T Cells In MM
· A global proteomic perspective.
T
l
ce lll
t
an i
tigens th
the
l
on y i
s
i
gn fi
ifi
t
can
l
ce lll l
u ar difference
between the peripheral blood of MGUS and MM.
· Expanded T cell clones
Prognostic significance
significance. Impact
Impact of
of thalidomide
thalidomide.
· Treg cells
Impact on immune modulation
· Trogocytosis and T cells in MM
High incidence in MM. Possible deletion of clones.
DotScan
- 160 dot proteomic micro-array
- Disease-specific signatures
- Cross-validated discriminant
analysis of cell surface antigens
Normal
MM
MGUS
Representative histograms of cell binding intensity of a single sample.
Anti
tigens with
ith the hi h
g
t
es ranking for diff
dif erenti
ti t
a i
tion
CD28 (reduced in MM)
CD8 and CD57 (high in MM)
C2
CD25 (reduced after thalidomide),
Flow Validation
CD95 (reduced in MGUS)
reinforcing the importance of immunomodulatory
mechanisms in both MM and MGUS.
Overall prediction success, sensitivity
andi
d spec fi
ifi i
c t
ity of di
discriminant analysis
· 100% success in predicting samples in normal (n=11)
and MM (n=
(n 24) groups .
· No specific signature detectable for 10-year MM survivors
· Only misclassifications were in the MGUS group.
MGUS (n=14) diagnosed an average of 7 years previously.
4 MGUS predicted to be normal have normal blood parameters
4/5 MGUS predicted as MM now have anaemia and/or renal failure
2. Clonal T cell populations
il
in myeloma
Tl
T ce l
ll l
c ones in MM
MM bl
blood
Originally detected by Southern blot
Brown et al. Leukemia (1997)
() 11:1312
T cell clones detected
83 patients
p
No T cell clones detected
T cell clones = Good prognosis
Additional studies
Throughout Disease (n=144)
At Diagnosis (n=45)
1.0
Detected at least once
Never detected
rvival
2
=5 7;
.
p<0 0
. 1
01
Su 0.5
2=7.1; p<0.008
0
100
100
200
Months
Raitakari et al (2003) Haem Oncol 21: 33
"Late" (CD28-CD27-) differentiated cytotoxic
T celllls are associtd
iated
i
w th
ith a survival d
a
t
van age
n=28
n=22
Sze et al (2003) Blood 102:927a
Survival advantage of patients with clonal
lT
late stage T
l
ce l
lls- Li
Longitudi
dinal
d
stu y
2000
CD28
CD57+
Majority of T cells - "late"
Serum IgG
IgG = 30g/L
Stable disease
Monoclonal
CD27
2003
Depletion of CD57+ popn.
CD57+
CD28
Majority of T cells -
"intermediate"
Serum IgG = 70g/L
Progressive disease
CD27
T Cell Clones are now
detected by Flow cytometry
BetaMark Analysis.
TCR V Repertoire
24 V families
4 colour flow cytometry
CD3
CD4
CD8
Flow cytometry allows....
· Immunophenotyping.
CD8+ CD57+ CD28-
CD8 CD57 CD28
perforin +ve
PE-
Cytotoxic suppressor T cells
andedp
ex
· Quantitation.
Vb
Represent up to 30%
CD57 - FITC
Perforin RED613
pp
of total T cells
30
Nov 97
25
Mar 98
Oct 98
nt 20
e
Mar 99
· Sequential studies.
15
Present for long
Perc 10
periods
5
periods
0
1
2
3
.1
.2
.3
7
8
9
1
2
.1
.6
4
6
7
8
0
1
2
3
5
5
5
1
1
3
3
1
1
1
1
2
2
2
2
1
1
Sze et al. Blood (2001) 98:2917
Br J Haem (2000) 110:203
TCR V
family
Are the T cell expansions truly clonal?
