Biology of Blood and Marrow Transplantation
Volume 14, Issue 1 , Pages 99-109, January 2008

Depletion of Alloreactive Donor T Lymphocytes by CD95-Mediated Activation-Induced Cell Death Retains Antileukemic, Antiviral, and Immunoregulatory T Cell Immunity

Department of Medicine III, Hematology and Oncology, Johannes Gutenberg-University School of Medicine, Mainz, Germany

Received 6 June 2007; accepted 2 October 2007.

Article Outline

Abstract 

In allogeneic hematopoietic stem cell transplantation (AHSCT) graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect are closely but not invariably linked. Thus, harnessing donor lymphocyte mediated GVL immunity and separating it from GVHD is of particular interest. Based on results obtained in murine models we have explored the CD95-mediated activation-induced cell death (AICD) strategy to selectively deplete alloreactivity in human donor T lymphocytes in vitro. Following stimulation of CD3+ T cells isolated from HLA-A∗0201-positive donors with HLA or minor histocompatibility antigen mismatched hematopoietic or nonhematopoietic cells in the presence of agonistic anti-CD95 antibody, we achieved efficient and selective allodepletion across major and minor histocompatibility mismatched barriers. Residual alloreactivity was in the range of 10% and 25% using hematopoietic cells and primary keratinocytes as alloantigen-presenting cells, respectively. CD8+ T cells specific for HLA-A∗0201-associated cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Wilms tumor 1 peptide epitopes were retained at significant numbers within the allodepleted donor lymphocyte subsets. Additionally, CD4+ FoxP3+ regulatory T cells persisted after the allodepletion procedure. Our results show that AICD induced by an agonistic anti-CD95 antibody might be useful to generate allodepleted donor lymphocyte products with preserved beneficial immune functions for patients undergoing AHSCT.

Key Words: Lymphocyte graft engineering, T cell depletion, Allodepletion

 

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Introduction 

Donor-derived T cells, either present in the stem-cell graft or administered as donor lymphocyte infusion (DLI), support engraftment [1], improve immunity particularly to viral infections [2], and confer potent graft-versus-leukemia (GVL) responses after allogeneic hematopoietic stem cell transplantation (AHSCT) 3, 4, 5. However, donor T cells are also central mediators of graft-versus-host disease (GVHD) 6, 7, which remains a major complication in AHSCT. Nonselective removal of donor T cells effectively controls GVHD, but concurrently increases the risk for opportunistic infections and abrogates therapeutically desired immune responses toward leukemic cells. There has been accumulating evidence suggesting that the GVL effect is closely but not inseparably linked with the development of GVHD 8, 9 and distinct subsets of allogeneic T lymphocytes can confer GVL reactivity in the absence of GVHD 10, 11, 12. This is further supported by in vitro studies demonstrating that donor T cells at the clonal level can recognize antigens with either ubiquitous or hematopoiesis-restricted tissue expression 13, 14, 15. Therefore, separation and expansion of allogeneic T cell specificities devoid of graft-versus-host (GVH) reactivity to specifically promote GVL immune responses remains of central interest for improving the therapeutic outcome in AHSCT.

Various conceptually different strategies have been developed to eliminate alloreactive donor T lymphocytes following exposure to allogeneic stimulator cells, either in vivo by inducible “suicide” genes expressed in genetically modified allogeneic T lymphocytes [16], or ex vivo by photodynamic purging [17], by concomitant costimulatory blockade [18], by fluorescence-activated cell sorting [19], or by targeting activation-induced antigens such as CD25 20, 21, 22, CD69 23, 24, HLA-DR [25], and CD137 [26]. Of those, CD25-based approaches already demonstrated in vivo efficacy in terms of GVHD reduction and graft survival in haploidentical and HLA-identical AHSCT 27, 28, 29. However, a considerable number of these studies did not include a detailed in vitro analysis on the persistence of antileukemic T cells after the depletion maneuver. In addition, as CD4+ CD25high regulatory T (Treg) cells have been suggested to play an important role as suppressive regulators of GVH reactivity [30], their retainment within allodepleted T cell subsets has not been addressed in most selective allodepletion (SD) strategies.

In the present study we extended our previously reported experiments on the depletion of murine alloreactive T cells by CD95/CD178-mediated activation-induced cell death (AICD) [31] to human donor-recipient pairs with complete or partial HLA match. We demonstrate that substantial CD95-induced allodepletion can be achieved in human T cells using either hematopoietic cells or primary keratinocytes as alloantigen-presenting cells. Residual antiviral and antileukemic T cells were preserved at significant numbers within the allodepleted cell subsets. Furthermore, naturally occurring CD4+ CD25+ FoxP3+ Treg cells did not appear to be as susceptible to CD95-mediated AICD as activated T cells, suggesting that Treg cells might be largely retained by SD via the CD95/CD178 pathway.

