Fermat's Library | Characterisation and induction of tissue-resident gamma delta T-cells to target hepatocellular carcinoma annotated/explained version.

ARTICLE

Characterisation and induction of tissue-resident

gamma delta T-cells to target hepatocellular

carcinoma

Nekisa Zakeri

, Andrew Hall

, Leo Swadling

, Laura J. Pallett

, Nathalie M. Schmidt

Mariana O. Diniz

, Stephanie Kucykowicz

, Oliver E. Amin

, Amir Gander

, Massimo Pinzani

Brian R. Davidson

, Alberto Quaglia

& Mala K. Maini

✉

Immunotherapy is now the standard of care for advanced hepatocellular carcinoma (HCC), yet

many patients fail to respond. A major unmet goal is the boosting of T-cells with both strong

HCC reactivity and the protective advantages of tissue-resident memory T-cells (T

Here, we show that higher intratumoural frequencies of γδ T-cells, which have potential for

HLA-unrestricted tumour reactivity, associate with enhanced HCC patient survival. We

demonstrate that γδ T-cells exhibit bona ﬁde tissue-residency in human liver and HCC, with

γδT

showing no egress from hepatic vasculature, persistence for >10 years and superior

anti-tumour cytokine production. The Vγ9Vδ2 T-cell subset is selectively depleted in HCC but

can efﬁciently target HCC cell lines sensitised to accumulate isopentenyl-pyrophosphate by

the aminobisphosphonate Zoledronic acid. Aminobisphosphonate-based expansion of per-

ipheral Vγ9Vδ2 T-cells recapitulates a T

phenotype and boosts cytotoxic potential. Thus,

our data suggest more universally effective HCC immunotherapy may be achieved by com-

bining aminobisphosphonates to induce Vγ9Vδ2T

capable of replenishing the depleted

pool, with additional intratumoural delivery to sensitise HCC to Vγ9Vδ2T

-based targeting.

https://doi.org/10.1038/s41467-022-29012-1

OPEN

Division of Infection & Immunity, Institute of Immunity & Transplantation, University College London, London, UK.

Institute for Liver & Digestive Health,

Royal Free London NHS Foundation Trust, London, UK.

Division of Surgery, University College London, London, UK.

Department of Cellular Pathology,

Royal Free London NHS Foundation Trust and UCL Cancer Institute, London, UK.

✉

email: m.maini@ucl.ac.uk

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1234567890():,;

epatocellular carcinoma (HCC) is the third most common

cause of cancer-related death worldwide

1,2

, often pre-

senting at an advanced stage when treatment options are

limited. Immunotherapy aims to induce T-cells with efﬁcient

tumour targeting and durable immune surveillance capacity. The

potential for immunotherapy to reduce the high mortality of

HCC has been exempliﬁed by the response to the combination of

anti-programmed death ligand-1 (PD-L1) and anti-vascular

endothelial growth factor (VEGF), which has now become

standard-of-care in advanced disease; however, this combination

still only achieves responses in around one third of patients

3,4

underscoring the urgent need for additional immunotherapeutic

approaches that can deliver more consistent and sustained

responses. A major reason for the failure of existing checkpoint

inhibitors is the scarcity of T-cells capable of recognising

expressed tumour antigens in many individuals, such that

insufﬁcient tumour-speciﬁc responses are available for

boosting

5–7

. A further limitation of existing immunotherapies is

that endogenously boosted or adoptively transferred T-cells may

fail to inﬁltrate and survive within the tumour and/or maintain

functionality in this immunosuppressive environment

2,4,8

. Here,

we present data supporting a novel strategy for expanding γδ

T-cells with the potential to lyse HCC and to persist long-term,

retaining functionality within the tumour niche.

γδ T-cells can play a critical role in tumour immuno-

surveillance, exempliﬁed by the favourable prognostic signature

of tumour-inﬁltrating γδ T-cells across many human cancer

types

9–13

, and an increased susceptibility to cancer in γδ T-cell

deﬁcient mice

14–16

. γδ T-cells are attractive effector cells for

cancer immunotherapy, due to their MHC-unrestricted antigen

recognition and lack of dependence on cancer neo-antigens

17,18

The main subtypes in humans, Vδ1 and Vδ2 T-cells, both

recognise stress-induced molecules including MHC-Class 1

chain-related protein A/B and UL16-binding proteins, expressed

at variable levels on tumour cells

11,17–20

. Additionally, the pre-

dominant γδ T-cell subset in blood, Vγ9Vδ2 T-cells, can easily be

expanded on a clinical scale for adoptive cell transfer to target

phosphorylated intermediates of the isoprenoid biosynthesis

pathway (isopentenyl pyrophosphate, IPP), upregulated in

transformed cells and presented via butyrophilin 2A1 and 3A1

transmembrane proteins

20,21

. Early phase clinical trials have

demonstrated adoptive cell transfer of in vitro expanded blood

Vγ9Vδ2 T-cells to be safe and well-tolerated in several cancer

types

18,22–24

, but their overall efﬁcacy remains low.

Accumulating evidence suggests that exploiting features of

tissue-resident memory T-cells (T

) may help to uncover novel

strategies for more effective cancer immunotherapies

25–27

are phenotypically and functionally distinct sentinels in the liver,

capable of long-lived retention, and well positioned for rapid and

potent front-line immunosurveillance

28–30

. αβ T

are marked

by the expression of the tissue retention molecules CD69 (a

negative regulator of sphingosine-1-phosphate-mediated T cell

egress), and the integrins CD103 (binding E-cadherin on epi-

thelial cells) and/or CD49a (binding collagen IV)

25–27

. The

control of tumours has been shown to be highly dependent on αβ

in murine models and human studies of various cancers

including HCC

27,31–40

, but the contribution of γδ T

has not

yet been investigated in HCC. Although most studies of T-cell

tissue-residence have focused on αβCD8

, the Vδ1 subset of γδ

T-cells are generally considered to be tissue-resident, supported

by recent data conﬁrming expression of tissue retention/homing

markers and distinct T cell receptor (TCR) clones by Vδ1 T-cells

in human liver

. By contrast, Vγ9Vδ2 T-cells are classically

regarded as the circulating γδ T-cell subtype, and have yet to be

shown to be capable of acquiring a functionally intact T

phenotype in the liver or HCC.

In this study, we ﬁnd that γδ T-cell inﬁltration of HCC

associates with a favourable prognosis, assessed both by tumour

size and patient survival. We demonstrate that Vγ9Vδ2 T-cells

are selectively reduced in frequency in the blood, livers, and

tumours of patients with HCC, but display the capacity to acquire

an intratumoural γδ T

(CD69

CD49a

or CD69

CD103

)

phenotype. Intratumoural γδ T-cells with a T

phenotype show

enhanced capacity to maintain the production of cytokines

favouring their survival and their anti-tumour potential, in the

liver and within HCC tumour-inﬁltrating lymphocytes (TIL). The

bona ﬁde nature of the γδ T

phenotype is conﬁrmed by a lack

of hepatic vasculature egress and by persistence of CD69

CD49a

γδ T

progeny for more than a decade in human leukocyte

antigen (HLA)-mismatched liver allografts. Therefore, we develop

a rationale for use of the aminobisphosphonate Zoledronic acid

(ZOL) to optimise the anti-tumour efﬁcacy of Vγ9Vδ2 T-cells

that can become locally resident; we show that its local applica-

tion to HCC cell lines allows IPP accumulation for speciﬁc lysis

by liver/HCC-inﬁltrating Vγ9Vδ2 T-cells, whilst its use for

in vitro clinical-scale expansion of peripheral Vγ9Vδ2 T-cells

induces a liver-homing T

proﬁle.

Results

Compartmentalisation of Vδ1 and Vδ2 T-cells with a tissue-

resident phenotype in human liver. To examine the potential for

the two major subsets of human γδ T-cells, Vδ1 and Vδ2, to

acquire tissue-residence, we ﬁrst compared their relative abun-

dance in human liver (tumour-free liver tissue from surgical

resections or explants) to paired peripheral blood samples (gating

strategy Supplementary Fig. 1a). Vδ1 T-cells are regarded as

prototypic tissue γδ T-cells and were accordingly signiﬁcantly

enriched in the liver compared to their very low frequencies in

matched blood samples (Supplementary Fig. 1b). Vδ2 T-cells,

whilst more frequent in blood than Vδ1, were also detectable in

the liver, comprising up to 5% of intrahepatic CD45

leukocytes

(Supplementary Fig 1b). Intrahepatic Vδ2 T-cells consisted pri-

marily of Vγ9

Vδ2 T-cells (henceforth referred to as Vγ9Vδ2),

with a very low frequency of the recently described Vγ9

−

Vδ2

sub-population

(Supplementary Fig. 1c).

