Immunology

In vitro-established alloantigen-specific CD8+ CTLs mediate graft-versus-tumor activity in the absence of graft-versus-host disease

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Abstract

Mature donor-derived T cells in allogeneic bone marrow (BM) transplants mediate the graft-versus-tumor (GVT) effect by recognizing alloantigens on leukemic cells. However, alloantigen reactivity towards non-malignant tissues also induces graft-versus-host disease (GVHD). Defining T-cell subpopulations that mediate the GVT effect in the absence of GVHD induction remains a major challenge in allogeneic BM transplantation. In this study, we show that in vitro-generated alloantigen-specific CD8+ cytotoxic T cells (CTLs) established by weekly stimulation with alloantigen-expressing antigen-presenting cells did not induce GVHD in two major histocompatibility complex-mismatched BM transplantation models, where induction of lethal GVHD is dependent on the presence of either CD4+ or CD8+ T cells. Despite their strong alloantigen specificity, transplantation of CTLs did not induce the expression of GVHD-associated cytokines IFN-γ and TNF-α or clinical or histological signs of GVHD, and lead to a survival rate of above 90%. However, transplantation of unstimulated CD8+ T cells, which were not primed by the alloantigen in vitro, induced GVHD in both the transplantation models. Although CTLs were impaired in GVHD induction, they efficiently eradicated Bcr–Abl-transformed B-cell leukemias or mastocytomas. Thus, in vitro-derived CTLs might be useful for optimizing anti-tumor therapy in the absence of GVHD induction.

Introduction

Allogeneic bone marrow transplantation (BMT) is a curative treatment modality for hematopoietic malignancies, such as acute and chronic leukemias and lymphomas. Mature donor T cells in the allograft support engraftment, promote early T-cell immunity of the recipient and mediate the graft-versus-leukemia (GVL) effect. However, these donor T cells are also responsible for the induction of graft-versus-host disease (GVHD) by attacking recipient tissue, such as the liver, skin, and bowel, leading to significant morbidity and mortality.1 Although depletion of mature T cells from the allogeneic donor graft significantly reduces the risk of GVHD, it also abrogates the beneficial effect of GVL and delays immune reconstitution of the host, thereby supporting opportunistic infections.2 Therefore, the major challenge in allogeneic BMT is the identification of T-cell subsets that are able to exhibit an anti-tumor effect without causing GVHD.

Peripheral T cells are broadly separated into naïve T cells (CD44, CD62L+, CCR7+) and antigen-stimulated T cells containing effector and memory T cells. Depending on the markers CCR7 and CD62L, memory T cells can be separated into effector/memory T cells (TEM: CD44+, CCR7, CD62L) and central/memory T cells (TCM: CD44+, CCR7+, CD62L+). While TEM display immediate effector functions and preferentially home to inflamed tissues and the spleen, TCM differentiate into effector cells after antigen re-encounter and migrate to secondary lymphoid organs and the bone marrow (BM).3 These T-cell subsets contribute differently to the pathogenesis of GVHD. CD4+ TEM from non-alloantigen-primed donors fail to induce GVHD4, 5, 6 because of an impaired cytotoxic immune response towards the alloantigen.7 However, in vivo priming of CD4+ T cells with recipient alloantigen induces alloantigen-specific CD4+ TEM with GVHD-mediating capacities.8 Such in vivo-derived alloantigen-specific memory T cells are long-lived and cause GVHD-associated host tissue injury.9 The role of different CD8+ T-cell subsets for GVHD induction is less well characterized. CD8+ TCM are reported to induce GVHD and mediate GVL,10 whereas a mixture of CD4+ and CD8+ TCM does not cause GVHD7 in the same donor/host strain combination. In vitro priming with host-derived dendritic cells induces CD44hiCD8+ memory cells that are unable to induce GVHD in a major histocompatibility complex (MHC)-identical but minor histocompatibility antigen (miHA)-mismatched transplantation model. In contrast, CD44loCD8+ donor T cells also induced by host-derived dendritic cells exhibit a naïve-like phenotype and are potent inducers of GVHD. Although homing and proliferation was indistinguishable between CD44loCD8+ and CD44hiCD8+ T cells, the inability of CD44hiCD8+ T cells to induce GVHD was associated with increased CD95-mediated apoptotic death in vivo.11 Recent reports characterized postmitotic CD8+CD44loCD62Lhi cells induced in a miHA-mismatched transplantation model as ‘host-reactive CD8+ memory stem cells’, which are able to self-renew and have the capacity to generate all populations of memory and effector cells.12 Transplantation of naïve, non-alloantigen-primed T cells into non-syngeneic recipients, however, always induces severe GVHD.5, 7, 10 After transfer, naïve T cells immediately migrate to secondary lymphoid organs, followed by massive expansion and subsequent infiltration into GVHD target organs.6, 13 Allogeneic priming in the host enables transplanted naïve T cells not only to induce GVHD but also to mediate an efficient anti-tumor response. Despite their lack of GVHD-inducing capacity, memory T cells can also eradicate tumor cells, indicating that GVHD induction and tumor elimination are not only dependent on antigen recognition of target cells but might additionally depend on microenvironment, cell numbers and proliferative capacities.4, 10, 11

