cells were further cultured with varying concentrations of anti-CD137—immunotoxin or control immunotoxin complexes at 37°C for 48 h. Cultures were pulsed with 1 μCi/well of [³H]thymidine (American Radiolabeled Chemicals, St. Louis, MO, USA) 8 h before harvest and processing. Thymidine incorporation was measured by liquid scintillation counting (PerkinElmer, Waltham, MA, USA).

Mixed Lymphocyte Reaction (MLR)

Responder splenocytes (2 × 10⁶ cells/well) were obtained from naive C57BL/6 (H-2^b) or BDF1 (H-2^b/d) mice in which acute GVHD was induced by injections of Balb/c (H-2^d) or C57BL/6 (H-2^b) mouse T-cells, respectively. In some experiments, splenocytes (2 × 10⁷ cells/ml) were isolated from BDF1 (H-2^b/d) mice with acute GVHD and labeled with 5 μM of 5,6-carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Waltham, MA, USA). Cells were then cocultured with irradiated (3,000 rad) stimulator BDF1 (H-2^b/d) splenocytes. Apoptosis of unlabeled alloreactive T-cells by anti-CD137—saporin immunotoxin was analyzed as described above. Division rates of CFSE-labeled CD4⁺ or CD8⁺ T-cells were analyzed by FACS after staining with anti-CD4 or anti-CD8 mAb. For a one-way mixed leukocyte reaction (MLR), responder human PBMCs were stimulated with irradiated (3,000 rad) stimulatory PBMCs for 5 days. After depleting CD137-expressing T-cells 5-day MLR culture with anti-CD137 (4785)-mouse IgG—saporin complexes, cells were rested for 5 days in the presence of interleukin-2 (IL-2) and restimulated with the irradiated first-party PBMCs. In some experiments, responder human PBMCs were cultured in the presence of irradiated stimulator PBMCs for 15 days, and CD137 expression was analyzed on CD4⁺ and CD8⁺ T-cells by FACS.

Measurement of Cytokine Levels

Cytokine levels in culture supernatants were quantified using a cytometric bead array kit (CBA; BD Biosciences) on a FACS Canto II equipped with Diva software and CBA software (BD Biosciences).

Cytotoxicity Assay

Splenocytes were obtained from BDF1(H-2^b/d) mice with acute GVHD to be used as effector cells. P815 (H-2K^d) cells were used as target cells and EL-4 (H-2K^b) cells as a negative control. P815 and EL-4 cells were incubated with 5 μM or 0.5 μM of carboxyfluorescein succinimidyl ester (CFSE), respectively, at 37°C for 10 min. Cells were cocultured at effector/target ratios of 1:1, 2:1, and 4:1 in a 24-well culture plate for 48 h. Percent lysis of target cells was measured by FACS. CFSE-labeled target cells were incubated without effector cells for 48 h as a control for nonspecific lysis. The specific percent lysis was calculated using the following formula: % of specific lysis = 100% × [1 − (% P815/% EL-4)_{with effectors}/ (% P815/% EL-4)_{without effectors}].

Induction of Acute GVHD

Splenocytes (5 × 10⁶ cells/ml) of C57BL/6 mice were cultured with irradiated BDF1 splenocytes (5 × 10⁵ cells/ml) in RPMI-1640 containing 100 IU/ml of recombinant human interleukin-2 (IL-2) (Peptron), 10% FBS and antibiotics (1,000 units/ml of penicillin and 1,000 μl/ml of streptomycin), in a T-75 flask for 5 days. To deplete alloreactive T-cells, cells were harvested and washed twice with cold PBS. After confirmation of CD137 expression and activation status of CD4⁺ and CD8⁺ T-cells by FACS, cells were treated with 1 μg/ml of rat IgG—saporin or anti-CD137—saporin immunotoxin per 2 × 10⁶ cells and cultured in T-100 flasks for an additional 2 days. Cultured cells were washed twice with cold PBS and resuspended in PBS. Dead cells were eliminated with greater than 90% efficiency before transplantation using dead cell removal microbeads according to the manufacturer's instructions (Miltenyi Biotech). Irradiated BDF1 mice (750 rad) received 1) T-cell depleted bone marrow (BM) (1 × 10⁷ cells) and donor T-cells (2 × 10⁶ cells) after allodepletion with anti-CD137—saporin immunotoxin or 2) control immunotoxin as described above or 3) BM alone.

