Upregulation of CD1d and ULBP3 on B cells from healthy donors and chronic lymphocytic leukaemia patients does not prime them for killing by γδ T cells

Julie David; Amy Walsh; Ke Sin Seow; Ellen Walsh; Stefan Elekes; Rohit Upadhyay; Nawal A B Taher; Samiha Al Siyabi; Ashanty M Melo; Carmel Waldron; Elisabeth Vandenberghe; Anthony M McElligott; Derek G Doherty

doi:10.1371/journal.pone.0346684

. 2026 Apr 20;21(4):e0346684. doi: 10.1371/journal.pone.0346684

Upregulation of CD1d and ULBP3 on B cells from healthy donors and chronic lymphocytic leukaemia patients does not prime them for killing by γδ T cells

Julie David ^1,², Amy Walsh ¹, Ke Sin Seow ¹, Ellen Walsh ^1,³, Stefan Elekes ¹, Rohit Upadhyay ¹, Nawal A B Taher ^1,^¤, Samiha Al Siyabi ¹, Ashanty M Melo ¹, Carmel Waldron ², Elisabeth Vandenberghe ², Anthony M McElligott ², Derek G Doherty ^1,^*

Editor: Xiaosheng Tan⁴

PMCID: PMC13095099 PMID: 42008532

Abstract

Despite advances in treatment, chronic lymphocytic leukaemia (CLL) remains an incurable disease. Vδ1⁺ γδ T cells are reported to expand in CLL patients and to kill leukemic cells, placing them as candidates for immunotherapy. However, their cytotoxic efficacy was limited and required specific stimulatory conditions. Since some γδ T cells recognise lipids presented by CD1d, we examined if inducing CD1d expression and presentation of CD1d-restricted lipids could promote Vδ1, Vδ2 or Vδ3 T cell killing of B cells from CLL patients and healthy donors. Lines of γδ T cells containing Vδ1, Vδ2 and Vδ3 T cells and enriched cultures of CD19⁺ B cells were generated from peripheral blood using magnetic bead separation. γδ T cell subset frequencies and CD1d and ULBP3 expression levels on B cells were determined using flow cytometry. CD1d expression was induced on B cells by treatment with all trans retinoic acid (ATRA) and its analogue AM580. ATRA-treated B cells were co-cultured with γδ T cell lines in the absence or presence of lipids and cytotoxicity was assessed by measuring CD107a externalisation or propidium iodide staining using flow cytometry. Vδ1 and Vδ3 T cell frequencies were significantly higher in CLL patients compared to age-matched healthy donors. CD1d and ULBP3 expression was lower on CLL cells compared to healthy B cells but was restored by treatment with ATRA or AM580. Although co-culturing CLL cells with γδ T cells led to decreased cell viability, CD1d and ULBP3 upregulation by healthy and CLL B cells did not elicit cytolytic degranulation or cytokine production by Vδ1, Vδ2 or Vδ3 T cells, even after presentation of CD1d-restricted lipid antigens. This study suggests that human Vδ1 T cell cytotoxicity against CLL B cells may be disease stage-dependent and requires B cell priming and selective activation of specific γδ T cell subsets.

Introduction

Chronic lymphocytic leukaemia (CLL) is a B cell malignancy characterized by the expansion of a mature population of CD23⁺ CD5⁺ B cells [1]. The five-year survival rate is highly variable ranging from 20% to 90%, depending on genetic factors and disease risk status [2]. CLL has an incidence rate of approximately 4.7 per 100,000 people, making it the most common haematological malignancy in the USA and Europe, with Ireland having one of the highest incidence rates [3,4]. Although treatment for CLL has evolved significantly in recent years, with chemotherapy being replaced by novel targeted agents including the Bruton-tyrosine kinase inhibitor ibrutinib and the B-cell lymphoma-2 antagonist venetoclax [5–8], CLL remains largely incurable [9]. The highly heterogenous nature of the disease, the development of drug-resistant subclones, and the toxicity of current therapeutic regimes illuminates the need for immune-based therapies.

γδ T cells show promise as cellular therapies for CLL. γδ T cells are innate T cells that recognise non-peptide antigens without the need for major histocompatibility complex (MHC) presentation, and they contribute to immunity against tumours by direct killing and rapidly releasing cytokines that promote the activation of other anti-tumour effector cells [10,11]. γδ T cells comprise 1–5% of the entire T cell population in human blood and consist of 3 major subsets – Vδ1, Vδ2 and Vδ3 – based on their T cell receptor (TCR) δ-chain usage. Vδ2 T cells are the predominant subset in blood, while Vδ1 and Vδ3 T cells predominate in tissues, such as the gut, liver and adipose tissue [10–14]. The TCR of Vδ2 T cells recognises pyrophosphate intermediates presented by MHC class I-like antigen-presenting molecules, known as butyrophilins [14–15], whereas the Vδ1 and Vδ3 TCRs recognise a number of stress-inducible molecules expressed by virus-infected and tumour cells and lipid antigens presented by CD1c and CD1d molecules [16–19].

In addition to the TCR, all γδ T cell subsets express the activating receptor NKG2D, which can directly bind the stress-inducible molecules MIC-A and MIC-B and UL16-binding proteins (ULPBs), commonly upregulated on tumour cells [20–22]. Many γδ T cells also express natural cytotoxicity receptors (NCRs) which recognise ligands on tumour cells [12,14]. Upon stimulation through the TCR, NKG2D, NCRs, or cytokine receptors, all three subsets of γδ T cells display innate cytotoxic activity with the rapid production of Th1-type cytokines, such as interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α), and the lytic molecules perforin and granzyme B [10–12,14].

A number of studies have revealed that the Vδ1 and Vδ3 subsets of γδ T cells are expanded in subgroups of patients with CLL [23–26]. Vδ1 T cells expanded from peripheral blood have been shown to display specific cytotoxicity towards B cells from CLL patients and CLL-derived cell lines in vitro [24,25,27], however, this cytotoxicity appears to be limited to particular properties of both the effector Vδ1 T cells and the target CLL cells. Poggi et al. [24] found that CLL cells required activation with pokeweed mitogen to make them susceptible to killing by Vδ1 T cells and this killing was mediated by engagement of NKG2D by ULBP3. Correia et al. [27] found that Vδ1 T cells expanded under conditions that promoted the expression of NCRs, particularly NKp30, mediated direct cytotoxic killing of CLL cells isolated from patients. Vδ2 T cells from healthy donors but not from CLL patients were also demonstrated to kill CLL cells in vitro [28], however, other studies found that Vδ2 T cells did not kill CLL cells [24,27]. Vδ1 T cells have also been shown to inhibit tumour growth and to prevent tumour cell dissemination in xenograft models of CLL [29].

Since Vδ1, Vδ2 and Vδ3 T cells all can kill CLL cells under certain conditions, several groups have attempted to treat CLL cells to make them more susceptible to lysis by γδ T cells. Poggi et al. [24] treated CLL cells with the retinoic acid receptor-α (RAR-α) agonist all-trans retinoic acid (ATRA) to upregulate ULBP3 and MIC-A, which made them susceptible to killing by Vδ1 T cells. RAR-α agonists can also induce CD1c and CD1d expression on tonsillar B cells [30], and we reported that ATRA and its analogue AM580 can restore CD1d expression on CLL cells and healthy B cells, and these cells were capable of presenting glycolipid antigens to invariant natural killer T (iNKT) cells, resulting in cytotoxic killing of CLL cells [31]. CD1d is an MHC class I-like antigen-presenting molecule that presents phospholipid and glycolipid antigens to T cells [10,32]. CD1d is expressed across multiple B cell subsets in humans [33]. CD1d expression varies among B cell disorders [34,35] and is downregulated on malignant cells in CLL [31,34–37]. In addition to iNKT cells, Vδ1 and Vδ3 T cells can also recognise and respond to lipid antigens presented by CD1d and subsequently mediate a potent anti-tumour response [17–19].

The present study aimed to sensitise CLL cells for killing by γδ T cells through the upregulation of CD1d and ULBP3 on the surface of CLL cells and the presentation of CD1d-binding lipids to γδ T cells. By understanding the conditions under which γδ T cells can target CLL cells, we aim to evaluate the potential of Vδ1, Vδ2 and Vδ3 T cell-based immunotherapies for CLL, providing an alternative or adjunct to traditional treatments.

Materials and methods

Subjects and ethical statement

Anticoagulated blood samples were obtained from patients with CLL who presented to the Department of Haematology, St. James’s Hospital, Dublin. This project was approved by St James’s and Tallaght Hospitals Joint Research Ethics committee, and written informed consent was obtained from all participants. Healthy control samples were acquired either from consenting volunteers or from anticoagulated whole blood packs kindly provided by the Irish Blood Transfusion Service (IBTS) at St. James’s Hospital. Peripheral blood mononuclear cells (PBMC) were prepared by standard density gradient centrifugation over Lymphoprep (STEMCELL Technologies) and cryopreserved in the Trinity/St. James’s Hospital Haematology Biobank until needed.

Antibodies and flow cytometry

PBMC or γδ T cell lines were stained with a dead cell stain (DCS; Fixable Viability Dye eFluor 506; eBiosciences) or propidium iodide (Miltenyi Biotec), human Fc blocking agent (Miltenyi Biotec) and the following mAbs: CD3 (clone HIT3A), CD19 (HIB19), CD1d (51.1), CD107a (H4A3), IFN-γ (4S.B3), TNF-α (Mab11) CTLA-4 (BNI3), TIM-3 (F38-2E2) and Vδ2 (B6) were purchased from Biolegend, CD5 (UCHT2) and Vδ1 (REA173) were purchased from Miltenyi Biotech; ULBP3 (166510) and LAG-3 (polyclonal IgG) were purchased from R&D Systems; PD-1 (EH12.1) was purchased from BD Pharmingen; and Vδ3 (P11.5B) was purchased from Coulter Immunotech. Following staining, the cells were acquired on a BD FACSCanto II flow cytometer (BD Biosciences) and analysed using FlowJo v10 software. The gating strategies used to measure CD1d and ULBP expression by B cells and CD107a expression by γδ T cell subsets are shown in S1 Fig. CLL cell viability was measured as uptake of propidium iodide (PI) by CD19⁺/CD5⁺ cells. Incubation with 0.18% (w/v) Triton-X 100 was used as a positive control of CLL cell death.

Enrichment of peripheral B cells

Flow cytometry confirmed that B cells were highly enriched in the CLL patients but accounted for less than 10% of lymphocytes from healthy donors, therefore, B cells were enriched from healthy donor PBMC only, resulting in similar frequencies to those found in PBMC from CLL patients (S1C Fig). B cells were enriched from 2x10⁸ PBMC by positive magnetic bead selection using CD19 Microbeads, followed by selection using MACS MS Cell Separation column according to the manufacturer’s instructions (Miltenyi Biotec). The enriched B cells were counted and plated in complete RPMI medium (RPMI 1640 with Glutamax containing 25 mM HEPES, 50 µg/ml streptomycin, 50 U/ml penicillin and 10% heat-inactivated foetal bovine serum (FBS)) in 96-well round bottom cell culture plates.