We next investigated if Vδ1 and Vγ9Vδ2 T-cells could express

a tissue-resident phenotype in human liver, assessing the

prototypic markers CD69, CD103, and CD49a. We found that

subpopulations of both Vδ1 and Vγ9Vδ2 T-cells isolated from

human liver expressed combinations of tissue-retention markers

characteristic of T

, that could not be detected in paired

peripheral blood (Fig. 1a, b). Vδ1 and Vγ9Vδ2 T-cells co-

expressing the tissue retention molecules CD69 with CD103 or

CD49a were compartmentalised in the liver and excluded from

blood (Fig. 1a, b). These tissue-resident γδ T-cell subsets were

primarily composed of CD27

−

CD45RA

−

effector memory cells

(Supplementary Fig. 1d), and consisted of distinct

CD69

CD103

or CD69

CD49a

subsets, with a smaller sub-

population expressing all three tissue-retention markers (Fig. 1c).

CD69

CD49a

co-expression was more prevalent than

CD69

CD103

on intrahepatic Vδ1 and Vγ9Vδ2 T-cells (Fig.

1c). The proportion of intrahepatic T-cells expressing

CD69

CD49a

showed a strikingly similar range within the

cohort for Vδ1, Vγ9Vδ2, and αβCD8

T-cells (mean

22.2 ± 18.6%, 21.1 ± 15.2%, 20.1 ± 12.3%, respectively, Supple-

mentary Fig. 1e), with a signiﬁcant correlation between expres-

sion levels on Vγ9Vδ2 and αβCD8

T-cells within individuals

(Supplementary Fig. 1e), suggesting shared determinants of these

residency markers. The percentage of CD69

CD103

expression

on intrahepatic Vδ1 and Vγ9Vδ2 T-cells demonstrated a weak

inverse correlation with patient age (Supplementary Fig. 1f). No

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sex related differences in Vδ1 and Vγ9Vδ2T

expression were

observed (Supplementary Fig. 1f).

To further assess Vδ1 and Vγ9Vδ2 T-cells tissue-homing

capability, we measured expression of the prototypic liver

chemokine receptors CXCR6 and CXCR3. On ex vivo staining

of bulk intrahepatic lymphocytes (IHL), we found that both Vδ1

and Vγ9Vδ2T

(CD69

CD49a

or CD69

CD103

) showed

increased expression of CXCR6 and CXCR3 in comparison to

their non-T

counterparts (CD69

−

CD103

−

or CD69

−

CD49a

−

;

Fig. 1d, e). Ligands for these chemokine receptors are expressed

by hepatic sinusoidal endothelium, promoting homing, tethering

and/or transmigration into the parenchyma

43,44

. Consistent with

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ﬁrm tethering or transmigration beyond the vasculature, we

observed that Vδ1 and Vγ9Vδ2T

were less likely to be ﬂushed

out of the liver in perfusates from healthy donor liver allografts

than to be isolated from liver tissue digests (healthy background

liver from resected colorectal cancer liver metastases, CRCLM)

(Fig. 1f). However, the isolation of a clear population of CD49a

expressing Vδ1 and Vγ9Vδ2 T-cells from perfusates (Fig. 1f),

albeit at a lower frequency than in liver tissue, is in line with the

demonstration that αβCD8

can patrol the liver sinusoidal

vasculature

28,45

In keeping with classical features of αβ T-cell tissue-residency,

intrahepatic Vδ1 and Vγ9Vδ2T

showed reduced expression of

the endothelial homing receptor CX3CR1, and lacked expression

of the lymph node homing receptor CD62L (L-selectin)

(Supplementary Fig. 1g, h). In addition, γδ T

demonstrated a

transcription factor proﬁle characteristic of αβ T

with higher

Blimp-1 and lower Eomes expression than their non-T

counterparts

28,46

(Supplementary Fig. 1i). Vδ1 and Vγ9Vδ2

also had a trend towards higher expression of Tcf1

(Supplementary Fig. 1i), a transcription factor conferring stem

cell-like longevity on T-cells

Long-lived hepatic retention and replenishment of Vδ1 and

Vγ9Vδ2T

. The absence of Vδ1 and Vγ9Vδ2 T-cells with a

tissue-resident phenotype (CD69

CD103

or CD69

CD49a

)

in peripheral venous blood points to an inability of these cells to

egress from the liver. However, it is difﬁcult to deﬁnitively con-

clude from this that they are completely compartmentalised in

the liver, since γδ T

leaving the liver vasculature at low fre-

quencies would be difﬁcult to detect following dilution in the

peripheral circulation. We, therefore, sampled hepatic venous

blood (draining directly from the liver) and portal venous blood

(entering the liver), obtained from patients with cirrhosis

undergoing transjugular intrahepatic portosystemic shunt pro-

cedures, in order to examine for potential low-level egress of γδ

from the liver or gut/spleen, respectively. γδ T-cell frequency

appeared higher in hepatic venous blood compared to portal

venous blood (Fig. 2a). γδ T-cells expressing CD69

CD103

CD69

CD49a

could not be detected in hepatic venous blood or

portal venous blood (Fig. 2b), providing further evidence to

support the tissue-compartmentalisation of intrahepatic Vδ1 and

Vγ9Vδ2T

subsets, with no low-level egress from the liver, gut

or spleen.

In addition to tissue compartmentalisation, another feature of

bona ﬁ de T

is their long-lived retention within organs, critical

for sustained immune surveillance of cancer. To investigate

whether Vδ1 and Vγ9Vδ2 T-cells with a tissue-resident

phenotype had this capacity, we tracked their longevity using

donor and recipient HLA-mismatched liver allografts. Utilising

HLA-speciﬁc monoclonal antibody staining, we were able to

differentiate between the original donor-derived (liver-resident)

γδ T-cells and the recipient-derived (blood-derived) γδ T-cells in

two liver allografts, explanted 7 and 11 years following their initial

transplantation (Fig. 2c, Supplementary Fig. 2a, patient char-

acteristics in Supplementary Table 1). By both of the time points

examined, the majority of the intrahepatic γδ T-cell population

had been replaced with recipient-derived γδ T-cells (Fig. 2c).

However, a small population of donor-derived γδ T-cells,

constituting around 3% of the total intrahepatic γδ T-cell pool

had persisted within the liver allografts, including in the explant

obtained 11 years following transplantation (Fig. 2c). No donor-

derived γδ T-cells were detected in the peripheral blood of the

recipients, arguing against systemic chimerism accounting for

intrahepatic persistence (Supplementary Fig. 2b). Of note, the

proportion of Vδ1 and Vγ9Vδ2 T-cells was similar between the

recipient-derived and donor-derived γδ T-cell populations,

suggesting these equilibrate according to liver-speciﬁc factors

(Fig. 2c).

The donor-derived intrahepatic γδ T-cell population persisting

in liver allografts displayed a predominant CD69

CD49a

phenotype, with little CD103 co-expression, indicating that

CD49a expression, in particular, marked the subset of γδ

T-cells capable of long-term retention and survival or renewal

in the liver (pan-γδ in Fig. 2d, Vδ1 and Vγ9Vδ2 T-cell subsets in

Supplementary Fig. 2c, d). Furthermore, the larger recipient-

derived γδ T-cell fraction was capable of acquiring high CD69

expression, with a small proportion co-expressing CD49a or

CD103 (Fig. 2d, Supplementary Fig. 2c, d). This demonstrates

that recipient γδ T-cells inﬁltrating into the liver from the

peripheral circulation were able to partially acquire a de novo

phenotype to replenish the intrahepatic γδ T

cell pool.

Although limited by the scarce availability of these valuable

samples, our data revealed the potential for a small population of

long-lived or self-renewing CD69

CD49a

γδ T

to persist in

the human liver for more than a decade, whilst being partially

replenished from the peripheral circulation.