In two different MHC-mismatched BMT models characterized by their dependence on either CD4+ or CD8+ T cells for lethal GVHD induction, we could show for the first time that in vitro-generated CD8+ cytotoxic T cells (CTLs), which exhibit strong alloantigen cytotoxicity due to repetitive alloantigen stimulation in vitro, do not cause GVHD. However, a significant anti-tumor response towards hematopoietic tumors such as mastocytomas or B-cell-derived leukemias was induced. Thus, alloantigen-specific CD8+ CTLs generated by repetitive antigen stimulation in vitro might present a clinically applicable T-cell population for allogeneic BMT, promoting anti-tumor activity and immune reconstitution with a reduced risk of GVHD induction.

Materials and methods

Mice and BMT

Female C57BL/6 (B6; H-2b), B6D2F1 (H-2bxd) (Harlan–Winkelmann, Borchen, Germany), DBA/2 (H-2d) (Charles River, Kissleg, Germany), B6. C-H2-Kbm1/ByJ (B6.bm1; Kbm1) (breeding pairs from the Jackson Laboratory (Bar Harbor, ME, USA) and bred at the University of Ulm) were used at an age of 6–10 weeks. On day −1, recipient mice were lethally irradiated with 14 Gy split into 2 doses, 3 h apart from a 137Cs source and were reconstituted on day 0 with 5 × 106 TCD BM (T-cell-depleted BM) cells through tail vein injection. Mature T cells from BM were depleted by incubation with hybridoma supernatant 30-H12 (anti-Thy-1.2) and subsequent lysis by Low-TOX-M rabbit complement (Cerdarlane, Ontario, Canada). TCD BM was co-injected either with 2 × 107 total spleen cells, 5 × 106 unstimulated CD8+ T cells isolated from spleens by anti-CD8a microbeads and subsequent passage over LS columns (Miltenyi, Bergisch Gladbach, Germany) or with 5 × 106 alloantigen-specific CTLs. Purity of T-cell populations was 94%. Alloantigen specificity of transplanted CTLs was determined at the day of transplantation in chromium-release assays. Weight loss, decreased activity, skin lesions, fur ruffling and hunched posture were scored on a scale from 0 to 2 in the recipient mice every 2 days, and the GVHD score was determined by summation of the five criteria.14 Animals dying during the experiment remained included in the calculation until the end of the experiment, with their final body weights and GVHD scores at the day of death. Mice were killed when more than 20% of their original weight was lost or if they became moribund, and survival was analyzed using Kaplan–Meier method and Log Rank Statistics. At the day of BMT, 2 × 103 P815 (H-2d) or 125 or 40 GFP (green fluorescent protein)–Bcr–Abl-transformed B-ALL (H-2d)15 cells were injected i.v.