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Material and Methods 

Cell Lines 

The human mutant cell line T2 (174×CEM.T2) and the human CML cell line K562 transfected with full-length cDNA encoding the HLA-A∗0201 allele [32] were cultured in RPMI 1640 medium (Cambrex Bio Science, Verviers, Belgium) supplemented with L-glutamine (2 mM/L), penicillin (100 U/mL), streptomycin (100 μg/mL) (Gibco BRL, Grand Island, NY) and 10% fetal calf serum (FCS) (Cambrex Bio Science). Cell cultures were kept in a water-saturated atmosphere with 5% CO2 at 37°C.

Peptides 

HLA-A∗0201-associated peptide epitopes used herein were Epstein-Barr virus (EBV) BMLF1 peptide 280-288 (GLCTLVAML), cytomegalovirus (CMV) pp65 peptide 495-503 (NLVPMVATV), and a peptide derived from human Wilms tumor antigen 1 (WT1) 126-134 (RMFPNAPYL). They were synthesized on solid phase using Fmoc chemistry, purified by reversed-phase high-performance liquid chromatography (HPLC), and characterized by mass spectrometry.

Generation of Human Primary Keratinocytes, B Cells, and Dendritic Cells 

Primary keratinocyte cultures were generated from 5-6 mm2 skin samples derived from skin or foreskin of patients undergoing surgery. Briefly, the epidermis was separated from dermis after overnight incubation at 4°C in 2.4 U/mL Dispase II (Roche, Mannheim, Germany) in phosphate-buffered saline (PBS) containing 2.5% Gentamycin (Invitrogen-Gibco, Karlsruhe, Germany) and trypsinized for 30 minutes at 37°C to obtain a single cell suspension. Keratinocytes (KCs) were then cultured at 37°C, 5% CO2 in MCDB 153 (Biochrom AG, Berlin, Germany)/keratinocyte-SFM medium (Invitrogen-Gibco) mixture (50:50, v/v) supplemented with 25 μg/mL bovine pituitary extract (Invitrogen-Gibco) and 100 μg/mL recombinant epidermal growth factor (Invitrogen-Gibco). Confluent cultures of KCs were split 1:1 and were expanded up to 5 to 6 culture passages. In some experiments KCs were pretreated with 500 IU/mL interferon-gamma (IFN-γ) (ImmunoTools, Friesoythe, Germany) over 3 days prior to use.

Peripheral blood B cells were stimulated using NIH-3T3 cells stably transfected with human CD40 ligand (CD40L) [33]. CD40L cells were irradiated (100 Gy) and seeded in 6-well plates (Greiner, Nürtingen, Germany) at 4 × 105 cells/mL in Dulbecco's Modified Eagle Medium/F-12 Nutrient (Gibco BRL) mixture (50:50, v/v) supplemented with L-glutamine (2 mM/L), penicillin (100 U/mL), streptomycin (100 μg/mL) (Gibco BRL) and 10% FCS (Cambrex Bio Science). After 18 to 24 hours, CD40L cells were cocultured with 2 × 106/mL peripheral blood mononuclear cells (PBMCs) in Iscove's Modified Dulbecco Medium (Invitrogen-Gibco) supplemented with 10% (v/v) human AB plasma, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 U/mL Interleukin (IL)-4 (ImmunoTools), 5 μg/mL insulin (Sigma Aldrich, Deisenhofen, Germany), and 500 ng/mL cyclosporine A (Sigma, St. Louis, MO). Cultures were replated onto freshly irradiated CD40L cells in the presence of cyclosporine A and IL-4 every 3 to 4 days. The number of B cells was monitored weekly using a CD19 monoclonal antibody (mAb) (Beckman Coulter, Miami, FL) and flow cytometry. Usually, B cell purity exceeded 90% after 2 to 3 weeks.