Distinct functional proﬁle of hepatic γδ T

. To further assess

the suitability of liver γδ T

for immunotherapeutic targeting, we

assessed their functional potential by ex vivo analysis of freshly

isolated IHL. Both Vδ1 and Vγ9Vδ2T

(CD69

CD49a

CD69

CD103

) tended to express higher levels of the T-cell

activation marker HLA-DR than their non-resident counterparts

(Fig. 3a, Supplementary Fig. 3a). However, Vδ1 and Vγ9Vδ2T

displayed markedly reduced cytotoxic potential, demonstrated by

lower expression of the serine protease granzyme B on direct

ex vivo staining (Fig. 3b, Supplementary Fig. 3b). By contrast, both

Vδ1 and Vγ9Vδ2T

were signiﬁcantly more capable of rapid

production of the pro-survival cytokine IL-2 than their non-T

counterparts, with a striking mean of 65% and 74% of Vδ1 and

Fig. 1 Compartmentalisation of Vδ1 and Vɣ9Vδ2 T-cells with a tissue-resident phenotype in human liver. a Representative ﬂow cytometry plots and

summary data of CD69

CD103

or CD69

CD49a

co-expression on Vδ1 and Vɣ9Vδ2 T-cells from intrahepatic lymphocytes (IHL) of tumour-free liver

tissue compared to paired peripheral blood mononuclear cells (PBMC) (n = 35–44; p < 0.0001). b t-distributed stochastic neighbour embedding (t-SNE)

was applied to ﬂow cytometry expression data (concatenated PBMC n = 3 and IHL n = 5) of Vδ1 (blue) and Vɣ9Vδ2 (red) T cells (top row); plots coloured

by CD69, CD49a and CD103 expression on Vδ1 T-cells (middle row) and Vɣ9Vδ2 T-cells (bottom row). c Frequencies of CD69

CD103

, CD69

CD49a

and CD69

CD49a

CD103

intrahepatic Vδ1 and Vɣ9Vδ2 T-cells (n = 40; Vδ1 p = 0.02, p = 0.004, p < 0.0001; Vɣ9Vδ2 p = 0.02, p = 0.0008;

p < 0.0001). d Frequencies of CXCR6-expressing CD69

CD103

or CD69

CD49a

Vδ1 and Vɣ9Vδ2T

(n = 17; Vδ1 p < 0.0001; Vɣ9Vδ2 p = 0.002,

p = 0.02). e Frequencies of CXCR3-expressing CD69

CD103

(n = 14) or CD69

CD49a

(n = 9–11) Vδ1 and Vɣ9Vδ2T

(Vδ1 p = 0.001, p = 0.004;

Vɣ9Vδ2 p = 0.008, p = 0.02). f Frequencies of Vδ1 and Vɣ9Vδ2T

in IHL from healthy liver tissue (disease-free margins of CRCLM) (n = 24) compared

to healthy donor liver transplant perfusates (n = 17, Vδ1 p < 0.0001, p = 0.03; Vɣ9Vδ2 p < 0.001, p = 0.0096). Each symbol represents a study participant,

with error bars showing the mean ± SEM (a, c–f); two-tailed p-values were determined using Wilcoxon matched-pairs signed rank test (a, d, e),

Kruskal–Wallis test (ANOVA) followed by Dunn’s post-hoc multiple comparisons test (c), Mann–Whitney test (f). *p < 0.05; **p < 0.01; ***p < 0.001,

****p < 0.0001.

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Vγ9Vδ2T

, respectively, producing IL-2 after just 4 h of sti-

mulation (Fig. 3c, Supplementary Fig. 3c). The majority of Vδ1

and Vγ9Vδ2T

also produced the anti-tumour cytokine IFN-γ

rapidly upon stimulation (Fig. 3d, Supplementary Fig. 3d). PD-1

expression was higher on Vδ1andVγ9Vδ2T

compared to

their non-T

counterparts ex vivo (Fig. 3e, Supplementary Fig.

3e); however, PD-1

high

Vδ1 and Vγ9Vδ2 T-cells maintained their

capacity to produce IFN-γ upon stimulation (Fig. 3f), congruent

with the ﬁnding that PD-1-expressing αβCD8

do not

represent a functionally exhausted population

28,35

. Furthermore,

no signiﬁcant increase in intrahepatic Vγ9Vδ2 T-cell IFN-γ or

Granzyme B expression was observed using anti-PD-L1 blockade

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in combination with γδ-TCR stimulation in vitro (Supplementary

Fig. 3f).

Taken together, assessing the functional potential of Vδ1 and

Vγ9Vδ2T

isolated from the human liver directly ex vivo or

after 4 h stimulation showed they are activated and skewed

towards IL-2 and IFN-γ rather than cytotoxic effector function.

γδ T-cell counts in HCC associate with tumour size and patient

survival. Having established a signature of γδ T-cells with clas-

sical tissue-residency features in human liver, we next investi-

gated whether γδ T-cells could exert a role in HCC. First, we used

immunostaining to compare the absolute numbers of γδ T-cells

within HCC and paired background liver tissue from patients

undergoing curative surgical resection (n = 28, patient char-

acteristics, Supplementary Table 2). γδ T-cells could be visualised

inﬁltrating HCC (Fig. 4a, Supplementary Fig. 4a) but their

numbers were signiﬁcantly reduced compared to paired tumour-

free liver tissue (Fig. 4b). This trend was unchanged by the pre-

sence or absence of underlying cirrhosis (Supplementary Fig. 4b).

The γδ/CD3

T-cell ratio was similarly reduced within HCC, due

to preservation of total CD3

T-cell numbers between HCC and

background liver tissue (Fig. 4b).

Importantly, higher intratumoural γδ T-cell numbers were

associated with both smaller HCC tumour size at resection

(maximum diameter ≤ 3 cm) (Fig. 4c), and enhanced 3-year

overall patient survival following surgical resection (Fig. 4d). This

ﬁnding was speciﬁc to intratumoural γδ T-cells, with no

associations observed between total CD3

T-cell or γδ T-cell

numbers within background liver and HCC tumour size or 3-year

overall patient survival (Fig. 4c, d, Supplementary Fig. 4c–e).

Consistent with this, analysis of the Cancer Genome Atlas

database using the Gene Expression Proﬁling Interactive Analysis

2 (GEPIA2) web server

, demonstrated a γδ-TCR gene signature

(TRDC, TRGC1, TRGC2) to be signiﬁcantly associated with

overall survival in a larger cohort of HCC patients (n = 364), with

higher Vγ9Vδ2 and non-Vγ9Vδ2 γδ-TCR gene expression

signatures both associated with increased patient survival

(Supplementary Fig. 4f, g)

48,49

Vγ9Vδ2 T-cells are selectively depleted, but can acquire tissue-

residence, within HCC TILs. To further deﬁne the depletion of

γδ T-cells within HCC, we quantiﬁed Vδ1 and Vγ9Vδ2 T-cell

subsets in blood, tumour-free liver and tumour tissue from

patients with HCC compared to colorectal cancer liver metastases

(CRCLM), by ﬂow cytometry (patient characteristics, Supple-

mentary Tables 3 and 4). No signiﬁcant differences in the fre-

quencies of Vδ1 T-cells were seen, but Vγ9Vδ2 T-cells were

signiﬁcantly reduced in the blood, liver and tumour of HCC

compared to CRCLM patients (Fig. 5a). The reduction of Vγ9Vδ2

T-cells in HCC was likely attributable to the background liver

cirrhosis in these donors, since we found a similar depletion in

cirrhotic livers without HCC (Supplementary Fig. 5a), supported

by recent literature describing increased activation and

subsequent apoptosis of liver sinusoidal Vγ9Vδ2 T-cells in

cirrhosis

Vδ1 T-cells had an increased proportion of terminally

differentiated effector memory (TEMRA, CD27

−

CD45RA

) cells

(Fig. 5b, Supplementary Fig. 5b), and demonstrated higher

expression of the T-cell activation markers HLA-DR, CD38 and

CD25 on direct ex vivo staining compared to Vγ9Vδ2 T-cells

within HCC and paired tumour-free liver (Fig. 5c, Supplementary

Fig. 5c). Despite their reduced frequency, Vγ9Vδ2 T-cells

inﬁltrating background liver in HCC patients were better able

to produce IFN-γ following 4 h stimulation than Vδ1 T-cells (Fig.

5d). Vγ9Vδ2 T-cells within HCC TILs also maintained high IFN-

γ expression upon stimulation in the majority of HCC samples

(Fig. 5d), although they had reduced granzyme B expression

directly ex vivo (Fig. 5e).

In view of the long-lived retention of CD49a-expressing γδT

we had demonstrated in the liver, we examined whether γδ

T-cells inﬁltrating into HCC could acquire this residency proﬁle.

Both Vδ1 and Vγ9Vδ2 T-cells were able to acquire a

phenotype in HCC and CRCLM Fig. 5f, g). Co-expression

of CD49a was higher than CD103 on intrahepatic and

intratumoural Vδ1 and Vγ9Vδ2T

subsets (Supplementary

Fig. 5d). Using the GEPIA2 web server to interrogate the Cancer

Genome Atlas database

, a combined CD69

CD103

and γδ-

TCR gene signature showed a favourable prognostic association

with overall survival in HCC, not detected with an equivalent

CD69

CD103

αβCD8

TCR gene signature (Supplementary

Fig. 5e), although the T

gene signature in the analysis may have

been contributed to by other cell types preventing deﬁnitive

conclusions.

Overall, Vδ1 T-cells appeared higher in frequency and tended

to be more activated with higher cytotoxic potential within HCC,

while Vγ9Vδ2 T-cells, despite being selectively reduced in

frequency in HCC, maintained high capacity for IFN-γ produc-

tion and an equivalent ability to acquire an intratumoural

CD69

CD49a

or CD69

CD103

phenotype.

Anti-tumour potential of Vγ9Vδ2T

against ZOL-sensitised

HCC cell lines. While both Vδ1 and Vγ9Vδ2 T-cells may con-

tribute to immunosurveillance of HCC, we focused on therapeutic

augmentation of the depleted Vγ9Vδ2 T-cell subset, for which

ligands and clinical expansion protocols are better validated.

