Open Access

Identification of a public CDR3 motif and a biased utilization of T-cell receptor V beta and J beta chains in HLA-A2/Melan-A-specific T-cell clonotypes of melanoma patients

  • Federico Serana1,
  • Alessandra Sottini1,
  • Luigi Caimi1,
  • Belinda Palermo2,
  • Pier Giorgio Natali2,
  • Paola Nisticò2 and
  • Luisa Imberti1Email author
Journal of Translational Medicine20097:21

https://doi.org/10.1186/1479-5876-7-21

Received: 03 March 2009

Accepted: 24 March 2009

Published: 24 March 2009

Abstract

Background

Assessment of T-cell diversity, besides giving insights about the molecular basis of tumor antigen recognition, has clinical implications since it provides criteria for evaluating antigen-specific T cells clinically relevant for spontaneous and vaccine-induced anti-tumor activity. Melan-A is one of the melanoma antigens most frequently recognized by peripheral and tumor-infiltrating lymphocytes in HLA-A2+ melanoma patients. Many clinical trials involving anti-tumor vaccination have been conducted using modified versions of this peptide.

Methods

We conducted an in-depth characterization of 210 T-cell receptor beta chain (TRB) clonotypes derived from T cells of HLA-A2+ melanoma patients displaying cytotoxic activity against natural and A27L-modified Melan-A peptides. One hundred and thirteen Melan-A-specific clonotypes from melanoma-free subjects, 199 clonotypes from T-cell clones from melanoma patients specific for melanoma antigens other than Melan-A, and 305 clonotypes derived from T cells of HLA-A2+ individuals showing unrelated specificities, were used as control. After sequence analysis, performed according to the IMGT definitions, TRBV and TRBJ usage, CDR3 length and amino acid composition were compared in the four groups of clonotypes.

Results

TRB sequences of Melan-A-specific clonotypes obtained from melanoma patients were highly heterogeneous, but displayed a preferential usage of few TRBV and TRBJ segments. Furthermore, they included a recurrent "public" amino acid motif (Glycine-Leucine-Glycine at positions 110-112-113 of the CDR3) rearranged with dominant TRBV and TRBJ segments and, in one case, associated with a full conservation of the entire TRB sequence.

Conclusion

Contrary to what observed for public anti-Melan-A T-cell receptor alpha motifs, which had been identified in several clonotypes of both melanoma patients and healthy controls, the unexpectedly high contribution of a public TRB motif in the recognition of a dominant melanoma epitope in melanoma patients may provide important information about the biology of anti-tumor T-cell responses and improve monitoring strategies of anti-tumor vaccines.

Background

T-cell receptor (TR) plays a central role in the immune response, interacting with peptide antigens (Ags) and with major histocompatibility complex (MHC) molecules. TR alpha (TRA) and beta subunits are comprised of a variable (V) and a constant (C) amino acidic region. The TRBV region, referred according to the ImMunoGeneTics (IMGT) database [1], is encoded by V, diversity (D), and joining (J) gene segments. The juxtaposition of these segments [2], the lack of precision during V(D)J gene rearrangement and the removal and/or addition of non-template encoded nucleotides at V(D)J junctions [3], create a region of hypervariability known as complementarity-determining region 3 (CDR3).

Despite the potentially vast T-cell repertoire, restrictions of TR composition, known as TR bias, are commonly observed [4]. These TR constraints include the preferential usage of one TRV or TRJ region without conserved CDR3, the selection of conserved amino acids (up to five) or 'motifs' at the same CDR3 specific positions, and the selection of clonal TR sequences with identical CDR3 [4]. The different individual responses to discrete Ags are manifested in terms of personal, or "private", and shared, or "public", motifs in the TR sequences [4]. A private TR repertoire describes a situation in which T cells of distinct subjects responding to the same peptide-MHC complex have no significant overlaps in their TR sequences. In contrast, TR repertoires are defined public when Ag-specific T cells in several individuals use the same TR motifs, either in the TRA or TRB chains. To date, TRA and TRB public motifs have been described in human T-cell responses directed against viral peptides [4], while, in the anti-melanoma Ag response, only public TRA motifs have been reported [57]. However, TRA constraints, in particular within TRAV12-1 (previously defined Vα2 or TCRAV2.1) T cells, were observed not only in melanoma patients [57], but also in cord blood, thymocytes and PBL of non tumor-bearing controls [5], as well as in several subjects with vitiligo [8, 9]. On the contrary, no public TRB motifs were identified in the sequences of Melan-A-specific T cells of melanoma patients and controls [58, 1019]. The unreported identification of public TRB in anti-melanoma Ag response may be related to the use of different methodological approaches employed to obtain T-cell lines or clones and to analyze CTL activity, as well as to prepare, characterize and analyze TR sequences. Another explanation can be the low number of patients analyzed in different studies. To bypass these limitations we took advantage, in the present study, of the availability of several published and unpublished TRB sequences obtained from a number of melanoma patients in order to study different aspects of TRB chain structural constraints imposed by the melanoma Ag MART1/Melan-A (hereafter reported as Melan-A). This differentiation Ag is a membrane-embedded protein of 118 amino acids expressed both by melanocytes and melanoma cells. Among the melanoma-associated Ags identified so far, Melan-A has received particular attention because of its immune dominance in HLA-A2+ patients. A large number of T-cell clones generated from HLA-A2+ patients are cross-reactive against either the natural nonamer/decamer Melan-A peptide (26/27–38) or the Alanine-to-Leucine substituted heteroclitic Melan-A A27L peptide [20, 21]. Here, we identified several melanoma/HLA-A2-restricted TRB clonotypes (sequences showing different CDR3 in a given individual), and, after the definition of a common TR nomenclature, numbering and CDR3 designation, we studied in details their molecular features.

Methods

The TRB sequences analyzed in this study were obtained either from previously reported or still unpublished studies. The rationale underlying selection of the 4 groups of TR sequences was to take into account three characteristics of the TR clonotypes which may generate biases in the selection of CDR3 region, i.e. Melan-A specificity, HLA-restriction and categories of individuals analyzed. Two hundred and ten Melan-A-specific clonotypes [[57, 1018] and manuscript in preparation], sequenced starting from T-cell lines or clones obtained from PBL and/or tumor-infiltrating lymphocytes (TIL) of melanoma patients ("Mel/M-A" group; Table 1), were compared with 113 Melan-A-specific clonotypes ("Ctrl/M-A" group) from healthy controls and from a subject with vitiligo [5, 8, 19], 199 clonotypes specific either for melanoma Ags other than Melan-A peptide or with undetermined specificity ("Mel/noM-A" group) obtained from T cells of melanoma patients [2241], and 305 clonotypes prepared from HLA-A2+ melanoma-free patients ("Ctrl/HLA-A2+" group) selected because sequenced from T-cell lines and clones displaying CTL activity against unrelated Ags [4254]. One hundred and seventy clonotypes of the Mel/M-A group and 85 from the Ctrl/M-A group were specific for the HLA-A2-restricted A27L-modified Melan-A peptide and their CTL activity was evaluated using a multimer-based approach [[5, 6, 8, 1214, 1719], and manuscript in preparation], by competition assay [15], or by analyzing the production of IL-2 in response to HLA-A2 Melan-A-expressing melanoma cell lines [7]. The remaining 40 clonotypes derived from cells of melanoma patients displayed CTL activity against natural Melan-A peptide, as demonstrated by 51Cr release assay [10, 11, 16]. Twenty-eight clonotypes of the Ctrl/M-A group, although specific for Melan-A peptide, were obtained from HLA-A2-negative healthy controls. Details on type of treatment, including vaccination, the starting material (peripheral blood or TIL), the experimental procedures used to obtain T-cell lines and clones or to analyze CTL activity, as well as the methodologies for TR sequencing are specified in the references included in Table 1. Before analysis, sequences available only in nucleotide form were translated into their amino acidic counterparts. All sequences analyzed in this study are supplied in the supplemental tables (additional file 1, 2, 3 and 4) showing, respectively, the clonotypes from Mel/M-A, Ctrl/M-A, Mel/noM-A and Ctrl/HLA-A2+ groups. In order to obtain uniformed information, TRBV gene family and CDR3 amino acid positions were named and numbered according to the IMGT indications http://imgt.cines.fr[55].
Table 1

