Neuroblastoma killing properties of Vδ2 and Vδ2-negative γδT cells following expansion by artificial antigen-presenting cells

Abstract

Purpose: The majority of circulating human γδT lymphocytes are of the Vγ9Vδ2 lineage, and have T-cell receptor (TCR) specificity for nonpeptide phosphoantigens. Previous attempts to stimulate and expand these cells have therefore focused on stimulation using ligands of the Vγ9Vδ2 receptor, whereas relatively little is known about variant blood γδT subsets and their potential role in cancer immunotherapy.

Experimental design: To expand the full repertoire of γδT without bias toward specific TCRs, we made use of artificial antigen-presenting cells loaded with an anti γδTCR antibody that promoted unbiased expansion of the γδT repertoire. Expanded cells from adult blood donors were sorted into 3 populations expressing respectively Vδ2 TCR chains (Vδ2(+)), Vδ1 chains (Vδ1(+)), and TCR of other δ chain subtypes (Vδ1(neg)Vδ2(neg)).

Results: Both freshly isolated and expanded cells showed heterogeneity of differentiation markers, with a less differentiated phenotype in the Vδ1 and Vδ1(neg)Vδ2(neg) populations. Expanded cells were largely of an effector memory phenotype, although there were higher numbers of less differentiated cells in the Vδ1(+) and Vδ1(neg)Vδ2(neg) populations. Using neuroblastoma tumor cells and the anti-GD2 therapeutic mAb ch14.18 as a model system, all three populations showed clinically relevant cytotoxicity. Although killing by expanded Vδ2 cells was predominantly antibody dependent and proportionate to upregulated CD16, Vδ1 cells killed by antibody-independent mechanisms.

Conclusions: In conclusion, we have demonstrated that polyclonal-expanded populations of γδT cells are capable of both antibody-dependent and -independent effector functions in neuroblastoma.

Figure 1. Expansion of diverse populations γδT cells from healthy donors and neuroblastoma patients

Expansion of γδT cells from PBMC using immobilized anti-γδTCR antibody (A) and from pure preparations of γδT cells using aAPC, B1-coated aAPC, immobilised B1 or IPP (B). γδT subsets as revealed by flow cytometry after expansion are shown in (C) (representative of >6 donors), (D) and (F). γδT cells isolated from thawed healthy-donor PBMC varied in their expansion potential (E), whereas γδT cells isolated from fresh blood of neuroblastoma patients, taken at point of diagnosis, were more consistent (G). Error bars represent SEM and each data point represents an individual donor.

Figure 2. Joining region diversity and Vγ/Vδ chain usage in fresh PBMC and expanded γδT cells from the same donors

Heat maps demonstrating variable and joining gene segment usage, as revealed by next generation RNA sequencing, in gamma and delta chain T-cell receptors in PBMC populations of healthy donors, before and after expansion using IPP or aAPC+B1. Relative frequency of V and J pairings is shown in blue (low abundance), through to red (high abundance). PBMC donor 1 (A) demonstrates a dominance of Vγ9Vδ2, which is reinforced following a 7-day expansion with IPP and IL2. PBMC donor 2 (B) demonstrates more diversity prior to expansion using aAPC and B1 and there is greater gamma chain diversity in the Vδ1+ subset than in the Vδ2+ subset. In the Vδ1^negVδ2^neg population sorted from donor 3 (C) there is marked diversity in both delta and gamma chain joining segment and V segment usage in both PBMC and expanded cells.

Figure 3. Memory phenotypes of un-stimulated γδT cells from healthy donors or neuroblastoma patients at the point of diagnosis

The representative FACS plots (A, D) show the total T-cell population from a neuroblastoma patient. Memory phenotype of Vδ1 and Vδ2 γδT cells from healthy donors (B) and neuroblastoma patients (C) (n=6) using CD45RA and CD62L staining. Memory phenotype of Vδ1 and Vδ2 γδT cells using CD27 and CD45RA from a different set of healthy donors (E) and some of the same neuroblastoma patients (F) (n=4) is also shown. Error bars represent SEM.

Figure 4. Memory and exhaustion markers in γδT cells after expansion with aAPC+B1

(A) Expression of CD27, CD45RA and CD62L as measured by flow cytometry in Vδ1, Vδ2 and Vδ1^negVδ2^neg γδT cells following 28 days of expansion using aAPC+B1; data derived from 8 donors and error bars represent SEM. (B) Expression of the PD-1 in αβT cells and γδT cells from the same donors at baseline and after 14 days of expansion using weekly stimulation with either aAPC+B1 (γδT cells) or CD3/CD28 Dynabeads (αβT cells) data derived from 3 donors and error bars represent SEM. MFI= Mean fluorescence intensity

Figure 5. Differential cytotoxic profile of Vδ1, Vδ2 and Vδ1−/Vδ2− γδT cells against neuroblastoma cell lines

(A) Antibody independent killing of GD2+ neuroblastoma cell lines by polyclonal populations of Vδ1+ and Vδ2+ γδT cells; n=11 for Kelly data; n= 3 for LAN1 data.. (B and C) Cytotoxicity of IPP expanded Vδ2+ cells is significantly enhanced by target opsonisation with ch14.18; raw data shown in B (n=3) and antibody dependent and independent components of killing shown in C (n=16 for Kelly and n= 3 for LAN1 and SKNAS) . (D) Comparative antibody dependent and independent killing of Kelly cells by Vδ2+ γδT cells expanded with IPP (n=9) or aAPC (n=7). (E) . Vδ1+, Vδ2+ and Vδ1^negVδ2^neg show different patterns of antibody dependent and independent cytotoxicity (n= between 5 and 11 for Kelly and n=3 or 4 for LAN1). (F) Interferon gamma and Tumor necrosis factor-alpha secretion as determine by cytokine bead array following co-culture of IPP expanded Vδ2+ with opsonized and non-opsonized Kelly cells (n=3). (G) Granzyme B in supernatant of expanded Vδ1+ and Vδ2+ γδT cells co-cultured with γδT cell sensitive GD2+ tumor cell lines Kelly and LAN1, expressed as fold change of –ve control (γδT cell insensitive cell line with irrelevant antibody)..

Figure 6. FcγR staining correlates with cytotoxicity and differentiation phenotype

Expression of Fcγ receptors CD16 (A), CD32 (B) and CD64 (C) in Vδ1+ and Vδ2+ γδT cells over a 3 week expansion period (data from 6-7 donors). (D) Mean fluorescence intensity of CD16 surface staining from sorted populations (n=7). (E) CD16 expression correlates with the ability of γδT cells to exert ADCC and (F), in Vδ2+ cells CD16 is inversely correlated with CD62L expression.