Assessment of Graft-Versus-Leukemia (GVL) Effect

C57BL/6 mice were injected with 1 × 10⁷ cells/mouse of EL-4 lymphoma cells via tail vein. Seven days later, GVHD was induced in irradiated C57BL/6 mice (850 rad) by receiving 1) T-cell depleted BM (1 × 10⁷ cells) and donor Balb/c cells as described above.

Histopathological Analysis

Organs were harvested, fixed in 10% formaldehyde (Sigma-Aldrich), and embedded in paraffin. Sections with 5-μm thickness were stained with H&E (Sigma-Aldrich). Slides were coded and evaluated by a single investigator (H.J.C.) blinded to sample identity, using a semiquantitative system for pathological markers of GVHD (24).

Statistical Analysis

GraphPad Prism 4 (GraphPad Inc., La Jolla, CA, USA) was used to analyze and present the data. Differences between two or more than three experimental groups were analyzed by the Mann—Whitney or Kruskal—Wallis tests followed by a post hoc Dunn's test, respectively. The log rank test was used to compare survival between experimental groups.

Internalization of Anti-CD137 mAb After Binding to Cell Surface CD137

An effective immunotoxin should exhibit high specificity and affinity to a cell surface target antigen and be internalized by the cell after binding to that antigen (25). Since dendritic cells internalize streptavidin—CD137L fusion protein after binding of the protein to receptor (42), we evaluated the internalization of anti-CD137 mAb following its interaction with cell surface CD137. Anti-CD137 mAb remained bound to the cell membrane of CD137-expressing CTLL-R8 cells when observed immediately after incubation at 4°C for 30 min (Fig. 1A). After removal of unbound anti-CD137 mAb and further incubation of the stained cells at 37°C, the anti-CD137 mAb was completely translocated into EEA-1⁺ endosomes within 4 h. We showed that anti-CD137 mAb bound to CD137 molecules in the cell membrane was also translocated into endosomes in EL-4 cells expressing CD137—EGFP fusion protein, which was consistent with internalization of the anti-CD137—CD137 complexes by the cells (Fig. 1B). We also found that activated CD4⁺ and CD8⁺ T-cells internalized anti-CD137 mAb after binding of the mAb to their receptors (Fig. 1C, D). We confirmed the specificity of anti-CD37 mAb by showing that 1) isotype control antibody did not bind to and translocate cell-surface CD137 into endosome (Fig. 1A—D); and 2) anti-CD137 mAb could not be internalized into activated CD137-deficient CD4⁺ and CD8⁺ T-cells purified from CD137 KO mice (Fig. 1D). These findings provide evidence that anti-CD137 mAb may facilitate entry of cytotoxic agents into CD137-expressing cells.

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Figure 1.

Internalization of anti-CD137 mAb after binding to cell surface CD137. CD137-expressing mouse cytotoxic T-lymphocyte cell line derived from (Balb/c × C57BL/6)F1 mice that were immunized with Balb/c leukemia RL male 1 (CTLL-R8) cells, CD137—EGFP-transfected mouse thyoma (EL-4) cells, and purified CD4⁺ and CD8⁺ T-cells activated with anti-CD3 mAb for 24 h, were incubated with anti-CD137 mAb for 30 min on ice. After removal of nonbound antibodies, cells were cultured at 37°C for 4 h. Cells were harvested and stained with anti-EEA-1 mAb (except for CD137—EGFP-transfected EL-4 cells) and analyzed for the subcellular localization of CD137 using confocal microscopy. (A) CTLL-R8 cells stained with anti-CD137—PE and anti-EEA—FITC. Isotype IgG—PE or −FITC was used as a negative control for anti-CD137—PE or anti-EEA—FITC, respectively. (B) CD137—EGFP-transfected EL-4 cells stained with anti-CD137—PE or isotype IgG—PE (upper panels) or unconjugated rat IgG or anti-CD137 mAb (lower panels). (C) Purified CD4⁺ and CD8⁺ T-cells stained with anti-CD137—PE and anti-EEA—FITC. Isotype IgG—PE or −FITC was used as a negative control for anti-CD137—PE or anti-EEA—FITC, respectively. (D) WT and CD137^-/- CD4⁺ and CD8⁺ T-cells stained with anti-CD137—PE or isotype IgG—PE and anti-CD4—FITC or anti-CD8—FITC. Experiments were repeated at least two times with similar results.