Characteristics of the TR clonotypes analyzed in this study

 

number of

        

Clono type

seq a

subjects/patients (patients ID) b

HLA

vaccination

 

source

type of sequenced cells

specificity c

TRBV selection

references

Mel/M-A

47

90

5 (8,22, 15, 30, 38)

A2

modified Melan-A

pre/postd

PBL

T-cell clones

Melan-A*

no

in preparation

 

6

6

1 (VER)

A2

no

 

PBL

T-cell clones

Melan-A*

no

5

 

26

27

3 (M199, M180, M138)

A2

no

 

TIL

T-cell clones

Melan-A *

no

6

 

11

11

10 (Mela01, 02, 03, 04, 05, 06, 10, 13, 15, 16)

A2

modified Melan-A CTL clones

pre

PBL

T-cell clones

Melan-A***

no

7

 

2

17

2 (-)e

A2

no

 

TIL

CTL lines

Melan-A****

4, 28

10

 

30

119

3 (1, 2, 3)

A2

no

 

PBL/TIL

CTL lines

Melan-A****

7, 20, 29, 12, 5

11

 

18

54

2 (LAU 181,203)

A2

no

 

TIL

CD8-sorted cells

Melan-A*

27, 30

12

 

7

7

3 (NW28, 29, 30)

A2

Melan-A, Tyrosinase, gp100

pre/post

PBL

CD8-sorted cells

Melan-A*

no

13

 

27

50

1 (-)

A2

no

 

PBL/TIL

T-cell clones

Melan-A*

no

14

 

9

10

3 (SK9-AV, M77, LB373)

A2

no

 

PBL/TIL

T-cell clones

Melan-A**

no

15

 

8

9

5 (8959, LB39, AV, 501, 9742)

A2

no

 

PBL/TIL

T-cell clones

Melan-A****

no

16

 

12

27

1 (LAU444)

A2

modified Melan-A

pre/post

TIL/PBL

CD8-sorted cells

Melan-A*

6, 28

17

 

7

17

1 (LAU337)

A2

Melan-A

post

PBL

T-cell clones

Melan-A*

no

18

 

210

444

         

Ctrl/M-A

53

53

3 (HD421, HD009, T12)

A2

NA

 

PBL/Thymocytes

T-cell clones

Melan-A*

no

5

 

32

37

1 (PSA)

A2

NA

 

PBL

T-cell clones

Melan-A*

no

8

 

28

28

4 (HD001, HD002, HD010, CB886)

Various A2-

NA

 

PBL

CD8-sorted cells

Melan-A*

no

19

 

113

118

         
 

4

8

1 (-)

A2, A24

peptide-pulsed DCf

post

PBL/TIL

-

-

no

22

Mel/noM-A

1

-

Patient 1

A11, A32

IL-7+ autologous melanoma cells

pre/post

PBL/TIL/DTH

-

-

27

23

 

2

2

1 (FON)

A2, A29

no

 

TIL

T-cell clones

autologous melanoma

no

24

 

4

9

1 (MZ2)

Cw16

MNNG-treated melanoma cells

pre/post

PBL

T-cell clones

BAGE, MAGE1

no

25

 

7

140

1 (-)

-

no

 

TIL/PBL/Skin

-

-

14, 29, 23

26

 

9

40

1 (-)

B14

no

 

TIL/Tissue

T-cell clones/lines/TIL

-

6

27

 

25

42

6 (20113, 20297,20254, 20249, 20360, 20063)

-

DNP-modified melanoma cells

post

TIL in metastases

-

-

no

28

 

6

38

1 (til 620)

-

no

 

TIL

T-cell colture

Melan-A/gp100

20, 19, 13

29

 

52

87

4 (1, 2, 5, 6)

A2

no

 

TIL, PBL, normal skin

-

-

27, 9, 20, 29, 28, 7

30

 

11

42

1 (2)

 

autologous stem cells after CTX

pre/post

PBL

-

-

2

31

 

3

3

2 (BON, MAR)

A2, A25

no

 

TIL

-

-

28, 2, 24

32

 

3

3

1 (MZ2)

A1

autologous melanoma cells

 

PBL

T-cell clones

MAGE1

no

33

 

5

10

1 (9742)

A2

no

 

PBL/TIL

T-cell clones, PBL-PHA

autologous melanoma

no

34

 

19

38

1 (JB)

A1, A28

DNP-modified melanoma cells

post

TIL

-

autologous melanoma

27

35

 

4

15

2 (1200, 501)

A1, A2

no

 

TIL

bulk/CTL microcultures

A1/A2+ melanoma cells

no

36

 

22

172

3 (1622, 1464, 1214)

A24, 26; A3, 11, A24

no

 

Tissue

-

-

6, 27, 28, 24, 10

37

 

1

1

1 (0831)

A2

no

 

TIL

-

-

8

38

 

16

100

5 (1, 2, 3, 4, 6)

-

no

 

Tissue

-

-

4, 28, 25, 29

39

 

1

15

1 (LB256)

A2

no

 

PBL

T-cell clones

gp100

no

40

 

4

192

1 (1803)

A1

no

 

TIL

bulk + cultures

-

20

41

 

199

957

         

Ctrl/HLA-A2+

41

46

15 (BD, CL, DD, DP, HL, JE, JM, JN, JW, KD, KE, MO, MP, NM, SW)

A2

NA

 

PBL

T-cell clones

M58-66 (flu)

19

42

 

56g

606

12 (PB1, PB2, PB3, PB4, RA1, RA2, RA3, RA4, RA5, RA11, RA14, RA15)

A2

NA

 

PBL/SFL

T-cell clones/CD8-sorted lines

GLC/A2 (EBV)

2, 20, 29, 9, 14

43

 

9

9

2 (FM, JM)

A2

NA

 

PBL

T-cell lines

M57-68 (flu)

no

44

 

42

-

5 (B, F, M, P, T)

A2

NA

 

PBL

CTL/CD8-sorted population

GL9 (EBV)

no

45

 

79

-

9 (D, F, H, K, M, N, P, R, S)

A2

NA

 

PBL

CTL/CD8-sorted cells

NV9 (CMV)

 

45

 

33

43

4 (BMT, HD, RA, KT)

A2

NA

 

PBL/SFL

T-cell clones

pp65 (NLV/A2, HCMV)

no

46

 

5

92

3 (003, 065, 868)

A2

NA

 

PBL

T-cell lines/clones/CD8-sorted cells

GAG (HIV), POL(HIV)

28, 5, 12

47

 

1

7

1 (HEU)

A2

NA

 

TIL

T-cell clones

lung cancer antigen

no

48

 

3

31

1 (HEU)

A2

NA

 

TIL/PBL

T-cell clones

alpha-actinin-4

no

49

 

14

28

2 (-, 5H13)

A2

NA

 

PBL

T-cell clones

mHag HA-2

no

50

 

9

15

1 (2)

A2

NA

 

