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. 2026 Apr 2;10(10):3572–3586. doi: 10.1182/bloodadvances.2025017700

BCL2 overexpression in donor lymphocytes reduces the risk of graft-versus-host disease

José Antonio Bejarano-García 1, Melanie Nufer 1, María José Palacios-Barea 1, Beatriz Guijarro-Albaladejo 1, Belén Sierro-Martínez 1, Ángela Bella 1, Cristian Robles-Benítez 1, Estefanía García-Guerrero 1, Inmaculada Carrasco-Brocal 1, Maribel Lara-Chica 1, Luzalba Sanoja-Flores 1, Nicolas Valerdiz Mendendez 2, Teresa Caballero-Velázquez 1,3, Alfonso Rodríguez-Gil 1, José Antonio Pérez-Simón 1,3,∗
PMCID: PMC13196513  PMID: 41915889

Key Points

  • •

    BCL2 overexpression in T cells is related to a lower risk of GVHD.

  • •

    BCL2 inhibitors should be used with caution after allogeneic hematopoietic stem cell transplantation.

Visual Abstract

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Abstract

Graft-versus-host disease (GVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. BCL2 overexpression has been related to a higher risk of GVHD and glucocorticoid resistance. In this study, we aimed to evaluate the role of BCL2 in GVHD pathophysiology. Remarkably, we observed that the pharmacological inhibition of BCL2 increased the risk of GVHD in murine models. In addition, we used a GVHD model with C57BL/6 donor mice that constitutively overexpress human BCL2 gene in hematopoietic cells (Vav-BCL2). Interestingly, the overexpression of BCL2 in donor lymphocytes was related to a lower risk and severity of GVHD. In vitro, the BCL2 inhibitor venetoclax significantly hampered resting T-cell viability, but it did not affect activated lymphocytes, at least in part because of reduced BCL2 interacting mediator of cell death–BCL2 binding after activation. Moreover, BCL2 overexpressing T cells had compromised functionality. Overexpression of BCL2 had no deleterious effect on graft-versus-leukemia activity. Finally, in vitro studies showed that exposure to steroids enriched the populations of T cells displaying higher BCL2 expression levels. Therefore, our study shows that high expression of BCL2 decreases the risk and severity of GVHD.

Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option for many patients with hematologic malignancies. Unfortunately, its results in the long term are hampered by transplant related complications, such as graft-versus-host disease (GVHD), which impairs survival and quality of life.

GVHD occurs because of the cytotoxic effect of alloreactive T cells and B cells from the donor against normal host tissues. The 3 sequential phases that take place during acute GVHD (aGVHD) are (1) the release of proinflammatory cytokines and damage-associated molecular pattern signals because of conditioning before transplantation, (2) host's antigen-presenting cells (APCs) coming into contact with donor's lymphocytes, leading to activation and expansion of alloreactive lymphocytes, and (3) activated lymphocytes migrating to the target tissues, where they can cause tissue damage through the release of proinflammatory cytokines, granzymes, and perforins.1, 2, 3 Similarly, the pathophysiology of chronic GVHD (cGVHD) has also been divided into 3 phases, including (1) an inflammatory phase which causes aGVHD and dysregulation of innate immune response; (2) a dysfunctional adaptive immune response primarily involving T cells and B cells, where donor B cells become activated, differentiate into plasma cells (PC), release allo- and auto-antibodies, and cause tissue damage by activating monocytes and macrophages; and (3) a phase in which activated macrophages release growth factors and induce proliferation of fibroblasts.2,4, 5, 6

Therefore, T-cell and B-cell activation is the cornerstone in the development of GVHD, both in its acute and its chronic forms and, accordingly, the different pathways involved in lymphocyte activation, proliferation, and survival are key players in the development of this complication. In this regard, the B-cell lymphoma 2 (BCL2) family of proteins control the intrinsic mitochondrial pathway of apoptosis, which is necessary to maintain homeostasis and proper functioning of the immune system. The BCL2 family of proteins consists of 3 subgroups, with proapoptotic and antiapoptotic functions. Among them, the proapoptotic proteins called BCL2 Homology 3 (BH3)-only, such as BCL2 interacting mediator of cell death (BIM), bind proapoptotic effectors such as BCL2-Antagonist/Killer (BAK) and BCL2-Associated X (BAK), which dimerize in the mitochondrial membrane and form pores through which cytochrome C is released so that signaling for apoptosis is activated. Finally, antiapoptotic proteins, such as BCL2 bind BIM and prevent it from exerting its antiapoptotic function.7

Although the function of the intrinsic mitochondrial pathway has been widely studied, the role of the BCL2 family proteins in the pathophysiology of GVHD remains incompletely understood. Some studies have suggested an increased BCL2 expression in T lymphocytes from patients who develop GVHD (especially in its severe forms) compared with transplanted patients without GVHD or healthy controls.8 Similarly, another study reported heightened BCL2 expression in circulating or infiltrating lymphocytes in the skin of patients with steroid-refractory GVHD, suggesting the possibility of using BCL2 as a potential target for the treatment of corticosteroid-refractory GVHD.9 However, the possible role of BCL2 in the pathophysiology of GVHD remains elusive.

In this study, we investigated the role of BCL2 in GVHD following allo-HSCT and evaluated the functional implications of BCL2 overexpression and pharmacological blockade in animal models of aGVHD and cGVHD. The results suggest that increased BCL2 expression is a consequence rather than a cause of clinical GVHD and highlight the detrimental effects of BCL2 inhibition in experimental GVHD models, which should be carefully considered in the context of clinical studies.