Histopathology

The liver, skin (ear), intestine and spleen were fixed in 4% formalin, paraffin embedded, sectioned and stained with hematoxylin and eosin. Slides were coded and examined by a pathologist who was blinded for the experimental history of the animals. Histopathology of GVHD in colon and liver was graded according to Chen et al.16 Cutaneous GVHD was graded according to keratinocytic vacuolization/loss of cell cohesion, epithelial apoptosis and lymphocytic infiltration with 0 (normal), 1 (mild), 2 (moderate) and 3 (severe).

Generation of alloantigen-specific T cells

Spleen cells from B6 mice were stimulated at 1:1 ratio, with irradiated spleen cells from B6 or B6.bm1 mice in α-minimum essential medium, 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol (basal medium) at 37 °C, 7.5% CO2. After 1 week, cells were re-stimulated weekly at different effector:stimulator ratios in basal medium supplemented with 5% v/v of 0.5 M methyl-α-D-mannopyranoside and 5% ConA supernatant. CD8+ T cells with a purity of 94% were isolated using anti-CD8a microbeads and LS columns (Miltenyi).

Chromium-release assay

Chromium-release assay was described previously.17 Assays were set up in triplicates and were performed at day 5 after T-cell stimulation. Spleen cells were activated with 25 μg/ml lipopolysaccharide (LPS) (Sigma-Aldrich, Munich, Germany) for 3–4 days and used as target cells.

Flow cytometry

In all, 2 × 105 cells/sample were monitored for GFP expression and analysis was done on an LSR II flow cytometer (Becton Dickinson, Heidelberg, Germany).

Western blot analysis

Western blot analysis was described previously.18 The following antibodies were used: Granzyme B, TNF-α (Cell Signaling, Beverly, MA, USA), CD95L (BD Biosciences, Heidelberg, Germany), perforin (Kamiya, Seattle, WA, USA), TRAIL (R&D Systems, Minneapolis, MN, USA), β-actin (Sigma-Aldrich, St Gallen, Schweiz).

Analysis of plasma cytokines

Blood from the tail vein was collected in a 1:2 dilution in 3.2% citrate buffer. After centrifugation, plasma samples were stored at −80 °C, and were analyzed simultaneously by Immumed (Munich, Germany) using Bio-Plex technology.

Results

In vitro-generated cytotoxic alloantigen-specific T cells do not induce GVHD in a CD4+-dependent parent → F1 BMT model

Alloantigen-specific CTLs represent effector T cells that are able to lyse alloantigen-expressing target cells without further activation. H-2d-specific effector T cells were generated by repetitive weekly stimulation of spleen cells from C57BL/6 (B6, H-2b) mice with allogeneic, irradiated spleen cells of DBA/2 (H-2d) mice (Supplementary Figure 1A). Lysis of H-2d-expressing target cells (P815) was induced by isolated CD8+ T cells after the first round of stimulation (1. stim.) and cytotoxicity increased with further rounds of stimulation, whereas H-2b-expressing cells (EL4) were not recognized. Unstimulated CD8+ T cells did not exhibit alloantigen-specific cytotoxicity (Figure 1a). H-2d alloantigens were also recognized on primary LPS blasts of DBA/2 origin (Supplementary Figure 1B). As CD4+ T cells contributed less than 10% to the total T-cell population after the second stimulation, they were not further analyzed. Development of alloantigen-specific cytotoxicity was associated with increased expression of perforin, granzyme B and TNF-related apoptosis-inducing ligand in T cells after four rounds of stimulation, whereas CD95L and TNF-α expression was similar compared with unstimulated CD8+ T cells (Figure 1b). During the course of alloantigen activation, CD8+ CTLs acquired an effector/memory phenotype characterized by CD44+CD122+CCR7CD62L cells. They expressed the activation markers CD25 and CD69, the IL-7R and homing receptors PSGL-1, CXCR3, α4β7 integrin, but lacked CCR9 (Supplementary Figure 2).