PBL

CD8-sorted cells

19-kDa M. tuberculosis

no

51

 

6

9

1 (-)

A2

NA

 

TIL

T-cell clones

various tumor epitopes

no

52

 

2

29

1 (LB37)

A2

NA

 

PBL

CD8-sorted cells

mutated malic enzyme

no

53

 

5

24

2 (MS2, MS7)

A2

NA

 

PBL

T-cell culture

TALpep

no

54

 

305

939

         

a Number of sequences from which the clonotypes have been selected.

b Abbreviations: CTL: Cytotoxic T Lymphocytes; CTX: Chemotherapy; DNP: Dinitrophenyl; DTH: delayed-type hypersensitivity site: ID: Identification number; MNNG: N-methyl-N'-nitro-N-nitrosoguanidine; NA: not applicable; Seq: sequences; SFL: synovial fluid lymphocytes; TIL: Tissue infiltrating lymphocytes.

c CTL specificity against modified Melan-A analyzed by *multimers; ** competition assay; *** production of IL-2 in response to HLA-A2 Melan-A-expressing melanoma cell lines **** or CTL specificity against natural Melan-A analyzed by 51Cr release assay.

d Clonotypes identified either in pre or in post vaccination.

e -: data not available.

f MAGE-4, MAGE-10, GnTV, gp100, Melan-A, FluMP, FluBNP-pulsed dendritic cells.

g Identical clonotypes are included if found in different patients.

Bold: total number of clonotypes and of sequenced TRBV chains in each group

Statistical analysis

To analyze TRBV or TRBJ segment usage, the 95% confidence intervals of the respective proportions were calculated. "Preferentially used" were defined those segments whose lower limit of the respective 95% confidence interval was higher than the mean percentage of TRBV or TRBJ transcripts usage, obtained by arbitrarily hypothesizing a uniform distribution of all segments. When proportions were compared, Fisher's exact test was employed, while the differences between the means of CDR3 length distributions in the four groups of clonotypes were evaluated by Kruskal-Wallis test and Dunn's post-hoc test. Results were considered significant for p < 0.05.

Results

Preferential TRBV and TRBJ usage in HLA-A2/Melan-A restricted response in melanoma patients

We first investigated whether clonotypes identified in HLA-A2+ melanoma patients with CTL specificity against Melan-A (Mel/M-A group) had a preferential usage of particular TRBV chains and whether these preferential TRBV were also predominantly utilized in the control (Ctrl/M-A, Mel/noM-A and Ctrl/HLA-A2+ groups) clonotypes. As shown in Figure 1A, multiple transcripts covering the majority of the TRBV families were observed in the 4 groups of clonotypes, although some TRBV segments were preferentially used. In particular, while TRBV6 and TRBV27 were highly represented in all groups of clonotypes, TRBV4 was overrepresented in response to melanoma Ags but not to unrelated Ags, TRBV19 was preferentially used in clones of HLA-A2+ control individuals, and TRBV28 appeared to be preferentially selected only by Melan-A-specific CTL. TRBV usage comparison among the 4 groups suggested that the proportion of clonotypes using TRBV27 chains was higher in Mel/M-A, Ctrl/M-A and Mel/noM-A sequences compared to Ctrl/HLA-A2+ clonotypes (p = 0.03; p = 0.004; p < 0.001), while TRBV28 was significantly more frequent in Mel/M-A clonotypes than in Mel/noM-A and Ctrl/HLA-A2+ groups (p = 0.001 and p < 0.001).
Figure 1

TRB segments usage. TRBV (A) and TRBJ (B) segments usage and CDR3 length (C) in clonotypes prepared from Melan-A-specific CTL lines and/or clones of melanoma patients (Mel/M-A), clonotypes from Melan-A-specific CTL of healthy controls and of a patient with vitiligo (Ctrl/M-A), clonotypes of melanoma patients specific for melanoma Ags other than Melan-A or with unknown specificity (Mel/noM-A), clonotypes from HLA-A2+ subjects derived from T lymphocytes specific for Ags unrelated to melanoma (Ctrl/HLA-A2+). The sequences analyzed here are those reported in Table 1. As indicated in Table 1, in some papers a pre-selection of cells bearing some specific TRBV segments was done before sequencing. * TRBV and TRBJ chains preferentially used within clonotype groups. The TRB nomenclature used throughout the paper is that of Lefranc et al [1]; the nomenclature reported in parenthesis is that of Arden et al [49]. (aa): amino acids.

Among Mel/M-A clonotypes there was a high number of clonotypes bearing the TRBJ2-1, TRBJ2-7 and TRBJ1-5 segments (Figure 1B). However, the first two TRBJ chains, however, were highly utilized also in other groups of clonotypes (Figure 1B), and had also been frequently observed among peripheral blood T-cells from healthy individuals [56].

The mean CDR3 length was highly similar (p = NS) in Mel/M-A, Ctrl/M-A and Mel/noM-A groups (mean ± SD: 12.37 ± 1.29, 12.32 ± 1.43 and 12.35 ± 1.71, respectively), but significantly lower in Ctrl/HLA-A2+ clonotypes (11.95 ± 1.50) in respect to Mel/M-A (p < 0.01) and Mel/noM-A sequences (p < 0.05). Furthermore, the majority of Mel/M-A and Ctrl/M-A CDR3 were 12 amino acid long (32.9% and 31.9% respectively), while most of CDR3 of Mel/noM-A and Ctrl/HLA-A2+ sequences were 13- and 11-amino acid-long, respectively (Figure 1C).

Collectively, the present analysis demonstrated that in melanoma patients there is a biased T-cell response to Melan-A, which is characterized by TR clonotypes using preferentially TRBV28 and TRBJ1-5 segments and containing a 12-amino acid-long CDR3.

Public TRB CDR3 motif within HLA-A2/Melan-A-restricted clonotypes of melanoma patients