Anti-CD137—Saporin Immunotoxin Can Kill Activated T-Cells and FoxP3⁺CD4⁺ Regulatory T-Cells

We conjugated anti-CD137 mAb to saporin, a ribosome-inactivating protein derived from the plant Saponaria officinalis (46), to assess the cytotoxic potential of anti-CD137—saporin immunotoxin. The addition of anti-CD137—saporin immunotoxin to splenocytes that expressed CD137 following polyclonal activation by anti-CD3 mAb induced apoptosis in both CD4⁺ and CD8⁺ T-cells in a dose-dependent manner (Fig. 2A). Given that rat IgG—saporin had a marginal effect on the apoptosis of activated CD4⁺ and CD8⁺ T-cells and that anti-CD137—saporin immunotoxin did not induce apoptosis in activated CD137-deficient CD4⁺ and CD8⁺ T-cells (Fig. 2A), anti-CD137—saporin immunotoxin seemed to specifically deplete CD137-expressing activated T-cells. To assess the nonspecific and off-target cytotoxic effect of anti-CD137—saporin immunotoxin on activated T-cells, we included unconjugated rat IgG or anti-CD137 antibodies as controls. The nonspecific cytotoxicity (unconjugated rat IgG vs. rat IgG—saporin) in CD4⁺ and CD8⁺ T-cells was 7.4% and 15.6%, respectively (Fig. 2B), whereas the specific cytotoxicity of anti-CD137—saporin immunotoxin (rat IgG—saporin vs. anti-CD137—saporin) on the respective subset was 117.6% and 94.2% (Fig. 2B). Since unconjugated anti-CD137 mAb decreased apoptosis of both CD4⁺ and CD8⁺ T-cells, their off-target cytotoxicity (unconjugated rat IgG vs. unconjugated anti-CD137) was −14.4% to CD4⁺ T-cells and −14.9% to CD8⁺ T-cells (Fig. 2B). These results indicated that the specificity of anti-CD137—saporin immunotoxin on activated T-cells was high. Three additional observations supported this interpretation: first, activated CD8⁺ T-cells that produced IFN-γ were significantly reduced in anti-CD137—saporin-treated splenocytes compared with rat IgG—saporin-treated splenocytes, and this reduction was not evident in anti-CD137—saporin-treated activated CD137-deficient CD8⁺ T-cells (Fig. 2C); second, a thymidine-incorporation assay showed that anti-CD137—saporin immunotoxin, but not rat IgG—saporin, depleted proliferating WT T-cells, but not CD137-deficient T-cells, in response to anti-CD3 mAb in a dose-dependent fashion (Fig. 2D); and third, treatment of activated T-cells with FITC-conjugated anti-CD137—doxorubicin immunotoxin resulted in apoptosis mainly in FITC-positive cells (Fig. 2E).

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Figure 2.

