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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.

Serana F, Sottini A, Caimi L, Palermo B, Natali PG, Nisticò P, Imberti L - J Transl Med (2009)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Diagnostics Department, Spedali Civili di Brescia, 25123 Brescia, Italy. federico.serana@gmail.com

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.

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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.
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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.

Mentions: 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].


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.

Serana F, Sottini A, Caimi L, Palermo B, Natali PG, Nisticò P, Imberti L - J Transl Med (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2667493&req=5

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.
Mentions: 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].

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Diagnostics Department, Spedali Civili di Brescia, 25123 Brescia, Italy. federico.serana@gmail.com

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.

Show MeSH
Related in: MedlinePlus