Methods

In vitro cytotoxicity assay

A total of 1 × 104 A20-Luc cells were seeded in white 96-well plates together with splenocytes in different target-to-effector ratios ranging from 1:0 to 1:20. The final volume per well was 230 μL, including luciferin (LK10000, OZ Biosciences, San Diego, CA) at 0.4 mg/mL. Tumor cell viability was monitored by bioluminescence using the CLARIOstar system (BMG Labtech, Ortenberg, Germany).

Mouse models of GVHD

aGVHD models were made by tail vein injection of 5 × 106 bone marrow (BM) cells and either 5 × 106 total splenocytes or 3 × 106 T cells purified by autoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) using the Pan T-cell Isolation Kit II, mouse (130-095-130, Miltenyi Biotec) from C57BL/6 mice (H2b, donors) into BALB/c mice (H2d, recipients) previously irradiated at doses of 800 cGy (400 cGy + 400 cGy, 3 hours apart). cGVHD models were made by injecting 5 × 106 BM cells and 20 × 106 to 50 × 106 splenocytes from B10D2 (H2d, donors) or F1 B10D2-Vav-BCL2 (H2d H2b, donors) mice into BALB/c (H2d, recipients) irradiated at doses of 1000 cGy (500 cGy + 500 cGy, 3 hours apart). cGVHD score was adapted from Rodríguez-Gil et al,10 including loss of weight (>5%, 0 points; >5%, 0.5; >10%,1; >15, 1.5; >20%; 2; and >25%, 2.5). Syngeneic transplant models were made by injecting 5 × 106 BM cells and 5 × 106 splenocytes from BALB/c mice (H2d, donors) into BALB/c mice (H2d, recipients) irradiated at doses of 1000 cGy (500 cGy + 500 cGy, 3 hours apart). Mice used were aged 8 to 12 weeks old.

Mouse model of GVL effect

The graft-versus-leukemia (GVL) model was made by injecting 1 × 106 A20-Luc (H2d) cells with or without 5 × 106 total BM cells and 5 × 106 total splenocytes from Vav-BCL2 donor mice into BALB/c mice irradiated at doses of 800 cGy (400 cGy + 400 cGy, 3 hours apart). Tumor measurement was determined using an IVIS Lumina III in vivo imaging system (PerkinElmer, Waltham, MA), as previously described.10

Histopathology score

The histopathological scoring was performed using validated methods based on existing literature.11, 12, 13 Briefly, the presence and severity of intestinal GVHD were assessed adapting the Lerner grading system as follows: grade 1, presence of apoptosis in the intestinal epithelium; grade 2, apoptosis with isolated crypt loss; grade 3, apoptosis with the loss of two or more contiguous crypts; and grade 4, mucosal ulceration.

In vivo drug treatment

On the one hand, animals were treated with venetoclax by oral gavage at doses of 50 or 100 mg/kg per day in cycles of 5 days of treatment and 2 days of rest. Venetoclax was reconstituted at 500 mg/mL in dimethyl sulfoxide and dissolved in vehicle (50% Phosal 60, 30% Poly-Ethilene Glycol 400, and 10% ethanol) at 20 mg/mL.

Co-IP and western blotting

For coimmunoprecipitation (co-IP), 25 μL of protein A/G (protein A/G PLUS-Agarose, sc-2003, Santa Cruz) was mixed with the anti-BCL2 antibody (15071S, Cell Signaling, Danvers, MA) for 2 hours at 4°C with rotation. After this, it was mixed with 1500 μg of protein and incubated overnight at 4°C with rotation. The next day the sample was diluted in Laemmli buffer and elution was carried out by thermal shock for 5 minutes at 100°C. Western blot was performed with the Bio-Rad Trans-Blot Turbo system as previously described.14 Antibodies used for western blots were anti-BCL2 (15071S, Cell Signaling), anti-BIM (AB17003, Millipore, Burlington, MA), anti-GAPDH (G9545, Sigma, Saint Louis, MO), peroxidase-conjugated donkey anti-rabbit (711-035-152, Jackson, Ely, UK), and anti-mouse (715-035-150, Jackson). Secondary antibodies were detected using western blotting luminol reagent (sc-2048, Santa Cruz, Dallas, TX).

See the supplemental Methods for T-cell and B-cell activation, cells lines, mouse strains, Peripheral Blod Mononuclear Cells (PBMNCs) isolation, cell culture, murine bone marrow-derived dendritic cells (BMDCs) and splenocytes coculture, flow cytometry assays, metabolism assays, statistical analysis, and data and materials availability.

This study was approved by the ethics committee of the Virgen del Rocío University Hospital and the Regional Ministry of Agriculture, Livestock, and Fisheries of the Andalusian Government (approval code, 05-07-2019-117).

Results

BCL2 inhibition increases the risk and severity of GVHD in murine models

Firstly, we analyzed the effects of venetoclax on the hematopoietic system of healthy C57BL/6 mice. Animals were treated with oral-gavage administration of venetoclax at doses of 50 and 100 mg/kg per day,15 for 28 days. A decrease in white blood cell counts was observed, which was mainly because of a decrease in both T and B lymphocytes. Importantly, hematopoietic stem cells (HSCs) were not affected at the analyzed doses and regulatory T-cell to conventional T-cell ratio (Treg:Tcon) increased (Figure 1A). Red blood cells were not affected, and similar results were observed in spleen (supplemental Figure 1). No deleterious effect was observed in terms of survival (Figure 1B) or weight loss upon treatment with venetoclax (Figure 1C). Therefore, as expected, venetoclax exerted a lympholytic effect without other significant toxicities at the doses tested in healthy animals.