Dendritic cells (DCs) were generated according to a protocol of Romani et al. [34] with minor modifications. Briefly, PBMCs isolated from buffy coats of healthy unrelated donors by Ficoll-Paque (Biochrom AG) density gradient centrifugation were incubated at 6.6 × 106 cells/mL in X-VIVO-15 medium (Cambrex Bio Science) on plastic dishes for 2 hours. The nonadherent cells were removed and the adherent fraction was cultured in X-VIVO-15 medium in the presence of 1000 U/mL granulocyte/macrophage colony stimulating factor (GM-CSF) (Leukomax; Sandoz, Munich, Germany) and 1000 U/mL IL-4. 50% (v/v) of culture medium supplemented with cytokines was renewed on day 3, and DCs were maturated on day 6 using a cytokine cocktail containing IL-4 (1000 U/mL), GM-CSF (1000 U/mL), IL-1ß (10 ng/mL; R&D Systems, Wiesbaden, Germany), IL-6 (1000 U/mL; Strathmann AG, Hamburg, Germany), Tumor necrosis factor (TNF)-α (10 ng/mL; PromoCell, Heidelberg, Germany), and Prostaglandine (PGE)2 (1 μg/mL; Pharmacia, Erlangen, Germany). On day 7 to 8, DCs were harvested and used for allogeneic mixed lymphocyte cultures (MLCs).

Isolation of Primary CD3+ T Cells from PBMCs 

CD3+ T lymphocytes or the CD3+ CD8+ subset were positively isolated from PBMCs by immunomagnetic separation using a T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). Purity of the enriched T cells always exceeded 98% as determined by flow cytometry.

Stimulation and Depletion of Alloreactive T Cells via AICD 

CD3+ or CD3+ CD8+ T cells isolated from fresh or thawed PBMCs were first polyclonally activated by immobilized anti-CD3 mAb (3 μg/mL, Orthoclone OKT® 3, Janssen-Cilag, Neuss, Germany) and soluble anti-CD28 mAb (1 μg/mL, clone YTH 913.12, Serotec, Düsseldorf, Germany) for 3 days followed by 5 to 7 days of culture in cytokine-free X-VIVO-20 medium in 6-well plates (Greiner). Prestimulated responder cells (105/well) were then challenged with either irradiated (30 Gy) PBMCs, KCs, B cell blasts, or DCs from HLA mismatched or matched donors at responder/stimulator ratios of 1:5, 1:2.5, 1:1, and 10:1, respectively, in MLCs in serum free X-VIVO 20 medium in the absence or presence of agonistic anti-CD95 mAb (clone CH-11 [IgM]; Immunotech, Marseille, France) in 96-well plate or bulk culture MLCs for 4 to 5 days. The isotype control mAb was also from Immunotech.

Efficacy of depletion in MLCs carried out in 96-well plates was determined functionally by measuring proliferative responses using 3H-thymidine (3HTdR) (0.5 μCi/well; Amersham-Life Science, Braunschweig, Germany) uptake on day 4 and 5 for the last 16 to 18 hours of culture. Accordingly, efficiency and specificity of the depletion procedure performed in bulk culture MLCs was examined by determining the proliferative response of allodepleted T cells (105/well) against primary (first party) or irrelevant (third party) donor PBMCs in HLA mismatch pairs and against first party or third party DCs in the minor histocompatibility antigen (HAg)-mismatch pairs, respectively, in 96-well MLCs. Radionuclide incorporation in responder cells was measured on a β-plate liquid scintillation counter (Wallace, Turku, Finland). Results represent means of triplicates ± SD.

Immunophenotyping 

Expression of cell surface antigens was examined using the following murine anti-human mAb and corresponding isotype-matched control mAb in direct immunofluorescence staining: anti-CD3-FITC, anti-CD3-PE, anti-CD4-FITC, anti-CD4-PE, anti-CD8-PE, anti-CD69-PE (all from Immunotech) and anti-CD25-PE (Miltenyi). Briefly, 1 × 105 viable cells were incubated with mAb for 15 minutes at 4°C followed by 2 washings with PBS containing 0.5% FCS.

Analyses of CD4+ CD25+ FoxP3+ regulatory T (Treg) cells before and after allodepletion was performed using a regulatory T cell staining kit (NatuTec, Frankfurt/Main, Germany) according to the manufacturer's instructions. Briefly, PBMCs were first surface labeled with CD4-PE and CD25-APC mAb, respectively, followed by fixation and permeabilization. After blocking with 2% rat serum, cells were counterstained with anti-human FoxP3-FITC mAb (clone PCH101).

For all flow cytometry analyses, 10.000 events were collected after gating of viable lymphocytes by FSC and SSC signal list mode data in Cellquest Pro software on a FACS-Calibur instrument (Becton Dickinson, Heidelberg, Germany).

