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PHENOTYPIC AND FUNCTIONAL ALTERATIONS
OF GAMMA/DELTA T CELLS IN MULTIPLE MYELOMA
Massimo Massaia
Divisione di Ematologia dell'Universita' di Torino,
Laboratorio di Ematologia Oncologica, CeRMS

indolent
overt
progressive
MGUS
myeloma
myeloma
myeloma
osteoclast
activation
angiogenic
switch
infections
immune
failure
tumor
escape
Adapted from Nature Reviews - Cancer 2. 177-189, 2002

immunosurveillance cell players
NK
NKT
dendritic cells
T cells
Treg
B cells
T cells
tumor cell
GN/GE/M

V9/V2 T cells:
· first line defense against pathogens
(non-peptidic compounds like phosphoantigens)
· spontaneous antitumor activity in vitro
(MM and NHL cell lines)
· involved in immunosurveillance in vivo
(2M-/-; PF-/- mice)
· activated and expanded by nBPs
(Kunzman et al., N Engl J Med 1999; 340: 737-8)

Total amounts of circulating V9/V2 T cells
10
80
8
60
µl
cellsT
6
2
40
cells/
4
T
9/V
2
20
V
2
%
9/V
V
0
0
Controls (n = 20)
MM (n = 44)

BPs
etidronate
clodronate
nBPs
pamidronate
ibandronate
zoledronate

IL-2
Zol + IL-2
90% controls
Responders
CD3
56% MM
(R)
TCR
10% controls
Non-Responders
CD3
44% MM
(NR)
TCR

Identification of four subsets of human V9/V2 Tcells
Dieli et al., J. Exp. Med. 192, 1645; 2003.
Backgated on V2+ T cells
Central Memory
Naive
(CD27+ CD45RA-)
(CD27+ CD45RA+)
proliferation high
proliferation high
cytokines low
CD27
cytokines low
perforin low
perforin low
Effector Memory
CD45RA
Late Effector
(CD27- CD45RA-)
(CD27- CD45RA+)
proliferation low
proliferation low
cytokines high
cytokines low
perforin low
perforin high

80
30
naive
lls
25
central memory
µl
e
60
effector memory
c2
TEMRA
20
9V 40
15
counts/
V
10
Total
20
percent
5
0
0
N
RNR
NN
R
R
R
NR
memory
naive
CD27-PE
effector
late effector
CD45RA-FITC

Effective Zol-induced antitumor activity in R and NR MM patiens
60
100x103
75
+
-
45
75x103
50
30
50x103
inhibition
inhibition 25
cells/wellT
15
25x103
percent
percent
0
0
0
RNR
RNR
R
NR
R: responders
NR: non-responders
Zol + IL-2
Percent inhibition: 1 - ---------------------
IL-2

Questions:
1) Has the skewed V9/V2 T-cell subset distribution any clinical implication?
2) Does it reflect myeloma cell recognition?
· sex :
P = 0,006 (M > F in NR)
· age:
NS
· 2M:
NS
· stage:
NS
·WBC:
NS
· T-cell counts: NS
·% BMPC:
NS

20 CLL patients
11 R
9 NR
IgVH available in 9 pts
IgVH available in 7 pts
9/9 mutated
6/7 unmutated
Loss of naive/central memory V9/V2 T cells
is associated with the IgV mutational status

V9V2 T-cell differentation pathway
Central
Effectort
Late
Naive
Memory
Memory
Effector
· stress antigens
(i.e, MIC-A/MIC-B)
tumor cell
· mevalonate metabolites

Acetyl CoA + Acetoacetyl CoA
mevalonate pathway
HMG CoA
HMG CoA
Reductase
Mevalonate
Mevalonate
Pyrophosphate
V9V2
Isopentenyl
Pyrophosphate
Geranyl
Pyrophosphate
Zoledronate
FPP-synthase
Farnesyl
Pyrophosphate
Ubiquinone
Geranyl-Geranyl
Pyrophosphate
Squalene
Protein
Farnesylation
Protein Geranyl-
Geranylation
Cholesterol

mevalonate pathway
Acetyl CoA + Acetoacetyl CoA
HMG CoA
HMG CoA
Mevastatin
Reductase
Mevalonate
Mevalonate
Pyrophosphate
Isopentenyl
Pyrophosphate
Geranyl
Pyrophosphate
FPP-synthase
Farnesyl
Pyrophosphate
Ubiquinone
Geranyl-Geranyl
Pyrophosphate
Squalene
Protein
Farnesylation
Protein Geranyl-
Geranylation
Cholesterol

mevalonate pathway
Acetyl CoA + Acetoacetyl CoA
HMG CoA
HMG CoA
Mevastatin
Reductase
Mevalonate
Mevalonate
Pyrophosphate
V9V2
Isopentenyl
Pyrophosphate
Geranyl
Pyrophosphate
Zoledronate
FPP-synthase
Farnesyl
Pyrophosphate
Ubiquinone
Geranyl-Geranyl
Pyrophosphate
Squalene
Protein
Farnesylation
Protein Geranyl-
Geranylation
Cholesterol

Zoledronate (Zol) enhances the sensitivity of myeloma cells
to V9/V2 T cells via the mevalonate pathway
80
75x103
80
60
60
50x103
40
inhibition
40
cells/well
inhibition
T
20
25x103

20
percent
percent
0
0
0
-20
RNR
RNR
Zol Zol
+
Mev
R: responders
Zol-pulsed MCL
NR: non-responders
Percent inhibition: 1 - -----------------------------
untreated MLC

Facts:
· Skewed distribution of V9/V2 T cells in approx 50% of MM pts at diagnosis
· Activation of V9/V2 T-cell effector functions by nBPS
· Immunosensitivity of myeloma cells to V9/V2 T cells is enhanced by nBPs
· Immunomodulation by nBPs is mediated by the mevalonate pathway
Thoughts:
· Is the loss of naive/memory V9/V2 associated with disease evolution?
· Is the mevalonate pathway of tumor cells a target of immune recognition?
· Is the mevalonate pathway a target of immune therapeutic intervention?

LABORATORY OF HEMATOLOGY ONCOLOGY
Michela
Barbara
Muraro
Nushack
Giorgia
Francesca
Sara
Myriam
Barbara
Francesca
Marta
Matta
Pantaleoni Mariani Foglietta
Castella
Fiore
Coscia

Torino Myeloma Team (TMT):
Antonio Palumbo
Benedetto Bruno
Paola Omede'
Patrizia Falco
Alessandra Bertola
Gabriele Aitoro
Maria Teresa Ambrosini
Federica Cavallo
Ilaria Avonto
Tiziana Marangon
Maria Teresa Petrucci (Rome)
Mario Boccadoro