1. Flow Cytometric TCR V Beta Analysis for Determination of T cell Clonality and Its Clinical Application
Prashant R. Tembhare, MD
Flow Cytometry Unit
Laboratory of Pathology
NCI/NIH, USA 1

2. Flow cytometric diagnosis of T-CLPD
Altered expression of pan T cell markers like
CD7, CD5, CD3, CD2
Subset restriction CD4+ or CD8+
Increased expression of few markers like CD25,
Aberrant Expression of antigens like CD10, CD30, CD103
Helpful in diseases with high tumor cell count

3. Problems in routine FCM
Altered expression of pan T-cell markers – Viral infections like IM, CMV, others
Subset restriction CD4+ or CD8+ may be seen in Viral infections like IM, HIV, Autoimmune diseases
Increased expression of few markers like CD25
- Increased T regulatory cells
Aberrant Expression of antigens like CD10, CD30 – limited to few NHL only
- CD30+ positive T cells can be activated T cells

4. Need of T cell Clonality
Confirmation & isolation of clonal cell proliferation
Follow up of treatment – with low cell count / partially treated
Minimal Residual Disease
Low cellularity specimens like CSF, FNA deep LNs/organs
staging
PB - Sezary cells
Morphology and IHC is not typical and helpful
LGLL
T cell clonality usually established by molecular techniques

5. Limitations of Molecular Techniques
Mainly qualitative
Time Consuming
Needs Specific set up
Amplification of background T cells may interfere
Clonal peak can not define Cell type and does not help in classification of CLPD
Highly Sensitive but less specific for malignancy

6. T cell Receptor (TCR) complex
T Cell Receptor complex
T cell membrane
Science :

7. α β polypeptides formation
Thymus
SCIENCE. 272, 21 ;1996:

8. TCR Vβ repertoire The a TCR – chromosome 7.
65 Vβ genes – 46 are functional
Vβ gene segments -26 subfamilies by cDNA
-75% sequence homology at the DNA level.
Palindromic & random additions and deletions
Unique V-D-J = 91 – Vβ segments
24 Vβ segments usage – 70% T cells
Each individual TCR-Vβ segment is expressed in only a small percentage of T cells.
SCIENCE. 272, 21 ;1996: Immunogenetics. 1995;42(6):451-3

9. Expanded T Cell Population - Vβ Analysis
Clonal T-cell expansions are expected to express a single V domain
FCA demonstration of a V restricted population is evidence of a T cell clone
There are V -specific antibodies now that recognize 70% of all individual V domains –
- TCR V beta Repertoire
V restricted T cell populations can be quantitatively monitored during and after therapy

10. Anti-TCR Vβ Ab-fluorochrome for each TCR-Vβ segment -
identify a Vβ specific T cell subset
Vb 7.1
FITC+PE
Vb 5.3 PE PE
FITC+PE
FITC
Vb 3
FITC

11. TCR Vβ Repertoire kit 3 antibodies in 2 colors (FITC, PE and FITC+PE)
The IOTest Beta Mark TCR-Vβ Repertoire kit – Beckman Coulter
24 Vβ-specific antibodies - recognize 70% of all individual Vβ domains
8 tube panel ( 8 X 3 = 24)
3 antibodies in
2 colors (FITC, PE and FITC+PE)
Cocktail
Mix A
Mix B C D E F G H
FITC
Vβ 3
Vβ 16
Vβ 20
Vβ 8
Vβ 12
Vβ 21.3
Vβ 14
Vβ 7.2 PE
Vβ 5.3
Vβ 9
Vβ 18
Vβ 13.1
Vβ 5.2
Vβ 23
Vβ 11
Vβ 13.2
FITC+PE
Vβ 7.1
Vβ 17
Vβ 5.1
Vβ 13.6
Vβ 2
Vβ 1
Vβ 22
Vβ 4
PerCP
CD3
APC
CD4 or CD8*

12. TCR Vβ Analysis – selection of T cells

13. Vβ Repertoire usage in T cells from reactive lymphoid tissues
% Vb usage in lymphocyte subsets Mean + 2 SD
TCR Vβ Family