Having demonstrated that Vγ9Vδ2T

were well-adapted for

long-lived function in the HCC niche, we next investigated if they

had the potential to mediate speciﬁc anti-tumour functionality.

Intrahepatic Vγ9Vδ2 T-cells isolated from fresh human liver

tissue exhibited minimal IFN- γ and TNF-α production in

response to co-culture with HepG2 or HuH7 human hepatoma

cell lines (Fig. 6a, b, Supplementary Fig. 6a). We hypothesised

that insufﬁcient isopentenyl pyrophosphate (IPP) production by

the tumour cell lines may be limiting Vγ9Vδ2 TCR activation.

Therefore, based on previous studies

51–53

, we pre-treated HepG2

and HuH7 cells for 16–18 h with the aminobisphosphonate ZOL,

which inhibits the enzyme farnesyl pyrophosphate synthase

(FPPS) in the mevalonate pathway, promoting upstream

Fig. 2 Long-lived hepatic retention and replenishment of Vδ1 and Vɣ9Vδ2-T

.aRepresentative ﬂow cytometry plot and summary data of total γδ

T-cells (as percentage of total CD3

T-cells) in peripheral, hepatic and portal venous blood obtained from cirrhotic patients undergoing transjugular

intrahepatic portosystemic shunt procedures (n = 4, p = 0.04). b Representative ﬂow cytometry plot and summary data demonstrating absence of

CD69

CD49a

or CD69

CD103

γδT

in peripheral, hepatic, portal venous blood (n = 4 matched samples). c Flow cytometry plot for the identiﬁcation

of donor human leukocyte antigen (HLA) A2

and recipient HLA A2

−

derived γδ T-cells from two explants obtained 7 years or 11 years following HLA-

mismatched liver transplantation. Vδ1 and Vɣ9Vδ2 T-cell frequencies within recipient-derived and donor-derived γδ T-cell subsets. d CD69

CD49a

and

CD69

CD103

expression on donor-derived (HLA A2

) and recipient-derived (HLA A2

−

) γδ T-cell subsets (n = 2). Error bars, mean ± SEM (b). Two-

tailed p-values were determined by Friedman test with Dunn’s post-hoc test for multiple comparisons (a, b).

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Fig. 3 Distinct functional proﬁle of hepatic γδT

.a–e Representative ﬂow cytometry plots and summary data of ex vivo functional proﬁle of intrahepatic

CD69

CD49a

Vδ1 and Vγ9Vδ2T

compared to non-T

(CD69

−

CD49a

−

) counterparts. a HLA-DR expression (n = 13; Vδ1 p = 0.008; Vγ9Vδ2

p = 0.03). b unstimulated Granzyme B expression (n = 17; Vδ1 p = 0.0005; Vγ9Vδ2 p = 0.0002). c IL-2 expression following 4 h PMA and Ionomycin

stimulation (n = 10; Vδ1 p = 0.004; Vγ9Vδ2 p = 0.03). d IFN-γ expression after 4 h PMA and Ionomycin stimulation (n = 10; Vδ1 p = 0.01; Vγ9Vδ2

p = 0.02). e unstimulated programmed death-1 (PD-1) expression (n = 11; Vδ1 p = 0.007; Vγ9Vδ2 p = 0.008). f IFN-γ expression by PD-1

high

and PD-1

low

intrahepatic Vδ1 and Vγ9Vδ2 T-cells after 4 h PMA and Ionomycin stimulation (n = 8; p value non-signiﬁcant). Each symbol represents a study participant,

with bars showing the mean (a–f); two-tailed p-values were determined using Wilcoxon matched-pairs signed rank test (a–f). *p < 0.05; **p < 0.01;

***p < 0.001.

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Fig. 4 γδ T-cell counts in HCC are associated with tumour size and patient survival. (a–d) Immunohistochemistry staining of paired background liver and

tumour tissue obtained from patients with hepatocellular carcinoma (HCC) undergoing surgical resection. Cell counts performed from ﬁve randomly

selected high-power ﬁelds (x20 objective magniﬁcation) per sample (n = 28 paired samples). a Representative multispectral analysis with non-γδ CD3

(green) and γδ

(red) T-cells as pseudo-colourised images in liver and HCC tumoural tissue, replicated across ﬁve high-power (x20) ﬁelds per sample.

b Absolute γδ T-cell and non-γδ CD3

T-cell numbers (calculated per mm

) and γδ/CD3

T-cell ratio in paired liver and HCC tumours (n = 28 paired

samples; p < 0.0001, p = 0.0006). c Intratumoural γδ T-cell counts and non-γδ CD3

T-cell counts (per mm

) in small HCC tumours with a maximum

diameter of ≤3 cm compared to HCC tumours >3 cm in diameter (n = 28; p = 0.03). d HCC intratumoural γδ T-cell counts and non-γδ CD3

T-cell counts

(per mm

) according to 3-year patient survival outcomes (overall survival data available n = 27, 22/27 survived, 5/27 died; p = 0.048). Kaplan Meier

graphs of overall survival (years post resection) split on the median intratumoural γδ T-cell (p = 0.009) or non-γδ CD3

T-cell count from 27 HCC

tumours. Two-tailed p-values were determined using Wilcoxon matched-pairs signed rank test test (b) or Mann–Whitney test (c, d), Kaplan Meier graphs

with Log-rank test (d). Error bars represent mean ± SEM. ns Not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

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accumulation of IPP within the tumour cell to trigger Vγ9Vδ2

TCR activation

. After removal of ZOL, hepatoma cell lines were

again co-cultured with IHL and were then able to trigger a sig-

niﬁcant increase in Vγ9Vδ2 T-cell effector function (IFN-γ, TNF-

α, IL-2 expression, Fig. 6c, Supplementary Fig. 6a). This activation

was speciﬁctoVγ9Vδ2 T-cells, with no effect on liver Vδ1or

αβCD8

T-cell function (Supplementary Fig. 6b). To conﬁrm

that the increase in Vγ9Vδ2 T-cell effector function was depen-

dent on tumour cell IPP expression, we additionally treated ZOL-

exposed hepatoma cell lines with Mevastatin, an inhibitor of the

mevalonate pathway, able to block tumour cell IPP production,

prior to the co-culture with IHL (Fig. 6d)

51,54

. Mevastatin

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signiﬁcantly reduced the function of co-cultured intrahepatic

Vγ9Vδ2 T-cells (IFN-γ and TNF-α), supporting a role for IPP-

mediated triggering (Fig. 6d).

Importantly, CD69

CD49a

Vγ9Vδ2T

isolated from the

human liver displayed a greater increase in effector function

(IFN-γ, IL-2 expression) compared to their non-resident

(CD69

−

CD49a

−

)Vγ9Vδ2 T-cell counterparts when co-cultured

with ZOL-treated hepatoma cell lines (Fig. 6e). We observed a

similar response using TILs isolated directly from four HCC

tumours. Co-culturing HCC TILs with plated HepG2 cells

in vitro, resulted in no measurable Vγ9Vδ2 T-cell effector

response (Fig. 6f). However, ZOL pre-treatment of the hepatoma

cell lines increased IFN-γ expression by the intratumoural

Vγ9Vδ2 T-cells, predominantly by the Vγ9Vδ2T

subset

(Fig. 6f, g). Collectively, these results suggest that low IPP

expression by HCC tumour cells could limit the local activation of

Vγ9Vδ2 T-cells by HCC. Thus, by sensitising HCC tumour cells

directly with ZOL ﬁrst, we could signiﬁcantly enhance the anti-

tumour function of Vγ9Vδ2 T-cells, in particular Vγ9Vδ2T

within HCC tumours and adjacent liver tissue.

ZOL expands de novo Vγ9Vδ2T

from blood, targeting

ZOL-sensitised HCC cell lines. Enhancing the function of resi-

dual endogenous Vγ9Vδ2T

is a promising novel immu-

notherapeutic strategy, however, its clinical efﬁcacy may be

limited by the low cell counts and reduced cytotoxicity of Vγ9Vδ2

in HCC livers and tumours. To expand a large population of

Vγ9Vδ2 T-cells suitable for immunotherapy, we used a well-

established clinical protocol (see Methods), treating healthy

donor PBMC with ZOL and IL-2 over 10 days

55–59

.Vγ9Vδ2

T-cells expanded with this protocol showed an unexpected de

novo induction of a CD69

CD49a

phenotype (mean

80.3 ± 17.9%), with varying levels of CD103 co-expression (Fig.