Figure 1
figure1

In vitro-generated alloantigen-specific CTLs do not induce GVHD in a CD4+-dependent MHC class I and II-mismatched parent → F1 BMT model. (a) Alloantigen-specific cytotoxicity of CD8+ CTLs from different rounds of stimulation with alloantigen-expressing spleen cells was determined by lysis of P815 (H-2d) and EL4 (H-2b) target cells in a 51Cr-release assay. Values represent the mean of triplicates. (b) Expression of cytotoxic molecules and death ligands in unstimulated B6-derived splenic CD8+ T cells (u) or H-2d-specific CTLs (4. stim.). β-Actin served as a loading control. (cv) Lethally irradiated B6D2F1 recipient mice were reconstituted with TCD BM (BM) from B6 mice without or with B6-derived total spleen cells (SC), B6-derived unstimulated splenic CD8+ T cells (CD8) or H-2d-specific CTLs (4. stim.). GVHD scores and body weights (c) and survival (d) were determined. Surviving animals/total animals treated are indicated in the survival curve in brackets. Expression of IFN-γ and TNF-α was determined in plasma from peripheral blood on days 6 and 13 post transplantation (e). On day 16 and 55 after transplantation, paraffin sections of the liver and colon of animals of each treatment group were analyzed for histological signs of GVHD (fu). Magnification: × 20/0.5 or × 40/0.75 NA objective. Data shown are representative for one animal out of six analyzed per group. Pathological scores were determined from 4 to 5 mice per group (v). Data represent values from three (c, d) or two (e) independent experiments, or are representative for one experiment (a, b) of the three analyzed.

The capacity of allogeneic CTLs to induce GVHD was tested in a parent → F1 BMT model with a full mismatch for MHC class I and II molecules (B6 → B6D2F1). Lethally irradiated B6D2F1 mice were reconstituted with TCD BM alone or together with (1) unseparated, total spleen cells from B6 mice, (2) unstimulated splenic CD8+ T cells of B6 mice or (3) CD8+ H-2d-specific CTLs (4. stim.). Induction of GVHD was determined by survival rates and by a clinical scoring system.14 Owing to the effect of lethal irradiation, a slight increase of GVHD scores and decrease in body weights were observed in all treatment groups between day 3 and 10. While all mice transplanted with TCD BM totally recovered and survived, mice injected with TCD BM and spleen cells developed severe GVHD, with a mortality of 64%, a GVHD score of 5 and a significant body weight reduction. Importantly, allogeneic CTLs did not cause any signs of clinical GVHD, and survival rate was 100% (Figures 1c and d). As lethal GVHD in the B6 → B6D2F1 model is dependent on the presence of CD4+ T cells,19 transplantation of unstimulated CD8+ T cells alone did not result in severe signs of clinical GVHD. Increased plasma levels of TNF-α, and IFN-γ associated with experimental acute GVHD, were detected in mice reconstituted with spleen cells and unstimulated CD8+ T cells, but not in mice injected with CTLs (Figure 1e). At day 16, mice transplanted with TCD BM plus spleen cells showed marked GVHD in gut and the liver. In the large intestine, crypt hyperplasia, goblet cell reduction, mononuclear inflammatory infiltrate of the lamina propria, increased numbers of intraepithelial lymphocytes and enhanced apoptosis of crypt epithelium were observed (Figure 1j). The liver displayed dense inflammation of the portal field, bile duct injury and focal endothelialithis. The lobular parenchyma contained scattered leukocytes and numerous apoptotic hepatocytes (Figure 1k). Mice receiving TCD BM plus unstimulated CD8+ T cells also had GVHD in gut and the liver, albeit at a lesser degree (Figures 1n and o). At day 55, GVHD was still observed in both groups, but considerably less pronounced than at day 16 (Figures 1l, m, p and q). Interestingly, alloantigen-specific CTLs did not induce any signs of histological GVHD (Figures 1r–u) comparable to animals repopulated with TCD BM alone (Figures 1f–i), also depicted in pathological scores (Figure 1v), showing that in vitro-generated CTLs do not induce GVHD despite their alloantigen reactivity.