Anti-CD137—immunotoxin kills activated T-cells. Splenocytes of WT, CD137^-/-, TCR specific for OVA peptide transgenic (OT-I), or OT-I-CD137^-/- mice were stimulated with anti-CD3 mAb (0.5 μg/ml) or OVA peptide (1 μg/ml) for 24 h. After confirmation of CD137 expression on CD4⁺ T- and CD8⁺ T-cells, cells were cultured at varying concentrations of anti-CD137—saporin immunotoxin for 48 h or anti-CD137—doxorubicin—FITC for 24 h. (A) Apoptosis was analyzed by staining cells with anti-annexin V mAb. *p < 0.05; **p < 0.01 between the indicated group and the other two groups at the indicated time points. (B) Unconjugated anti-CD137 or rat IgG was included in experimental group. Note that unconjugated anti-CD137 mAb increases cell survival, suggesting that it does not have off-target toxicity in CD4⁺ and CD8⁺ T-cells. (C) The absolute number of interferon (IFN)-γ⁺ CD8⁺ T-cells was counted by gating IFN-γ-expressing CD8⁺ T-cells from splenocytes after further stimulation with phobol 12-myristate 13-acetate (PMA) plus inomycin for 5 h. **p < 0.01 between the two groups. (D) Thymidine incorporation assay for WT and CD137^-/- T-cells. **p < 0.01; ***p < 0.001 between the indicated group and the other three groups at the indicated time points. (E) Anti-CD137—doxorubicin—FITC-treated cells were harvested and stained with anti-CD8 and anti-annexin V mAbs and apoptotic cells were analyzed by fluorescence-activated cell sorting (FACS). (F) FACS analysis of apoptotic OT-I and OT-I-CD137^-/- T-cells. (G) Thymidine incorporation assay for OT-1 and OT-I-CD137^-/- T-cells. Experiments were repeated at least two times with similar results. **p < 0.01; ***p < 0.001 between the indicated group and the other three groups at the indicated time points.

The anti-CD137—saporin immunotoxin also showed lethal specificity for OVA-specific OT-I T-cells expressing CD137 when applied following a 12-h stimulation with OVA_257—264 peptide. OT-I cells not expressing CD137 (OT-I-CD137^-/- T-cells) did not undergo apoptosis under these conditions (Fig. 2F). Rat IgG—saporin did not affect apoptosis of either antigen-activated WT or CD137-deficient OT-I T-cells compared with untreated cells (Fig. 2F). We confirmed the cytotoxic specificity of anti-CD137—saporin immunotoxin on CD137-expressing OT-I T-cells using a proliferation assay (Fig. 2G). These findings further support use of the anti-CD137—saporin immunotoxin to selectively deplete CD137-expressing activated T-cells.

Various subsets of lymphoid cells express CD137. We analyzed CD137 expression of subsets of splenocytes and examined whether the CD137-expressing subsets could be deleted by anti-CD137—saporin immunotoxin. We found that only forkhead box P3-positive (FoxP3⁺)CD4⁺ regulatory T-cells expressed cell-surface CD137 (Fig. 3A). Incubation of splenocytes with anti-CD137—saporin immunotoxin deleted FoxP3⁺CD4⁺ regulatory T-cells in a dose-dependent manner (Fig. 3B).

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Figure 3.

Anti-CD137—saporin immunotoxin can kill FoxP3⁺CD4 regulatory T-cells. (A) Splenocytes were isolated from C57BL/6 mice. Cells were stained with anti-CD137 mAb and antibodies raised against immune cell markers: CD3, CD4, CD25, and forkhead box P3 (Foxp3) for regulatory T-cells; CD3 and CD8 for CD8⁺ T-cells; CD3 and CD4 for CD4⁺ T-cells; CD3 and NK1.1 for NK and NKT cells; CD11b for monocytes; CD11c for dendritic cells. (B) Splenocytes (2 × 10⁶ cells) were treated with various concentrations of rat IgG—saporin or anti-CD137—saporin and incubated for 48 h, and harvested cells were stained with anti-CD3, anti-CD4, anti-CD25, and anti-Foxp3 mAbs, and the extent of depletion of regulatory T-cells was analyzed by FACS.

Depletion of Alloreactive T-Cells by Anti-CD137—Saporin Immunotoxin in an MLR Assay

We established an in vitro MLR system using C57BL/6 T-cells as responder cells and irradiated BDF1 splenocytes as stimulator cells. In this system, CD137 expression was observed in only a small proportion of CD4⁺ and CD8⁺ T-cells until 3 days of culture. Thereafter, the percentage of T-cells expressing CD137 gradually increased, reaching maximum proportions of 13% and 35% in CD4⁺ and CD8⁺ T-cells, respectively, by 9 days, and remaining at those levels throughout the culture period (Fig. 4A). Addition of anti-CD137—saporin immunotoxin to the 5-day MLR cultures resulted in depletion of most of the CD137-expressing CD4⁺ and CD8⁺ T-cells (Fig. 4B). Concurrently, anti-CD137—saporin immunotoxin depleted most of the CD44^hiCD62L^lo CD4⁺ and CD8⁺ T-cells (Fig. 4B), confirming that the CD137-expressing T-cells were activated effector T-cells.