7a, b). These de novo CD69

CD49a

+ /- CD103

Vγ9Vδ2T

displayed a CD27

low

CD45RA

low

effector memory phenotype

(Supplementary Fig. 7a), with downregulation of CD62L (Sup-

plementary Fig. 7b). ZOL-induced Vγ9Vδ2T

recapitulated

other features of Vγ9Vδ2T

analysed directly from the liver,

with even higher levels of the liver-homing chemokine receptors

CXCR6 and CXCR3 (Fig. 7c) and greater capacity for IFN-γ

expression, although with lower PD-1 expression in comparison

to ex vivo isolated intrahepatic Vγ9Vδ2T

(Fig. 7d, Supple-

mentary Fig. 7c). Crucially, in vitro induced Vγ9Vδ2T

had

enhanced cytotoxic potential (ex vivo granzyme B expression)

compared to Vγ9Vδ2T

isolated directly from the liver (Fig.

7d).

ZOL inhibits FPPS within the mevalonate pathway of dendritic

cells and monocytes in PBMCs, leading to accumulation of IPP to

trigger Vγ9Vδ2 TCR activation

60–63

, pointing to this as a putative

mechanism for de novo Vγ9Vδ2T

induction. To further

explore the role of TCR activation as a potential driving factor for

Vγ9Vδ2 T-cell tissue-residency, we stimulated Vγ9Vδ2 T-cells

within PBMCs using a plate-bound anti-γδTCR antibody.

Continuous TCR stimulation (7-days) induced Vγ9Vδ2 T-cell

activation (increased CD38), accompanied by preferential de

novo CD49a and CD103 expression on the CD38-expressing

fraction (Supplementary Fig. 7d), accumulating in a time-

dependent manner (Supplementary Fig. 7e), and accompanied

by signiﬁcantly higher levels of CXCR6 and CXCR3 expression

(Supplementary Fig. 7f). IL-2 without TCR stimulation did

induce a degree of CD49a expression on Vγ9Vδ2 T cells,

although to a signiﬁcantly lower intensity (Supplementary Fig.

7d, e). Exposure to other prototypic hepatic cytokines, in

particular combined IL-15 and TGF-β, also induced de novo

expression of CD49a, and to a much lower extent CD103, on

Vγ9Vδ2 T-cells, and therefore may be additional factors driving

the induction of γδ T

within the liver and HCC (Supplemen-

tary Fig. 7g).

We next co-cultured the ZOL and IL-2 expanded blood

Vγ9Vδ2 T-cells with human hepatoma cell lines, HepG2 and

HuH7, to examine for an anti-HCC response. ZOL pre-treatment

of the hepatoma cell lines was again required in order to evoke

substantial IFN-γ and TNF-α expression by the expanded blood

Vγ9Vδ2T

(Fig. 7e, Supplementary Fig. 7h), which was

reduced in a dose-dependent manner by the addition of

Mevastatin, conﬁrming the dependence on tumour cell IPP

expression (Fig. 7f, Supplementary Fig. 7i). Notably, ZOL-

induced blood Vγ9Vδ2T

exhibited higher IFN-γ and

Granzyme B expression than ex vivo isolated liver Vγ9Vδ2T

following co-culture with hepatoma cell lines (Fig. 7g). The

addition of anti-PD-L1 blockade did not further increase

expanded Vγ9Vδ2T

effector function towards ZOL pre-

treated HepG2 cells (IFN-γ, TNF-α) (Fig. 7h). Critically, de novo

induced Vγ9Vδ2T

were able to lyse HCC cell lines that had

been sensitised by ZOL, assessed by the ToxiLight

biolumi-

nescent cytotoxicity assay measuring adenylate kinase in the

culture medium released following cell death (Fig. 7i).

Overall, these data indicate that a de novo T

phenotype with

enhanced anti-tumour functionality can be successfully recapitu-

lated and expanded in vitro from peripheral blood Vγ9Vδ2T-

cells, with liver-homing/retention potential for adoptive cell

transfer. However, to enhance their anti-tumour function and

maximise tumour cell lysis, prior treatment of HCC tumour cells

with ZOL is required to upregulate IPP expression for Vγ9Vδ2

TCR activation in situ.

Discussion

HCC is now known to be partially amenable to immunotherapy,

but new approaches are urgently needed to tackle the dual tol-

erogenic inﬂuences of the liver and tumour microenvironments.

T-cells specialised for tissue residence are adapted to combat local

tolerising effects, and are able to maintain long-term, rapid

Fig. 5 Vγ9Vδ2 T-cells are selectively depleted, but can acquire tissue-residence, within HCC TILs. (a–g) Flow cytometry analysis of paired peripheral

blood mononuclear cells (PBMC), intrahepatic lymphocytes (IHL) from tumour-free liver tissue, and tumour-inﬁltrating lymphocytes (TIL) from patients

undergoing surgical resection or liver transplantation for hepatocellular carcinoma (HCC) compared to patients undergoing surgical resection for colorectal

cancer liver metastases (CRCLM). a Total frequencies of Vδ1 (above) and Vγ9Vδ2 (below) T-cells in PBMC, IHL and TILs of CRCLM (n = 23) compared to

HCC (n = 26) (expressed as a percentage of total CD3

T-cells; Vγ9Vδ2 PBMC p = 0.0007, IHL p = 0.0002, TIL p = 0.02). b Representative ﬂow

cytometry plot and summary data of CD27-CD45RA + terminally differentiated effector memory (TEMRA) Vδ1 and Vγ9Vδ2 T-cells in paired liver and

HCC TILs (n = 10; IHL p = 0.002, TIL p = 0.001). c ex vivo HLA-DR (n = 12; IHL p = 0.02, TIL p = 0.05) and CD38 expression (n = 8; IHL p = 0.02) by Vδ1

and Vγ9Vδ2 T-cells within HCC IHL and TILs. d IFN-γ expression by Vδ1 and Vγ9Vδ2 T-cells within HCC IHL (n = 12) and TILs (n = 10) after 4 h PMA and

Ionomycin stimulation (IHL p = 0.03).eUnstimulated Granzyme B expression by Vγ9Vδ2 T-cells in HCC IHL and TILs (n = 11; TIL p = 0.008).

f CD69

CD49a

expression on Vδ1 and Vγ9Vδ2 T-cells in HCC IHL (n = 23) and TILs (n = 21). g CD69

CD49a

expression on Vγ9Vδ2 T-cells in HCC

compared to CRCLM IHL (n = 22) and TILs (n = 13). Each symbol represents a study participant, error bars indicate mean ± SEM; two-tailed p-values were

determined using Mann–Whitney test (a, g), or Wilcoxon paired test (b–f). *p ≤ 0.05; **p < 0.01; ***p < 0.001.

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functionality at the site of disease. In the case of αβCD8

T-cells,

tissue-residence is emerging as a critical feature for protection

against tumours

25,31–37,39

γδ T-cells are promising alternative

candidates for cancer immunotherapies but their capacity for

tissue residency in HCC is unknown. Here, we show that both

major subsets of human γδ T-cells (Vδ1 and Vγ9Vδ2) are able to

acquire phenotypic and functional features of tissue residence in

human HCC. We conﬁrm the bona ﬁde T

status of

CD69

CD49a

γδ T-cells by demonstrating their lack of egress

into hepatic vasculature, long-lived persistence in human liver

allografts, and rapid production of cytokines favouring survival

and non-cytolytic functionality, similar to intrahepatic αβ CD8

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28–30

. CD49a expression has similarly been described to

promote αβCD8

survival in the human lung, mediated in

part through engagement with its ligand collagen IV

Theprotectivecapacityofγδ T-cells against other cancer types

has been demonstrated in mice and humans

9–16

; here, we show this

also applies to HCC, with higher intratumoural γδ T-cell frequencies

associating with smaller tumour size and longer patient survival. It

will be important to extend this to much larger cohorts to assess for

potential prognostic value as a clinical biomarker. Whilst the

endogenous intratumoural γδ T-cell response may be partially

limitedbyregulatoryand/orpro-inﬂammatory inﬂuences

65–67

,our

ﬁndings reveal the potential to exploit the capacity of both Vδ1and

Vγ9Vδ2 T-cells to acquire tissue-residenc e within HCC. Here, we

concentrated on exploring the immunotherapeutic potential of

Vγ9Vδ2 T-cells since they were selectively depleted in HCC but can

be readily expanded in vitro, have a well-deﬁned tumour ligand in

theformofIPPandshowedthecapacitytoacquireT

features.

Although intratumoural Vγ9Vδ2 T-cells maintained efﬁcient pro-

duction of IFN-γ, a cytokine critical to the orchestration of tumour

immunity

, they had reduced cytotoxic potential, lower expression

of T-cell activation markers, and poor recognition of HCC cell lines.