In vitro-generated alloantigen-specific CTLs do not induce GVHD in a CD8+-dependent single MHC class I-disparate BMT model

Inability of in vitro-generated CTLs to induce GVHD in the B6 → B6D2F1 model might be because of the requirement of CD4+ T cells in the transplant for the induction of severe GVHD. Therefore, we tested the GVHD-inducing capacity of CTLs in a single MHC class I-disparate model (B6 → B6.bm1), where lethal GVHD is dependent on the presence of donor-derived CD8+ T cells.20 CD8+ H-2bm1-specific CTLs were established by repetitive stimulation of B6 spleen cells with allogeneic B6.bm1 spleen cells, following the protocol described in Supplementary Figure 1A. CTLs after four rounds of stimulation lysed LPS-activated spleen cells of B6.bm1 mice, while LPS blasts of B6 mice were not recognized (Figure 2a). They exhibited a phenotype similar to H-2d-specific CTLs (Supplementary Figure 2) and expressed cytotoxic molecules and death ligands (Figure 2b). To test their GVHD-inducing capacity, lethally irradiated B6.bm1 mice were reconstituted with B6-derived TCD BM alone or together with unstimulated splenic CD8+ T cells of B6 mice or CD8+ H-2bm1-specific CTLs (4. stim.). While reconstitution with unstimulated CD8+ T cells mediated severe weight loss and elevated GVHD scores, the transfer of H-2bm1-specific CTLs induced no signs of clinical GVHD comparable to animals transplanted with TCD BM alone (Figure 2c). Out of 15 animals transplanted with CTLs, 14 animals survived until day 60 after transplantation. One animal died at day 10 for undefined reasons, but without any histological signs of GVHD (data not shown). However, 80% of animals reconstituted with unstimulated CD8+ T cells died because of GVHD induction (Figure 2d). They exhibited increased plasma levels of IFN-γ and TNF-α, while no increase was detected in mice reconstituted with CTLs comparable to animals transplanted with TCD BM alone (Figure 2e). The liver of mice reconstituted with unstimulated CD8+ T cells showed leukocytic infiltration of the portal fields, venolar endothelialitis, bile duct lesions and apoptosis of hepatocytes. The skin displayed vacuolar degeneration of the basal epidermis, lymphocytic infiltration at the dermo-epidermal junction and keratinocyte apoptosis (Figures 2j–m). GVHD was less severe in the intestine but increased numbers of intraepithelial lymphocytes, and epithelial cell apoptosis was detected (data not shown). However, mice transplanted with CTLs did not show signs of histological GVHD comparable to animals transplanted with TCD BM alone (Figures 2f–i, n–q and r). Lack of GVHD-mediating capacity of CTLs could not be overcome by injecting a fivefold increased CTL number or CTLs activated only for two rounds of allogeneic stimulation (Supplementary Figures 3 and 4). Taken together, we could show in a CD4+ and CD8+ T-cell-dependent GVHD model that in vitro-established alloantigen-specific CTLs do not induce GVHD.