The amino acid composition of TRB hypervariable regions of Melan-A-specific CTL from melanoma patients were subsequently analyzed in detail. Serine, Glycine, Alanine and Glutamine were by far the most frequently used residues in the IMGT-defined CDR3, and were almost equally represented in all groups of analyzed sequences (Figure 2A). However, while Alanine, Serine, and Glutamine were abundantly present because of their occurrence at positions 105, 106, 107 and 114 in the majority of canonical TRBV and TRBJ chains, Glycine, as reported for murine [57] and human sequences [56], was clearly predominant in the region created by N-D-N recombination events. Furthermore, in the N-D-N region of Mel/M-A and Ctrl/M-A sequences there was an increased Leucine usage (Figure 2A), and Glycine and Leucine were overrepresented at CDR3 positions 110, 112 and 113 (Figure 2B). Moreover, the overall percentage of non-polar amino acids at these CDR3 positions in the clonotypes carrying 12-amino acid-long CDR3s, which were the most commonly represented among the Melan-A-specific T-cell clones, was significantly higher in the Mel/M-A group (75%) compared to Ctrl/M-A (62%, p = 0.017), Mel/M-A (52%, p < 0.001) and Ctrl/HLA-A2+ (38%, p < 0.001) groups. This indicates that non-polar amino acids may be important for Melan-A-peptide-TR interaction. Furthermore, we found a public clonotype identified in two laboratories from cells of two melanoma patients: one was sequenced in our laboratory starting from a T-cell clone (ID 16) obtained from patient 22 [manuscript in preparation], the other from a T-cell clone (ID 27) obtained in the laboratory of Trautmann et al [6] employing melanoma-infiltrating lymphocytes of patient M180 (Figure 3). Both sequences contained identical 12-amino acid-long CDR3s, created by the joining of TRBV28 and TRBJ1-5 segments and containing a Glycine-Leucine-Glycine stretch at positions 110-112-113 of the CDR3. This motif was recurrent among other sequences derived from several patients, since it was found in 27 additional clonotypes sequenced in different laboratories and obtained from 15 melanoma patients. This peculiar motif rearranged only with members of TRBJ1 cluster, because 19 out of 29 clonotypes were joined with TRBJ1-5 segments, 7 with TRBJ1-1, 2 with TRBJ1-2 and one with TRBJ1-6 (Figure 3). TRBV usage was also restricted in these clonotypes since 16 of them were TRBV28, 7 were TRBV30 and 2 were TRBV20. The recurrent motif was found in Melan-A-specific CTL isolated from PBL and from tumor sites of HLA-A2+ melanoma patients, independently of the stage of disease and of the methodological approaches used for T-cell cloning. The same motif was identified in two Melan-A T-cell clones derived from cells of healthy donors [5, 19], but not in the remaining 504 clonotypes sequenced from T-cell lines or clones with specificity for other Ags. Similarly, the Glycine-Leucine-Glycine motif at position 110-112-113 was absent in the 219 clonotypes identified analyzing 353 sequences randomly obtained from CD8+ lymphocytes of healthy subjects (data not shown). Furthermore, no common motifs were found when Melan-A-specific sequences of melanoma patients were compared using particular BV or BVBJ combinations. Of clinical relevance, the Glycine-Leucine-Glycine motif was detected in lymphocytes obtained from untreated patients, representing spontaneous anti-tumor responses, as well as from patients having undergone vaccination with the natural or modified peptides (Figure 3). Interestingly, one clonotype sequenced in our laboratory (ID 4) was detected both in samples prepared before and after the vaccination [58]. Furthermore, all but one clonotype containing the Glycine-Leucine-Glycine motif were sequenced from T-cell clones whose specificity was identified using modified Melan-A peptide/multimers. The specificity of the remaining clone for natural Melan-A peptide was established by the analysis of the ability of Melan-A-transfected COS-7 cells to stimulate IFN-γ release. This last clonotype (ID 1E2), identified by Cole et al [10], bore TRBV28 and TRBJ1-1 chains and differed only by the amino acid at position 109 (Figure 3) from ID 57, ID CTL01 and ID 6E4 clonotypes [6, 7, 18], which were sequenced starting from 3 melanoma patients. Furthermore, the same motif was present, at slightly different positions of the CDR3, in 7 other Melan-A-specific clonotypes [5, 7, 10, 19], but never in non-Melan-A clonotypes. While the Glycine-Leucine-Glycine stretch is composed exclusively by non-polar or frankly hydrophobic amino acids, all the amino acids at position 114 and several of those at position 109 were hydrophilic (Figure 3). Finally, we looked for very similar sequences at the same CDR3 positions because it is conceivable that these sequences adopt equivalent structures in the recognition complex. We found a Glycine-Valine-Glycine stretch in 8 clonotypes, 5 of which were identified in melanoma patients [[4, 12, 14, 30] and manuscript in preparation] and 3 in controls [3, 5].
Figure 2

Amino acid frequency. Amino acid frequency in the entire IMGT-defined CDR3 (A) and in the position 110, 112 and 113 of the CDR3 (B) in the indicated groups of sequences.

Figure 3

Public motifs in Melan-A-specific clonotypes. Aminoacidic composition and sequence alignments of public CDR3 of Melan-A-specific clonotypes found in melanoma patients. aPBL: peripheral blood lymphocytes; bTIL: tumor infiltrating lymphocytes; cNA: ID not available; dm: modified Melan-A A27L; eClonotype 4 was obtained from one T- clone was obtained before and one after vaccination; fX: amino acid not available; gn: natural Melan-A. In dark gray: amino acids identical to the consensus sequences; in light gray: other preferentially used amino acids at the given position; in bold: amino acids belonging to N-D-N region; in the boxes: hydrophilic amino acids at position 109 and 114.

Since previous studies focusing on the analysis of shared TR amino acid sequences in humans did not address the extent to which TRB nucleotides are shared among public amino acid stretches, we identified the N-D-N regions of the 22 available nucleotide sequences of clonotypes with Glycine-Leucine-Glycine at position 110, 112 and 113. As summarized in Table 2, all N-D-N regions were different, with the only exception of those of ID D/a and ID 30 sequences, in which, however, the Adenine at the extreme 3'V region must be ascribed to the TRBV segment in clone ID D/a and to the D region in clone ID 30. Finally, the alignment of the 22 nucleotide sequences with the TRBV, TRBJ and TRBD germline gene segments allowed us to calculate the germline contribution and the number of nucleotide deletions (the so-called "nibbling") and additions during the VDJ recombination process. The exonucleolytic nibbling was highly heterogeneous: at 3' V end varied from 0 to 7 nucleotides, at 5' J end ranged from 4 to 9, at 3' D from 0 to 9 and at 5' D from 0 to 7. Similarly, N-addition was highly different at both sites ranging from 0 to 9 nucleotides at N1 and from 0 to 6 at N2 position. Finally, also TRBD region length is diverse since it varies from 3 to 8 nucleotides.
Table 2

Nucleotide composition of available N-D-N regions of public Melan-A-specific clonotypes of melanoma patients

Clone ID

3' V region

N1

P

D region

N2

5' J region

TRBV

TRBJ

References

D/a

GCCAGCAGTTTA..

  

....CAGGGG..

CTGGGG

.......CAGCCCCAGCAT   

28

1–5

14

30

GCCTGGAGTGT   

  

...ACAGGGG..

CTGGGG

.....TATGGCTACACC     

30

1–2

in preparation

50B,55

GCCAGCAGCTT...

CACTGGGCT

 

......GGGG..

 

.......CAGCCCCAGCAT   

7

1–5

14

17

GCCAGCA.......

 TCTGGG  

 

....CAGGG...

CTCGGG

.......CAGCCCCAGCAT   

28

1–5

in preparation

814S1

XXXXXXA....   a

  CGAAT  

 

....CAGGGG..

CTCGGG

.......CAGCCCCAGXXX   

30

1–5

12

82899S32

XXXXXX.....   

GGGCAAAT 

 

.......GGGGC

TCGGG 

.......CAGCCCCAGXXX   

30

1–5

12

A/5

AGTGCTAG...   

 TGTGCC  

 

.......GGGGC

TCGGG 

.......CAGCCCCAGCAT   

20

1–5

14

25

GCCAGCAG......

   ACA   

 

...ACAGGGG..

TTGGG 

.........TTCACCCCTCCAC

28

1–6

in preparation

814S2

XXXXXXAG...   

 CCCGGT  

 

.....AGGG...

TTGGG 

......TCAGCCCCAGXXX   

30

1–5

12

82899S26

XXXXXXAGT..   

   CC    

 

....CAGGGGGC

TCGG  

......TCAGCCCCAGXXX   

30

1–5

12

42

GCCAGCAGT.....

   GT    

 

...ACAGGGG..

CTCGG 

......TCAGCCCCAGCAT   

28

1–5

in preparation

D/b

GCCAGTAGTAT...

  

..GACAGGG...

CTAGGG

.......CAGCCCCAGCAT   

19

1–5

14

6

AGTGC.......  

 GCCCGAT 

 

...ACAGGG...

CTTGGC

.......CAGCCCCAGCAT   

20

1–5

in preparation

4

GCCAGCAG......

 ATACCA  

 

GGGAC.......

TAGGA 

.......CAGCCCCAGCAT   

28

1–5

in preparation

39

GCCTGGAGTGT   

   CC    

 

....CAGGG...