Figure 1.

Figure 1.

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Study of the effect of venetoclax in healthy mice and in murine models of aGVHD. (A) Hematopoietic populations in control C57BL/6 animals after treatment with venetoclax. (Top to bottom) Total white blood cells quantified by hematocytometer; B lymphocytes (CD19+), T lymphocytes (CD3+) and T regulatory lymphocytes to conventional T lymphocytes ratio (Tregs:Tcon) in peripheral blood measured by flow cytometry; HSCs in BM measured by flow cytometry. n = 5 mice per group. (B) Survival and (C) body weight of C57BL/6 control animals after treatment with venetoclax. n = 5 mice per group. (D) Survival and (E) body weight after treatment with venetoclax in murine model of syngeneic bone marrow transplantation (BMT) (BALB/c into BALB/c). n = 10 mice per group from 2 independent experiments. (F) Survival curves after venetoclax used from day +5 as treatment of aGVHD (C57BL/6 into BALB/c). n = 8 mice per group (D-F) was 100 mg/kg per day. The groups were compared with the Student t or Mann-Whitney U tests for unpaired samples and 2-way analysis of variance (ANOVA) depending on the normality of the data calculated by the Shapiro-Wilk and Kolmogorov-Smirnov tests; log-rank test was used for survival curves. The dotted vertical line indicates the day of the start of treatment. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

Taking into account that conditioning regimen favors cytokine release and inflammation, we proceeded to verify whether venetoclax increased its toxicity profile in the context of syngeneic HSCT (BALB/c into BALB/c, strain used as recipients in GVHD models). Again, these animals were not affected in terms of survival (Figure 1D) or loss of body weight (Figure 1E). Once its safety was confirmed in control animals, we proceeded to use venetoclax in murine models of GVHD. In models of aGVHD (C57BL/6 into BALB/c), animals that received venetoclax daily starting on day +5 showed a dramatic increase in mortality (Figure 1F).

A similar approach was performed in a cGVHD model (B10D2 into BALB/c), in which the animals were treated from day +28 after transplant, the day on which signs of GVHD do appear. No significant increase in mortality (supplemental Figure 2A) or cGVHD score was observed in the group treated with venetoclax (supplemental Figure 2B).

In summary, these results show that venetoclax is safe in healthy controls and in the context of syngeneic-HSCT, but unexpectedly increased the incidence of aGVHD in the context of allo-HSCT, whereas in cGVHD, which is driven by distinct pathogenic mechanisms, the impact was minimal.

Venetoclax depletes resting lymphocytes but spares activated lymphocytes, associated with altered BIM–BCL2 binding upon activation

The differential effect of venetoclax on syngeneic and allogeneic transplantation suggested potential dependencies on immune cells. Therefore, we compared the effect of venetoclax in resting or activated T cells. For this purpose, peripheral blood mononuclear cells (PBMCs) from healthy human donors were used. T lymphocytes were activated with anti CD3/CD28 antibodies for 72 hours and treated with venetoclax for 24 hours. Viability assays demonstrated that nonactivated CD4 and CD8 T cells are highly sensitive to venetoclax and become comparatively more resistant upon activation (Figure 2A-B). These results confirm previous studies16 and extend their implications to various stages along T-lymphocyte maturation in a time and dose-dependent manner (supplemental Figure 3). Furthermore, proliferation assays demonstrated that venetoclax does not affect the proliferation of activated T lymphocytes (Figure 2C-D), underscoring the dissimilar effects of venetoclax depending on T-cell activation.

Figure 2.

Figure 2.

Figure 2.

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In vitro study of the effect of venetoclax on activated human lymphocytes and the interaction between BIM and BCL2. (A-B) Flow cytometry study of the effect of venetoclax on the viability of activated T lymphocytes in vitro. Viable cells were determined as annexin-V and 7AAD negative population. Representative cytometry dot plots of the different cases (A) and quantification (B) are shown. n = 6 to 8 healthy donors from at least 3 independent experiments. (C-D) Flow cytometry study showing the effect of venetoclax on the proliferation of CD4 (C) and CD8 (D) T lymphocytes activated in vitro. The proliferation index was calculated with the specific proliferation tool of the FlowJo software. n = 6 to 8 healthy donors from at least 3 independent experiments. (E-F) Study by co-IP and western blot of the difference in the binding of BIM and BCL2 in T lymphocytes before and after activation. Western blot (E) of the inputs (left) and after IP of BCL2 (right) and the quantification of BIM/BCL2 binding relativized to the nonactivation condition (F) are shown. T cells were purified by autoMACS and HL60 and Daudi cell lines were used as positive and negative controls for BCL2 expression, respectively. n = 3 from 3 independent experiments. For these assays (A-F) T lymphocytes were activated for 72 hours with anti-CD3/CD28 and treated with venetoclax (50nM and 500nM) for 24 hours when appropriate. (G-H) Flow cytometry study of the effect of venetoclax on the viability of activated B lymphocytes differentiated into PCs in vitro. B lymphocytes were activated from PBMCs with CpG for 7 days and treated with venetoclax (50nM and 500nM) for 24 hours. Representative flow cytometry dot plots of the different cases (G) and quantification (H) are shown. n = 5 healthy donors from at least 3 independent experiments. Groups were compared with Student t or Mann-Whitney U tests for paired samples depending on the normality of the data calculated by the Shapiro-Wilk and Kolmogorov-Smirnov tests. Asterisks show statistically significant differences. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

The most widely accepted explanation for resistance to BH3 mimetics is the balance in the relative expression of proapoptotic and antiapoptotic proteins of the BCL2 family.16, 17, 18 Therefore, we investigated the BIM/BCL2 axis in activated and nonactivated T lymphocytes. For this purpose, we immunoprecipitated BCL2 in purified T lymphocytes before and after activation and measured its association with BIM. Although BCL2 was still present after activation by T-cell receptor (Figure 2E), BIM–BCL2 binding was reduced by almost 50% upon activation (Figure 2E-F), suggesting that the BIM/BCL2 balance changes upon T-cell activation.