Figure 2
figure2

In vitro-generated alloantigen-specific CTLs do not induce GVHD in a CD8+-dependent, MHC class I-mismatched B6 → B6.bm1 BMT model. (a) Alloantigen-specific cytotoxicity of B6-derived CD8+ T cells after four rounds of stimulation with B6.bm1 spleen cells was determined on B6- and B6.bm1-derived LPS blasts in a 51Cr-release assay. Values represent the mean of triplicates. (b) Expression of cytotoxic molecules and death ligands in unstimulated B6-derived splenic CD8+ T cells (u) or H-2bm1-specific CTLs (4. stim.). β-Actin served as a loading control. (cr) Lethally irradiated B6.bm1 recipient mice were reconstituted with TCD BM (BM) from B6 mice without or with B6-derived splenic, unstimulated CD8+ T cells (CD8) or alloantigen-specific CTLs (4. stim.). GVHD scores and body weights (c) and survival (d) were determined. Surviving animals/total animals treated are indicated in the survival curve in brackets. Expression of IFN-γ and TNF-α was determined in plasma from peripheral blood on days 6 and 13 post transplantation (e). On day 16 and 61 after transplantation, paraffin sections of the liver and skin from animals of each treatment group were analyzed for histological signs of GVHD (fq). Magnification: × 20/0.5 NA objective. Data shown are representative for one animal out of six analyzed per group. Pathological scores (r) were determined from 5 to 6 mice per group. Data represent mean values from two independent experiments (ce) or are representative for one experiment (a, b) out of three analyzed.

In vitro-generated alloantigen-specific CTLs induce an efficient graft-versus-tumor (GVT) effect

In order to analyze whether in vitro-generated CTLs can induce anti-tumor cytotoxicity, we transplanted lethally irradiated B6D2F1 mice with TCD BM (B6) in the absence or presence of (1) unseparated spleen cells (B6), (2) unstimulated CD8+ T cells (B6) or (3) H-2d-specific CTLs together with P815 (H-2d) mastocytoma cells. P815 cells, which preferentially home to liver and spleen, were detected by histology or by the increase in liver and spleen weights. Out of 10 mice receiving TCD BM and P815 cells, 8 mice died of tumor burden between day 22 and 30 (Figure 3a). No infiltrating tumor cells were found in the liver and spleen of the two surviving mice at the end of the experiment (Supplementary Figures 5C and D). At the day of death, P815-bearing mice exhibited significantly increased liver and spleen weights, (Figure 3b) and histology showed multifocal, extensive tumor infiltrates in both organs (Supplementary Figures 5A and B). All other treatment groups receiving tumor cells in combination with immunocompetent cells showed no increase in liver and spleen weights (Figure 3b), and no tumors by histology (Supplementary Figures 5E–P). Mice reconstituted with TCD BM and spleen cells in the absence or presence of P815 died between day 12 and 43 due to GVHD, indicated by elevated score levels, severe weight loss (Supplementary Figure 5Q) and histopathology of liver (Supplementary Figures 5F and H). Notably, 73 % of mice reconstituted with CTLs survived, were tumor free until the end of the experiment and did not develop clinical or histological GVHD (Supplementary Figures 5M–P and Q). Out of 11 mice transplanted with unstimulated CD8+ T cells and P815 cells, 9 mice survived but histological GVHD was detected (Supplementary Figures 5J and L). Maintenance of alloantigen-specific cytotoxicity of transplanted CTLs was confirmed by lysis of P815 cells by CTLs re-isolated at day 14 from spleens of transplanted mice (Supplementary Figure 6). As BMT is a curative therapy for leukemias, the anti-tumor effect of CTLs was tested on the growth of Bcr–Abl-transformed B-ALL cells established by transformation of BM with MSCV MIG-p185 Bcr–Abl/GFP-expressing retroviral construct.15 The aggressive nature of the tumor induced death of animals between day 15 and 20 when injected with low tumor cell numbers (125 cells per mouse=L125 or 40 cells per mouse=L40). Co-transplantation of CTLs efficiently prevented growth of L40, and six out of seven animals survived tumor free. Only one animal died at day 23 because of leukemia development. Growth of L125 was delayed in mice reconstituted with TCD BM and CTLs, but only one animal stayed tumor free until the end of the experiment (Figure 3c). All mice receiving TCD BM, spleen cells and L40 or L125 died before day 40 because of GVHD induction (Figure 3d), and were free of tumor cells (Supplementary Figure 7). Slightly elevated score levels in mice transplanted with TCD BM plus CTLs and L125 indicated tumor-dependent weight loss and fur ruffling. Tumor cytotoxicity of CTLs towards L40 was confirmed by the absence of GFP+ tumor cells in BM, spleen, lymph nodes and thymus at day 61, whereas mice transplanted with TCD BM and L40 exhibited tumor growth in all organs already at day 13 (Figure 3e). Taken together, our experiments show that in vitro-generated CD8+ alloantigen-specific CTLs exhibit efficient cytotoxicity towards tumors of different origin in the absence of GVHD-inducing capacity.