Treatment of B cells with RAR-α agonists

B cells from healthy donors and CLL patients were treated for 24–72 hours with ATRA, AM580 or retinol (Sigma-Aldrich) at 30, 300 and 3000 ng/mL. Cells were cultured in a 5% CO₂ incubator at 37˚C and subsequently analysed for CD1d and ULBP3 expression by flow cytometry.

Generation of primary cultures of γδ T cells

Primary cultures of γδ T cells, which contained Vδ1, Vδ2, and Vδ3 T cells, were generated from 2x10⁸ PBMC isolated from healthy donors. Following treatment with human FcR blocking reagent (Miltenyi Biotec), the PBMC were incubated with a CliniMACS TCR α/β-Biotin mAb followed by anti-biotin Microbeads, as per the supplier’s instructions (Miltenyi Biotec) and the αβ TCR⁺ T cells were then depleted using a MACS LD Cell Separation column. The αβ T cell-depleted fraction was then resuspended at a density of 7.5x10⁶ cells/mL in ‘γδ T cell medium’ (RPMI Glutamax medium containing 10% FBS, 25 mM HEPES, 150 µg/ml streptomycin, 50 U/ml penicillin, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 1% non-essential amino acids and 1% essential amino acids) and stimulated with 1 μg/mL anti-CD3 antibody (clone OKT3, BioLegend). Cells were cultured in 96-well round-bottom plates at 37˚C and 5% CO₂. After 24 hours, and every 4–5 days thereafter, the medium was replaced with fresh medium containing 100 U/mL IL-2 and 70 ng/mL of IL-15 (Miltenyi Biotec). Cells were cultured for up to 60 days and the purities and relative frequencies and phenotypes of Vδ1, Vδ2 and Vδ3 T cells were assessed by flow cytometry. Ten lines of γδ T cells were used in the present study. At the time of use in experiments, Vδ1 T cells accounted for 13.9–51% (mean 29.2%) of the cells, whereas Vδ2 T cells accounted for 18.5–82.0% (mean 45.7%) and Vδ3 T cells accounted for 1.3–25.0% (mean 8.3%). The remaining cells were Vδ1^- Vδ2^- Vδ3^- γδ T cells and no αβ T cells, B cells or NK cells were present.

Analysis of cytolytic degranulation by γδ T cells

Cytotoxic responses of γδ T cells were recorded through flow cytometric measurement of cell-surface CD107a expression, a marker of cellular degranulation. CD19⁺ B cells from healthy donors or PBMC from CLL patients and healthy controls, were plated in 96-well, round bottom microtitre plates at densities of 0.25x10⁶ cells per well and treated for 24–72 hours with various concentrations of RAR-α agonists, as described above. Equal numbers of expanded γδ T cells were added. Phorbol myristate acetate (50 ng/mL; Sigma-Aldrich) and ionomycin (1 µg/ml; Sigma Aldrich) or anti-CD3 and anti-CD28 mAbs (1 μg/mL each) were added to positive control wells containing γδ T cells alone. Fluorochrome labelled anti-CD107a antibody was added to each well. After 1 hr, monensin (BioLegend) at a final concentration of 5 nmol/well was added and left to incubate for a further 3 hr. The cells were then stained for analysis by flow cytometry.

Priming CLL cells for cytotoxicity by γδ T cells using glycolipids

B cells were also treated with 5 glycolipids, that have been shown by us (S2 Fig, ref [38]) and others [17,39–41] to bind to CD1d and activate T cells. The structures of these glycolipids are shown in Fig 1. Sulfatide, lyso-sulfatide, cardiolipin and tetramyristoyl cardiolipin (TMC) were purchased from Avanti Polar Lipids and α-galactosylceramide (GC) was obtained from Funakoshi. The glycolipids were prepared in dimethylsulphoxide (DMSO) to a stock concentration of 1 mM and stored at -20^oC. Following thawing at room temperature, glycolipids were vortexed for 1 minute, heated at 80^oC for 2 minutes and sonicated for 10 minutes before diluting them in medium and adding them to the B cells or PBMC at 100 ng/mL. After incubating the glycolipid-treated cells for 16 hours, equal numbers of expanded γδ T cells were then added into each well and the CD107a degranulation assay was carried out as described above.

Fig 1 — Sulfatide derives from galactosylceramide (GalCer) via esterification of a sulphate group to 3-hydroxyl of the galactose moiety. Lyso-sulfatide lacks the fatty acid chain of sulfatide. Cardiolipin consists of 2 phosphate residues and 4 fatty acyl chains. Tetramyristoyl cardiolipin (TMC) is a synthetic analogue of cardiolipin.

Statistical analysis

Statistical analysis was carried out using GraphPad PRISM 8.0.2 for Windows. Mann-Whitney U test was used to compare unpaired, independent samples. Wilcoxon signed-rank test was used to compare paired, dependent samples. One-way ANOVA and post hoc Dunnett’s multiple comparison test were used to compare the means of three or more groups. Repeated measures Two-Way ANOVA and post hoc Tukey’s test was used to compare group means across matched samples. Statistical significance was reported as p < 0.05.

Results

Characteristics of the CLL patients

Eighteen patients with CLL were recruited for this study. The patient demographics are shown in Table 1. Of the total patient population, 72% were male. The participant’s age ranged from 36–83 years (median, IQR 24), with 44% of the cohort being ≥ 65 years. Of the sixteen tested, three individuals (18.75%) in the cohort tested positive for the expression of CD38, a well-classified negative prognosticator. Of the twelve individuals in which CD49 expression was tested (Patients 6–15, 17–18 in Table 1], six (50%) were positive. Of the nine individuals in which the immunoglobulin heavy chain variable (IGHV) gene region mutational status was determined (Patients 1, 2, 3, 6, 7, 10, 11, 14 and 15 in Table 1], four patients (~44%) and five patients (~55%) had IGHV-mutated and -unmutated disease, respectively. Of the thirteen individuals in which TP53 mutational status was determined (Patients 1, 2, 3, 6, 7, 9, 10, 11, 12, 14, 15, 17 and 18 in Table 1], one patient (~8%) had TP53 mutated disease. Chromosomal abnormalities were measured in eleven patients by FISH (fluorescent in situ hybridisation), in which 36.7% carried the Del13q aberration. One case of trisomy 12 (0.9%) (Patient 5) and another of Del11q (0.9%) (Patient 11) was detected, with no aberrations found in 45% of patients. Approximately 60% of patients received treatment prior to sample biobanking.

Table 1. Patient demographics and disease characteristics.

Patient	Sex	Age	Flow cytometry	IGHV/TP53 status	FISH	Pre-treated?	Treatment¹
1	M	78	CD38+	UM/UM	No aberrations	Y	Retuximab, cyclophosphamide x6
2	F	54	CD38-	MU/UM	Del 13q	N	n/a
3	M	59	NT	MU/UM	Del 13q	Y	Retuximab-bendamustine
4	F	83	CD38-	NT	NT	Y	Chlorambucil
5	M	80	CD38-	NT	Trisomy 12	N	n/a
6	M	54	CD38- CD49d+	UM/UM	No aberrations	Y	FCR, retuximab-bendamustine, ibrutinab
7	F	57	CD38- CD49d-	MU/UM	Del 13q	N	n/a
8	F	71	CD38- CD49d+	NT	No aberrations	Y	FCR x4, retuximab-bendamustine x4, ibrutinib
9	M	74	CD38- CD49d+	NT/MU	NT	Y	FCR x6, zanubrutinib
10	M	49	CD38- CD49d+	UM/UM	No aberrations	N	n/a
11	M	55	CD38- CD49d-	UM/UM	Del 11q	N	n/a
12	M	83	CD38 + CD49d-	NT/UM	NT	Y	FCR x6, IV-Ig, azathioprine + retuximab x4
13	M	73	CD38- CD49d+	NT	Del 13q	N	n/a
14	M	51	CD38- CD49d-	MU/UM	No aberrations	Y	Venetoclax + obinutuzumab
15	M	36	CD38 + CD49d-	UM/UM	NT	Y	FCR x4, ibrutinib
16	F	74	NT	NT	NT	N	n/a
17	M	68	CD38- CD49d-	NT/UM	NT	N	n/a
18	M	48	CD38- CD49d+	UM/UM	NT	Y	Venetoclax + obinutuzumab

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F, female; FCR, fludarabine, cyclophosphamide and rituximab; FISH, fluorescent in situ hybridisation; IGHV, immunoglobulin heavy chain; IV-Ig, intravenous immunoglobulin; M, male; MU, mutated; N, no; n/a, not applicable; NT, not tested; UM, unmutated; Y, yes.

¹No patients were on treatment at the time of study.

²Patient 12 underwent Richter transformation from underlying CLL to an anaplastic large cell lymphoma requiring new treatment with each relapse. Patient 12 also developed immune thrombocytopenia.

Sixteen age-matched healthy donors were recruited for phenotypic comparisons with the CLL patients. Blood samples from a further 27 anonymous healthy donors were obtained for cell enrichment and functional studies.

Reduced CD1d expression by B cells and increased Vδ1 and Vδ3 T cell frequencies in CLL patients

PBMC were isolated from 16 healthy donors and 13 patients with CLL and stained with mAbs specific for CD3, CD19, Vδ1, Vδ2 and Vδ3 and analysed by flow cytometry (S1 Fig). Fig 2 shows that the frequency of Vδ1 and Vδ3 T cells, as percentages of total T cells, was significantly increased in CLL patients compared to age-matched healthy donors (Fig 2A,C). The frequencies of Vδ2 T cells were similar in CLL patients and age-matched healthy controls (Fig 2B). As expected, B cells were significantly expanded within PBMC of the CLL patients (Fig 2D). Phenotypic analysis of B cells revealed that B cells from CLL patients expressed significantly lower levels of CD1d, as measured by median fluorescence intensity (MFI) (Fig 2E). ULBP3 expression by B cells was also lower in CLL patients compared to control subjects, but this difference was not significant (Fig 2F).

Fig 2 — PBMC were isolated from healthy donors and patients with CLL. PBMC from healthy donors were enriched for CD19⁺ B-cells using magnetic bead separation. Cells were stained with mAbs specific for CD3, CD19, Vδ1, Vδ2, Vδ3, CD1d and ULBP3 and analysed by flow cytometry. Scatter bar plots depict the frequencies of T cells from healthy donors and CLL patients that expressed Vδ1 **(A)**, Vδ2 (B) and Vδ3 **(C)** TCRs, the percentages of lymphocytes that are B cells **(D)**, and the mean fluorescence intensities of CD1d (E) and ULBP3 (F) expression on CD19⁺ B-cells. The data in **(A)**, (B) and **(C)**, show means ± standard deviation (SD) of 13 CLL patients and 11-14 age-matched control subjects. The data in (D) show means ± SD of 8 CLL patients and 8 healthy donors. The data in (E) show means ± SD of 14 CLL patients and 16 control subjects. The data in (F) show means ± SD of 6 healthy subjects and 4 CLL patients. Statistical significance was determined using the non-parametric Mann-Whitney U test and statistical differences are indicated.