We expanded these studies to B cells, which are key drivers of cGVHD pathogenesis.2,4 For this purpose, PBMCs from healthy donors were differentiated into PCs using CpG for 7 days19 and were treated with venetoclax for 24 hours. We observed that the number of PCs (identified as a CD19low/+ CD27+ CD38+ population) was not affected after treatment (Figure 2G-H), indicating that, once activated, they become insensitive to venetoclax, adding further evidence to previous studies.16,17

BCL2-overexpressing T cells exhibit compromised functionality

To explain the underlying mechanisms involved in BCL2 overexpression in T-cell biology, we performed ex vivo assays to measure key mechanisms related to T-lymphocyte function using C57BL/6-background mice that constitutively overexpress the human BCL2 gene in hematopoietic cells (Vav-BCL2).20 Taking into account that reactive oxygen species (ROS) can act as second messengers that influence T-lymphocyte function21 and considering that BCL2 acts as a regulator of ROS at the mitochondrial level,22 we decided to determine the levels of ROS by flow cytometry (supplemental Figure 4A-B). The results showed that CD4 Vav-BCL2 T cells, both resting and activated, exhibited decreased levels of ROS compared with wild type (WT) (Figure 3A), whereas no significant differences were detected for CD8 T cells (supplemental Figure 4C). In addition, we addressed the metabolic activity of both WT vs Vav-BCL2 cells. We found that BCL2 promotes a highly efficient mitochondrial profile characterized by a significantly increased coupling efficiency (Figure 3B) and a reduced proton leak (Figure 3C) compared with WT cells. These parameters indicate that the electron transport chain is more tightly coupled to ATP synthesis, minimizing the superoxide formation.21,23,24 Although the activation of T cells drastically increased the overall energy demand, Vav-BCL2 cells fulfill this requirement by significantly boosting their mitochondrial ATP production (mitoATP; Figure 3C) without the proportional increase in oxidative stress (ROS) that usually accompanies high metabolic rates in WT cells. This confirms that BCL2-driven metabolic reprogramming optimizes energy output while maintaining a superior redox balance.

Figure 3.

Figure 3.

Figure 3.

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In vitro studies of the functionality of BCL2-overexpressing T cells. (A) Study of ROS levels in WT and Vav-BCL2 CD4 T lymphocytes by flow cytometry. Freshly obtained splenocytes without activation (resting) or activated with anti CD3/28 for 48 hours were used. Activation was detected by the CD25+ marker. N = 3 mice. (B-D) Metabolic analysis of WT and Vav-BCL2 resting freshly isolated T cells from spleen (top row) and after 48 hours of activation with Concanavalin A (bottom row). Quantification of the coupling efficiency (B), proton leak (C) and total ATP production rate (D), showing the contribution of mitochondrial (mitoATP) and glycolytic (glycoATP) pathways. N = 3 mice. (E-F) Flow cytometry analysis of tumor necrosis factor-α production after 24 hours of activation with concanavalin A in CD4 (E) and CD8 (F) T lymphocytes from WT or Vav-BCL2 mice. For both cases, the quantification (left) and a representative dot plot for each genotype (right) are shown. n = 6 mice from 2 independent experiments. (G-H) Flow cytometry analysis of the proliferation of CD4 (G) and CD8 (H) T lymphocytes from WT or Vav-BCL2 mice activated in vitro with Concanavalin A for 48 hours. The quantification (left) and a representative proliferation histogram for each genotype (right) are shown. Cell proliferation was assessed using Tag-it Violet staining prior to activation. n = 3 mice. (I-J) Flow cytometry analysis of activation and proliferation of CD4 (I) and CD8 (J) T lymphocytes from WT or Vav-BCL2 mice (C57BL/6), activated in vitro for 3 days with BM-derived allogeneic DCs from BALB/c mice. The quantification of the percentage of activated (CD25+) dividing T cells (left) and a representative proliferation histogram for each genotype (right) are shown. Cell proliferation was assessed using Tag-it Violet staining prior to activation. n = 7 mice from 3 independent experiments. The groups were compared with the 2-way ANOVA and Student t or Mann-Whitney U tests for unpaired samples depending on the normality of the data calculated by the Shapiro-Wilk and Kolmogorov-Smirnov tests. Asterisks show statistically significant differences. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

Next, we investigated the effect of BCL2 overexpression on the production of proinflammatory cytokines and T-cell proliferation following direct activation by the T-cell receptors, using concanavalin A. Compared with WT lymphocytes, CD4+ and CD8+ lymphocytes overexpressing BCL2 exhibited reduced tumor necrosis factor-α production (Figure 3E-F) and slower proliferation (Figure 3G-H). Furthermore, to mimic the physiological activation that occurs in the context of GVHD, WT and Vav-BCL2 lymphocytes were cocultured with allogeneic dendritic cells differentiated from BM cells of BALB/c mice (supplemental Figure 5). Similarly to what was observed with concanavalin A, Vav-BCL2 exhibited a reduced proportion of activated-divided T cells in both CD4 (Figure 3I) and (Figure 3J) its subsets.