Figure 3
figure3

In vitro-generated CTLs mediate GVT activity. (a, b) Lethally irradiated B6D2F1 recipient mice were reconstituted with TCD BM (BM) from B6 mice without or with B6-derived total spleen cells (SC), B6-derived unstimulated splenic CD8+ T cells (CD8) or H-2d-specific CTLs (4. stim.) in the absence or presence of P815 cells. Survival (a) was monitored for 55 days. Surviving animals/total animals treated are indicated in brackets. Spleen and liver weights (b) were determined either the day mice were killed because of their moribund state or at the end of the experiment. **P<0.001, n.s.=not significant. (ce) Lethally irradiated B6D2F1 recipient mice were reconstituted with TCD BM (BM) from B6 mice without or with B6-derived total spleen cells (SC) or H-2d-specific CTLs (4. stim.). Leukemia was induced by i.v. injection of 125 (L125) or 40 (L40) Bcr–Abl-transformed B-ALL cells. Survival (c) and GVHD sores (d) were monitored for 61 or 58 days following transplantation. The number of surviving animals/total animals treated is indicated in brackets (c). BM, spleen, lymph nodes and thymus were analyzed for the presence of GFP+ B-ALL cells by flow cytometry in mice reconstituted with TCD BM and co-injected with 40 B-ALL cells (BM+L40) the day they started to become moribund (day 13) and in mice reconstituted with TCD BM and CTLs and co-injected with 40 B-ALL cells (CTL+L40) at the end of the experiment (day 61) (e). Data are representative for one animal out of three analyzed. Data represent values from one (c, d) or two (a, b) independent experiments.

Discussion

One of the major aims in allogeneic BMT is the identification of T-cell populations able to exert a GVL effect without causing GVHD. To our knowledge, we show for the first time in a CD4+ and CD8+ T-cell-dependent MHC-mismatched BMT model that in vitro-generated CTLs do not induce GVHD but mediate an efficient anti-tumor effect, indicating that adoptive transfer of such effector cells might provide a treatment strategy for tumor eradication in the context of allogeneic BMT.