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Figure 4.

Anti-CD137—immunotoxin depletes alloreactive T-cells in an in vitro MLR. (A) C57BL/6 mouse splenocytes were stimulated with irradiated (C57BL/6 × DBA/2)F1 (BDF1) mouse splenocytes for 15 days. Cells were harvested at the indicated time points and stained with anti-CD137 and anti-CD4 or anti-CD8 mAb. Percentages of CD137-expressing CD4⁺ and CD8⁺ T-cells are presented here. (B) Splenocytes of C57BL/6 mice were cultured with irradiated BDF1 mouse splenocytes for 5 days and further cultured in the presence of 1 μg/ml of anti-CD137—saporin or rat IgG—saporin immunotoxin for 2 days. Cells were stained with combinations of anti-CD137 and anti-CD4 or anti-CD8, or anti-CD62L and anti-CD44 plus anti-CD4 or anti-CD8 mAb. Representative dot plots are shown in the upper panels, and percentages of activated T-cells are shown in the lower panels. Experiments were repeated at least two times with similar results. ***p < 0.001 between the indicated two groups.

HSCT After Allodepletion with Anti-CD137—Saporin Immunotoxin Prevents Acute GVHD

We tested the effect of depleting alloreactive T-cells from donor T-cells using anti-CD137—saporin immunotoxin for the induction of GVHD in the C57BL/6 (H-2K^b) → BDF1 (H-2K^b/d) acute GVHD model. As shown in Figure 5A—C, the transfer of donor T-lymphocytes after depletion of alloreactive donor T-cells from an MLR reaction with anti-CD137—saporin immunotoxin did not induce evident GVHD, as determined based on body weight, survival rate, and histopathology of GVHD target organs, and the severity of GVHD was comparable to that in recipients of BM cells only. In contrast, mice that received donor T-cells after allodepletion with rat IgG—saporin exhibited severe GVHD (Fig. 5A—C).

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Figure 5.

Transfer of donor T-cells after allodepletion with anti-CD137-saporin immunotoxin does not induce acute GVHD. After depletion of alloreactive T-cells with anti-CD137-saporin immunotoxin in an in vitro MLR, residual donor T-cells (2 × 10⁶ cells /mouse) were transferred with T-cell-depleted BM cells to irradiated (750 cGy) BDF1 mice. (A) Changes in body weight, (B) survival times (n = 15 for BM only, n = 15 for rat IgG-SAP, and n = 18 for anti-CD137-SAP; ***p < 0.001 between the indicated group and the other two groups), and (C) histopathological scoring (**p < 0.05; ***p < 0.001 between the rat IgG-saporin- and anti-CD 137-saporin-treated groups) of target organs at 50 days after disease induction confirm GVHD. (D, E) Splenocytes were isolated from mice with acute GVHD and stimulated with BDF1 mouse splenocytes for 5 days after labeling with CFSE. Percentages of dividing CD4⁺ and CD8⁺ T-cells are shown (D). Cytokine levels in culture supernatants were measured using a CBA kit (E). **p < 0.05; ***p< 0.001 between the indicated groups. NS, not significant. (F) A cytotoxic T-lymphocyte assay was performed using splenocytes isolated from mice with GVHD. The splenocytes were incubated with CFSE^low-EL4 (H-2K^b) and CFSE^high-P815 (H-2K^b) cells for 48 h, and their cytolytic activity against allogeneic cells was analyzed by counting the surviving P815 cells. Samples were harvested from n = 5-7 mice per group on day 50 after donor cell transfer (for experiments shown in C-F). GVHD experiments were repeated at least two times (n = 7-10 mice per group), and representative results are shown.