Firstly, we showed that the anti-tumour function of existing endo-

genous intrahepatic and intratumoural Vγ9Vδ2T

could be

enhanced in an IPP-dependent manner by pre-treating HCC cell

lines with the aminobisphosphonate ZOL, as previously shown for

circulating Vγ9Vδ2T-cells

. However, this would not tackle the

depleted numbers of Vγ9Vδ2 T-cells we observed in HCC. Next, we,

therefore, explored the expansion of Vγ9Vδ2 T-cells with the

combination and doses of ZOL and IL-2 previously optimised for

adoptive cell transfer. We discovered that this clinically-validated

protocol induced a de novo T

phenotype on blood Vγ9Vδ2

T cells, with increased liver-homing chemokine receptor expression

and even higher expression of the retention molecules CD69/

CD49a/CD103 than endogenous γδT

, as well as enhancing

cytotoxicity. Analogous to the endogenous Vγ9Vδ2 T-cell response,

these peripheral induced Vγ9Vδ2T

were able to efﬁciently

recognise and lyse HCC cells pre-sensitised with aminobispho-

sphonates to increase IPP accumulation.

Thus, Vγ9Vδ2 T-cell-based immunotherapy holds potential as

an alternative treatment strategy for HCC patients resistant or

intolerant to immune checkpoint inhibition, removing the need

for tumour-antigen speciﬁcity due to the uniform, MHC-

unrestricted responsiveness of Vγ9Vδ2 T-cells to phospho-

antigen (IPP) stimulation. The adoptive cell transfer of ex vivo

ZOL-expanded blood Vγ9Vδ2 T-cells has demonstrated a good

safety proﬁle in several early phase clinical trials

55–59

, including a

recent phase one trial in patients with advanced HCC

. How-

ever, to date, clinical efﬁcacy has remained low. Systemic ZOL

therapy can expand Vγ9Vδ2 T-cells in vivo, but the high afﬁnity

of ZOL for bone mineral and its rapid renal clearance limits

systemic availability of ZOL for tumour cell uptake

, likely

contributing to the insufﬁcient anti-tumour responses

observed

22,71–73

. In contrast, local intraperitoneal or intratu-

moural administration of ZOL to tumours has demonstrated

early promise in small proof-of-concept studies of other tumour

types

52,53,57,74

. Our data suggest that aminobisphosphonates

should be tested in a combination strategy for HCC, to overcome

two key limiting factors: their direct delivery to the tumour can

enhance IPP expression to increase in situ activation of Vγ9Vδ2

T-cells, whilst their use for large-scale expansion of blood

Vγ9Vδ2 T-cells can expand a population with enhanced cyto-

toxicity and the tissue-retention properties of T

to replenish

depleted numbers within the tumour.

Methods

Study approval. This study was approved by the local ethics board Brighton and

Sussex (Research Ethics Committee reference number 11/LO/0421) and complies

with the Declaration of Helsinki. All study participants provided written informed

consent before inclusion. All storage of samples obtained complied with the

requirement of the Human Tissue Act 2004 and the Data Protection Act 1998.

Sample collection. All uses of human samples have been approved. Surgically

resected tumour-free liver tissue and tumours of HCC or CRCLM, explants from

patients with HCC or cirrhosis without HCC undergoing liver transplantation, and

perfusates from healthy donor liver allografts, together with paired blood samples,

were obtained through the Tissue Access for Patient Beneﬁt initiative at The Royal

Free Hospital (approved by the University College London–Royal Free Hospital

BioBank Ethical Review Committee; Research Ethics Committee reference number

11/WA/0077). Formalin ﬁxed parafﬁn-embedded tissue sections for immunohis-

tochemistry were obtained from back ground liver and tumours of HCC (approval

by the Royal Free Hospital Ethics Committee reference number 07/Q0501/50).

Blood samples were obtained from patients with cirrhosis undergoing transjugular

intrahepatic portosystemic shunt procedures (approved by the University College

London–Royal Free Hospital BioBank Ethical Review Committee; Research Ethics

Committee reference number 16/WA/0289). For comparison, peripheral blood

samples from healthy control individuals were included within the study (approved

by the South East Coast Research Ethics Committee; Research Ethics Committee

reference number 11/LO/0421).

Explanted liver tissue was obtained from two patients undergoing re-

transplantation where there was an HLA class I mismatch between the initial

allograft donor and the recipient (approved by the University College

London–Royal Free Hospital BioBank Ethical Review Committee; Research Ethics

Committee reference number 11/WA/0077). HLA phenotype was determined by

HLA-haplotyping PCR by A. Nolan (National Health Service, Lond on, UK) or

MRC Weatherall Institute of Molecular Medicine Sequencing Facility (Oxford,

UK). Clinical details of the transplant recipients and donors are included in

Supplementary Table 1.

Human PBMC, IHL and TIL cell isolation. Peripheral blood mononuclear cells

(PBMC) were isolated from heparinised blood by density centrifugation using

Pancoll (Pan Biotech) and was centrifuged for 20 min at 800 g with slow accel-

eration and deceleration. PBMCs were used fresh where possible or were frozen in

10% DMSO (Sigma-Aldrich) in heat-inactivated FBS (Sigma-Aldrich) and stored

in accordance with the Human Tissue Act.

To isolate intrahepatic lymphocytes (IHL) and tumour-inﬁltrating lymphocytes

(TIL), liver or tumour tissue was cut into small pieces and incubated for 30 min at

C in 0.01% collagenase IV (Thermo Fisher Scientiﬁc) and 0.001% DNAse I

Fig. 6 Anti-tumour potential of Vγ9Vδ2T

against ZOL-sensitised HCC cell lines. a Schema of untreated or Zoledronic acid (ZOL) pre-treated (5μM,

16–18 h) adherent HepG2 cells co-cultured with intrahepatic lymphocytes (IHL) (n = 11) or HCC tumour-inﬁltrating lymphocytes (TIL) (n = 4) (E:T 2:1 ratio;

0.6 × 10

IHL or TILs to 0.3 × 10

HepG2 cells, 6 h co-culture, all conditions performed in duplicate or triplicate). b IFN-γ, TNFα expression by Vγ9Vδ2T-

cells in IHL unstimulated or after co-culture with untreated HepG2 cells (n = 11 IFN-γ, n = 8 TNF-α). c IFN-γ, TNFα, IL-2 expression by Vγ9Vδ2 T-cells in

IHL: unstimulated, directly treated with 5 μM ZOL, or after co-culture with untreated or ZOL pre-treated HepG2 cells (n = 11 IFN-γ p = 0.0004,

p = 0.0008; n = 8 TNFα p = 0.0006, p = 0.004; n = 7 IL-2 p = 0.007, p = 0.02). d IFN-γ (n = 11), TNF-α (n = 8) expression by Vγ9Vδ2T-cells in IHL after

co-culture with ZOL pre-treated HepG2 cells with or without 100 µM Mevastatin (Mev) treatment (IFN-γ p = 0.008, TNF-α p = 0.03). e Representative

ﬂow cytometry plot of IFNγ expression and summary data of IFNγ

and IL-2 expression by CD69

CD49a

Vγ9Vδ2T

compared to CD69

−

CD49a

−

Vγ9Vδ2 non-T

, following co-culture of IHL with ZOL pre-treated HepG2 cells (g = 8, IFNγ p = 0.02, IL-2 p = 0.03). f IFN-γ expression by Vγ9Vδ2T-

cells in HCC TILs unstimulated, or after co-culture with untreated or ZOL pre-treated HepG2 cells (n = 4). g Representative ﬂow cytometry plot of IFN-γ

expression by CD69

CD49a

Vγ9Vδ2T

and CD69

−

CD49a

−

Vγ9Vδ2 non-T

in HCC TILs after co-culture with ZOL pre-treated HepG2 cells. Each

symbol represents a study participant; error bars represent the mean ± SEM. Two-tailed p-values were determined using Wilcoxon paired test (b, d, e), or

Friedman test with Dunn’s post-hoc multiple comparisons test (c, f). *p < 0.05; **p < 0.01; ***p < 0.001.

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(Sigma-Adrich) followed by further mechanical disruption via GentleMACS

(Miltenyi Biotech), and ﬁltration through a 70 μm cell strainer (BD Bioscience) to

achieve a single cell solution. Parenchymal cells were removed by 400 g

centrifugation on a 30% Percoll gradient (GE Healthcare) and lymphocytes were

isolated via density centrifugation as described above. IHL and TILs were used

immediately after isolation.

Flow cytometry: cell surface, intracellular and cytokine staining. Multi-

parametric ﬂow cytometry was used for phenotypic and functional analysis of

PBMC, IHL and TILs. Cells were stained with a ﬁxable Live/Dead dye (Invitrogen)

before incubation with saturating concentrations of surface mAbs diluted in 50%

Brilliant Stain buffer (BD Biosciences) and 50% PBS (Invitrogen) for 15 min at

4 °C. Cells were ﬁxed and permeabilized for further functional assessment with

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Cytoﬁx/Cytoperm (BD Biosciences) according to the manufacturer’s instructions.