CTAGG 

......TCAGCCCCAGCAT   

30

1–5

in preparation

NA

XXXXXXAGT..   

   CAT   

 

....CAGGG...

ATTGGG

....CTATGGCTACXXX     

30

1–2

12

16

GCCAGCA.......

  CCCT   

 

..GACAGGG...

CTTGGA

.......CAGCCCCAGCAT   

28

1–5

in preparation

6E4

GCCAGCAGTTT...

   TCT   

C

GGG.........

TTGGG 

....CACTGAAGCTTTC     

28

1-1

18

40

GCCAGCAGTTTA..

  

....CAGGG...

TTGGGG

.....ACTGAAGCTTTC     

28

1-1

in preparation

B/22

GCCAGCAGT.....

   CA    

 

...ACAGGG...

TTTGGG

......TCAGCCCCAGCAT   

28

1–5

14

41

GCCAGCAG......

   CCA   

 

...ACAGGGG..

CTCGG 

....CACTGAAGCTTTC     

28

1-1

in preparation

B/9

GCCAGCAGTTT...

   TCA   

 

GGGAC.......

TCGG  

......TCAGCCCCAGCAT   

28

1–5

14

aNucleotides not available

Discussion

T-cells recognize peptide Ags in the context of MHC molecules through their TR, and during chronic infections, autoimmunity and alloreactivity a preferential use of particular TRA or TRB regions has been observed [4]. Therefore much effort has been put into the characterization also of tumor Ag-specific TRs. Several data demonstrated a major role of TRAV than TRBV chains in TR-Ag recognition, due to the higher number of contacts of this chain with peptides [59], and, accordingly, a preferential usage of a TRAV chain has been observed in Melan-A-specific T cells from melanoma or vitiligo patients and healthy donors [59]. However, this has not been considered a result of TR repertoire narrowing due to affinity focusing during Ag-driven immune responses, but to reflect a structural constraint already present in the pre-immune TR repertoire [5, 9]. Differently from TRAV, the TRBV repertoire of Melan-A-specific T lymphocytes appears to be large and diverse in terms of clonal composition and TRBV region usage, as multiple clonotypic transcripts, covering the majority of the TRBV families, have been identified in HLA-A2+ patients [57, 14, 17]. Conversely, other authors reported that the recognition of melanoma Ags involved the use of T lymphocytes bearing specific TRBV chains, such TRBV5, TRBV9, TRBV19, TRBV27, and TRBV28 [16, 18, 23, 30, 35]. The different results are likely due to intrinsic limitations imposed by the limited number of patients analyzed and by the fact that the mature TR repertoire is influenced not only by the coding potential of TR VDJ regions, but also by the immunological history of the individuals. To clarify this issue, we analyzed several HLA-A2/Melan-A-specific clonotypes derived from 40 melanoma patients and we compared their features with those found in 103 other individuals including 8 subjects of Ctrl/M-A group, 36 of Mel/noM-A group and 59 of Ctrl/HLA-A2+ group. This comparative analysis indicated that T cells reacting with melanoma Ags utilize preferentially TRBV27 chain, but this segment is also predominant in clonotypes with unrelated specificity derived from HLA-A2+ individuals. On the contrary, TRBV28 chain is significantly more represented in HLA-A2+/Melan-A-specific T-cell clones obtained from melanoma patients and controls. It is of note that TRBV27 and TRBV28 chains (previously defined TCRBV14S1 and TCRBV13S1, respectively) were expressed at very low percentage when PBL of healthy individuals were analyzed by cytofluorimetry using a panel of TRBV subfamily-specific mAbs covering about 65% of TR-expressing cells [60]. Although we cannot exclude that anti-TRBV27 and anti-TRBV28 mAbs may not recognize well these TRBV chains, the overexpression of these segments in the clonotypes that we have analyzed strongly suggests that these TRBV segments are important for melanoma Ag recognition, with TRBV28 being preferentially involved in the interaction between TR and Melan-A.

Looking in depth at the peculiar features of TR-Melan-A interaction, we found a biased utilization of TRBJ1-5 segment and a 3-amino acid-long Glycine-Leucine-Glycine public motif occurring in several clonotypes of melanoma patients. Further biases were the frequent association of this public motif with TRBV28 and TRBJ1-5 segments and the lack of rearrangement with members of TRBJ2 cluster. The finding of this public motif demonstrates that the discrepancy between the anti-viral and anti-melanoma Ag responses is only apparent and supports our hypothesis that the lack of common TRB constraints among patients analyzed in different studies [58, 1018] is likely due to the paucity of individuals studied and to the diverse technical approaches employed for the sequence analysis. Indeed, Mandruzzato et al [14] have previously identified the Glycine-Leucine-Glycine stretch, but they could not appreciate the frequency of this feature since they studied a single melanoma patient. Clones carrying recurrent motifs were present at low frequency in each patient, exception made for two patients from whom 9 and 28 clones with the same Glycine-Leucine-Glycine-containing TR were isolated [12, 18], while during viral infections, public clonotypes are very frequent not only within the population, but are also sequenced in a large number in the same patient [4, 4346]. This is not surprising since most of these studies were carried out in the context of chronic, most likely lifelong, viral infections, i.e. EBV infection, where exposure to Ags is continuous and a selective pressure on T cells remains constantly high.

There is not a general rule that could account for the occurrence of public T-cell responses. Some public TRB motifs have been made from near-germline recombination events, involving only few nucleotides deletion from V, D and J germline and no or minimal random nucleotide additions [61, 62] but the extent of exonucleolytic nibbling and the substantial number of nucleotide additions in the public anti-Melan-A TR stretch exclude that its public nature is generated by near-germline rearrangements.

Looking at the biochemical structure of the public motif identified, one may speculate that the Glycine-Leucine-Glycine stretch positioned in the central region of the CDR3, which is surrounded by hydrophilic residues, can favour the interaction with the antigenic Melan-A peptide, which has a similar central Glycine-Isoleucine-Glycine motif, with the large non polar side chain of the Isoleucine protruding extensively from the molecular surface [63]. The relevance of this and of other structural affinities in the two sequences, such as the potential interactions between the hydrophilic residues flanking their central positions, might be assessed with more confidence when further data on the recently crystallized TR-Melan-A-MHC complex [64] will be available, and the spatial relationships between Melan-A and CDR3 amino acids will became clearer.

Conclusion

The finding of a conserved amino acid motif in the CDR3, together with the selective use of certain TRBJ and TRBV segments, indicates an important role of the TRB chain in fine-tuning TR affinity of Melan-A-specific T cells of melanoma patients and argues against the hypothesis that high affinity TRs against self-Ags, like Melan-A, are removed during selection in the thymus or, alternatively, by tumor-induced deletion of dominant TR clonotypes [65].

Further studies are needed to elucidate the clinical relevance of these melanoma-associated clones, which were found not only in T-cell clones isolated from PBL but also from tumor sites, thus suggesting some lymph-node homing properties of the T cells bearing the public motif. However, whatever the function of these clonotypes is, the occurrence of this public CDR3 sequence may have implications for the tracking of tumor Ag-specific T cells in different clinical settings. In particular, sensitive molecular approaches targeting TRBV28+TRBJ1-5+ cells bearing Glycine-Leucine-Glycine motif could be designed to immune-monitor Melan-A-specific responses in melanoma patients and to investigate whether the presence of this specific motif can provide prognostic information, contributing to the design of efficient anti-melanoma vaccines.

Declarations

Acknowledgements

This work was supported by grants from ISS-ACC, Ricerca Finalizzata 2007 Fasc. N.ACC5/2, Italian Ministry of Health, Ricerca Finalizzata 2007 Fasc.7OAF4, ISS-ACC, Ricerca Finalizzata 2007 Fasc. N.ACC2/R2.6.