We analyzed the alloreactivity of T-cells from mice that received BM cells only or donor T-cells after allodepletion with anti-CD137—saporin immunotoxin or rat IgG—saporin. On day 50 after GVHD induction, splenocytes were isolated from each group of mice and restimulated with irradiated BDF1 splenocytes. In comparison to CD4⁺ and CD8⁺ T-cells from mice that received donor T-cells after depletion of alloreactive T-cells with rat IgG—saporin, those of mice that received T-cells after depletion of alloreactive T-cells by anti-CD137—saporin immunotoxin showed lower proliferation in response to allostimulation, as seen in mice that received BM cells only (Fig. 5D). In accordance with lower immunoresponsiveness, the allostimulated T-cells of mice that received allodepleted donor T-cells or BM cells only produced significantly lower levels of IFN-γ and TNF-α (Fig. 5E). In addition, T-cells from mice that received anti-CD137—saporin-treated donor T-cells showed significantly lower cytotoxic activity than did T-cells from mice that received rat IgG—saporin-treated donor T-cells (Fig. 5F).

HSCT After Allodepletion with Anti-CD137—Saporin Immunotoxin Does Not Impair a GVL Effect

To test the effect of allodepletion by the anti-CD137—saporin immunotoxin on the GVL effect following BM transplantation, we used an EL-4 lymphoma model that has been well established by our and other laboratories (23). Recipient C57BL/6 mice were inoculated intravenously with 1 × 10⁷ of EL4 cells 7 days before receiving lethal total body irradiation (850 rad). Recipients were then injected with Balb/c BM cells with or without donor T-cells that were depleted of alloreactive T-cells using anti-CD137—saporin or treated with rat IgG—saporin in an in vitro MLR. All mice that received BM cells only died subsequent to severe body weight loss within 20 days after BMT (Fig. 6A, B). Histological analysis of multiple organs confirmed that the mice died of EL-4 lymphoma (Fig. 6C). Mice that received donor T-cells after allodepletion with rat IgG—saporin experienced a transient recovery of body weight and then a severe loss of body weight, and most (seven out of eight mice) died by 70 days after GVHD induction. This pattern of body weight change and histological findings indicated that the high mortality in this group of mice was due to severe GVHD (Fig. 6B, C). Mice that received donor cells after allodepletion with anti-CD137—saporin immunotoxin displayed less severe GVHD as determined based on body weight, survival rate, and histopathology (Fig. 6A—C). At 150 days after GVHD induction, the surviving mice that received donor cells after allodepletion with anti-CD137—saporin immunotoxin were evaluated by gross observation and histological analysis (Fig. 6C). All four of these mice were free of EL-4 lymphoma in the lung, liver, kidney, spleen, and colon (Fig. 6C). In contrast, the mice that survived for 20 days with GVHD presented tumor foci in multiple organs. Since there was no GVHD-related mortality in recipient mice that received donor T-cells after allodepletion with anti-CD137—saporin immunotoxin in the C57BL/6 → BDF1 GVHD acute model (Fig. 5A), it seems that the GVL effect was completely preserved in 50% of recipient mice in the Balb/c → C57BL/6 acute GVHD model (Fig. 6A). Taken together, these results provide evidence that the donor T-cell pool contained a significant portion of T-cells that retained antileukemia activity after depletion of alloreactive T-cells with anti-CD137—saporin immunotoxin.

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Figure 6.

Transfer of donor T-cells after allodepletion with anti-CD137—saporin immunotoxin does not impair a GVL effect. Mice were injected with EL-4 cells (1 × 10⁷ cells/mouse) through the tail vein and 7 days later lethally irradiated (850 rad) C57BL/6 mice received T-cell-depleted Balb/C bone marrow cells (1 × 10⁷ cells/per mouse) and T-cells (2 × 10⁶ cells) after allodepletion with anti-CD137—saporin immunotoxin. (A) Survival times, (B) changes of body weight, and (C) histological analysis of various organs (n = 5—8 per group). Experiments were repeated with similar results.