γδ T cells do not spontaneously degranulate in response to B cells from CLL patients or healthy donors

We next investigated if γδ T cells from healthy donors could kill B cells from CLL patients and healthy donors. Lines of γδ T cells were generated from 5 healthy donors under conditions that promote the growth of Vδ1, Vδ2 and Vδ3 T cells (S1B Fig). The γδ T cell lines were co-cultured with equal numbers of PBMC from 9 CLL patients and B cells enriched from PBMC from 10 healthy donors in the presence of an anti-CD107a mAb. Cells were then stained with mAbs specific for CD3, CD19, Vδ1, Vδ2 and Vδ3 and the expression of the degranulation marker CD107a by individual Vδ1, Vδ2 and Vδ3 T cells was analysed by flow cytometry. As seen in Fig 3A, no γδ T cell subset displayed increased degranulation in response to B cells from CLL patients or healthy donors. The expression of inhibitory molecules on γδ T cell lines at Day 24 was evaluated, directly before their use in cytotoxicity assays. As seen in Fig 3B, High expression of PD-1 and CTLA-4 was observed on Vδ2 and Vδ3 T cells from two γδ T cell lines (GDTC 1, GDTC 2). Moderate expression of TIM-3 was observed on Vδ2 and Vδ3 T cells from two γδ T cell lines (GDTC 1, GDTC 2), while LAG-3 was lowly expressed on all three lines. These results suggest that γδ T cells do not spontaneously degranulate in response to CLL cells or healthy B cells under standard culture conditions, and this may be partially due to functional exhaustion of long-term expanded γδ T cell lines.

Fig 3 — PBMC from 9 CLL patients and B cells enriched from PBMC from 10 healthy donors were co-cultured with lines of expanded γδ T cell lines from healthy donors in the presence of an anti-CD107a mAb. Cells were then stained with mAbs specific for CD3, CD19, Vδ1, Vδ2 and Vδ3 and analysed by flow cytometry. **(A)** Scatter bar plots depict the mean fluorescence intensities (MFI) of CD107a expression by gated Vδ1 (left panels), Vδ2 (centre panels) and Vδ3 (right panels) T cells cultured alone or with B cells from healthy donors (n = 10) (upper panels) and CLL patients (n = 9) (lower panels). These data show means ± standard deviation. MFIs of CD107a expression by γδ T cell subsets cultured alone were compared to when cultured with B cells using the using the non-parametric Wilcoxon matched pairs signed rank test and no significant differences were found. **(B)** Flow cytometry analysis of inhibitory receptor expression by γδ T cell lines (rows) and subsets (columns) at Day 24 in culture. Cells were stained with mAbs specific for CD3, CD19, Vδ1, Vδ2, Vδ3, PD-1, CTLA-4, TIM-3 and LAG-3 and analysed by flow cytometry.

The viability of CLL cells decreases in co-culture with γδ T cells and this is not enhanced by ATRA

The viability of CLL cells in co-culture with γδ T cells was next assessed at varying E:T ratios. Lines of γδ T cells were generated from 3 healthy donors under conditions that promote the growth of Vδ1, Vδ2 and Vδ3 T cells (S1B Fig). Ex vivo CLL PBMC were plated and co-cultured with equal numbers (1:1), three-fold (3:1) or six-fold (6:1) as many γδ T cells for 4 hr. Cells were then harvested, stained with propidium iodide and mAbs specific for CD19 and analysed by flow cytometry. Contrasting with previous results, the % dead CLL cells increased upon co-culture with expanded γδ T cell lines, and this became more pronounced at higher E:T ratios (Fig 4.). The previous lack of γδ-mediated cytotoxic degranulation suggests that this drop in viability may reflect either non-degranulating cytotoxic mechanisms, cytotoxicity mediated by γδ T cells other than Vδ1, Vδ2 and Vδ3 T cells, or may be due to highly stressful co-culture conditions.

Fig 4 — PBMC from 3 CLL patients were treated for 72 hr with medium alone or 30 ng/mL ATRA. The cells were then co-cultured with expanded γδ T cell lines from healthy donors for 4 hr (n = 3). Viability of CLL cells was measured as PI uptake by CD19⁺/CD5⁺ cells by flow cytometry. Scatter bar plots compare the % dead (± SD) (PI⁺) CLL cells with and without treatment with ATRA. Data were compared using repeated measures Two-way ANOVA and post hoc Tukey’s multiple comparison test and significant differences (P < 0.05) are indicated.

Upregulation of CD1d and ULBP3 on B cells from CLL patients and healthy controls by ATRA does not sensitise them for killing by γδ T cells

We hypothesised that treatment of B cells from CLL patients with RAR-α agonists would induce upregulation of CD1d and ULBP3, which would provide stimulatory signals for TCR- and NKG2D-mediated γδ T cell activation, respectively, resulting in B cell killing [24,31]. PBMC from 5 CLL patients and B cells enriched from PBMC from 10–13 healthy donors were treated for 72 hr with 30, 300 or 3,000 ng/mL ATRA, AM580 and retinol. The cells were then stained with mAbs specific for CD19, CD1d and ULBP3 and analysed by flow cytometry. Fig 5A,C shows that treatment with ATRA and AM580 led to a significant increase in CD1d expression on both healthy donor B and CLL B cells, whereas retinol had no effect on the expression of either marker. Upregulation of CD1d and ULBP3 by ATRA appeared to follow a dose dependence, while upregulation by AM580 did not. Fig 5B shows that treatment with ATRA also led to a significant increase ULBP3 expression on healthy B cells. This upregulation was not achieved by AM580 or retinol stimulation.

Fig 5 — PBMC from 5 CLL patients and B cells enriched from PBMC from 10-13 healthy donors were treated for 72-96 hr with ATRA, AM580 and retinol at the indicated concentrations. The cells were subsequently stained with mAbs specific for CD1d or ULBP3 and analysed by flow cytometry. Scatter bar plots depict the geometric median fluorescence intensities (± SD) of CD1d (n = 13) (A) and ULBP3 (n = 10) (B) expression on B cells from healthy donors following treatment with 30, 300 and 3000 ng/ml ATRA (left panels), AM580 (centre panels) and retinol (right panels). (C), Scatter bar plots depict the geometric MFI (± SD) of CD1d expression on B cells from CLL patients. Data were compared using One-way ANOVA and post hoc Dunnett’s multiple comparisons test and significant differences (P < 0.05) are indicated.

We next investigated if treatment of B cells with RAR-α agonists would make them susceptible to killing by Vδ1, Vδ2 or Vδ3 T cells through upregulation of CD1d and ULBP3 on their cell surface. Enriched B cells from five healthy donors and PBMCs from five CLL patients were treated for 72 hr with 30 ng/mL RAR-α agonists. The cells were then co-cultured with equal numbers of expanded γδ T cell lines from healthy donors in the presence of an anti-CD107a mAb. Cells were then stained with mAbs specific for CD3, CD19, Vδ1, Vδ2 and Vδ3 and analysed by flow cytometry. Fig 6 A shows that treatment of B cells from CLL patients or healthy donors with any of the RAR-α agonists at 30 ng/mL did not trigger cytolytic degranulation by Vδ1, Vδ2 or Vδ3 T cells. Thus, although ATRA and AM580 can induce the expression of stimulatory ligands on B cells, this did not lead to cytolytic degranulation by any of the subsets of γδ T cells. The production of two key pro-inflammatory cytokines, IFN-γ and TNF-α, by γδ T cell subsets was next assessed in response to CLL cells treated with or without ATRA. Although Vδ1, Vδ2 and Vδ3 T cells produced both cytokines in response to PMA and ionomycin stimulation, no difference in IFN-γ or TNF-α production, and therefore no non-cytolytic functionality, was observed in co-culture conditions with or without pre-treatment of ATRA.

Fig 6 — (A) Enriched B cells from 5 healthy donors and PBMCs from 5 CLL patients were treated for 72 hr with RAR-α agonists at 30 ng/mL. The cells were then co-cultured with expanded γδ T cell lines from healthy donors in the presence of an anti-CD107a mAb for 4 hr (n = 5). Cells were then stained with mAbs specific for CD3, CD19, Vδ1, Vδ2 and Vδ3 and analysed by flow cytometry. Scatter bar plots depict the mean (± SD) fluorescence intensities (MFI) of CD107a expression by gated Vδ1 (left panels), Vδ2 (centre panels) and Vδ3 (right panels) T cells cultured alone or with the treated B cells. **(B)** PBMCs from 3 CLL patients were treated for 72 hr with medium alone or ATRA at 30 ng/mL. The cells were then co-cultured with expanded γδ T cell lines from 3 healthy donors for 4 hr. The γδ T cells were analysed for IFN-γ and TNF-α production by flow cytometry. Percent positivity of IFN-γ and TNF-α expression by Vδ1 (left panel), Vδ2 (centre) and Vδ3 (right) T cells was compared using a One-way or Two-way ANOVA and post hoc Tukey’s multiple comparison test and significant differences (P < 0.05) are indicated.

Addition of CD1d-binding glycolipids to B cells from healthy donors and CLL patients does not sensitise them for killing by Vδ1, Vδ2 and Vδ3 T cells

It was hypothesised that the addition of CD1d-binding glycolipids may sensitise B-cells from healthy donors and CLL patients to TCR-mediated killing by Vδ1, Vδ2 and Vδ3 γδ T-cells. Five different glycolipids; α-GC, sulfatide, lyso-sulfatide, cardiolipin and TMC were vortexed, heated, sonicated and added to enriched B-cells from healthy donors or CLL cells + /- ATRA and left overnight. The cells were then co-cultured with equal numbers of expanded γδ T cell lines generated from healthy donors in the presence of anti-CD107a mAb. Cells were then stained with mAbs specific for cell surface expression of CD3, CD19, Vδ1, Vδ2 and Vδ3 T cells and analysed by flow cytometry. Fig 7 shows that the addition of glycolipid antigens does not induce killing of healthy donor B cells or CLL cells by any γδ T-cell subset.