Cells were incubated with saturated concentrations of mAbs diluted in a 0.1%

saponin-based buffer (Sigma-Aldrich) for 20 min at 4 °C for the detection of

intracellular proteins. All samples were acquired on Fortessa X20 ﬂow cytometer

using FACSDiva software v8.0 (BD biosciences) and analysed using FlowJo v.9

(Tree Star; BD Biosciences). Full details on monoclonal antibodies used can be

found in Supplementary Table 5.

t-Distributed Stochastic Neighbour Embedding (tSNE) analysis. The dimen-

sion reduction algorithm tSNE was applied to concatenated ﬂow cytometry data

(~2500 cells per samples) from 5 IHL and 3 PBMC samples using default para-

meters (iterations, 1,000; perplexity, 30; and eta 4076, vantage point tree KNN

algorithm) in FlowJo. tSNE was applied to expression data for Vδ1, Vδ2, Vγ9,

CD69, CD49a, CD103, CXCR3, CXCR6, HLA-DR, NKG2D, after pre-gating on

single, live, CD45 + , CD3 + , γδ-TCR + lymphocytes. Manual gating of Vδ1, Vδ2,

Vγ9, CD69, CD103, and CD49a were used to colour subpopulations within the

plots in Fig. 1b.

In vitro stimulation. Prior to intracellular cytokine staining, PBMC, IHL or TILs

were plated in a 96-well plate (0.4 x 10

cells per well) in complete RPMI (cRPMI;

RPMI-1640 containing 10% FBS, 100 U/ml penicillin/streptomycin, 1× non-

essential amino acids, 1× essential amino acids, and β-mercaptoethanol; Invitro-

gen) and stimulated with 50 ng/ml phorbol myristate acetate (PMA) (Sigma-

Aldrich) and 500 ng/ml Ionomycin (Sigma-Aldrich) for 4 h with 1 μg/ml brefeldin

A (Sigma-Aldrich) added, at 37 °C in a humidiﬁed atmosphere with 5% CO

. After

stimulation, γδ T-cells were stained for cytokine production as above and analysed

by ﬂow cytometry.

For assessment of the impact of anti-PD-L1 receptor blockade, IHL were plated

in 200μl cRPMI in a 96-well plate (0.4 x 10

cells/well) with or without anti-PD-L1

blocking antibody (Invitrogen, 2.5 μg/ml or 5 μg/ml) and plate-bound anti γδ-TCR

monoclonal antibody stimulation (Biolegend, 4 μg/ml), with 1 μg/ml brefeldin A

(Sigma-Aldrich) added. After 16 h, cells were removed from stimulation, stained

for cytokine production and analysed by ﬂow cytometry.

Immunohistochemistry staining of CD3

and γδ T-cells. The immunostaining

protocol was ﬁrst optimised on formalin-ﬁxed parafﬁn-embedded tonsil tissue as a

positive control, and subsequently liver tissue. Formalin-ﬁxed parafﬁn-embedded

sections obtained from paired HCC and background liver tissue (n = 28) were

placed through xylene and alcohol to distilled water, placed in 1 L of pH 9.0 Tris

EDTA buffer and then microwaved for 20 min at 640 W before cooling and rinsing

in running tap water. The slides were soaked in Tris Buffered Saline (TBS) with

0.04% Tween-20 for 5 min, Bloxall (Vector – SP-6000-100) blocking solution was

applied for 10 min, washed in TBS and blocked with normal horse serum (MP-

7402 – Vector Laboratories) for 10 min. Slides were incubated for 1 h at room

temperature in a combined cocktail of rabbit anti human CD3 (SP7 clone,

ab16669-Abcam) at 1:100 and mouse anti human TCR-δ (H41 clone, sc-100289-

Santacruz) at 1:100 in TBS. After washing in TBS Tween for 5 min, the slides were

incubated for 25 min in ImPRESS horse anti-mouse HRP reagent (MP-7402-

Vector Laboratories), washed in TBS Tween, developed with ImmPACT DAB

(SK4105-Vector Laboratories), washed in TBS, and blocked with normal horse

serum (MP5401–Vector Laboratories) for 10 min. Slides were incubated for 25 min

in ImPRESS horse anti-rabbit HRP reagent (MP5401-Vector Laboratories), washed

in TBS Tween, developed with fast red alkaline phosphatase substrate

(ab64254=Abcam), washed in TBS Tween, counterstained with Mayers haema-

toxylin and then rinsed in running tap water. Slides were allowed to air-dry to

dehydrate, clear in xylene and mounted. All relevant negativ e controls and single

stains were included to exclude the possibility of cross reaction between CD3 and

TCR-δ antibodies and detection kits.

Immunohistochemistry imaging and quantiﬁcation of CD3

and γδ T-cells.

Five ﬁelds were selected at random from each of the background liver (n = 28) and

HCC tumour (n = 28) slides using a graticule and a random number generator,

and enlarged to x20 objective magniﬁcation. Images were taken using a Akoya

Mantra 2

microscope and multispectral camera which captures an absorbance

spectrum for each pixel. Spectral libraries were set up from single stained slides for

each of DAB, Fast Red and Haematoxylin to identify the chromogen for TCR-γδ,

CD3 and nuclei, respectively. The images were then spectrally unmixed using the

spectral libraries to identify each chromogenic signal. Cells were identiﬁed using

automatic segmentation with the software available from Akoya, inForm

, using

the multispectral data to identify cells based on morphological parameters and

chromogen expression. Automatic cell counts of CD3

positive cells and

CD3

TCR-γδ

cells were carried out on each image and segmented images were

reviewed for accuracy of detection. Overall CD3

and CD3

TCR-γδ

cell counts

and a count per mm

were obtained.

Expansion of Vγ9Vδ2 T-cells . PBMC from healthy controls were plated in a 24-

well plate (0.2 × 10

cells/well) and incubated with 1000 IU/ml IL-2 (PeproTech) in

the presence or absence of 5 μM Zometa (Novartis), in cRPMI at 37 °C for 3-days.

On day 3 the PBMCs were removed, washed in cRPMI and re-plated in a 24-well

plate (0.2 × 10

cells/well) with fresh cRPMI media containing 1000IU/ml IL-2.

Every 2–3 days a media change was performed with 1000 IU/ml IL-2 added and the

cells were split as required. On day 9–12, the cells were stained for Vγ9Vδ2 T-cell

frequency and phenotype and analysed by ﬂow cytometry. CountBright

absolute

counting beads (ThermoFisher) were used to determine total cell counts to conﬁrm

Vγ9Vδ2 T-cell expansion.

For comparisons of cytokine production post-expansion, following the Vγ9Vδ2

T-cell expansion protocol as described, PBMC were rested overnight in cRPMI and

then plated in a 96-well plate (0.4 x 10

cells/well) and stimulated using 50 ng/ml

PMA (Sigma-A ldrich) and 500 ng/ml Ionomycin (Sigma-Aldrich) for 4 h at 37 °C

with 1 μg/ml brefeldin A (Sigma-Aldrich). After stimulation, cells were stained for

cytokine production and analysed by ﬂow cytometry.

In vitro γδ-TCR activation. PBMC from healthy contro ls were plated in 200 μl

cRPMI in a 96-well plate (0.3 x 10

cells/well) with IL-2 (20 IU/ml) added with or

without additional plate-bound anti-γδTCR monoclonal antibody stimulation

(Biolegend, 4μg/ml). PBMC were removed at 4 h, 24 h, 3 days, and 7 days of culture

and stained for CD38, CXCR6, CXCR3 and γδ T

marker expression.

In vitro cytokine exposure . PBMC from healthy controls were plated in a 96-well

plate (0.3 x 10

cells/well) and cultured in 200 μl cRPMI with combinations of IL-2

(20 IU/ml), IL-7 (50 ng/ml), IL-15 (50 ng/ml), TGF-β (50 ng/ml). On day 7 PBMC

were stained for cell surface γδ T

marker (CD69, CD103, CD49a) expression.