IFN-γ Enzyme Linked Immunospot (ELISpot) Assay 

IFN-γ ELISpot assays were performed as recently described [26]. Briefly, CD3+ or CD8+ T cells at 105/well and APCs at 105/well (DCs at 104/well) were seeded in ELISPOT plates in serum-free X-VIVO 20 medium. T cells without APCs served as controls. Peptides were added at final concentration of 10-20 μg/mL directly into the wells or were loaded onto stimulator cells for 2 hours prior to addition of T cells. After 20 hours of incubation at 37°C, IFN-γ spots were visualized and counted using an Axioplan 2 microscope combined with the computer-assisted image analysis system KS ELISpot 4.1 (Carl Zeiss Vision, Hallbergmoos, Germany). Results represent means of triplicate wells. Depletion efficacy was calculated by comparing ELISpot results from allodepleted T cells compared with those from undepleted controls.

Statistics 

All values are indicated as means ± SD. Statistical analysis of data was performed by the Mann-Whitney U-test. A value of P < .01 was considered statistically significant.

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Results 

Human Alloreactive T Cells Are Efficiently Depleted by CD95-Mediated AICD 

Based on our previous results obtained in murine bone marrow transplantation models [31] we explored the CD95-mediated induction of apoptosis in human activated alloreactive T lymphocytes in vitro. Because resting human T lymphocytes are resistant to AICD [35], purified CD3+ T cells derived from PBMCs of healthy donors were first activated by incubation with anti-CD3 and anti-CD28 mAbs. Preactivated responders were then cocultured with irradiated major or minor histoincompatible allogeneic stimulator cells in bulk mixed lymphocyte cultures (MLCs) over 5 days either in the presence or absence of agonistic anti-CD95 mAb.

Following stimulation of T cells with HLA-incompatible DCs (Figure 1A) or CD40L-activated B cells (Figure 1B) in the presence of anti-CD95 mAb, the proliferative alloimmune response was reduced to a mean of 6.3% ± 9.3% and 10.6% ± 18.1%, respectively, compared to undepleted controls. This decrease of alloreactivity was dependent on the dose of anti-CD95 mAb showing a maximum depletion efficiency at 200 ng/mL. In contrast, the isotype matched control Ab did not affect the alloimmune response (Figure 1A and B).

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  • Figure 1 

    Depletion of alloreactive T lymphocytes by CD95-mediated AICD. Purified CD3+ donor T cells were first activated with immobilized anti-CD3 and soluble anti-CD28 mAbs for 3 days followed by 5 days of culture in cytokine-free medium. Activated T cells were then stimulated in allogeneic MLCs with HLA-mismatched (A) DCs, (B) B cell blasts, or (C) primary human keratinocytes (KCs) for 5 days in the presence of agonistic anti-CD95 or isotype control mAbs at indicated concentrations. In some experiments KCs were pretreated with 500 IU/mL IFN-γ over 3 days. (D) Following MLCs, allodepleted T cells were challenged with PBMCs of the original stimulator cell donors (first-party) or with irrelevant (third-party) PBMCs in MLCs to determine the specificity of the depletion procedures. Proliferative responses were measured by [3H]-thymidine uptake for the last 18 h of incubation on days 3, 4, or 5 of culture. T cells stimulated with autologous DCs, B cells, or medium (for KCs) were used as controls (auto control). Data are given as means ± SD of triplicates and are shown for 1 representative of 5 donor-recipient pairs. P-values were analyzed between the most relevant experimental groups. Values <.01 are considered statistically significant. Asterisk means that the difference between 2 experimental groups was not significant.

Furthermore, primary human keratinocytes (KCs) established from skin biopsies of surgery patients were successfully used to stimulate alloreactive T lymphocytes in HLA-mismatched donor-recipient pairs. Mean residual alloreactivity was 25% ± 9.5% following allodepletion (Figure 1C). Pretreatment of KCs with IFN-γ did not significantly affect the allodepletion results. These data confirmed that depletion of alloreactive T cells can be achieved using nonprofessional APCs of epithelial origin within the SD approach [25].

Residual reactivity of allodepleted donor lymphocytes was then determined against original stimulator PBMCs (first party) and found to be strongly reduced (mean of 7.0% ± 13.5%) compared to undepleted controls (Figure 1D). However, the strength of alloimmune responses to HLA-irrelevant third-party PBMCs remained unaffected after the depletion maneuver (Figure 1D).

To exclude preferential allodepletion of the CD4+ subset within the CD3+ T cell responder population, purified CD8+ T lymphocytes were stimulated with HLA-incompatible DCs in the presence of anti-CD95 Ab (Figure 2). Again, alloreactivity by CD8+ responder cells was reduced to a mean of 22.8% ± 9.4% when compared to undepleted controls as determined by proliferative responses (Figure 2A) or IFN-γ secretion levels of residual allodepleted donor CD8+ T cells stimulated against primary stimulator DCs (Figure 2B). Moreover, allodepletion to given HLA-mismatch antigens was specific and did not significantly affect the anti-third-party alloimmune response (Figure 2C). Taken together, these results suggested that both CD4+ and CD8+ alloreactive donor T lymphocytes are effectively eliminated by CD95-mediated SD.