Fig 7 — Enriched B cells from 5 healthy donors and PBMCs from 4 CLL patients were treated with or without 100 ng/mL ATRA for 72 hours and 5 different glycolipids were sonicated and added and then incubated for an additional 24 hours. The cells were then co-cultured with expanded γδ T cell lines from healthy donors in the presence of an anti-CD107a mAb (n = 5). Cells were then stained with mAbs specific for CD3, CD19, Vδ1, Vδ2 and Vδ3 and analysed by flow cytometry. Scatter bar plot indicates the mean (±SD) fluorescence intensities (MFI) of CD107a expression by gated Vδ1 (top row), Vδ2 (centre row), and Vδ3 (bottom row) T-cells. A negative control (T cells alone) and positive control (PMA & Ionomycin, anti-CD3/anti-CD28) were included (n = 4). MFIs were compared using One-way ANOVA and post hoc Dunnett’s multiple comparison test and no significant differences were found.

Discussion

γδ T cells have emerged as promising candidates for cancer immunotherapy due to their ability to recognize a broad range of tumour-associated ligands, their potent cytotoxicity, efficient tumour infiltration, and their capacity to stimulate adaptive immune responses [10–14,42]. Unlike conventional CD8⁺ T cells, γδ T cells are not MHC-restricted, meaning they can potentially be used as allogeneic ‘off-the-shelf’ therapies for multiple patients without inducing graft-versus-host disease. Multiple clinical trials are currently exploring the therapeutic potential of γδ T cells – including those expressing chimeric antigen receptors (CAR-γδ T cells) – for various cancers, including CLL [11,43].

A number of studies have reported expansions of Vδ1, Vδ2, and/or Vδ3 T cells in patients with CLL [23–26]. These cells can be induced to kill CLL B cells under certain stimulatory conditions [24,25,27], making them candidates for cell-based therapy. However, the cytotoxic efficacy of these cells in CLL was limited and required specific stimulatory conditions. In the present study, we found that the frequencies of both peripheral blood Vδ1 and Vδ3 T cells, as percentages of total T cells, were significantly increased in CLL patients compared to those in age-matched healthy donors. Two patients with CLL demonstrated abnormally high levels of Vδ1 T cells (8.3 and 14.2%, respectively), one of which harboured a trisomy 12 chromosomal aberration. This finding may support previous statements describing a correlation between disease severity and Vδ1 T cell frequencies. Vδ2 T cell frequencies were similar in both groups. Importantly, when γδ T cells (including Vδ1, Vδ2, and Vδ3 subsets) were co-cultured with CLL B cells, there was no observable cytolytic activity, as indicated by a lack of CD107a externalisation, or pro-inflammatory cytokine production. The expansion of Vδ1 and Vδ3 T cells observed in this cohort is consistent with previous reports, however, these cells lack intrinsic cytotoxic activity against CLL cells, as has also been described [24,25,27]. As discussed below, this discrepancy may stem from differences in patient populations, disease stages or methodological approaches.

To investigate why γδ T cells failed to recognize or kill CLL cells, the expression of two key ligands - CD1d and ULBP3 – on B cells was analysed. Confirming previous reports [31,34–37], we found that both ligands are expressed at markedly lower levels on B cells from CLL patients compared to B cells from healthy donors. CD1d can present glycolipid antigens to the TCRs of Vδ1 and Vδ3 T cells [10,17–19], whereas ULBP3 binds to NKG2D, a stimulatory receptor found on all γδ T cell subsets [20,21]. The observed downregulation may represent an immune evasion strategy used by CLL cells, to limit the antigenic exposure of CLL cells to T cells, similar to the downregulation of MHC class I seen in other cancers [44]. One mechanism might be the overexpression of lymphoid enhancer-binding factor-1 (LEF-1), a transcriptional repressor of CD1d known to be upregulated in CLL patients [45].

We attempted to prime CLL cells for killing by treating them with ATRA and its analogue AM580, which have been shown by us and others to induce both CD1d and ULBP3 expression on B cells. ATRA has been used clinically to sensitize CLL cells to fludarabine-induced apoptosis [46] and is a standard therapy in acute promyelocytic leukaemia [47]. Consistent with prior findings [24,30,31], we found that treatment with ATRA and AM580 (but not retinol) significantly increased CD1d expression on both healthy and CLL B cells. Additionally, treatment with 3000 ng/mL ATRA resulted in significant upregulation of ULBP3 on healthy donor B cells. However, despite upregulation of these ligands, γδ T cells still failed to degranulate in response to treated CLL cells, suggesting that ligand expression alone is not sufficient to induce cytotoxicity. Interestingly, the viability of CLL cells was seen to decrease upon co-culture with γδ T cells, an observation which was more pronounced at greater E:T ratios. This drop in viability may reflect either non-degranulating cytotoxic mechanisms, such as those associated with death receptor ligation, or cytotoxicity mediated by γδ T cells other than Vδ1, Vδ2 and Vδ3 T cells (which typically account for 1–5% of cells in the γδ T cell lines).

Alternatively, the drop in viability may be the result of the highly stressful co-culture conditions employed, which could reflect nutrient deficiency, local hypoxia, lactate accumulation or harsh conditions reflected in physically crowded co-culture conditions [48–50]. The substantial cell density needed for high E:T ratios and stressful culture conditions as a result may explain this loss of viability independent of γδ T-cell cytotoxicity. CLL cells are metabolically fragile and notoriously sensitive to oxidative stress and changes in pH and nutrient availability, all of which are heavily impacted by co-culture conditions with high E:T ratios. Given that ATRA significantly upregulates both CD1d and ULBP3 on CLL cells, and given the viability of CLL cells did not change between ATRA-treated and -untreated cells at any E:T ratio, this supports that upregulation of CD1d and ULBP3 on B cells from healthy donors and chronic lymphocytic leukaemia patients does not prime them for killing by γδ T cells.

CD1d-dependent killing requires the presentation of lipid or glycolipid antigens to T-cells [10,17,18,31–33]. It was originally hypothesised that CLL cells would produce an endogenous tumour-associated lipid antigen that would bind to CD1d and stimulate γδ T-cell cytotoxicity. Since this was not the case, and because iNKT cell agonist glycolipid α-galactosylceramide is required to prime CLL cells for killing by iNKT cells [31], we next tested five synthetic glycolipids for their ability to induce γδ T cell killing of untreated or ATRA-treated B cells. These glycolipids – α-galactosylceramide, sulfatide, lyso-sulfatide, cardiolipin, and TMC – have previously been shown to activate T cells, including γδ T cells, via CD1d presentation [17,38–41]. However, none of these glycolipids succeeded in priming B cells for killing by Vδ1, Vδ2 or Vδ3 T cells even after treatment with ATRA to upregulate CD1d expression.

Several methodological differences may explain the lack of cytotoxicity observed. Firstly, the variable patient characteristics may produce different findings. Our study cohort included eighteen CLL patients with variable clinical backgrounds and considerable diversity in disease severity and treatment history. Some had high-risk genetic abnormalities such as TP53 mutations or 11q deletions with unmutated IGHV genes [51,52]. Over half had received treatment prior to sample collection, including a patient with relapsed disease or Richter transformation. Chemotherapy-induced mutations and immune evasion mechanisms in these patients may have impacted the functionality and expression of ligands on CLL cells, thus influencing γδ T cell recognition and activity. Interestingly, Poggi et al. [24] reported that patients with high-risk CLL did not exhibit expansions of cytotoxic Vδ1 T cells and showed resistance to ULBP3 induction by ATRA. The high proportion of patients with adverse prognostic markers in our study may explain the lack of Vδ1 T cell cytotoxicity observed.

The failure of γδ T cells to kill ATRA-treated B cells may reflect differences in γδ T cell line and CLL cell treatments in different studies. Poggi et al. [24] reported that Vδ1 T cells from CLL patients were only capable of lysing CLL cells if first treated with pokeweed mitogen or 10 μg/mL ATRA to upregulate MIC-A or ULBP3. This concentration of ATRA is much higher than that used in our study (30 ng/mL). The current study used mixed polyclonal γδ T cell lines derived from healthy donors, contrasting with the CD4^- CD8^- individual Vδ1 and Vδ2 T cell lines generated by Poggi et al. [24]. Indeed, CD1d-dependent cytotoxicity mediated by iNKT cells against CLL cells was limited to CD8⁺ iNKT cells, whereas their CD4⁺ and CD4^-CD8^- counterparts did not exhibit cytotoxic activity [24] and the same might be true for Vδ1 T cells. Correia and co-workers [27] reported that purified NKp30⁺ Vδ1 T cell lines expanded with IL-2 and phytohemagglutinin, exhibited cytotoxicity against leukemic cell lines and patient-derived CLL cells, but NKp30^- Vδ1 T cells were not cytotoxic. The expression of NKp30 was not investigated in the present study. Siegers et al. [25] expanded Vδ1 T cells from isolated pan-γδ T cell lines with concanavalin A for 6–8 days and these Vδ1 T cells were cytotoxic towards two CLL cell lines that do not express ULBP3 or MIC-A/B but expressed TRAILR1. These authors reported that concanavalin A was superior to PHA or anti-CD3 (OKT3; as utilised in our study protocol) as a γδ T cell mitogen. Additionally, γδ T cell cytotoxicity may be disease-stage dependent, as a recent study has demonstrated that expanded γδ T cells display reduced cytotoxic potential against increasing CLL cell burden [53].

The γδ T cells used in the present study contained variable numbers of Vδ1, Vδ2 or Vδ3 T cells and it is possible that each population can influence each other when in culture. Data from Siegers et al. [25] suggests that activated Vδ2 T cells can inhibit Vδ1 T cells. Therefore, it may be worth selecting for and expanding Vδ1 T cell lines, rather than using polyclonal pan γδ lines. Another difference between our γδ T cell lines and those used by Poggi et al. [24], is that theirs’ were derived from CLL patients and not healthy donors. Lines generated from patients with CLL have likely been exposed to circulating CLL cells in vivo, which may influence their cytotoxic potential or re-invoke a memory response upon secondary exposure to antigens [54,55]. The E:T ratios of 20:1, 10: and 5:1 used in other studies [24,25,27] also differ from the E:T ratio of 1:1 used in our study and may explain why no significant killing was observed. Moreover, functional exhaustion of long-term expanded γδ T cell lines, as seen by moderate expression of PD-1, CTLA-4, and TIM-3 on Vδ1 and Vδ3 subsets, may in part contribute to the lack of cytotoxicity observed against CLL cells in this study.

Our study was limited by a small sample size and highly variable patient cohort. Future studies should include larger, stratified cohorts, and compare γδ T cells expanded using different methods. The use of purified Vδ1, Vδ2 or Vδ3 T cell cultures may prove more effective in the killing of primary CLL cells.