Authors’ Affiliations

(1)
Diagnostics Department, Spedali Civili di Brescia
(2)
Immunology Laboratory, Regina Elena Cancer Institute

References

  1. Lefranc MP, Giudicelli V, Ginestoux C, Bodmer J, Müller W, Bontrop R, Lemaitre M, Malik A, Barbié V, Chaume D: IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 1999, 27: 209-212. 10.1093/nar/27.1.209.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Pannetier C, Cochet M, Darche S, Casrouge A, Zöller M, Kourilsky P: The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments. Proc Natl Acad Sci USA. 1993, 90: 4319-4323. 10.1073/pnas.90.9.4319.PubMed CentralView ArticlePubMedGoogle Scholar
  3. Cabaniols JP, Fazilleau N, Casrouge A, Kourilsky P, Kanellopoulos JM: Most alpha/beta T cell receptor diversity is due to terminal deoxynucleotidyl transferase. J Exp Med. 2001, 194: 1385-1390. 10.1084/jem.194.9.1385.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Turner SJ, Doherty PC, McCluskey J, Rossjohn J: Structural determinants of T-cell receptor bias in immunity. Nat Rev Immunol. 2006, 6: 883-894. 10.1038/nri1977.View ArticlePubMedGoogle Scholar
  5. Dietrich PY, Le Gal FA, Dutoit V, Pittet MJ, Trautman L, Zippelius A, Cognet I, Widmer V, Walker PR, Michielin O, Guillaume P, Connerotte T, Jotereau F, Coulie PG, Romero P, Cerottini JC, Bonneville M, Valmori D: Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen. J Immunol. 2003, 170: 5103-5109.View ArticlePubMedGoogle Scholar
  6. Trautmann L, Labarrière N, Jotereau F, Karanikas V, Gervois N, Connerotte T, Coulie P, Bonneville M: Dominant TCR V alpha usage by virus and tumor-reactive T cells with wide affinity ranges for their specific antigens. Eur J Immunol. 2002, 32: 3181-3190. 10.1002/1521-4141(200211)32:11<3181::AID-IMMU3181>3.0.CO;2-2.View ArticlePubMedGoogle Scholar
  7. Vignard V, Lemercier B, Lim A, Pandolfino MC, Guilloux Y, Khammari A, Rabu C, Echasserieau K, Lang F, Gougeon ML, Dreno B, Jotereau F, Labarriere N: Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells. J Immunol. 2005, 175: 4797-4805.View ArticlePubMedGoogle Scholar
  8. Mantovani S, Gabelli S, Palermo B, Campanelli R, Brazzelli V, Borroni G, Martinetti M, Benvenuto F, Merlini G, della Cuna GR, Rivoltini L, Giachino C: Molecular and functional bases of self-antigen recognition in long-term persistent melanocyte-specific CD8+ T cells in one vitiligo patient. J Invest Dermatol. 2003, 121: 308-314. 10.1046/j.1523-1747.2003.12368.x.View ArticlePubMedGoogle Scholar
  9. Mantovani S, Palermo B, Gabelli S, Campanelli R, della Cuna GR, Gennari R, Benvenuto F, Lantelme E, Giachino C: Dominant TCR-alpha requirements for a self antigen recognition in humans. J Immunol. 2002, 169: 6253-6260.View ArticlePubMedGoogle Scholar
  10. Cole DJ, Weil DP, Shamamian P, Rivoltini L, Kawakami Y, Topalian S, Jennings C, Eliyahu S, Rosenberg SA, Nishimura MI: Identification of MART-1-specific T-cell receptors: T cells utilizing distinct T-cell receptor variable and joining regions recognize the same tumor epitope. Cancer Res. 1994, 54: 5265-5268.PubMedGoogle Scholar
  11. Cole DJ, Wilson MC, Rivoltini L, Custer M, Nishimura MI: T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes. Cancer Res. 1997, 57: 5320-5327.PubMedGoogle Scholar
  12. Dietrich PY, Walker PR, Quiquerez AL, Perrin G, Dutoit V, Liénard D, Guillaume P, Cerottini JC, Romero P, Valmori D: Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires. Cancer Res. 2001, 61: 2047-2054.PubMedGoogle Scholar
  13. Jäger E, Höhn H, Necker A, Förster R, Karbach J, Freitag K, Neukirch C, Castelli C, Salter RD, Knuth A, Maeurer MJ: Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1. Int J Cancer. 2002, 98: 376-388. 10.1002/ijc.10165.View ArticlePubMedGoogle Scholar
  14. Mandruzzato S, Rossi E, Bernardi F, Tosello V, Macino B, Basso G, Chiarion-Sileni V, Rossi CR, Montesco C, Zanovello P: Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient. J Immunol. 2002, 169: 4017-4024.View ArticlePubMedGoogle Scholar
  15. Romero P, Gervois N, Schneider J, Escobar P, Valmori D, Pannetier C, Steinle A, Wolfel T, Lienard D, Brichard V, van Pel A, Jotereau F, Cerottini JC: Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma. J Immunol. 1997, 159: 2366-2374.PubMedGoogle Scholar
  16. Sensi M, Traversari C, Radrizzani M, Salvi S, Maccalli C, Mortarini R, Rivoltini L, Farina C, Nicolini G, Wölfel T, Brichard V, Boon T, Bordignon C, Anichini A, Parmiani G: Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1. Proc Natl Acad Sci USA. 1995, 92: 5674-5678. 10.1073/pnas.92.12.5674.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Speiser DE, Baumgaertner P, Barbey C, Rubio-Godoy V, Moulin A, Corthesy P, Devevre E, Dietrich PY, Rimoldi D, Liénard D, Cerottini JC, Romero P, Rufer N: A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination. J Immunol. 2006, 177: 1338-1348.View ArticlePubMedGoogle Scholar
  18. Valmori D, Dutoit V, Schnuriger V, Quiquerez AL, Pittet MJ, Guillaume P, Rubio-Godoy V, Walker PR, Rimoldi D, Liénard D, Cerottini JC, Romero P, Dietrich PY: Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity. J Immunol. 2002, 168: 4231-4240.View ArticlePubMedGoogle Scholar
  19. Pittet MJ, Gati A, Le Gal FA, Bioley G, Guillaume P, de Smedt M, Plum J, Speiser DE, Cerottini JC, Dietrich PY, Romero P, Zippelius A: Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex. J Immunol. 2006, 176: 2330-2336.View ArticlePubMedGoogle Scholar
  20. Palermo B, Campanelli R, Garbelli S, Mantovani S, della Cuna GR, Necker A, Manganoni AM, Carella G, Rivoltini L, Lantelme E, Giachino C: Cytotoxic T-lymphocyte responses in melanoma through in vitro stimulation with the Melan-A peptide analogue A27L: a qualitative analysis. Melanoma Res. 2002, 12: 491-498. 10.1097/00008390-200209000-00011.View ArticlePubMedGoogle Scholar
  21. Dutoit V, Rubio-Godoy V, Pittet MJ, Zippelius A, Dietrich PY, Legal FA, Guillaume P, Romero P, Cerottini JC, Houghten RA, Pinilla C, Valmori D: Degeneracy of antigen recognition as the molecular basis for the high frequency of naive A2/Melan-a peptide multimer(+) CD8(+) T cells in humans. J Exp Med. 2002, 196: 207-216. 10.1084/jem.20020242.PubMed CentralView ArticlePubMedGoogle Scholar