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  • Figure 2 

    Depletion of alloreactive CD8+ T cells by CD95-mediated AICD. Purified CD8+ donor T lymphocytes were first activated as described in Figure 1. (A) T cells were then stimulated with HLA-mismatched DCs in the presence of agonistic anti-CD95 or isotype control mAbs at indicated concentrations and proliferative alloimmune responses were determined by [3H]-thymidine uptake for the last 18 hours of incubation on day 3 of culture. T cells stimulated with autologous DCs were used as controls (auto control). (B) Following MLCs with agonistic anti-CD95 mAb or isotype control mAb at indicated concentrations, 1 × 105 of allodepleted or untreated CD8+ T cells/well were challenged with original DCs as stimulators to examine IFN-γ secretion of residual effector cells by ELISpot assay. (C) IFN-γ secretion of 1 × 105 allodepleted or untreated CD8+ T cells/well after restimulation with first-party DCs or HLA-irrelevant third-party DCs as stimulators. Background in all figures is represented by proliferation or IFN-γ spots obtained from T cells cultured with autologous DCs (auto DC). Data are given as means ± SD of triplicates and are shown for 1 representative of 3 donor-recipient combinations. P-values are indicated as described in Figure 1.

The allodepletion results obtained in HLA-mismatched MLCs could be confirmed in donor-recipient pairs with complete genomic HLA match at high-resolution level. Coculture of donor T lymphocytes with HLA-identical DCs (Figure 3A) or B cells (Figure 3B) in the presence of agonistic anti-CD95 mAb resulted in a mean reduction of minor histoincompatibility driven alloreactivity to 10.5% ± 11.1% and 14.4% ± 9.2%, respectively. Moreover, analyses of residual immunity of allodepleted donor T cells to first-party and HLA-irrelevant third-party DCs showed a significant and specific decrease of reactivity towards the original stimulator cells (Figure 3C).

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  • Figure 3 

    Depletion of minor HAg mismatch reactive T lymphocytes via CD95-mediated AICD. Preactivated (see Figure 1) CD3+ donor T cells were stimulated in MLCs with (A) allogeneic DCs or (B) allogeneic B cell blasts generated from individuals with full HLA class I and class II match according to high-resolution typing. Agonistic anti-CD95 mAb was added at 200 ng/mL where indicated. (C) Following MLCs, allodepleted T cells were restimulated with original DCs (first-party) or irrelevant DCs (third-party) to determine depletion specificity. Proliferation was analyzed in triplicates as described in Figure 1. T cells stimulated with autologous DCs were used as controls (auto control). Data are from 1 representative of 3 donor-recipient pairs. P-values are indicated as described in Figure 1.

In summary, these results confirmed our previous findings in murine models [31] and strongly suggested that SD of human alloreactive donor lymphocytes by AICD is efficient and specific for given alloantigens across both HLA or minor histocompatibility antigen (H1g) barriers.

CMV and EBV-Specific CD8+ T Cells Are Retained after CD95-Mediated Allodepletion 

We analyzed the effect of the CD95-mediated selective allodepletion procedure on the frequency of antiviral CD8+ T cells. PBMCs isolated from HLA-A∗0201-positive healthy donors were first screened in IFN-γ ELISpot assays for memory CD8+ T cells recognizing HLA-A∗0201-restricted CMV-pp65 and EBV-BMLF1 peptide epitopes. Subsequently, T cells of donors with detectable antiviral T cell memory were stimulated with irradiated recipient PBMCs isolated from EBV and CMV-seronegative individuals that carried HLA-A∗0201 as the only matched HLA-class-I allele. After CD95-mediated allodepletion, frequencies of spot-forming antiviral T cells were determined within allodepleted cell subsets and were compared to undepleted allogeneic and autologous control populations, respectively (Figure 4).

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  • Figure 4 

    CMV and EBV-specific CD8+ T cells persist after CD95-mediated allodepletion. CD3+ T cells derived from HLA-A∗0201-positive healthy donors with previous CMV and EBV exposure were activated in HLA-A∗0201-matched allo-MLCs followed by anti-CD95-induced AICD. T cells obtained from autologous control PBMCs, or from allodepleted and untreated MLC populations were then stimulated with K562-HLA-A∗0201 transfectant cells alone (gray bars) or pulsed with the HLA-A∗0201-binding CMV-pp65495-503 and EBV-BMLF1280-288 peptides (black bars), respectively, in IFN-γ ELISpot assays. Controls (white bars) represented T cells that secreted IFN-γ spontaneously. Data are presented as means ± SD of duplicates or triplicates, respectively. One representative of 3 experiments performed for 3 different CMV-positive (A) and EBV-seropositive (B) donors is shown. P-values are indicated as described in Figure 1.