Clonality confirmed by TCR length and sequencing
· Purified TCR V expansions
· CDR3 length - Clonal (or biclonal)
()
· Clonal 1 sequence in PCR product
Sze et al Blood (2001) 98:2917
Br J Haem (2000) 110:203
Study Protocol
ALLG MM6 (
243)
n=
T
ARM 1
P
Z
Melphalan
R
2
R
200mg/m
REGISTERED
and ASCT
No PD after 6wk
P
ARM 2
Z
Spencer et al 2006
MM6 - Progression-Free Survival
by Arm
100
free
Thalidomide
No Thalidomide
ion
80
P=0.0005
progressng 60
survivi
40
ntage
perce
PFS
ARM 1
ARM 2
20
ated
1 year
90%
69%
m
2 year
66%
40%
Esti
3 year
39%
25%
00
3
6
9
1215
182124
273033
363942
4548
Months following randomisation
Number at risk
Thal.
114
112
106
106
98
83
68
55
43
29
22
15
10
7
3
0
0
No Thal.
129
127
118
105
87
72
49
37
28
21
16
15
8
7
3
1
0
Spencer et al 2006
Aims of MM6 T cell Study
· To verify the prognostic significance of clonal T cell
expansions with a different
different patient cohort.
· To investigate the immunomodulatory effects of
thalidomide on T cell expansions
The MM6 T cell study
Peripheral blood samples
- After induction & prior to transplant
- 8 months
months post randomisation
randomisation
- 12 months post randomisation
TCR V repertoire analysed
- detection of clonal expansions of
T cells
cells
MM6 T Cell Samples
· 221 peripheral blood samples
· 120
1
patients
104 pre-transplant and
117
11 after maintenance
· 15 Tr
T ial
r
Centres
· 42 Age-
Age matched control samples
samples
Progression Free Survival
ALLG M6 Patients studied for T
cell clones
1.0
e
Thalidomide
ref
No Thalidomide
ions
2
s 05
0.5
=75
7.5; p=0.006
006
e
n=120
ogr
Pr
0.0
0
10
20
30
40
50
60
Months
Median survival
Thal
T
i
hal dom
d
i
om de
d
36 mont
mon hs
ths
No Thalidomide
21 months
Progression Free Survival
ALLG MM6 - Presence of T cell
clones
1.0
T cel clones detected
e
No T cel clones detected
fren
2
=42
4. ;
2; p=00
p0. 4
04
ioe 0.5
n=120
reg
roP
0.0
0
102030405060
Months
Medi
M
an PFS
PFS
T cel s detected
32.1 months
No T cel s detected
24.1 months
Progression Free Survival
ALLG- MM6
1.0
Thal and T cel s
e
Th l
a
d
an No T
l
cells
ref
No Thal and T cel s
ion
No Thal and No T cel s
ss 05
0.5
e
2= 9.5; p=0.002
rogrP
0.0
0
10
20
30
40
50
60
Mon
Mo t
n hs
h
Median Survival
Thal and T cel s
40.1 months
Thal and No T cel s
28.3 months
No Thal and Tcells
21.3 months
No Thal and No T cel s
15.9 months
Results
93% of all T cell clones in MM6 patients were CD8+
All 24 TCR V families studied were over expressed in at least one patient
Ni
No si
l
ng e TCR V fi
fam lily was over-represented
48% patients had T cell clones prior to transplant
After 8 months
After 12 months
Thalidomide
76%
62%
Control
60%
47%
More than one clone/patient
Thalidomide
49%
Control arm
23%
2=6.8; p=0.01
There was a trend for patients to develop new T cell clones after thalidomide
(52%) compared with the control arm (40%)
3. T reg Cells in MM
· Exert strong suppression after T-cell
receptor
p
stimulation
· Expanded in patients with
with tumours
tumours ,
Hodgkins lymphoma and CLL
· Thymic origin CD4+CD25high FoxP3+
Treg Cells in Myeloma
· Do abnormalities of T reg cells explain
Immune dysfunction
Failure of active immunization
Expanded T cell
cell clones
clones
Hyperreactive T cells
Treg Cells in Myeloma
CD4+ CD25+FOXP3+
Beyer et
et al
al Blood
Blood (2006) 107:3940
- conflicting reports
- ? increased or decreased.