In conclusion, this study confirms that both Vδ1 and Vδ3 T cells are significantly expanded in the blood of CLL patients, and that CLL B cells downregulate key ligands (CD1d and ULBP3) required for γδ T cell activation. Although treatment with RARα agonists like ATRA and AM580 can restore these ligand levels, this alone does not render CLL cells susceptible to γδ T cell killing. Similarly, pulsing with synthetic glycolipids failed to activate cytotoxicity. These findings challenge the idea that RAR-α agonists or glycolipid-based priming can sufficiently activate γδ T cells against CLL and underscore the complexity of immune evasion in CLL. Nevertheless, the study highlights the potential of γδ T cells as immunotherapeutic agents, provided that optimal activation conditions and cytotoxic subsets can be identified. Despite not being a dominant endogenous anti-CLL mechanism, γδ T cell cytotoxicity warrants further investigation, given the resistance associated with anti-CD20 therapies and the advantage of being able to use γδ T cells as an allogeneic therapy.

Supporting information

S1 Fig. Flow cytometric analysis of B cells and γδ T cell subsets.

(TIF)

pone.0346684.s001.tif^{(255.9KB, tif)}

S2 Fig. Identification of CD1d-binding glycolipids with agonist T cell activity within human PBMC and purified Vδ1 T cells.

(TIF)

pone.0346684.s002.tif^{(224.4KB, tif)}

S1 Table. David et al.Raw data.

(XLSX)

pone.0346684.s003.xlsx^{(25.6KB, xlsx)}

Acknowledgments

The authors are grateful to the donors who participated in this study. We are indebted to the Irish Blood Transfusion Service for providing buffy coat packs as a source of cells for this study.

Abbreviations

α-GalCer: α-galactosylceramine
ATRA: all trans retinoic acid
CAR: chimeric antigen receptor
CLL: chronic lymphocytic leukaemia
Con A: concanavalin A
DCS: dead cell stain
E:T: Effector:target
FISH: fluorescent in situ hybridisation
FBS: foetal bovine serum
GDTC: γδ T cell
IBTS: Irish Blood Transfusion Service
IFN-γ: interferon-γ
IGHV: immunoglobulin heavy chain variable gene
iNKT: invariant natural killer T cell
LEF-1: lymphoid enhancer binding factor-1
MHC: major histocompatibility complex
MFI: mean fluorescence intensity
mAb: monoclonal antibody
NCR: natural cytotoxicity receptor
PBMC: peripheral blood mononuclear cell
PBS: phosphate buffered saline
PHA: phytohemagglutinin
RAR: retinoic acid receptor
TCR: T cell receptor
TMC: tetramyristoyl cardiolipin
TNF-α: tumour necrosis factor-α
ULBP: UL16-binding protein

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Bosch F, Dalla-Favera R. Chronic lymphocytic leukaemia: from genetics to treatment. Nat Rev Clin Oncol. 2019;16(11):684–701. doi: 10.1038/s41571-019-0239-8 [DOI] [PubMed] [Google Scholar]
2.International CLL-IPI working group. An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data. Lancet Oncol. 2016;17(6):779–90. doi: 10.1016/S1470-2045(16)30029-8 [DOI] [PubMed] [Google Scholar]
3.Wu S-J, Chiang C-J, Lin C-T, Tien H-F, Lai M-S. A nationwide population-based cross-sectional comparison of hematological malignancies incidences between Taiwan and the United States of America. Ann Hematol. 2016;95(1):165–7. doi: 10.1007/s00277-015-2513-x [DOI] [PubMed] [Google Scholar]
4.Waldron C, O’Brien D, Brophy S, Perera K, Crotty GM, Dunlea E, et al. Epidemiology of chronic lymphocytic leukaemia in an Irish subpopulation with total case ascertainment: an additional tool for health economic planning. Br J Haematol. 2022;196(5):e47–9. doi: 10.1111/bjh.17929 [DOI] [PubMed] [Google Scholar]
5.Hallek M, Shanafelt TD, Eichhorst B. Chronic lymphocytic leukaemia. Lancet. 2018;391(10129):1524–37. doi: 10.1016/S0140-6736(18)30422-7 [DOI] [PubMed] [Google Scholar]
6.Roberts AW, Davids MS, Pagel JM, Kahl BS, Puvvada SD, Gerecitano JF, et al. Targeting BCL2 with Venetoclax in Relapsed Chronic Lymphocytic Leukemia. N Engl J Med. 2016;374(4):311–22. doi: 10.1056/NEJMoa1513257 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Sharman JP, Coutre SE, Furman RR, Cheson BD, Pagel JM, Hillmen P, et al. Final Results of a Randomized, Phase III Study of Rituximab With or Without Idelalisib Followed by Open-Label Idelalisib in Patients With Relapsed Chronic Lymphocytic Leukemia. J Clin Oncol. 2019;37(16):1391–402. doi: 10.1200/JCO.18.01460 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Zhou H, Hu P, Yan X, Zhang Y, Shi W. Ibrutinib in Chronic Lymphocytic Leukemia: Clinical Applications, Drug Resistance, and Prospects. Onco Targets Ther. 2020;13:4877–92. doi: 10.2147/OTT.S249586 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Iyer P, Wang L. Emerging Therapies in CLL in the Era of Precision Medicine. Cancers (Basel). 2023;15(5):1583. doi: 10.3390/cancers15051583 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Godfrey DI, Uldrich AP, McCluskey J, Rossjohn J, Moody DB. The burgeoning family of unconventional T cells. Nat Immunol. 2015;16(11):1114–23. doi: 10.1038/ni.3298 [DOI] [PubMed] [Google Scholar]
11.Mensurado S, Blanco-Domínguez R, Silva-Santos B. The emerging roles of γδ T cells in cancer immunotherapy. Nat Rev Clin Oncol. 2023;20(3):178–91. doi: 10.1038/s41571-022-00722-1 [DOI] [PubMed] [Google Scholar]
12.Adams EJ, Gu S, Luoma AM. Human gamma delta T cells: Evolution and ligand recognition. Cell Immunol. 2015;296(1):31–40. doi: 10.1016/j.cellimm.2015.04.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Melo AM, Mylod E, Fitzgerald V, Donlon NE, Murphy DM, Foley EK, et al. Tissue distribution of γδ T cell subsets in oesophageal adenocarcinoma. Clin Immunol. 2021;229:108797. doi: 10.1016/j.clim.2021.108797 [DOI] [PubMed] [Google Scholar]
14.Tyler CJ, Doherty DG, Moser B, Eberl M. Human Vγ9/Vδ2 T cells: Innate adaptors of the immune system. Cell Immunol. 2015;296(1):10–21. doi: 10.1016/j.cellimm.2015.01.008 [DOI] [PubMed] [Google Scholar]
15.Karunakaran MM, Willcox CR, Salim M, Paletta D, Fichtner AS, Noll A, et al. Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing. Immunity. 2020;52(3):487-498.e6. doi: 10.1016/j.immuni.2020.02.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells. Science. 1998;279(5357):1737–40. doi: 10.1126/science.279.5357.1737 [DOI] [PubMed] [Google Scholar]
17.Bai L, Picard D, Anderson B, Chaudhary V, Luoma A, Jabri B, et al. The majority of CD1d-sulfatide-specific T cells in human blood use a semiinvariant Vδ1 TCR. Eur J Immunol. 2012;42(9):2505–10. doi: 10.1002/eji.201242531 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Uldrich AP, Le Nours J, Pellicci DG, Gherardin NA, McPherson KG, Lim RT, et al. CD1d-lipid antigen recognition by the γδ TCR. Nat Immunol. 2013;14(11):1137–45. doi: 10.1038/ni.2713 [DOI] [PubMed] [Google Scholar]
19.Mangan BA, Dunne MR, O’Reilly VP, Dunne PJ, Exley MA, O’Shea D, et al. Cutting edge: CD1d restriction and Th1/Th2/Th17 cytokine secretion by human Vδ3 T cells. J Immunol. 2013;191(1):30–4. doi: 10.4049/jimmunol.1300121 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Pende D, Rivera P, Marcenaro S, Chang C-C, Biassoni R, Conte R, et al. Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxicity. Cancer Res. 2002;62(21):6178–86. [PubMed] [Google Scholar]
21.Lazarova M, Steinle A. The NKG2D axis: an emerging target in cancer immunotherapy. Expert Opin Ther Targets. 2019;23(4):281–94. doi: 10.1080/14728222.2019.1580693 [DOI] [PubMed] [Google Scholar]
22.Salih HR, Antropius H, Gieseke F, Lutz SZ, Kanz L, Rammensee H-G, et al. Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood. 2003;102(4):1389–96. doi: 10.1182/blood-2003-01-0019 [DOI] [PubMed] [Google Scholar]
23.Bartkowiak J, Kulczyck-Wojdala D, Blonski JZ, Robak T. Molecular diversity of gammadelta T cells in peripheral blood from patients with B-cell chronic lymphocytic leukaemia. Neoplasma. 2002;49(2):86–90. [PubMed] [Google Scholar]
24.Poggi A, Venturino C, Catellani S, Clavio M, Miglino M, Gobbi M, et al. Vdelta1 T lymphocytes from B-CLL patients recognize ULBP3 expressed on leukemic B cells and up-regulated by trans-retinoic acid. Cancer Res. 2004;64(24):9172–9. doi: 10.1158/0008-5472.CAN-04-2417 [DOI] [PubMed] [Google Scholar]
25.Siegers GM, Dhamko H, Wang X-H, Mathieson AM, Kosaka Y, Felizardo TC, et al. Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells. Cytotherapy. 2011;13(6):753–64. doi: 10.3109/14653249.2011.553595 [DOI] [PubMed] [Google Scholar]
26.Simões C, Silva I, Carvalho A, Silva S, Santos S, Marques G, et al. Quantification and phenotypic characterization of peripheral blood Vδ1 + T cells in chronic lymphocytic leukemia and monoclonal B cell lymphocytosis. Cytometry B Clin Cytom. 2019;96(2):164–8. doi: 10.1002/cyto.b.21645 [DOI] [PubMed] [Google Scholar]
27.Correia DV, Fogli M, Hudspeth K, da Silva MG, Mavilio D, Silva-Santos B. Differentiation of human peripheral blood Vδ1+ T cells expressing the natural cytotoxicity receptor NKp30 for recognition of lymphoid leukemia cells. Blood. 2011;118(4):992–1001. doi: 10.1182/blood-2011-02-339135 [DOI] [PubMed] [Google Scholar]
28.de Weerdt I, Hofland T, Lameris R, Endstra S, Jongejan A, Moerland PD, et al. Improving CLL Vγ9Vδ2-T-cell fitness for cellular therapy by ex vivo activation and ibrutinib. Blood. 2018;132(21):2260–72. doi: 10.1182/blood-2017-12-822569 [DOI] [PubMed] [Google Scholar]
29.Almeida AR, Correia DV, Fernandes-Platzgummer A, da Silva CL, da Silva MG, Anjos DR, et al. Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinical-Grade Expansion/Differentiation and Preclinical Proof of Concept. Clin Cancer Res. 2016;22(23):5795–804. doi: 10.1158/1078-0432.CCR-16-0597 [DOI] [PubMed] [Google Scholar]
30.Allan LL, Stax AM, Zheng D-J, Chung BK, Kozak FK, Tan R, et al. CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling. J Immunol. 2011;186(9):5261–72. doi: 10.4049/jimmunol.1003615 [DOI] [PubMed] [Google Scholar]
31.Ghnewa YG, O’Reilly VP, Vandenberghe E, Browne PV, McElligott AM, Doherty DG. Retinoic acid induction of CD1d expression primes chronic lymphocytic leukemia B cells for killing by CD8+ invariant natural killer T cells. Clin Immunol. 2017;183:91–8. doi: 10.1016/j.clim.2017.08.002 [DOI] [PubMed] [Google Scholar]
32.Salio M, Silk JD, Jones EY, Cerundolo V. Biology of CD1- and MR1-restricted T cells. Annu Rev Immunol. 2014;32:323–66. doi: 10.1146/annurev-immunol-032713-120243 [DOI] [PubMed] [Google Scholar]
33.Zeng SG, Ghnewa YG, O’Reilly VP, Lyons VG, Atzberger A, Hogan AE, et al. Human invariant NKT cell subsets differentially promote differentiation, antibody production, and T cell stimulation by B cells in vitro. J Immunol. 2013;191(4):1666–76. doi: 10.4049/jimmunol.1202223 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Kotsianidis I, Nakou E, Spanoudakis E, Bouchliou I, Moustakidis E, Miltiades P, et al. The diagnostic value of CD1d expression in a large cohort of patients with B-cell chronic lymphoproliferative disorders. Am J Clin Pathol. 2011;136(3):400–8. doi: 10.1309/AJCP2F2DOXOTXHZA [DOI] [PubMed] [Google Scholar]
35.Chaudhry MS, Karadimitris A. Role and regulation of CD1d in normal and pathological B cells. J Immunol. 2014;193(10):4761–8. doi: 10.4049/jimmunol.1401805 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Zheng Z, Venkatapathy S, Rao G, Harrington CA. Expression profiling of B cell chronic lymphocytic leukemia suggests deficient CD1-mediated immunity, polarized cytokine response, altered adhesion and increased intracellular protein transport and processing of leukemic cells. Leukemia. 2002;16(12):2429–37. doi: 10.1038/sj.leu.2402711 [DOI] [PubMed] [Google Scholar]
37.Zaborsky N, Gassner FJ, Asslaber D, Reinthaler P, Denk U, Flenady S, et al. CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells. Oncotarget. 2016;7(31):49459–69. doi: 10.18632/oncotarget.10372 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Melo AM, Zhang L, Dockry ÉF, Petrasca A, Ghnewa YG, Breen EP, et al. Novel thioglycoside analogs of α-galactosylceramide stimulate cytotoxicity and preferential Th1 cytokine production by human invariant natural killer T cells. Glycobiology. 2018;28(7):512–21. doi: 10.1093/glycob/cwy035 [DOI] [PubMed] [Google Scholar]
39.Girardi E, Maricic I, Wang J, Mac T-T, Iyer P, Kumar V, et al. Type II natural killer T cells use features of both innate-like and conventional T cells to recognize sulfatide self antigens. Nat Immunol. 2012;13(9):851–6. doi: 10.1038/ni.2371 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Luoma AM, Castro CD, Mayassi T, Bembinster LA, Bai L, Picard D, et al. Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells. Immunity. 2013;39(6):1032–42. doi: 10.1016/j.immuni.2013.11.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Dieudé M, Striegl H, Tyznik AJ, Wang J, Behar SM, Piccirillo CA, et al. Cardiolipin binds to CD1d and stimulates CD1d-restricted γδ T cells in the normal murine repertoire. J Immunol. 2011;186(8):4771–81. doi: 10.4049/jimmunol.1000921 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Yazdanifar M, Barbarito G, Bertaina A, Airoldi I. γδ T Cells: The Ideal Tool for Cancer Immunotherapy. Cells. 2020;9(5):1305. doi: 10.3390/cells9051305 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Fisher JP, Heuijerjans J, Yan M, Gustafsson K, Anderson J. γδ T cells for cancer immunotherapy: A systematic review of clinical trials. Oncoimmunology. 2014;3(1):e27572. doi: 10.4161/onci.27572 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Taylor BC, Balko JM. Mechanisms of MHC-I Downregulation and Role in Immunotherapy Response. Front Immunol. 2022;13:844866. doi: 10.3389/fimmu.2022.844866 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Erdfelder F, Hertweck M, Filipovich A, Uhrmacher S, Kreuzer K-A. High lymphoid enhancer-binding factor-1 expression is associated with disease progression and poor prognosis in chronic lymphocytic leukemia. Hematol Rep. 2010;2(1):e3. doi: 10.4081/hr.2010.e3 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Fernández-Calotti PX, Lopez-Guerra M, Colomer D, Pastor-Anglada M. Enhancement of fludarabine sensitivity by all-trans-retinoic acid in chronic lymphocytic leukemia cells. Haematologica. 2012;97(6):943–51. doi: 10.3324/haematol.2011.051557 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Lo-Coco F, Cicconi L, Breccia M. Current standard treatment of adult acute promyelocytic leukaemia. Br J Haematol. 2016;172(6):841–54. doi: 10.1111/bjh.13890 [DOI] [PubMed] [Google Scholar]
48.Adekola KUA, Dalva Aydemir S, Ma S, Zhou Z, Rosen ST, Shanmugam M. Investigating and targeting chronic lymphocytic leukemia metabolism with the human immunodeficiency virus protease inhibitor ritonavir and metformin. Leuk Lymphoma. 2015;56(2):450–9. doi: 10.3109/10428194.2014.922180 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Liu J, Chen G, Pelicano H, Liao J, Huang J, Feng L, et al. Targeting p53-deficient chronic lymphocytic leukemia cells in vitro and in vivo by ROS-mediated mechanism. Oncotarget. 2016;7(44):71378–89. doi: 10.18632/oncotarget.12110 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Koczula KM, Ludwig C, Hayden R, Cronin L, Pratt G, Parry H, et al. Metabolic plasticity in CLL: adaptation to the hypoxic niche. Leukemia. 2016;30(1):65–73. doi: 10.1038/leu.2015.187 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Delgado J, Doubek M, Baumann T, Kotaskova J, Molica S, Mozas P, et al. Chronic lymphocytic leukemia: A prognostic model comprising only two biomarkers (IGHV mutational status and FISH cytogenetics) separates patients with different outcome and simplifies the CLL-IPI. Am J Hematol. 2017;92(4):375–80. doi: 10.1002/ajh.24660 [DOI] [PubMed] [Google Scholar]
52.Kwok M, Wu CJ. Clonal Evolution of High-Risk Chronic Lymphocytic Leukemia: A Contemporary Perspective. Front Oncol. 2021;11:790004. doi: 10.3389/fonc.2021.790004 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Zarobkiewicz M, Kowalska W, Szymańska A, Lehman N, Kowalczyk B, Tomczak W, et al. γδ T Are Significantly Impacted by CLL Burden but Only Mildly Influenced by M-MDSCs. Cancers (Basel). 2025;17(2):254. doi: 10.3390/cancers17020254 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Lalor SJ, McLoughlin RM. Memory γδ T Cells-Newly Appreciated Protagonists in Infection and Immunity. Trends Immunol. 2016;37(10):690–702. doi: 10.1016/j.it.2016.07.006 [DOI] [PubMed] [Google Scholar]
55.Comeau K, Paradis P, Schiffrin EL. Human and murine memory γδ T cells: Evidence for acquired immune memory in bacterial and viral infections and autoimmunity. Cell Immunol. 2020;357:104217. doi: 10.1016/j.cellimm.2020.104217 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0346684.r001