As demonstrated in a total of 3 HLA-mismatched donor-recipient pairs, CMV-pp65/A∗0201-reactive T cells persisted in allodepleted donor lymphocytes with a mean frequency of 71.9% ± 6.4% compared to undepleted controls (Figure 4A). Similarly, EBV-BMLF1-reactive CD8+ T cells were retained after allodepletion in all 3 HLA-mismatched donor-recipient pairs analyzed (97% ± 16.68%) (Figure 4B). Interestingly, some undepleted allogeneic control populations contained increased frequencies of antiviral CD8+ T cells following stimulation with recipient PBMCs devoid of any detectable reactivity to CMV-pp65 or EBV-BMLF1 peptides. This may likely represent bystander activation of antiviral memory T cells previously observed by us and others 22, 36 or be attributed to increased resistance to cell death through upregulation of Bcl-2 expression [37].

Allodepleted T Cell Subsets Contain WT1-Specific CD8+ T Cells 

In addition to antiviral T cell immunity, we analyzed whether allogeneic T cell specificities potentially conferring GVL immune responses would persist at significant frequencies following CD95-mediated SD. Because cytotoxic T cells specific for the HLA-A∗0201-binding peptide 126-134 encoded by the leukemia-associated WT1 antigen can be detected at low frequencies in PBMC of healthy donors [38], we investigated the preservation of WT1 peptide-specific T cells after allodepletion of anti-MHC mismatch responders. Following stimulation of purified CD3+ T cells from HLA-A2-positive WT1-reactive donors with HLA-A2-matched but HLA-B/C-mismatched stimulators in the presence of anti-CD95 mAb, remaining responders were challenged with WT1 peptide 126-134 loaded onto HLA-A∗0201-expressing TAP-deficient T2 cells. Because K562-HLA-A∗0201 transfectants present endogenously processed WT1 and thus elicit strong background reactivity [39], T2 cells were used as APCs in these experiments.

When compared with the frequency of WT1 peptide-specific T cells found in the undepleted control prior to allogeneic stimulation, WT1 reactivity in the allodepleted cell subset was detected at a mean frequency of 84.4% ± 9.0% (Figure 5). This suggested that donor T cells potentially conferring WT1-specific GVL reactivity can be retained at significant numbers after allodepletion by AICD. The apparently increased WT1 reactivity above background levels observed in this experiment might be explained by the simultaneous reduction of background reactivity against T2 cells following allodepletion.

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  • Figure. 5. 

    WT1-specific CD8+ T cells are retained after CD95-mediated allodepletion. T cells of WT1-positive, HLA-A∗0201-positive healthy donors were activated in HLA-A∗0201-matched allo-MLCs followed by depletion of alloreactivity via CD95-mediated AICD. CD8+ T cells from autologous control, untreated or allodepleted populations were stimulated with unloaded T2 cells (gray bars) or with T2 cells pulsed with the HLA-A∗0201-binding WT1 126-134 peptide (black bars) in IFN-γ ELISpot assay. Data are given as means ± SD of triplicates and are representative of 3 donor-recipient combinations totally analyzed. P-values are indicated as described in Figure 1.

CD4+ CD25+ FoxP3+ Regulatory T Cells Can Be Retained following CD95-Mediated SD 

As CD4+ CD25+ FoxP3+ Treg cells have been suggested to suppress GVHD-inducing T lymphocytes, the persistence of this cell type in donor lymphocyte products appears desirable [30]. Thus, we analyzed the frequency of Treg cells after SD using the agonistic anti-CD95 mAb.

Among CD3+ T cells isolated from fresh PBMCs and analyzed for CD4, CD25, and FoxP3 expression, 3.0% of all CD4+ T cells were positive for FoxP3 (Figure 6; data on CD25 FoxP3 costaining not shown). Upon primary stimulation with immobilized anti-CD3 and soluble anti-CD28 mAbs followed by a resting period in cytokine-free medium, the frequency of CD4+ FoxP3+ Treg cells remained in the same range (3.1%). Following allostimulation and CD95-mediated SD over the next 5 days, 2.6% of CD4+ responder cells stained positive for FoxP3 showing a comparable percentage of double positive cells as before MLCs (Figure 6). These data suggested that Treg cells defined by FoxP3 expression remained at reduced but clearly detectable frequencies after CD95-mediated SD.