- ? functional
functional or
or dysfunctional
dysfunctional
CD4+ FOXP3+
Prabhala et al Blood (2006) 107:301
4. Trogocytosis in MM
Acquisition by T cells of presentasome
CD40
CD40L
Ag
TCR
APC
MHC
T cell
CD80
CD28
T reg cells and trogocytosis
· Acquisition of MHC/CD80 complex
complex by
memory T cells can lead to apoptosis
· Naïve T cells following acquisition of
CD80, can
can function as APC s
Wiendl, H. Blood 2007;109:1796-1797
T reg cells and trogocytosis
· T cells acquire HLA-G1 from APC's and
become inhibitory regulatory cells
· Acquisition of MHC/CD80 complex by
memory T cells can lead to apoptosis
· Naïve T cells following acquisition of
CD80, can function as APC s
Wiendl, H. Blood 2007;109:1796-1797
Trogocytosis the transfer of cell membrane molecules
across the immunological synapse. Common in MM.
CD40
MHC
APC
La Maoult et al (2007) Blood 109:2010.
CD86 mRNA
mRNA in Monos but
CD80 and/or 86 expression detected
not in T cells
on CD3 cells in >30% of MM patients
T M
T
M T
M
T
M
T
M
90%
13
25
4
-ve
CD86
Patient
CD80 and CD86 Upregulation
Upregulation on B and T Cells
Cells
BC ll
d T
50
CD80
CD86
· B Cells and T
50
CD80
Cells cultured
in CD40L and
40
IL 2
- .
30
%
· CD80 and
ressionp
CD86
20
ex
upregulated on
B Cells.
10
Cells.
0
· No CD80 or 86
B Cells
T cells
B Cells
T cells
upregult
la i
tion on
Control
IL-2
T cells.
huCD40LT
huCD40LT+IL-2
CD80acq+ CD86acq+ and HLA-G1acq+ T cells
CD80
CD86
HLA-G1
30
40
30
lls
ells
ells
ce
c 30
c
e
20
20
sitiveo
20
P
positive
positive
1
80
10
86
-G 10
D
D
A
D
10
L
C
C
%
%
%H
0
0
0
CD4
CD4
CD8
CD8
CD4
CD4
CD8
CD8
CD3
CD3
Normal
MM
Normal
MM
Normal
MM
Normal
MM
Normal MM
Conclusions - T reg
· Conflicting published data on role in myeloma
· Trogocytosis ( CD80/MHC )and Treg
abnormalities may be responsible for immune
df
defects seen in T celllls in myeloma
· ?A
? Are th
the Treg celllls under
d
expresse
h
w en
clones are present?
Conclusions - Proteomic expression
· DotScan can be used to differentiate the monoclonal
gammopathies.
· Misclassifications occurred only for MGUS patients.
Apparent misclassification
misclassification correctly identified evolving
evolving disease.
· Thalidomide caused a significantly different expression pattern.
· Patients with a >10 year survival could not be identified by
discriminant analysis.
· Proteomic microarray expression patterns were validated by
trad
ta i
d ti
t on
o al
a flow
o
cyto
cyt m
o et
e ry.
ty
Conclusions - Clonal T cells
cells
· CD8+ T cell clones were of prognostic
pg
significance
g
in this
patient cohort of patients validating our previous
studies using Southern Blots.
· Thalidomide had a prognostic impact on both the patients
who had T cells clones and those who did not.
· The best prognosis was the Thal + T cells group.
· In patients who had T cell clones, those patients on
thalidomide developed multiple T cell clones.
Acknowledgements
Ross Brown
Funding
Alan Murray
Simon Cooper
IMF
Mila Dolotin
MMRF
Karieshma Kabani
Kabani
Anthony Rothe Trust
Trust
CINSW
Daniel Sze
NSW SCC
Shihong Yang
Y
Foundation IV
IV
Joy Ho
John Gibson
Cris dos Remedios
Jeremy Crisp
Larissa Belov