Taken together, these results demonstrate how BCL2 overexpression impairs different aspects of T lymphocyte functionality.

Overexpression of BCL2 in donor lymphocytes reduces GVHD in experimental models

Next, we evaluated the functional consequences of increased BCL2 expression in lymphocytes during GVHD pathogenesis. An aGVHD model was established using BALB/c mice as recipients of donor cells from Vav-BCL2 mice. All animals received 5 × 106 WT BM cells and 5 × 106 splenocytes. Then, 3 experimental groups were evaluated, where WT and Vav-BCL2 splenocytes were mixed in different proportions (Figure 4A). Percentages were adjusted by cell counting and flow cytometry (supplemental Figure 6A). Notably, donor lymphocytes overexpressing BCL2 dose-dependently improved the survival of GVHD mice (Figure 4B; supplemental Figure 6B). To further demonstrate the direct role of T cells in these results and to rule out the effect of any other population (such as APCs), similar experiments were conducted by infusing 1 × 106 isolated T cells (CD3+), with WT BM cells. Animals receiving isolated Vav-BCL2 lymphocytes exhibited prolonged survival compared with recipients of WT lymphocytes (Figure 4C). Moreover, animals that received Vav-BCL2 splenocytes exhibited lower histopathological scores in the gut, which is the main GVHD target tissue (Figure 4D). Together, these results show an overall protective role of BCL2 in GVHD. Finally, we employed an in vivo competitive aGVHD model specifically to compare the proliferative capacity of WT with Vav-BCL2 T cells by coinfusing both populations at a 1:1 ratio (supplemental Figure 6C). This experimental approach avoids the potential biases of in vitro assays that arise from time-dependent differences in cell viability, as BCL2 overexpression confers a survival advantage that can confound long-term culture readouts25 (supplemental Figure 7). Using this in vivo setting, we observed that WT T cells consistently outcompeted Vav-BCL2 T cells in peripheral blood expansion, thereby confirming the in vitro observations in a physiologically relevant context (Figure 4E; supplemental Figure 7).

Figure 4.

Figure 4.

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Study of the effect of BCL2 expression on the incidence of aGVHD. (A-B) aGVHD model using total splenocytes. Scheme of the experimental design (A): BALB/c recipient animals were transplanted with 5 × 106 WT BM cells and a pool of 5 × 106 WT and Vav-BCL2 splenocytes mixed in different proportions from C57BL/6 donors. Monitoring of the survival of the animals (B) is shown. Showed n from at least 3 independent experiments. (C) Survival curve of the murine model of aGVHD using isolated lymphocytes. BALB/c recipient animals were transplanted with 5 × 106 WT BM cells and 3 × 106 WT or Vav-BCL2 CD3+ cells purified by autoMACS. (D) Histopathological score of the small intestine (left) and large intestine (right) in murine models of aGVHD (C57BL/6 into BALB/c) derived from model using total splenocytes from WT or Vav-BCL2 donors on day 6 after transplantation. n = 10 from 2 independent experiments. (E) In vivo competitive expansion study of T lymphocytes (CD3+) from WT or Vav-BCL2 mice in peripheral blood using a murine model of aGVHD. Recipient animals (BALB/c) were transplanted with 5 ×106 WT CD45.2 BM cells and 5 × 106 splenocytes composed of a 50% WT-CD45.1 T-cell and 50% Vav-BCL2-CD45.2 T-cell mixture. n = 9 to 10 from 2 independent experiments. (F) Percentage of donor-derived (H2Kb+) CD8 T cells in mesenteric LNs at day +6 after transplantation. n = 5 to 6 mice per group. (G) Donor-derived (H2Kb+) CD4/CD8 T-cell ratio in mesenteric LNs at day +6 after transplantation. n = 5 to 6 mice per group. (H-I) Naïve and memory T-cell composition of donor cells, assessed in splenocytes at baseline and in mesenteric LNs at day +6 after transplantation in the aGVHD model, for CD4 (H) and CD8 (I) T cells. Analysis was performed using standard CD44 CD62L identification. n = 5 to 6 mice per group from 2 independent experiments. The groups were compared with Student t test or Mann-Whitney U and 1-way ANOVA depending on the normality of the data calculated by the Shapiro-Wilk and Kolmogorov-Smirnov tests; the log-rank test was used for survival curves. Asterisks show statistically significant differences. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

In the aGVHD model, total donor cell numbers did not differ between WT and Vav-BCL2 groups in mesenteric lymph nodes (LNs), spleen, or peripheral blood at day +6, corresponding to the peak of disease (supplemental Figure 8A-E). However, we observed a reduced representation of CD8 Vav-BCL2 T cells (Figure 3F) and a lower CD8:CD4 (Figure 3G) ratio specifically in LNs, the primary site of early donor T-cell migration and activation following transplantation. This difference was not detected in other tissues (supplemental Figure 8A-C,F). In addition, Vav-BCL2 donor mice exhibited a reduced pool of naïve CD4 and CD8 T cells at baseline, whereas terminal differentiation into memory subsets during aGVHD was preserved (Figure 3H-I). Consistently, expression of the activation markers CD25 and CD69 was comparable between WT and Vav-BCL2 T cells in GVHD (supplemental Figure 8A-C,F). Together, these in vivo readouts indicate that the attenuated GVHD phenotype is not driven by impaired T-cell activation or defective differentiation, but can be partially explained by a reduced early expansion of Vav-BCL2 T cells, as confirmed in the competitive in vivo assay, reflected by the decreased CD8 representation in mesenteric LNs and the smaller naïve T-cell pool in Vav-BCL2 donors.