Anti-Human CD137 mAbs Can Deliver Toxin Into the Cell and Kill Alloreactive T-Cells

After binding to cell surface CD137 receptors, anti-human CD137 mAb localized in the EEA-1⁺ endosomes of activated human CD4⁺ and CD8⁺ T-cells within 4 h, suggesting that the mAb was internalized through receptor-mediated endocytosis (Fig. 7A), as seen in mouse cells. We next tested whether anti-human CD137 mAbs could deliver toxin into activated T-cells and subsequently kill those cells. For this, we indirectly bound saporin to anti-CD137 mAbs using saporin-conjugated anti-mouse IgG (anti-mouse IgG—saporin). We activated purified human CD4⁺ and CD8⁺ T-cells with 0.5 μg/ml of OKT3 for 24 h. Treatment with anti-mouse IgG—saporin induced approximately twofold increased apoptosis of activated CD4⁺ and CD8⁺ T-cells following treatment with agonistic anti-human CD137 (4B4) or antagonistic anti-CD137 mAbs (4785) compared with control goat IgG—saporin and unconjugated anti-human CD137 mAbs (Fig. 7B). In our culture conditions, OKT3 induced apoptosis of a high portion of T-cells most presumably through activation-induced cell death (Fig. 7B). Since activation of T-cells accompanies their proliferation, we examined whether sequential treatment with 4B4 or 4785 and anti-mouse IgG—saporin would delete proliferating CD4⁺ and CD8⁺ T-cells following stimulation with allogeneic antigen-presenting cells in the presence of OKT3. In this experimental setting, anti-human CD137 and anti-mouse IgG—saporin complexes deleted approximately 47% of proliferating CD4⁺ T-cells and 25—29% of proliferating CD8⁺ T-cells (Fig. 7C). Finally, we tested the ability of anti-human CD137—anti-mouse IgG—saporin complexes to delete alloreactive T-cells in a one-way MLR. Human T-cells showed a similar kinetics of CD137 expression in this MLR setting, as shown in mouse T-cells (Figs. 4A and 7D). We sequentially added 4785 and anti-mouse IgG—saporin to the 5-day MLR cultures and restimulated the remaining responder T-cells with irradiated first-party simulator PBMCs after 5 days of resting. A thymidine incorporation assay showed that deletion of alloreactive T-cells by anti-human CD137 mAb from the primary MLR cultures resulted in a 4.5-fold reduction in proliferation of T-cells (Fig. 7E). These results suggest that anti-human CD137 mAbs physically linked to toxin can be predicted to deplete alloreactive human T-cells.

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Figure 7.

Anti-human CD137 mAbs can deliver toxin into activated human T-cells and induce cell death. (A) Purified CD4⁺ and CD8⁺ T-cells from PBMCs were stimulated with purified anti-CD3 mAb (OKT3; 0.5 μg/ml) for 24 h and incubated with anti-human CD137-PE for 30 min on ice. After removal of nonbound antibodies, cells were cultured at 37°C for 4 h. Cells were harvested and stained with anti-CD4-FITC (left panels), anti-CD8-FITC (middle panels), or anti-EEA-l-FLTC (right panels) and analyzed for the subcellular localization of CD137 using confocal microscopy. (B) PBMCs were stimulated with OKT3 (0.5 μg/ml) for 24 h and incubated with anti-human CD137 mAbs (4B4 or 4785) and 1 μg/ml of anti-mouse IgG—saporin or goat IgG—saporin on ice for 30 min. After removal of nonbound anti-human CD137 mAb, cells were cultured for 48 h. Harvested cells were stained with anti-CD8 and anti-annexin V mAb. Histograms for anti-annexin V staining in gated CD8⁺ T-cells are presented. (C) CFSE-labeled responder PBMCs were cultured with irradiated stimulator PBMCs and OKT3 (0.5 μg/ml) for 24 h. After staining with anti-CD137 mAbs plus anti-mouse IgG-saporin or anti-goat IgG-saporin, cells were cultured 48 h. Harvested cells were stained with anti-CD4 or anti-CD8 mAb. Histograms for CFSE dilution in gated CD4⁺ and CD8⁺ T-cells are shown. (D) Kinetics of CD137 expression on T-cells in a one-way MLR. Responder human PBMCs were stimulated with irradiated stimulator PBMCs for 15 days, and CD137 expression was analyzed on CD4⁺ and CD8⁺ T-cells by FACS. (E) A thymidine incorporation assay. After depleting CD137-expressing T-cells from 5-day MLR culture with anti-CD137 (4785)-mouse IgG-saporin complexes, cells were rested for 5 days in the presence of IL-2 and restimulated with the irradiated first-party PBMCs. *p < 0.05 between the two indicated groups.