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  • Figure 6 

    CD4+ FoxP3+ Treg cells are not affected by CD95-mediated allodepletion. The percentages of CD4+ FoxP3+ Treg cells were determined within fresh PBMCs (d0) and after a preactivation culture period over 6 days (d6) by flow cytometry. Following allodepletion in MLCs against HLA-mismatched PBMCs, CD4+ FoxP3+ T cells were again measured in the anti-CD95 Ab-treated and isotype control Ab-treated cell subsets, respectively (d11). Data of 1 representative of 3 donor-recipient combinations are shown.

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Discussion 

SD strategies aim at the removal of unwanted host-reactive T cells from the donor lymphocyte graft while preserving allogeneic specificities able to generate beneficial antitumor and antipathogen immune responses, respectively. Several approaches to selectively inhibit or deplete alloreactivity have been investigated in human in vitro systems and murine animal models 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. However, with some exceptions 20, 21, 22, 36, 40, a detailed in vitro analysis on the persistence of T cells with antiviral, immunoregulatory, and, in particular, potential antileukemic immune functions following allodepletion remains to be demonstrated.

Based on previous work in murine bone marrow transplantation (BMT) models [31] we explored an anti-CD95 in vitro protocol to selectively deplete human alloreactive T cells from the total pool of donor lymphocytes. Following stimulation of donor T cells with HLA-mismatched as well as HLA-matched recipient hematopoietic cells in the presence of agonistic anti-CD95 mAb, we demonstrated reduction of alloreactivity with a mean efficiency of ≥93% for HLA-mismatched and ≥89% for HLA-matched settings, respectively. In addition, restimulating residual donor cells with original (first-party) or irrelevant (third-party) stimulator cells revealed that the depletion was alloantigen specific. Moreover, significant reduction of CD8+ T cell-mediated alloimmune responses could be demonstrated, strongly suggesting that CD95-mediated SD does affect both CD4+ and CD8+ alloreactive donor T lymphocytes.

Polymorphic minor HAgs, expressed either ubiquitously or restricted to cells of the hematopoietic lineage, are not only regarded as main target structures for GVH reactivity, but have also been reported to be important for the induction of GVL immunity [15]. Thus, the retainment of donor T cells reactive to hematopoietic minor HAgs within allodepleted donor lymphocyte grafts would be beneficial for the induction of specific GVL responses. Skin biopsy-derived human KCs represent accessable cells of nonhematopoietic origin, which can trigger efficient T cell alloreactivity [25] and express a comprehensive set of ubiquitous and lineage-specific minor HAgs [41]. Using KCs as allogeneic stimulator cells we could demonstrate significant CD95-mediated allodepletion, suggesting that this SD strategy might be feasible for eliminating reactivity to ubiquitous or epithelial alloantigens, but simultaneously spare T cells specific for hematopoiesis-restricted minor HAgs.

Apart from severe GVHD, opportunistic infections, particularly caused by CMV and, albeit less frequently, by EBV, remain major causes of morbidity and mortality after AHSCT [42]. Both CD8+ CTLs as well as CD4+ T helper cells have been shown to play a central role in controlling CMV and EBV infections [42]. Following SD by CD95-mediated AICD, we observed in HLA-A∗0201-matched donor-recipient pairs that within allodepleted donor lymphocytes antiviral CD8+ T cells to both CMV and EBV are retained at frequencies comparable to levels detected prior to depletion. Similar to previous reports using CD25 and CD69-mediated SD approaches 20, 21, 22, 36, 40, reactivity to the EBV-BMLF1 peptide within alloantigen stimulated donor cells was found to be elevated. This observation might be attributed to bystander activation acquired during repetitive stimulations of alloreactive responders resulting in IFN-γ secretion of recently EBV-activated specificities, as T lymphocytes derived from healthy donors with reactivities to viruses most likely represent memory-type T cells. Moreover, virus-specific memory CD8+ T cells may have an increased resistance to cell death as they can upregulate antiapoptotic genes such as Bcl-2 [37].

There is increasing evidence that donor immunity toward hematopoiesis-restricted minor HAgs is important for generating clinically effective GVL immunity 15, 43. The situation is less clear for leukemia-associated antigens, which also have been identified as target structures of donor T cells. Using WT1 as a model for leukemia-associated antigens, we investigated the persistence of HLA-A∗0201-restricted WT1 peptide-specific CD8+ T cells following allodepletion in healthy donors with detectable WT1 reactivity 38, 39. We demonstrated that WT1-reactive T cells are largely retained at significant frequencies after CD95-mediated SD. Taken together, our experimental data suggest that allodepletion by CD95-mediated AICD can preserve donor lymphocytes specific for herpes virus and leukemia-associated antigens.