Decision Letter 0

Xiaosheng Tan

9 Dec 2025

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Reviewer #2: Yes

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Reviewer #1: Julie et al. present a study that provides valuable insights into the regulation of CD1d and ULBP3 on B cells from healthy donors and CLL patients and their inability to sensitize these cells to γδ T-cell–mediated killing. While the findings are clearly presented, addressing the following points would substantially strengthen the study’s scientific rigor, clarify mechanistic causality, and enhance its translational relevance.

Major Comments:

1. The cytotoxicity assays depend almost exclusively on CD107a surface expression as a surrogate for γδ T-cell degranulation. Although CD107a is a widely used marker, it does not always correlate with actual target-cell death, particularly in cases where immunological synapse formation or perforin-mediated killing is impaired. Incorporating direct cytotoxicity measurements—such as flow-based live/dead killing assays, or real-time imaging—across multiple effector-to-target ratios and time points would provide more definitive evidence regarding whether B cells are truly resistant to γδ T-cell–mediated killing.

2. All functional assays rely on long-term expanded γδ T-cell lines derived from healthy donors. Such expanded lines may exhibit altered receptor expression or activation thresholds, and allogeneic interactions do not accurately model autologous physiology. Including freshly isolated T cells and ideally autologous γδ T cells from the same individuals would help exclude artifacts arising from in vitro expansion or allogeneic mismatch and strengthen confidence in the negative findings.

3. RAR-α agonist treatment may induce additional immunomodulatory molecules on B cells beyond CD1d and ULBP3. Assessing inhibitory ligands such as PD-L1, or other immunosuppressive markers would clarify whether these pathways contribute to the lack of γδ activation. Furthermore, the study does not evaluate whether RAR-α agonists directly alter γδ T-cell viability, metabolic state, or cytokine production/proliferation. These controls are important to exclude unintended suppression or modulation of γδ T-cell function by the compounds.

4. Additional functional profiling would provide a more complete understanding of γδ T-cell activation status. Quantifying granzyme B, perforin, IFN-γ, TNFα, and activation markers such as CD69 or CD25 would help determine whether γδ T cells exhibit partial, non-cytolytic, or exhausted activation states not reflected by CD107a alone.

Reviewer #2: The manuscript reports CLL patients have reduced B-cell CD1d and increased Vδ1/Vδ3 frequencies and aims to test whether restoration of CD1d expression on B cells can sensitize CLL cells to γδ T-cell-mediated cytotoxicity. While this is an interesting and clinically relevant question, the current experimental evidence is not sufficient to conclude that γδ T cells fail to respond to CD1d-restored B cells. Further experiments and clarifications could strengthen the results. Please see my comments below.

Abstract

Authors claim that “This study reveals that human Vδ1 T cell cytotoxicity against CLL B cells may be disease stage dependent and requires B cell priming and selective expansion and activation of specific γδ T cell subsets.” However, no results or discussion are presented regarding how cytotoxicity varies with disease stage.

Introduction:

The introduction is clear and provides sufficient background to set the context and the relevance of this study.

Methods:

The authors report using an E:T ratio of 1:1 in their co-cultures. While this is acceptable, γδ T-cell-mediated responses often require higher ratios (e.g., 5:1 or 10:1) to detect degranulation and target killing, particularly against primary B cells.