Zoledronic acid treated hepatoma cell line co-culture. HepG2 or HuH7 cells

were plated at a density of 0.3 x 10

cells/well in a 24-well plate (Costar) and

Fig. 7 ZOL expands de novo Vγ9Vδ2T

from blood, targeting ZOL-sensitised HCC cell lines. a Representative ﬂow cytometry plots of CD69

CD103

and CD69

CD49a

expression on Vγ9Vδ2 T-cells pre- and post-Zoledronic acid (ZOL) and IL-2 based expansion. b Summary data of CD69

CD103

and

CD69

CD49a

expression on Vγ9Vδ2 T-cells pre- and post-ZOL and IL-2 based expansion (n = 15, p = 0.001). c % CXCR6 (left) and % CXCR3 (right)

expression on ex vivo intrahepatic Vγ9Vδ2T

(n = 11)

compared to de novo blood CD69

CD103

or CD69

CD49a

Vγ9Vδ2T

(induced T

, n = 8)

following ZOL/IL-2 expansion (CXCR6 p = 0.0001, p = 0.0008; CXCR3 p = 0.0002, p = 0.0002). d IFN-γ expression (left) by ex vivo intrahepatic

Vγ9Vδ2T

compared to induced blood Vγ9Vδ2T

after 4 h PMA and Ionomycin stimulation (n = 7, p = 0.001); unstimulated Granzyme B expression

(right) by intrahepatic Vγ9Vδ2T

(n = 11) compared to induced blood Vγ9Vδ2T

(n = 6, p = 0.002, p = 0.001). e Schema demonstrating expanded

blood Vγ9Vδ2T-cells co-culture with untreated or ZOL pre-treated (5 μM, 16–18 h) HepG2 cells (E:T 2:1 ratio; 0.6 × 10

expanded Vγ9Vδ2 T-cells to

0.3 × 10

HepG2 cells, 6 h co-culture, all conditions performed in triplicate). Representative ﬂow cytometry plot and summary data of IFN-γ and TNF-α

expression by ZOL/IL-2 expanded Vγ9Vδ2 T-cells: unstimulated, directly treated with ZOL 5 μM, or after co-culture with untreated or ZOL pre-treated

HepG2 cells (n = 10; IFN-γ p < 0.0001, p = 0.03, p = 0.01; TNF-α p < 0.001, p = 0.03, p = 0.01). f IFN-γ and TNF-α expression by ZOL/IL-2 expanded

Vγ9Vδ2 T-cells after co-culture with ZOL pre-treated HepG2 cells, with or without the addition of 100 µM Mevastatin (Mev) (n = 10; IFN-γ p = 0.004,

TNF-α p = 0.004). g IFN-γ and Granzyme B expression by expanded blood Vγ9Vδ2T

(n = 9) compared to ex vivo intrahepatic Vγ9Vδ2T

(n = 10)

after co-culture with ZOL pre-treated HepG2 cells (IFN-γ p = 0.03, GRZB p = 0.005). h IFN-γ and TNF-α expression by ZOL/IL-2 expanded Vγ 9Vδ2

T-cells after co-culture with ZOL pre-treated HepG2 cells, with or without the addition of anti-programmed death-ligand 1 (PDL-1) blockade (n = 6). i Lysis

of HepG2 cells and ZOL pre-treated HepG2 cells after co-culture with PBMCs containing ZOL-expanded Vγ9Vδ2 T-cells (n = 7, p = 0.02), measured using

Toxilight

cytotoxicity assay. Each symbol represents a study participant; error bars show mean ± SEM. Two-tailed p-values determined using Wilcoxon

paired test (b, f, i), Mann–Whitney test (c, d, g), or Friedman test with Dunn’s post-hoc test for multiple comparisons (e, h). *p < 0.05; **p < 0.01;

***p < 0.001; ****p < 0.0001.

ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-29012-1

14 NATURE COMMUNICATIONS | (2022) 13:1372 | https://doi.org/10.1038/s41467-022-29012-1 | www.nature.com/naturecommunications

incubated in GlutaMax DMEM media (Gibco) containing 10% FCS, 1% NEAA, 1%

Sodium Pyruvate, 1% PenStrep, for 16–18 h to adhere to the well, in the presence

or absence of Zometa (ZOL) 5 μM (Novartis) and/or Mevastatin 100 μM or 200 μM

(Sigma-Aldrich) and/or 5 μg anti-PD-L1 blocking antibody (Invitrogen). After

16–18 h incubation, the media was carefully removed and the adherent HepG2 cells

in each well were washed twice with PBS. PBMC containing ZOL-expanded blood

Vγ9Vδ2 T-cells, bulk IHL or TILs (0.6 x 10

cells/well, E:T ratio 2:1) were then

added to each well in cRPMI for 6 h at 37 °C with 1 μg/ml brefeldin A added in

culture. All conditions were performed in duplicate or triplicate and combined

before staining. Cells were removed, stained for Vγ9Vδ2 T-cell phenotype and

effector function and analysed by ﬂow cytometry.

HCC cell line cytotoxicity assay. HepG2 cells were plated (0.3 x 10

cells/well) in

a 24-well plate (Costar) and incubated in GlutaMax DMEM media containing 10%

FCS, 1% NEAA, 1% Sodium Pyruvate, 1% PenStrep for 16–18 h, in the presence or

absence of Zometa (ZOL) 5 μM (Novartis). After incubation, adherent HepG2 cells

were washed twice with PBS. ZOL treated PBMCs (containing expanded Vγ9Vδ2

T-cells) were subsequently added to each well in cRPMI for 6 h at 37 °C. The

culture supernatant was collected, frozen at −20 °C and thawed at time of analysis.

Control wells included ZOL Vγ9Vδ2-expanded PBMCs alone, HepG2 cells alone

and ZOL pre-treated HepG2 cells alone. 100% lysis positive controls included

HepG2 cells and ZOL pre-treated HepG2 cells resuspended in 0.1% Triton X-100

in PBS. ToxiLight

bioluminescent cytotoxicity assay (Lonza) was used to mea-

sure adenylate kinase (AK) release in the culture supernata nt as a marker of cell

death. As per manufacturer’s instructions, samples were mixed with the recon-

stituted AK detection agent, incubated for 5 min and then placed in a luminescence

compatible plate reader. Speciﬁc lysis was calculated as follows, referring to ZOL

Vγ9Vδ2-expanded PBMCs as effector cells and HepG2 cells as target cells: (RLU):

(RLU(Effector+Target)-RLU(Effector)-RLU(Target))/(RLU(100%lysis)-RLU(Ef-

fector)-RLU(Target))*100.

Statistical analyses. Statistical analyses were performed in Prism 7.0 (GraphPad)

using appropriate methods as indicated in the ﬁgure legends (Mann–Whitney test,

Wilcoxon matched-pairs signed rank test, Spearman rank correlation test,

Kruskal–Wallis test for unpaired non-parametric multiple comparisons, Friedman

test for paired non-parametric multiple comparisons, with Dunn’s post-hoc test for

multiple comparisons) with signiﬁcant differences marked on all ﬁ gures. All tests

were performed as two-tailed tests, and for all tests signiﬁcance levels were deﬁned

as: not signiﬁcant (ns) p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Reporting summary. Further information on research design is available in the Nature

Research Reporting Summary linked to this article.

Data availability

The raw data for Figs. 1–7 and Supplementary Figs. 1–7 are provided in the Source Data

ﬁle. The gene expression analysis data obtained from the Cancer Genome Atlas database

are publicly available through the Gene Expression Proﬁling Interactive Analysis 2

(GEPIA2) web server, http://gepia2.c ancer-pku.cn/#survival

. Source data are provided

with this paper.

Received: 29 May 2021; Accepted: 17 February 2022;

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Acknowledgements

This work was funded by a Wellcome Trust PhD Clinical Research Fellowship Award

(175479) to N.Z., and by a CRUK Accelerator award HUNTER, CRUK Immunology

project grant (26603) and Wellcome Trust Investigator Award (214191/Z/18/Z) to

M.K.M. A.Q. is supported by the National Institute for Health Research (NIHR) UCLH/

UCL Biomedical Research Centre (BRC). We are grateful to patients participating in the

study and to clinical staff who helped with participant recruitment and sample acqui-

sition, including NHS transplant coordinators, research nurses and the Tissue Access for

Patient Beneﬁt (TapB) project at The Royal Free Hospital (RFH) funded by RFH Charity

and UCLH/UCL BRC, as well as Jamie Evans in the Rayne Building Flow Cytometry

Core Facility who provided Fortessa X20 support. The illustrations in Figs. 2a, c, 6a, 7e

were created with BioRender.com, with a granted publication license.

Author contributions

N.Z. and M.K.M. conceived the project; N.Z., A.H., M.K.M. designed experiments; N.Z.

and A.H. generated data; N.Z., A.H., A.Q., M.K.M. analysed and interpreted data; N.Z.,

A.H., L.S., L.J.P., N.S., M.D., S.K., O.A., A.G., M.P., B.D., A.Q., M.K.M. provided or

processed essential patient samples and clinical data. N.Z. and M.K.M. prepared the

manuscript. All authors provided critical review of the manuscript.

Competing interests

Unrelated to the content of this manuscript, authors M.K.M. and N.M.S. have an

international patent application No.1917498.6 entitled Treatment of Hepatitis B Virus

(HBV) Infection ﬁled by applicant UCL Business Ltd. MP is co-founder and director of

Engitix Therapeutics Ltd, UK. The Maini lab has received unrestricted funding from

Gilead, Roche and Immunocore. The remaining authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s41467-022-29012-1.

Correspondence and requests for materials should be addressed to Mala K. Maini.

Peer review information Nature Communications thanks the anonymous reviewer(s) for

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ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-29012-1

16 NATURE COMMUNICATIONS | (2022) 13:1372 | https://doi.org/10.1038/s41467-022-29012-1 | www.nature.com/naturecommunications

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