  22. Berger TG, Haendle I, Schrama D, Lüftl M, Bauer N, Pedersen LØ, Schuler Thurner B, Hohenberger W, Straten PtP, Schuler G, Becker JC: Circulation and homing of melanoma-reactive T cells to both cutaneous and visceral metastases after vaccination with monocyte-derived dendritic cells. Int J Cancer. 2004, 111: 229-237. 10.1002/ijc.20238.View ArticlePubMedGoogle Scholar
  23. Carsana M, Tragni G, Nicolini G, Bersani I, Parmiani G, Anichini A, Sun YS, Möller P, Schadendorf D, Sensi ML: Comparative assessment of TCRBV diversity in T lymphocytes present in blood, metastatic lesions, and DTH sites of two melanoma patients vaccinated with an IL-7 gene-modified autologous tumor cell vaccine. Cancer Gene Ther. 2002, 9: 243-253. 10.1038/sj.cgt.7700435.View ArticlePubMedGoogle Scholar
  24. Dufour E, Carcelain G, Gaudin C, Flament C, Avril MF, Faure F: Diversity of the cytotoxic melanoma-specific immune response: some CTL clones recognize autologous fresh tumor cells and not tumor cell lines. J Immunol. 1997, 158: 3787-3795.PubMedGoogle Scholar
  25. Farina C, Bruggen van der P, Boël P, Parmiani G, Sensi M: Conserved TCR usage by HLA-Cw* 1601-restricted T cell clones recognizing melanoma antigens. Int Immunol. 1996, 8: 1463-1466. 10.1093/intimm/8.9.1463.View ArticlePubMedGoogle Scholar
  26. Ferradini L, Mackensen A, Genevée C, Bosq J, Duvillard P, Avril MF, Hercend T: Analysis of T cell receptor variability in tumor-infiltrating lymphocytes from a human regressive melanoma. Evidence for in situ T cell clonal expansion. J Clin Invest. 1993, 91: 1183-1190. 10.1172/JCI116278.PubMed CentralView ArticlePubMedGoogle Scholar
  27. Mackensen A, Ferradini L, Carcelain G, Triebel F, Faure F, Viel S, Hercend T: Evidence for in situ amplification of cytotoxic T-lymphocytes with antitumor activity in a human regressive melanoma. Cancer Res. 1993, 53: 3569-3573.PubMedGoogle Scholar
  28. Manne J, Mastrangelo MJ, Sato T, Berd D: TCR rearrangement in lymphocytes infiltrating melanoma metastases after administration of autologous dinitrophenyl-modified vaccine. J Immunol. 2002, 169: 3407-3412.View ArticlePubMedGoogle Scholar
  29. McKee MD, Clay TM, Diamond RA, Rosenberg SA, Nishimura MI: Quantitation of T-cell receptor frequencies by competitive polymerase chain reaction: dynamics of T-cell clonotype frequencies in an expanding tumor-infiltrating lymphocyte culture. J Immunother. 2000, 23: 419-429. 10.1097/00002371-200007000-00005.View ArticlePubMedGoogle Scholar
  30. Pisarra P, Mortarini R, Salvi S, Anichini A, Parmiani G, Sensi M: High frequency of T cell clonal expansions in primary human melanoma. Involvement of a dominant clonotype in autologous tumor recognition. Cancer Immunol Immunother. 1999, 48: 39-46. 10.1007/s002620050546.View ArticlePubMedGoogle Scholar
  31. Protheroe AS, Pickard C, Johnson PW, Craddock T, Shefta J, Short K, Lancaster F, Selby PJ, Henwood J, Boylston AW: Persistence of clonal T-cell expansions following high-dose chemotherapy and autologous peripheral blood progenitor cell rescue. Br J Haematol. 2000, 111: 766-773. 10.1046/j.1365-2141.2000.02427.x.PubMedGoogle Scholar
  32. Puisieux I, Even J, Pannetier C, Jotereau F, Favrot M, Kourilsky P: Oligoclonality of tumor-infiltrating lymphocytes from human melanomas. J Immunol. 1994, 153: 2807-2818.PubMedGoogle Scholar
  33. Romero P, Pannetier C, Herman J, Jongeneel CV, Cerottini JC, Coulie PG: Multiple specificities in the repertoire of a melanoma patient's cytolytic T lymphocytes directed against tumor antigen MAGE-1.A1. J Exp Med. 1995, 182: 1019-1028. 10.1084/jem.182.4.1019.View ArticlePubMedGoogle Scholar
  34. Sensi M, Salvi S, Castelli C, Maccalli C, Mazzocchi A, Mortarini R, Nicolini G, Herlyn M, Parmiani G, Anichini A: T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone. J Exp Med. 1993, 178: 1231-1246. 10.1084/jem.178.4.1231.View ArticlePubMedGoogle Scholar
  35. Sensi M, Farina C, Maccalli C, Lupetti R, Nicolini G, Anichini A, Parmiani G, Berd D: Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine. J Clin Invest. 1997, 99: 710-717. 10.1172/JCI119215.PubMed CentralView ArticlePubMedGoogle Scholar
  36. Shilyansky J, Nishimura MI, Yannelli JR, Kawakami Y, Jacknin LS, Charmley P, Rosenberg SA: T-cell receptor usage by melanoma-specific clonal and highly oligoclonal tumor-infiltrating lymphocyte lines. Proc Natl Acad Sci USA. 1994, 91: 2829-2833. 10.1073/pnas.91.7.2829.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Strohal R, Brna C, Mossbacher U, Fischer G, Pehamberger H, Stingl G: First comparative delineation of the T cell receptor repertoire in primary and multiple subsequent/coexisting metastatic melanoma sites. J Invest Dermatol. 1998, 111: 1085-1091. 10.1046/j.1523-1747.1998.00450.x.View ArticlePubMedGoogle Scholar
  38. Thor Straten P, Schøller J, Hou-Jensen K, Zeuthen J: Preferential usage of T-cell receptor alpha beta variable regions among tumor-infiltrating lymphocytes in primary human malignant melanomas. Int J Cancer. 1994, 56: 78-86.View ArticlePubMedGoogle Scholar
  39. Willhauck M, Scheibenbogen C, Pawlita M, Möhler T, Thiel E, Keilholz U: Restricted T-cell receptor repertoire in melanoma metastases regressing after cytokine therapy. Cancer Res. 2003, 63: 3483-3485.PubMedGoogle Scholar
  40. Zarour H, De Smet C, Lehmann F, Marchand M, Lethé B, Romero P, Boon T, Renauld JC: The majority of autologous cytolytic T-lymphocyte clones derived from peripheral blood lymphocytes of a melanoma patient recognize an antigenic peptide derived from gene Pmel17/gp100. J Invest Dermatol. 1996, 107: 63-67. 10.1111/1523-1747.ep12298177.View ArticlePubMedGoogle Scholar
  41. Zhou J, Dudley ME, Rosenberg SA, Robbins PF: Selective growth, in vitro and in vivo, of individual T cell clones from tumor-infiltrating lymphocytes obtained from patients with melanoma. J Immunol. 2004, 173: 7622-7629.PubMed CentralView ArticlePubMedGoogle Scholar
  42. Lehner PJ, Wang EC, Moss PA, Williams S, Platt K, Friedman SM, Bell JI, Borysiewicz LK: Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment. J Exp Med. 1995, 181: 79-91. 10.1084/jem.181.1.79.View ArticlePubMedGoogle Scholar
  43. Lim A, Trautmann L, Peyrat MA, Couedel C, Davodeau F, Romagné F, Kourilsky P, Bonneville M: Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire. J Immunol. 2000, 165: 2001-2011.View ArticlePubMedGoogle Scholar