Results:

While CD107a reflects degranulation, it does not necessarily demonstrate that target cells are being killed. Complement CD107a with direct granzyme B delivery and target death are recommended.

Besides the E:T ratio and complementary assays for CD107a, the negative results may also reflect (a) the need of subset purification/priming, Vδ1 T cells ranged from 13.9 to 51%, which means in some cases, Vδ1 could be too diluted to show changes. Vδ3 T cells are a minor population (1.3-25%), so CD107a negativity in this subset could be due to low numbers rather than true absence of degranulation. (b) the possibility that Vδ1/Vδ3-mediated cytotoxicity proceeds via non-degranulation pathways, which would not be captured by CD107a.

Line 308 states PBMC from 13 CLL patients were analyzed but Table 1 indicates there are 15 patients. Please explain.

Figure 3F, why is sample number in ULBP3 detection different from the sample number in CD1d figure?

Figure 5 does not show ULBP3 expression after treatment in CLL donors.

Figure 6 showed healthy donor T cells alone show even higher CD107a than the positive control (PMA/ionomycin), which requires explanation.

Discussion:

Discussion comparing these results to previous work with iNKT-mediated killing would help interpret the negative findings.

Since γδ-mediated killing is not a major mechanism in CLL, providing rationale for studying this mechanism, e.g., limitations of anti-CD20 therapies and potential additive effects of alternative cytotoxic pathways, would strengthen the significance.

Figures:

Gating strategy figure (Figure 1) should be moved to supplementary materials.

Figure 5, please keep colors consistent with other figures.

Figure 7, please label for Vδ1/2/3 T cells in figures to improve readability.

Figure legends appear misplaced.

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Reviewer #1: Yes: Jing JuJing JuJing JuJing Ju

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PLoS One. 2026 Apr 20;21(4):e0346684. doi: 10.1371/journal.pone.0346684.r002

Author response to Decision Letter 1

10 Mar 2026

Responses to all reviewer and editor comments have been uploaded in the file entitled 'Response to reviewers'

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pone.0346684.s005.docx^{(36.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0346684.r003

Decision Letter 1

Xiaosheng Tan

23 Mar 2026

Upregulation of CD1d and ULBP3 on B cells from healthy donors and chronic lymphocytic leukaemia patients does not prime them for killing by γδ T cells

PONE-D-25-60204R1

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Additional Editor Comments (optional):

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2. Is the manuscript technically sound, and do the data support the conclusions??>

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Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: While a few comments remain partially unresolved, most of the major concerns have been properly addressed. In my opinion, the revised manuscript meets the standards for acceptance.

Reviewer #2: I appreciate the authors’ detailed responses, revisions, and additional experiments, which have strengthened the manuscript and clarified several points. I believe the manuscript is now suitable for publication.

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PLoS One. doi: 10.1371/journal.pone.0346684.r004

Acceptance letter

Xiaosheng Tan

PONE-D-25-60204R1

PLOS One

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Flow cytometric analysis of B cells and γδ T cell subsets.

(TIF)

pone.0346684.s001.tif^{(255.9KB, tif)}

S2 Fig. Identification of CD1d-binding glycolipids with agonist T cell activity within human PBMC and purified Vδ1 T cells.

(TIF)

pone.0346684.s002.tif^{(224.4KB, tif)}

S1 Table. David et al.Raw data.

(XLSX)

pone.0346684.s003.xlsx^{(25.6KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0346684.s005.docx^{(36.1KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0346684.ref001] 1.Bosch F, Dalla-Favera R. Chronic lymphocytic leukaemia: from genetics to treatment. Nat Rev Clin Oncol. 2019;16(11):684–701. doi: 10.1038/s41571-019-0239-8 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref002] 2.International CLL-IPI working group. An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data. Lancet Oncol. 2016;17(6):779–90. doi: 10.1016/S1470-2045(16)30029-8 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref003] 3.Wu S-J, Chiang C-J, Lin C-T, Tien H-F, Lai M-S. A nationwide population-based cross-sectional comparison of hematological malignancies incidences between Taiwan and the United States of America. Ann Hematol. 2016;95(1):165–7. doi: 10.1007/s00277-015-2513-x [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref004] 4.Waldron C, O’Brien D, Brophy S, Perera K, Crotty GM, Dunlea E, et al. Epidemiology of chronic lymphocytic leukaemia in an Irish subpopulation with total case ascertainment: an additional tool for health economic planning. Br J Haematol. 2022;196(5):e47–9. doi: 10.1111/bjh.17929 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref005] 5.Hallek M, Shanafelt TD, Eichhorst B. Chronic lymphocytic leukaemia. Lancet. 2018;391(10129):1524–37. doi: 10.1016/S0140-6736(18)30422-7 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref006] 6.Roberts AW, Davids MS, Pagel JM, Kahl BS, Puvvada SD, Gerecitano JF, et al. Targeting BCL2 with Venetoclax in Relapsed Chronic Lymphocytic Leukemia. N Engl J Med. 2016;374(4):311–22. doi: 10.1056/NEJMoa1513257 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref007] 7.Sharman JP, Coutre SE, Furman RR, Cheson BD, Pagel JM, Hillmen P, et al. Final Results of a Randomized, Phase III Study of Rituximab With or Without Idelalisib Followed by Open-Label Idelalisib in Patients With Relapsed Chronic Lymphocytic Leukemia. J Clin Oncol. 2019;37(16):1391–402. doi: 10.1200/JCO.18.01460 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref008] 8.Zhou H, Hu P, Yan X, Zhang Y, Shi W. Ibrutinib in Chronic Lymphocytic Leukemia: Clinical Applications, Drug Resistance, and Prospects. Onco Targets Ther. 2020;13:4877–92. doi: 10.2147/OTT.S249586 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref009] 9.Iyer P, Wang L. Emerging Therapies in CLL in the Era of Precision Medicine. Cancers (Basel). 2023;15(5):1583. doi: 10.3390/cancers15051583 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref010] 10.Godfrey DI, Uldrich AP, McCluskey J, Rossjohn J, Moody DB. The burgeoning family of unconventional T cells. Nat Immunol. 2015;16(11):1114–23. doi: 10.1038/ni.3298 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref011] 11.Mensurado S, Blanco-Domínguez R, Silva-Santos B. The emerging roles of γδ T cells in cancer immunotherapy. Nat Rev Clin Oncol. 2023;20(3):178–91. doi: 10.1038/s41571-022-00722-1 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref012] 12.Adams EJ, Gu S, Luoma AM. Human gamma delta T cells: Evolution and ligand recognition. Cell Immunol. 2015;296(1):31–40. doi: 10.1016/j.cellimm.2015.04.008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref013] 13.Melo AM, Mylod E, Fitzgerald V, Donlon NE, Murphy DM, Foley EK, et al. Tissue distribution of γδ T cell subsets in oesophageal adenocarcinoma. Clin Immunol. 2021;229:108797. doi: 10.1016/j.clim.2021.108797 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref014] 14.Tyler CJ, Doherty DG, Moser B, Eberl M. Human Vγ9/Vδ2 T cells: Innate adaptors of the immune system. Cell Immunol. 2015;296(1):10–21. doi: 10.1016/j.cellimm.2015.01.008 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref015] 15.Karunakaran MM, Willcox CR, Salim M, Paletta D, Fichtner AS, Noll A, et al. Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing. Immunity. 2020;52(3):487-498.e6. doi: 10.1016/j.immuni.2020.02.014 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref016] 16.Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells. Science. 1998;279(5357):1737–40. doi: 10.1126/science.279.5357.1737 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref017] 17.Bai L, Picard D, Anderson B, Chaudhary V, Luoma A, Jabri B, et al. The majority of CD1d-sulfatide-specific T cells in human blood use a semiinvariant Vδ1 TCR. Eur J Immunol. 2012;42(9):2505–10. doi: 10.1002/eji.201242531 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref018] 18.Uldrich AP, Le Nours J, Pellicci DG, Gherardin NA, McPherson KG, Lim RT, et al. CD1d-lipid antigen recognition by the γδ TCR. Nat Immunol. 2013;14(11):1137–45. doi: 10.1038/ni.2713 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref019] 19.Mangan BA, Dunne MR, O’Reilly VP, Dunne PJ, Exley MA, O’Shea D, et al. Cutting edge: CD1d restriction and Th1/Th2/Th17 cytokine secretion by human Vδ3 T cells. J Immunol. 2013;191(1):30–4. doi: 10.4049/jimmunol.1300121 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref020] 20.Pende D, Rivera P, Marcenaro S, Chang C-C, Biassoni R, Conte R, et al. Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxicity. Cancer Res. 2002;62(21):6178–86. [PubMed] [Google Scholar]

[pone.0346684.ref021] 21.Lazarova M, Steinle A. The NKG2D axis: an emerging target in cancer immunotherapy. Expert Opin Ther Targets. 2019;23(4):281–94. doi: 10.1080/14728222.2019.1580693 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref022] 22.Salih HR, Antropius H, Gieseke F, Lutz SZ, Kanz L, Rammensee H-G, et al. Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood. 2003;102(4):1389–96. doi: 10.1182/blood-2003-01-0019 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref023] 23.Bartkowiak J, Kulczyck-Wojdala D, Blonski JZ, Robak T. Molecular diversity of gammadelta T cells in peripheral blood from patients with B-cell chronic lymphocytic leukaemia. Neoplasma. 2002;49(2):86–90. [PubMed] [Google Scholar]