Alloantigen-specific CD8+ CTLs established by repetitive stimulation with the alloantigen in vitro exhibited alloantigen-specific cytotoxicity and increased expression of granzyme B, perforin and TNF-related apoptosis-inducing ligand compared with unstimulated T cells. While TNF-α and perforin are indispensable for anti-tumor activity, CD95L, TNF-α and perforin are responsible for GVHD induction.21, 22, 23 The role of TNF-related apoptosis-inducing ligand for the GVT effect is controversially discussed.4, 24 Despite their allogeneic cytotoxicity and expression of death ligands and cytotoxic molecules, transplantation of CTLs did not cause any signs of GVHD in two different BMT models. They did not induce increased plasma levels of TNF-α, in contrast to mice repopulated with unstimulated T cells, probably indicating insufficient CTL infiltration into the gastrointestinal tract and therefore no release of inflammatory molecules, for example, LPS into the circulation, which subsequently stimulates TNF-α production of monocytes and macrophages. TNF-α and CD95L, however, are known to be important for the manifestation of GVHD, especially in the liver.21, 25 Allogeneic CTLs after the 4. stim. exhibited an effector/memory phenotype. Effector/memory CD4+ T cells were recently described as T cells that are unable to induce GVHD but mediate anti-tumor cytotoxicity.4, 5, 6, 26 These TEM, however, were isolated from spleens of donor mice previously not activated by the recipient alloantigens, and therefore most likely represent memory T cells induced by environmental and endogenous antigens or expanded by homeostatic proliferation, which are deficient in mediating an efficient alloresponse. However, in vivo priming of T cells with the recipient antigen induced alloantigen-specific CD4+TEM, which are responsible for the persistence of GVHD.8, 9 Zhang et al.11 reported that antigenic in vitro priming of T cells with host-derived dendritic cells induced CD8+CD44hi T cells that are unable to mediate GVHD but retain GVL activity in a MHC-identical, miHA-mismatched model, supporting our findings in two MHC-mismatched BMT models. However, postmitotic CD8+CD44loCD62Lhi T cells generated in the initial phase of an allogeneic response are potent GVHD –inducers, as they exhibit a phenotype of ‘memory stem cells’ with self-renewable, long-lived and multipotential capacity.12 Missing GVHD-inducing capacity of TEM might be explained by limited T-cell expansion in vivo in contrast to naïve T cells, which immediately migrate to secondary lymphoid organs, followed by massive proliferation and subsequent infiltration to GVHD target organs.6, 13 Lack of CCR7 and CD62L expression on CTLs prevents migration to secondary lymphoid organs and further antigenic restimulations, although GVHD induction is only prevented if T-cell entry to all secondary lymphoid organs is impaired.27 Expression of α4β7 integrins, PSGL-1 and CXCR3 of CTLs, however, might facilitate entry into GVHD target organs but also into inflamed tissues, such as tumors.28 Homing of CTLs into tumor sites was supported by the finding that the mastocytoma cell line P815 could be totally eradicated by CTLs, whereas the more aggressive B-ALL cells were only eradicated when injected with very low cell numbers. Thus, an anti-tumor effect of allogeneic CTLs in clinical settings might only be achieved if very low numbers of residual tumor cells need to be eliminated. Probably, tumor eradication requires lower threshold numbers of effector cells than GVHD induction. Additionally, tumors themselves might provide enough inflammatory signals to facilitate efficient tissue infiltration of CTLs and may, therefore, be more accessible for CTL-mediated killing than GVHD target organs. Lack of transplanted mature CD4+ T cells might also impair GVHD-inducing capacity of CTLs, although requirement for CD4+ T cell help in CTL organ infiltration is target organ dependent.29, 30 Despite the observation that allogeneic CTLs prevent GVHD and maintain an anti-tumor effect, several in vitro and in vivo trials showed that reducing the number of allogeneic T cells in the transplant decreases GVHD induction.31 We can speculate that repetitive antigen activation might induce effector cells, which are genetically distinct from T cells activated only once with the alloantigen as recently shown for murine effector T cells.32

In summary, we show that repetitive T-cell activation with alloantigen in vitro induces CTLs, which do not mediate GVHD in two different MHC-mismatched BMT models, but maintain anti-tumor cytotoxicity. Testing CTLs in a chimeric human/mouse xenogeneic GVHD model will further elucidate whether this stimulation protocol might be applicable in the clinic.

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Acknowledgements

We thank Maxi Weiswange for excellent technical assistance. This research was supported by the Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS R07/04).

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Correspondence to G Strauss.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Leukemia website

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Hartmann, N., Leithäuser, F., Albers, C. et al. In vitro-established alloantigen-specific CD8+ CTLs mediate graft-versus-tumor activity in the absence of graft-versus-host disease. Leukemia 25, 848–855 (2011) doi:10.1038/leu.2011.16

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Keywords

  • graft-versus-host disease (GVHD)
  • graft-versus-tumor (GVT) effect
  • alloantigen specificity
  • cytotoxic T cells (CTLs)
  • bone marrow transplantation models

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