Considering that venetoclax has pleiotropic effects beyond BCL2 inhibition,26 we next aimed to investigate whether the deleterious effect of venetoclax on aGVHD model was specifically because of BCL2 inhibition or to a different mechanism. For this purpose, we administered venetoclax from day −1 to +3 as, but using also Vav-BCL2 mice as donors. The favorable impact of infusing Vav-BCL2 lymphocytes was abolished upon venetoclax treatment; conversely, the deleterious effect of venetoclax on survival was partially compensated by infusing Vav-BCL2 lymphocytes (supplemental Figure 9). Overall, these results suggest that the deleterious effect of venetoclax on GVHD incidence and severity is BCL2 dependent.

Since the differential sensitivity of lymphocytes to BCL2 inhibitor did not only affect T cells but also B cells (which play a key role in cGVHD), we investigated the consequences of increased BCL2 expression using a cGVHD model carrying donor cells from Vav-BCL2 mice. For this purpose, F1 animals from the crossing of the C57BL/6Vav-BCL2 strain and the B10D2 strain were used as donors of BALB/c recipients (supplemental Figure 10A). In this way, donor cells (H2d/H2b) do not recognize recipient T cells (H2d) as MHC mismatched, but they differ in minor histocompatibility antigens, and donor animals overexpress BCL2 in T and B cells (supplemental Figure 10B). No impact in cGVHD severity was observed between animals receiving Vav-BCL2 cells as compared with WT (supplemental Figure 10C), suggesting a differential impact of BCL2 expression in aGVHD vs cGVHD.

BCL2 overexpressing T cells maintain their GVL potential

Once the reduced risk of GVHD associated with BCL2 overexpression in donor lymphocytes was confirmed in murine models, we sought to determine whether BCL2 overexpression affected the antitumor capacity of the lymphocytes and whether the GVL effect might be compromised.

To address this question, we employed a murine GVL model in which recipient animals were transplanted as previously described27 with A20-Luc cells with or without WT BM cells, accompanied or not with WT or Vav-BCL2 splenocytes. The results demonstrated that splenocytes overexpressing BCL2 were capable of controlling tumor growth, albeit slightly less effectively than WT splenocytes. However, this difference did not result in impaired survival as compared with those receiving WT splenocytes (Figure 5A-C).

Figure 5.

Figure 5.

Figure 5.

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Study of the effect of BCL2 overexpression on the antitumor capacity of mouse T lymphocytes. (A-C) GVL model: recipient animals (BALB/c) were transplanted with 1 × 106 A20-Luc cells (BALB/c), with or without 5 × 106 WT BM cells and 5 × 106 splenocytes from WT or Vav-BCL2 mice (C57BL/6). Tumor growth in vivo was monitored by bioluminescence using the IVIS-Lumina system. Representative images of tumor growth (A), quantification of tumor burden (photons/second) (B), and survival of recipient animals (C) are shown. n = 10 mice per group from 2 independent experiments. (D) In vitro cytotoxicity assay: preactivated WT and Vav-BCL2 T lymphocytes were cocultured with A20-Luc tumor cells in different target-to-effector cell ratio. Tumor cells growth measured by bioluminescence using CLARIOstar system is shown. n = 6 from 3 independent experiments. (E) Study of the cytotoxicity marker CD107a: freshly obtained splenocytes were cocultured with A20-Luc cells for 24 hours and the expression of the CD107a marker in CD4 (top) and CD8 (bottom) was measured by flow cytometry. n = 3 mice per genotype. The groups were compared with the 2-way ANOVA and Student t or Mann-Whitney U tests for unpaired samples depending on the normality of the data calculated by the Shapiro-Wilk and Kolmogorov-Smirnov tests; the log-rank test was used for survival curves. Asterisks show statistically significant differences. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

Similar findings were observed in vitro, where Vav-BCL2 lymphocytes exhibited slightly reduced antitumor activity (Figure 5D). This was correlated with lower expression of the cytotoxicity marker CD107a,28,29 as assessed by flow cytometry (Figure 5E).

GCs enrich the pool of BCL2 overexpressing T cells

Glucocorticoids (GCs) are the gold standard first line treatment for GVHD. It has been described that overexpression of BCL2 confers resistance to GC(26) and, as previously mentioned, a higher expression of BCL2 in T lymphocytes has been related to a higher incidence of GVHD8,9 and GC refractoriness.9 This is in contrast to our data, which indicates that BCL2 overexpression correlates with a lower risk of GVHD and a lower cytotoxic capacity of T cells. Therefore, we considered studying the role that BCL2 overexpression plays in the treatment of GVHD with GC.

Although it has been previously demonstrated that BCL2 overexpression confers resistance to GC in resting T cells,25 we extended upon this study by activating WT and Vav-BCL2 T lymphocytes and exposing them to methyl-prednisolone in vitro. Our findings confirmed that this resistance also occurs following T-cell activation (Figure 6A-B). This suggests that BCL2 may play a role in the resistance of activated human T cells to corticosteroids.

Figure 6.

Figure 6.