  44. Moss PA, Moots RJ, Rosenberg WM, Rowland-Jones SJ, Bodmer HC, McMichael AJ, Bell JI: Extensive conservation of alpha and beta chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc Natl Acad Sci USA. 1991, 88: 8987-8990. 10.1073/pnas.88.20.8987.PubMed CentralView ArticlePubMedGoogle Scholar
  45. Price DA, Brenchley JM, Ruff LE, Betts MR, Hill BJ, Roederer M, Koup RA, Migueles SA, Gostick E, Wooldridge L, Sewell AK, Connors M, Douek DC: Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses. J Exp Med. 2005, 202: 1349-1361. 10.1084/jem.20051357.PubMed CentralView ArticlePubMedGoogle Scholar
  46. Trautmann L, Rimbert M, Echasserieau K, Saulquin X, Neveu B, Dechanet J, Cerundolo V, Bonneville M: Selection of T cell clones expressing high-affinity public TCRs within Human cytomegalovirus-specific CD8 T cell responses. J Immunol. 2005, 175: 6123-6132.View ArticlePubMedGoogle Scholar
  47. Wilson JD, Ogg GS, Allen RL, Goulder PJ, Kelleher A, Sewell AK, O'Callaghan CA, Rowland-Jones SL, Callan MF, McMichael AJ: Oligoclonal expansions of CD8(+) T cells in chronic HIV infection are antigen specific. J Exp Med. 1998, 188: 785-790. 10.1084/jem.188.4.785.PubMed CentralView ArticlePubMedGoogle Scholar
  48. Echchakir H, Vergnon I, Dorothée G, Grunenwald D, Chouaib S, Mami-Chouaib F: Evidence for in situ expansion of diverse antitumor-specific cytotoxic T lymphocyte clones in a human large cell carcinoma of the lung. Int Immunol. 2000, 12: 537-546. 10.1093/intimm/12.4.537.View ArticlePubMedGoogle Scholar
  49. Echchakir H, Dorothée G, Vergnon I, Menez J, Chouaib S, Mami-Chouaib F: Cytotoxic T lymphocytes directed against a tumor-specific mutated antigen display similar HLA tetramer binding but distinct functional avidity and tissue distribution. Proc Natl Acad Sci USA. 2002, 99: 9358-9363. 10.1073/pnas.142308199.PubMed CentralView ArticlePubMedGoogle Scholar
  50. Heemskerk MH, Hoogeboom M, de Paus RA, Kester MG, Hoorn van der MA, Goulmy E, Willemze R, Falkenburg JH: Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region. Blood. 2003, 102: 3530-3540. 10.1182/blood-2003-05-1524.View ArticlePubMedGoogle Scholar
  51. Höhn H, Kortsik C, Nilges K, Necker A, Freitag K, Tully G, Neukirch C, Maeurer MJ: Human leucocyte antigen-A2 restricted and Mycobacterium tuberculosis 19-kDa antigen-specific CD8+ T-cell responses are oligoclonal and exhibit a T-cell cytotoxic type 2 response cytokine-secretion pattern. Immunology. 2001, 104: 278-288. 10.1046/j.1365-2567.2001.01307.x.PubMed CentralView ArticlePubMedGoogle Scholar
  52. Ito M, Shichijo S, Tsuda N, Ochi M, Harashima N, Saito N, Itoh K: Molecular basis of T cell-mediated recognition of pancreatic cancer cells. Cancer Res. 2001, 61: 2038-2046.PubMedGoogle Scholar
  53. Karanikas V, Colau D, Baurain JF, Chiari R, Thonnard J, Gutierrez-Roelens I, Goffinet C, Van Schaftingen EV, Weynants P, Boon T, Coulie PG: High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival. Cancer Res. 2001, 61: 3718-3724.PubMedGoogle Scholar
  54. Niland B, Banki K, Biddison WE, Perl A: CD8+ T cell-mediated HLA-A*0201-restricted cytotoxicity to transaldolase peptide 168-176 in patients with multiple sclerosis. J Immunol. 2005, 175: 8365-8378.View ArticlePubMedGoogle Scholar
  55. The International Immunogenetics Information System. [http://imgt.cines.fr/]
  56. Quiròs Roldan E, Sottini A, Bettinardi A, Alberini A, Imberti L, Primi D: Different TCRBV genes generate biased patterns of V-D-J diversity in human T cells. Immunogenetics. 1995, 41: 91-100.PubMedGoogle Scholar
  57. Candéias S, Katz J, Benoist C, Mathis D, Haskins K: Islet-specific T-cell clones from nonobese diabetic mice express heterogeneous T-cell receptors. Proc Natl Acad Sci USA. 1991, 88: 6167-6170. 10.1073/pnas.88.14.6167.PubMed CentralView ArticlePubMedGoogle Scholar
  58. Nisticò P, Capone I, Palermo B, Del Bello D, Ferraresi V, Moschella F, Aricò E, Valentini M, Bracci L, Cognetti F, Ciccarese M, Vercillo G, Roselli M, Fossile E, Tosti ME, Wang E, Marincola F, Imberti L, Catricalà C, Natali PG, Belardelli F, Proietti E: Chemotherapy enhances vaccine-induced antitumor immunity in melanoma patients. Int J Cancer. 2009, 124: 130-9. 10.1002/ijc.23886.View ArticlePubMedGoogle Scholar
  59. Rudolph MG, Wilson IA: The specificity of TCR/pMHC interaction. Curr Opin Immunol. 2002, 14: 52-65. 10.1016/S0952-7915(01)00298-9.View ArticlePubMedGoogle Scholar
  60. Benicchi T, Ghidini C, Re A, Cattaneo C, Casari S, Caimi L, Rossi G, Imberti L: T-cell immune reconstitution after hematopoietic stem cell transplantation for HIV-associated lymphoma. Transplantation. 2005, 80: 673-682. 10.1097/01.tp.0000168490.29862.b8.View ArticlePubMedGoogle Scholar
  61. Venturi V, Price DA, Douek DC, Davenport MP: The molecular basis for public T-cell responses?. Nat Rev Immunol. 2008, 8: 231-238. 10.1038/nri2260.View ArticlePubMedGoogle Scholar
  62. Cibotti R, Cabaniols JP, Pannetier C, Delarbre C, Vergnon I, Kanellopoulos JM, Kourilsky P: Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice. J Exp Med. 1994, 180: 861-872. 10.1084/jem.180.3.861.View ArticlePubMedGoogle Scholar
  63. Sliz P, Michielin O, Cerottini JC, Luescher I, Romero P, Karplus M, Wiley DC: Crystal structures of two closely related but antigenically distinct HLA-A2/melanocyte-melanoma tumor-antigen peptide complexes. J Immunol. 2001, 167: 3276-3284.View ArticlePubMedGoogle Scholar
  64. Yuan F, Georgiou T, Hillon T, Gostick E, Price DA, Sewell AK, Moysey R, Gavarret J, Vuidepot A, Sami M, Bell JI, Gao GF, Rizkallah PJ, Jakobsen BK: Crystallization and preliminary X-ray structural studies of a Melan-A pMHC-TCR complex. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007, 63: 758-760. 10.1107/S1744309107037244.PubMed CentralView ArticlePubMedGoogle Scholar
  65. Thor Straten P, Schrama D, Andersen MH, Becker JC: T-cell clonotypes in cancer. J Transl Med. 2004, 2: 11-10.1186/1479-5876-2-11.PubMed CentralView ArticlePubMedGoogle Scholar

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© Serana et al; licensee BioMed Central Ltd. 2009

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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