Finally, we explored the persistence of CD4+ CD25+ FoxP3+ Treg cells usually present at 1%-5% of total CD4+ cells in human peripheral blood [44]. Tregs have been phenotypically defined by constitutive coexpression of CD4 and the interleukin-2 receptor alpha chain (CD25) [45]. However, CD25 is also expressed by activated T cells, and thus represents an unreliable marker for Treg cells. FoxP3, encoding a forkhead/winged helix transcription factor, has been identified as a key regulatory gene required for the development of Treg cells, and therefore, has been used to define a naturally occurring CD4+ Treg population within PBMCs [46]. However, CD4+ CD25− T cells have also been reported to express FoxP3 and differentiate into Treg cells upon suboptimal activation conditions or in the presence of low doses of antigen [47].

In our study, the frequency of FoxP3+ cells detected within the CD4+ T cell population remained stable following short-term polyclonal activation and 3 day resting in the absence of exogenous IL-2. Moreover, the proportion of residual CD4+ FoxP3+ cells after CD95-mediated SD was largely comparable to the frequency detected in the control population on d0. Supported by the findings that in contrast to their naive CD4+CD25− counterparts Treg cells appeared to be resistant to IL-2 driven CD95-induced apoptosis following TCR-mediated activation 48, 49, this result suggested that Treg cells are not highly susceptible to allodepletion via the CD95/CD178 pathway. Nevertheless, our data should be interpreted with caution because of the following reasons: first, although the percentages of Treg cells might be preserved, the absolute numbers will most likely be reduced by the anti-CD95 depletion maneuver in vitro. It should be kept in mind that efficient suppression of GVHD in murine models required the adoptive transfer of a substantial excess of CD4+ Treg cells [30]. Second, there is the possibility that a procedure-related reduction of Treg cell numbers might have been masked by a simultaneous increase of FoxP3 expression levels on T cells that were activated by our in vitro protocol. This as well as potential pitfalls of Treg cell immunotherapy in humans [50] prevent us from overstating the relevance of the Treg cell data shown herein.

Most SD approaches are performed after primary MLC in which alloreactive donor T cells are activated by nonmalignant recipient cells over a few days. This also applies to SD technologies that use anti-CD69 immunomagnetic cell separation 23, 24, 36 or the currently described agonistic anti-CD95 antibody inducing AICD. The comparably short MLC period followed by SD should facilitate the clinical translation of such methods, as has already been successfully accomplished with similar anti-CD25 directed SD procedures 20, 21, 22, 27, 28, 29. In contrast, our recently described anti-CD137 approach is based on the enrichment of GVL reactivity by repetitive in vitro stimulations with leukemia cells over 2 to 3 weeks before SD [26]. Although the CD137-based method is considerably more complex and laborious compared to current anti-CD69/-CD25/-CD95 technologies, such leukemia-sensitized allodepleted T cell lines might be of considerable value for patients who require an immediately strong GVL effect, for example, because of ongoing or threatening leukemia relapse.

In conclusion, this study demonstrates that the removal of alloreactivity by CD95-mediated AICD is feasible, and results in efficient allodepletion of human donor lymphocyte products in vitro, while retaining substantial numbers of antiviral, antileukemic, and regulatory T cells. The data confirm our previous work on depleting alloreactivity in murine models via the CD95/CD178 pathway [31]. Nevertheless, it remains unclear from these results wether the observed allodepletion in the order of 1 − log will translate into significant GVHD reduction following adoptive transfer of manipulated DLI into humans in vivo, as suggested from our murine studies. Moreover, efficient allodepletion does not mean that residual antileukemic T cells will be of sufficient quantity and quality to prevent leukemia relapse. Expansion of leukemia-reactive T cell precursors might be accomplished by subsequent in vitro stimulations of allodepleted T cells with leukemia antigens. For the SD strategies based on CD95/CD178 and CD137, we are presently trying to fulfill all prerequisites that are necessary for future clinical testing.

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Acknowledgments 

Financial support for these studies was provided by a grant from the German Cancer Aid (Deutsche Krebshilfe; Project-No. 70-3344), by a grant from the MAIFOR program of Mainz University School of Medicine, and by a grant from the Stiftung Rheinland-Pfalz für Innovation. The authors gratefully acknowledge the technical assistance of Ms. C. Metz.

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PII: S1083-8791(07)00505-8

doi:10.1016/j.bbmt.2007.10.002

Biology of Blood and Marrow Transplantation
Volume 14, Issue 1 , Pages 99-109, January 2008

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