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Study of the effect of GC treatment on the expression of BCL2 of T lymphocytes. (A-B) In vitro study of the effect of methylprednisolone on the viability of activated CD4+ (A) and CD8+ (B) T lymphocytes from WT or Vav-BCL2 mice. Total splenocytes from WT and Vav-BCL2 mice were activated in vitro with anti-CD3/CD28 for 48 hours and subsequently treated with methylprednisolone (1mM) for 72 hours. n = 6 to 8 mice from at least 3 independent experiments. (C-D) Effect of the treatment of methyl-prednisolone on the viability of activated human CD4 (C) and CD8 (D) T lymphocytes from healthy donor PBMCs. Cells were activated with anti CD3/CD28 for 72 hours, treated with increasing doses of methylprednisolone (from 1μM to 1mM) for another 72 hours and viability was analyzed by flow cytometry with annexin V and 7AAD. n = 18 to 26 healthy donors from >3 independent experiments. (E-F) Quantification of the median fluorescence intensity (MFI) of BCL2 by flow cytometry in activated CD4 (E) and CD8 (F) human T cells after exposure to methyl-prednisolone for 72 hours. BCL2 MFI relative to untreated cells is shown. n = 4 from 2 independent experiments. The groups were compared with the 2-way ANOVA and Student t or Mann-Whitney U tests for unpaired samples depending on the normality of the data calculated by the Shapiro-Wilk and Kolmogorov-Smirnov tests. Asterisks show statistically significant differences. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

One of the mechanisms involved in GCs-induced apoptosis in T cells is related to the increased transcription of BIM.30 We confirmed this finding in our model. Activated T lymphocytes from healthy donors treated in vitro with methyl-prednisolone increased BIM levels in a dose-dependent manner (supplemental Figure 11). Next, we observed that GCs decrease lymphocytes viability in a dose-dependent manner, both in CD4 (Figure 6C) and CD8 (Figure 6D) subsets. Finally, BCL2 expression was measured by flow cytometry in activated GC-resistant T cells. BCL2 expression increased after treatment with GC in viable CD4 (Figure 6E) and CD8 (Figure 6F) T lymphocytes in a dose-dependent manner. Therefore, GCs enrich the pool of T cells with a greater expression of BCL2.

Discussion

BCL2 is an antiapoptotic protein belonging to the BCL2 family protein, which governs the intrinsic mitochondrial pathway of apoptosis,7,31 critical for maintaining immune system homeostasis.25 The reliance of immune cells on BCL2 for survival fluctuates based on their maturation and activation status.16,17,32,33 Our findings using venetoclax (ABT-199), a BCL2-specific inhibitor endorsed for the treatment of several hematological malignancies,18 alongside previous studies,15,16,34 indicate that resting T and B lymphocytes exhibit a high dependency on BCL2, which drastically diminishes following activation. The currently prevailing explanation for resistance to BH3 mimetics revolves around the relative expression of BCL2 family proteins.17,18 In this regard, our analysis reveals that not only the expression but the interaction between these proteins can dynamically change depending on the activation state of the lymphocytes. In this regard, using co-IP assays of BIM and BCL2, we demonstrated that despite activated lymphocytes not relying on BCL2 for their survival, BCL2 continues to be expressed, albeit with a 50% reduction in its binding to BIM. Therefore, lymphocytes become comparatively resistant to BCL2 inhibition upon activation because of the altered BIM–BCL2 interaction balance.

Although some studies have documented an upregulation in the expression of BCL2 among patients who develop GVHD8,9 and some of them even suggest a correlation between BCL2 expression and the severity of the disease,8 there is scanty information and a lack of preclinical models analyzing the direct role of BCL2 in the pathophysiology of GVHD.

In this study, we used the C57BL/6-VavP-BCL2-Tg (Vav-BCL2) mice strain as donors of mismatched recipients as a preclinical model of GVHD. Vav-BCL2 strain features the overexpression of the human BCL2 gene in all hematopoietic cells.20 Our findings in aGVHD models indicate that recipients receiving a higher proportion of lymphocytes overexpressing BCL2 exhibit enhanced survival in a dose-dependent manner and a reduced histopathological score. This difference is attributable to a lower risk and severity of aGVHD. Our data in the aGVHD model can be partially explained by a reduced early expansion of Vav-BCL2 T cells, as demonstrated in the competitive in vivo assay, and are further supported by the reduced representation of CD8 T cells in LNs, together with a smaller naïve T-cell pool in Vav-BCL2 donors, indicating that T-cell composition is selectively altered. Our in vitro results indicate that T lymphocytes overexpressing BCL2 show delayed proliferation and reduced proinflammatory cytokine production after activation, and diminished expression of the cytotoxicity marker CD107a as compared with WT lymphocytes. This aligns with previous studies demonstrating that BCL2 regulates calcium flux and inhibits nuclear factor of activated T cells translocation to the nucleus,35 a process that is critical for T-cell activation. Our findings also suggest a potential role of metabolism and ROS in these outcomes. ROS, acting as second messengers, are known to influence lymphocyte functionality21 by modulating the redox balance.22 Supporting that T lymphocytes overexpressing BCL2 displayed increased coupling efficiency, reduced proton leak, altered mitochondrial ATP production, and reduced ROS levels, consistent with the BCL2 role in the regulation of redox homeostasis. These findings highlight the interplay between metabolism, ROS and BCL2 in shaping lymphocyte responses.21,24,36

Despite these findings, T cells overexpressing BCL2 retained their GVL potential in vivo and overexpression on BCL2 had no impact in survival of GVL models. On the other hand, regarding cGVHD models, neither BCL2 overexpression nor BCL2 inhibition with venetoclax had a significant impact on disease development, suggesting a differential role of BCL2 in aGVHD vs cGVHD that warrants further investigation in the future.