[pone.0346684.ref024] 24.Poggi A, Venturino C, Catellani S, Clavio M, Miglino M, Gobbi M, et al. Vdelta1 T lymphocytes from B-CLL patients recognize ULBP3 expressed on leukemic B cells and up-regulated by trans-retinoic acid. Cancer Res. 2004;64(24):9172–9. doi: 10.1158/0008-5472.CAN-04-2417 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref025] 25.Siegers GM, Dhamko H, Wang X-H, Mathieson AM, Kosaka Y, Felizardo TC, et al. Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells. Cytotherapy. 2011;13(6):753–64. doi: 10.3109/14653249.2011.553595 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref026] 26.Simões C, Silva I, Carvalho A, Silva S, Santos S, Marques G, et al. Quantification and phenotypic characterization of peripheral blood Vδ1 + T cells in chronic lymphocytic leukemia and monoclonal B cell lymphocytosis. Cytometry B Clin Cytom. 2019;96(2):164–8. doi: 10.1002/cyto.b.21645 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref027] 27.Correia DV, Fogli M, Hudspeth K, da Silva MG, Mavilio D, Silva-Santos B. Differentiation of human peripheral blood Vδ1+ T cells expressing the natural cytotoxicity receptor NKp30 for recognition of lymphoid leukemia cells. Blood. 2011;118(4):992–1001. doi: 10.1182/blood-2011-02-339135 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref028] 28.de Weerdt I, Hofland T, Lameris R, Endstra S, Jongejan A, Moerland PD, et al. Improving CLL Vγ9Vδ2-T-cell fitness for cellular therapy by ex vivo activation and ibrutinib. Blood. 2018;132(21):2260–72. doi: 10.1182/blood-2017-12-822569 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref029] 29.Almeida AR, Correia DV, Fernandes-Platzgummer A, da Silva CL, da Silva MG, Anjos DR, et al. Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinical-Grade Expansion/Differentiation and Preclinical Proof of Concept. Clin Cancer Res. 2016;22(23):5795–804. doi: 10.1158/1078-0432.CCR-16-0597 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref030] 30.Allan LL, Stax AM, Zheng D-J, Chung BK, Kozak FK, Tan R, et al. CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling. J Immunol. 2011;186(9):5261–72. doi: 10.4049/jimmunol.1003615 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref031] 31.Ghnewa YG, O’Reilly VP, Vandenberghe E, Browne PV, McElligott AM, Doherty DG. Retinoic acid induction of CD1d expression primes chronic lymphocytic leukemia B cells for killing by CD8+ invariant natural killer T cells. Clin Immunol. 2017;183:91–8. doi: 10.1016/j.clim.2017.08.002 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref032] 32.Salio M, Silk JD, Jones EY, Cerundolo V. Biology of CD1- and MR1-restricted T cells. Annu Rev Immunol. 2014;32:323–66. doi: 10.1146/annurev-immunol-032713-120243 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref033] 33.Zeng SG, Ghnewa YG, O’Reilly VP, Lyons VG, Atzberger A, Hogan AE, et al. Human invariant NKT cell subsets differentially promote differentiation, antibody production, and T cell stimulation by B cells in vitro. J Immunol. 2013;191(4):1666–76. doi: 10.4049/jimmunol.1202223 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref034] 34.Kotsianidis I, Nakou E, Spanoudakis E, Bouchliou I, Moustakidis E, Miltiades P, et al. The diagnostic value of CD1d expression in a large cohort of patients with B-cell chronic lymphoproliferative disorders. Am J Clin Pathol. 2011;136(3):400–8. doi: 10.1309/AJCP2F2DOXOTXHZA [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref035] 35.Chaudhry MS, Karadimitris A. Role and regulation of CD1d in normal and pathological B cells. J Immunol. 2014;193(10):4761–8. doi: 10.4049/jimmunol.1401805 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref036] 36.Zheng Z, Venkatapathy S, Rao G, Harrington CA. Expression profiling of B cell chronic lymphocytic leukemia suggests deficient CD1-mediated immunity, polarized cytokine response, altered adhesion and increased intracellular protein transport and processing of leukemic cells. Leukemia. 2002;16(12):2429–37. doi: 10.1038/sj.leu.2402711 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref037] 37.Zaborsky N, Gassner FJ, Asslaber D, Reinthaler P, Denk U, Flenady S, et al. CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells. Oncotarget. 2016;7(31):49459–69. doi: 10.18632/oncotarget.10372 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref038] 38.Melo AM, Zhang L, Dockry ÉF, Petrasca A, Ghnewa YG, Breen EP, et al. Novel thioglycoside analogs of α-galactosylceramide stimulate cytotoxicity and preferential Th1 cytokine production by human invariant natural killer T cells. Glycobiology. 2018;28(7):512–21. doi: 10.1093/glycob/cwy035 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref039] 39.Girardi E, Maricic I, Wang J, Mac T-T, Iyer P, Kumar V, et al. Type II natural killer T cells use features of both innate-like and conventional T cells to recognize sulfatide self antigens. Nat Immunol. 2012;13(9):851–6. doi: 10.1038/ni.2371 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref040] 40.Luoma AM, Castro CD, Mayassi T, Bembinster LA, Bai L, Picard D, et al. Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells. Immunity. 2013;39(6):1032–42. doi: 10.1016/j.immuni.2013.11.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref041] 41.Dieudé M, Striegl H, Tyznik AJ, Wang J, Behar SM, Piccirillo CA, et al. Cardiolipin binds to CD1d and stimulates CD1d-restricted γδ T cells in the normal murine repertoire. J Immunol. 2011;186(8):4771–81. doi: 10.4049/jimmunol.1000921 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref042] 42.Yazdanifar M, Barbarito G, Bertaina A, Airoldi I. γδ T Cells: The Ideal Tool for Cancer Immunotherapy. Cells. 2020;9(5):1305. doi: 10.3390/cells9051305 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref043] 43.Fisher JP, Heuijerjans J, Yan M, Gustafsson K, Anderson J. γδ T cells for cancer immunotherapy: A systematic review of clinical trials. Oncoimmunology. 2014;3(1):e27572. doi: 10.4161/onci.27572 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref044] 44.Taylor BC, Balko JM. Mechanisms of MHC-I Downregulation and Role in Immunotherapy Response. Front Immunol. 2022;13:844866. doi: 10.3389/fimmu.2022.844866 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref045] 45.Erdfelder F, Hertweck M, Filipovich A, Uhrmacher S, Kreuzer K-A. High lymphoid enhancer-binding factor-1 expression is associated with disease progression and poor prognosis in chronic lymphocytic leukemia. Hematol Rep. 2010;2(1):e3. doi: 10.4081/hr.2010.e3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref046] 46.Fernández-Calotti PX, Lopez-Guerra M, Colomer D, Pastor-Anglada M. Enhancement of fludarabine sensitivity by all-trans-retinoic acid in chronic lymphocytic leukemia cells. Haematologica. 2012;97(6):943–51. doi: 10.3324/haematol.2011.051557 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref047] 47.Lo-Coco F, Cicconi L, Breccia M. Current standard treatment of adult acute promyelocytic leukaemia. Br J Haematol. 2016;172(6):841–54. doi: 10.1111/bjh.13890 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref048] 48.Adekola KUA, Dalva Aydemir S, Ma S, Zhou Z, Rosen ST, Shanmugam M. Investigating and targeting chronic lymphocytic leukemia metabolism with the human immunodeficiency virus protease inhibitor ritonavir and metformin. Leuk Lymphoma. 2015;56(2):450–9. doi: 10.3109/10428194.2014.922180 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref049] 49.Liu J, Chen G, Pelicano H, Liao J, Huang J, Feng L, et al. Targeting p53-deficient chronic lymphocytic leukemia cells in vitro and in vivo by ROS-mediated mechanism. Oncotarget. 2016;7(44):71378–89. doi: 10.18632/oncotarget.12110 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref050] 50.Koczula KM, Ludwig C, Hayden R, Cronin L, Pratt G, Parry H, et al. Metabolic plasticity in CLL: adaptation to the hypoxic niche. Leukemia. 2016;30(1):65–73. doi: 10.1038/leu.2015.187 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref051] 51.Delgado J, Doubek M, Baumann T, Kotaskova J, Molica S, Mozas P, et al. Chronic lymphocytic leukemia: A prognostic model comprising only two biomarkers (IGHV mutational status and FISH cytogenetics) separates patients with different outcome and simplifies the CLL-IPI. Am J Hematol. 2017;92(4):375–80. doi: 10.1002/ajh.24660 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref052] 52.Kwok M, Wu CJ. Clonal Evolution of High-Risk Chronic Lymphocytic Leukemia: A Contemporary Perspective. Front Oncol. 2021;11:790004. doi: 10.3389/fonc.2021.790004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref053] 53.Zarobkiewicz M, Kowalska W, Szymańska A, Lehman N, Kowalczyk B, Tomczak W, et al. γδ T Are Significantly Impacted by CLL Burden but Only Mildly Influenced by M-MDSCs. Cancers (Basel). 2025;17(2):254. doi: 10.3390/cancers17020254 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0346684.ref054] 54.Lalor SJ, McLoughlin RM. Memory γδ T Cells-Newly Appreciated Protagonists in Infection and Immunity. Trends Immunol. 2016;37(10):690–702. doi: 10.1016/j.it.2016.07.006 [DOI] [PubMed] [Google Scholar]

[pone.0346684.ref055] 55.Comeau K, Paradis P, Schiffrin EL. Human and murine memory γδ T cells: Evidence for acquired immune memory in bacterial and viral infections and autoimmunity. Cell Immunol. 2020;357:104217. doi: 10.1016/j.cellimm.2020.104217 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Upregulation of CD1d and ULBP3 on B cells from healthy donors and chronic lymphocytic leukaemia patients does not prime them for killing by γδ T cells

Julie David

Amy Walsh

Ke Sin Seow

Ellen Walsh

Stefan Elekes

Rohit Upadhyay

Nawal A B Taher

Samiha Al Siyabi

Ashanty M Melo

Carmel Waldron

Elisabeth Vandenberghe

Anthony M McElligott

Derek G Doherty

Roles

Abstract

Introduction

Materials and methods

Subjects and ethical statement

Antibodies and flow cytometry

Enrichment of peripheral B cells

Treatment of B cells with RAR-α agonists

Generation of primary cultures of γδ T cells

Analysis of cytolytic degranulation by γδ T cells

Priming CLL cells for cytotoxicity by γδ T cells using glycolipids

Fig 1. Glycolipids used in the present study.

Statistical analysis

Results

Characteristics of the CLL patients

Table 1. Patient demographics and disease characteristics.

Reduced CD1d expression by B cells and increased Vδ1 and Vδ3 T cell frequencies in CLL patients

Fig 2. Phenotypic analysis of PBMCs from CLL patients and healthy donors.

γδ T cells do not spontaneously degranulate in response to B cells from CLL patients or healthy donors

Fig 3. γδ T-cells do not degranulate in response to B cells from CLL patients or healthy donors.

The viability of CLL cells decreases in co-culture with γδ T cells and this is not enhanced by ATRA

Fig 4. Dose dependent reduction in CLL cell viability inγδ T cell co-cultures.

Upregulation of CD1d and ULBP3 on B cells from CLL patients and healthy controls by ATRA does not sensitise them for killing by γδ T cells

Fig 5. Upregulation of CD1d and ULBP3 on B cells from healthy controls and CLL patients using RAR-α agonists.

Fig 6. RAR-α agonist treatment of B cells from CLL patients and healthy donors did not sensitise them for killing by γδ T-cells.

Addition of CD1d-binding glycolipids to B cells from healthy donors and CLL patients does not sensitise them for killing by Vδ1, Vδ2 and Vδ3 T cells

Fig 7. Treatment of 100 ng/mL ATRA in addition of 5 different glycolipids; α-galactosylceramide (GC), sulfatide, lyso-sulfatide, cardiolipin and TMC to B cells from healthy donors and CLL patients did not sensitise them for cytolytic degranulation by γδ T cells.

Discussion

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Xiaosheng Tan

Roles

Author response to Decision Letter 1

Decision Letter 1

Xiaosheng Tan

Roles

Acceptance letter

Xiaosheng Tan

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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