14. Human Immunology. 64, 689–695 (2003).

15. TCR Vβ Repertoire usage in T cells : Normal Range
ALL CD3+ T cells
CD3+/CD4+ T cells
CD3+/CD8+ T cells
Mean
Range
V beta 1
3.53
(Normal )
3.32
(Normal )
4.24
(Normal )
V beta 2
8.3
(Normal )
9.36
(Normal )
5.43
(Normal )
V beta 3
4.68
(Normal )
4.37
(Normal )
4.44
(Normal )
V beta 4
1.91
(Normal )
2.03
(Normal )
1.9
(Normal )
V beta 5.1
5.45
(Normal )
6.71
(Normal )
3.22
(Normal )
V beta 5.2
1.33
(Normal )
(Normal )
1.12
(Normal )
V beta 5.3
1.08
(Normal )
1.09
(Normal )
0.92
(Normal )
V beta 7.1
2.56
(Normal )
1.93
(Normal )
3.39
(Normal )
V beta 7.2
1.47
(Normal )
(Normal )
2.44
(Normal )
V beta 8
(Normal )
4.81
(Normal )
4.06
(Normal )
V beta 9
3.13
(Normal )
4.07
(Normal )
3.47
(Normal )
V beta 11
1.04
(Normal )
0.87
(Normal )
(Normal )
V beta 12
1.66
(Normal )
1.82
(Normal )
1.29
(Normal )
V beta 13.1
3.83
(Normal )
4.03
(Normal )
3.42
(Normal )
V beta 13.2
2.8
(Normal )
2.81
(Normal )
3.34
(Normal )
V beta 13.6
1.86
(Normal )
(Normal )
1.6
(Normal )
V beta 14
3.49
(Normal )
2.59
(Normal )
5.74
(Normal )
V beta 16
(Normal )
0.95
(Normal )
0.8
(Normal )
V beta 17
5.15
(Normal )
5.46
(Normal )
5.06
(Normal )
V beta 18
1.49
(Normal )
1.92
(Normal )
0.57
(Normal )
V beta 20
2.52
(Normal )
2.6
(Normal )
2.31
(Normal )
V beta 21.3
2.38
(Normal )
2.46
(Normal )
2.39
(Normal )
V beta 22
3.84
(Normal )
4.26
(Normal )
3.17
(Normal )
V beta 23
0.85
(Normal )
0.48
(Normal )
1.34
(Normal )
Total
69.95
(Normal 69.95)
72.25
(Normal 72.25)
66.58
(Normal 66.58)

16. TCR Vβ Analysis: A case of ATLL

17. Total V beta usage
Normal
Direct Clonal
Indirect Clonal

18. TCR Vβ Analysis: A case of PTCL

19. What levels of expanded T-cell subset as a monoclonal T-cell population?
Suggestions in literature : Expression of a single TCR-Vβ
Lima M et al. Am J Pathol. 2001;159:
> 40% sensitivity 93% & specificity 80%
> 60% sensitivity 81% & specificity 100%
Beck RC et al. Am J Clin Pathol. 2003;120:
1.6 times of UNL
Morice WG et al. Am J Clin Pathol. 2004;121:
10-fold or > 50% of the T cells
40% to 49% suggestive
> 70% failed to react with panel of Ab
B Feng,et al. J Clin Pathol :
> 50% of a gated T cell subset; or
>10 times above its normal ranges T cell subsets; or
>70% of gated T cells failed to react to any of the TCR-Vb antibodies

20. Questions for TCR - V beta analysis
1 – At what level or % – clonal ?
2 – Gating strategy ?
3 – Can we use it in samples like BM, CSF, FNA ?
4 – Can we use it for MRD ?

21. NCI Study – 2007 to 2010
Patients & Samples
Total number of patients analyzed for screening 41
Peripheral Blood
Bone Marrow Asp- 03 +
Total samples
ATLL – 24
CTCL – 07
LGL
PTCL - 03
T-PLL - 01
Total number of patients followed up to monitor MRD and response to treatment 14
Peripheral Blood
Bone Marrow Asp- 02 +
Total samples
Concurrent molecular studies for TCRG gene rearrangement by PCR were performed in 36 cases.
Tembhare P et al. Am J Clin Pathol 2011;135:

22. Gating Strategy
Gating System -1 (GS-1)
Gating System -2 (GS-2)

23. Cut off levels for T cells Clonality
We found,
Using aberrant phenotype based gating strategy
i.e. GS-2
Demonstration of > 50% usage of single V beta domain OR
Failure to demonstrate > 70% of immunoreactivity
Tembhare P et al. Am J Clin Pathol 2011;135:

24. TCR-Vβ Monitoring of MRD T NHL
MRD - 61(48 PB, 7 FNA, 4 CSF and 2 BM)
Sequential samples - 14 patients - 3 years
Short immunophenotypic panels and
Single clone specific TCR-Vβ antibody cocktails
Number of tumor cells per micro liter of blood
Tembhare P et al. Am J Clin Pathol 2011;135:

25. Follow up

26. Number of cells at each interval

27. MRD by TCR-Vβ repertoire
The minimum level of clonal T cells cells/ul
Ranged from 0.8 cells/ul - 306,603 cells/ul
Median value of 1132 cell/ul

28. Immunophenotypic profile
TCR-Vβ Monitoring in Paucicellular specimens
FNAs and CSF:
Patients: 6 ATLL & 2 PTCL-NOS
Case
No 
Specimen 
 Immunophenotypic profile
TCR
V β
Family
% of Aberrant T Cells Vβ
Restricted
Cytology
Results
CD2
CD3
CD4
CD5
CD7
CD8
CD25
CD26
CD45
CD57 1
FNA +
+/- - ++
100 NA
94.8
Positive 2 +* 17
76.5
Suspicious
53.4
Atypical cells
76.1
60.0
60.4 3
13.1 90 4 84 5
CSF
72.4
Negative 7
86.5
93.3
91.9
92.6
76.2
Tembhare P et al. Am J Clin Pathol 2012;137:

29. Summary Sensitive method for detection of T cell clonality
Gating strategy –
aberrant immunophenotypes based – superior than T cell subset based.
Clonality - > 50% TCR-Vβ subset restriction
> 70% negative for all Vβ subsets TCR-Vβ Repertoire kit
Useful in low cellularity specimens such as FNA and CSF
Quantitative and highly sensitive MRD evaluation
Highly sensitive - treatment response & early relapse