Next, we explored the use of venetoclax after allo-HSCT and evaluated its effect on GVHD. Our findings confirm the safety of venetoclax in both healthy controls and in mice undergoing syngeneic HSCT. However, in animals undergoing allo-HSCT, BCL2 inhibition was associated with an increased mortality. These results in the allogeneic setting could extend beyond the direct inhibition of BCL2, as venetoclax is known to have additional effects on cellular metabolism,26 enhance T-cell cytotoxicity by increasing ROS levels,37 induce the direct activation of APCs38 and augments immune effector function.39 This may be because of the inhibition of BCL2 ability to suppress activation through the nuclear factor of activated T-cell pathway.35 Therefore, there is an emerging body of evidence suggesting that venetoclax can exacerbate immune responses, which aligns with the results observed exclusively in the allogeneic context. This is consistent with our findings using venetoclax in aGVHD models. In animals that received splenocytes overexpressing BCL2, which exhibited a dampened immune response and reduced aGVHD severity, the adverse impact of venetoclax on survival was significantly mitigated compared with mice that received WT splenocytes. Overall, these findings indicate that the detrimental effect of venetoclax on GVHD incidence and severity is largely dependent on its effect on immune response through BCL2.

Our preclinical results challenge the efficacy of pharmacological BCL2 inhibition as a therapeutic strategy for GVHD and question the assumed direct correlation between BCL2 expression and GVHD risk or severity. Although some studies highlight the safety of venetoclax in preventing relapse in high-risk patients with AML undergoing allo-HSCT without increasing GVHD risk,40, 41, 42, 43 these findings are based on its combination with 5-azacitidine. However, regarding the ability of 5-azacitidine to enhance regulatory T-cell populations44 and its potential benefits in managing GVHD,45 it may counterbalance the increased risk of GVHD (re)activation associated with venetoclax. Consequently, venetoclax monotherapy should be used with caution in the immediate after transplant setting. Finally, the literature has highlighted that BCL2 overexpression imparts resistance to corticosteroids,25 and patients refractory to corticosteroids exhibit heightened BCL2 expression in lymphocytes.9 Corticosteroids exert a direct influence on BCL2 family proteins, including the elevation of levels of the proapoptotic BCL2 pathway member BIM.31 Consequently, we sought to explore the interplay between corticosteroids and BCL2 expression in activated T lymphocytes. Our in vitro data demonstrate an enrichment in cells with more BCL2 expression following GC exposure. These observations are correlative and may reflect the preferential survival of BCL2-expressing cells under steroid treatment. Further studies will be required to better understand the relevance of this finding in clinical settings. Supporting this concept, recent studies suggest that corticosteroid refractoriness is mediated by a T-cell–independent mechanism.46,47 Then, BCL2 overexpression in GVHD patients seems to correlate with corticoid treatment, but our genetic model clearly demonstrates that BCL2 overexpression is not the direct cause of higher severity. Corticoid effects are pleiotropic, and other unexplored mechanisms may explain the higher reactivity of corticoid resistant lymphocytes in these patients.

In summary, these results raise questions about the appropriate consideration of BCL2 as a potential target in GVHD and show that BCL2 overexpression in T cells is related to a lower risk of GVHD after allo-HSCT.

Limitations of the study

This study has several limitations that warrant further investigation. First, the conditioning regimens and immune disparities largely differ from the clinical setting, where venetoclax has been administered to individuals at high risk of relapse. Together, these factors complicate direct comparison with our preclinical findings and highlight the need for further studies to better define the impact of venetoclax on GVHD development. Second, our preclinical experiments relied on a genetic model of human BCL2 overexpression in mice. Although this system provides a useful tool to explore the biological consequences of increased BCL2 activity, it remains uncertain whether the levels of BCL2 expression achieved in this model accurately reflect those that can occur under physiological conditions. Finally, the cGVHD model used in this study exhibited considerable variability, which limits the strength of the conclusions, therefore additional studies would be necessary to validate and extend these findings.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for J.A.B.-G. is Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, United Kingdom.

Acknowledgments

The authors thank Simon Mendez-Ferrer for reviewing the manuscript and providing valuable advice, and Andreas Strasser for providing the Vav-hBCL2-Tg mouse model.

This work was supported by the Spanish Foundation of Hematology and Hemotherapy and AbbVie Pharmaceuticals.

Authorship

Contribution: J.A.B.-G., A.R.-G., and J.A.P.-S. conceptualized the study; J.A.B.-G., M.N., M.J.P.-B., B.G.-A., B.S.-M., A.B.-C., C.R.-B., I.C.-B., M.L.-C., L.S.-F., N.V.M., E.G.-G., T.C.-V., A.R.-G., and J.A.P.-S. formulated the methodology; J.A.B.-G., B.G.-A., A.R.-G., T.C.-V., and J.A.P.-S. conducted investigation; J.A.B.-G. and J.A.P.-S. acquired funding; J.A.P.-S. was responsible for project administration; A.R.-G., T.C.-V., and J.A.P.-S. supervised the study; J.A.B.-G. wrote the original draft; and J.A.B.-G., A.R.-G., and J.A.P.-S. wrote, reviewed, and edited the manuscript.

Footnotes

This study does not include data deposited in public databases. Access to any additional data or methodologies not included in the manuscript is available from the corresponding author, José Antonio Pérez-Simón (josea.perez.simon.sspa@juntadeandalucia.es).

The full-text version of this article contains a data supplement.

Supplementary Material

Supplemental Figures, Methods and Table
BLOODA_ADV-2025-017700-mmc1.pdf (1.2MB, pdf)

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Associated Data

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Supplementary Materials

Supplemental Figures, Methods and Table
BLOODA_ADV-2025-017700-mmc1.pdf (1.2MB, pdf)

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