BONE MARROW FAILURE IN PAROXYSMAL NOCTURNAL HEMOGLOBULINURIA IS ASSOCIATED WITH EXPANSION OF ANTIGEN-SELECTED T CELLS: HIGH-THROUGHPUT EVIDENCE
(Abstract release date: 05/19/16)
EHA Library. Stalika E. 06/11/16; 135229; S473
Mrs. Evangelia Stalika
Contributions
Contributions
Abstract
Abstract: S473
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Room H5
Background
Paroxysmal nocturnal hemoglubinuria (PNH) is an acquired clonal disease of hematopoietic stem cells caused by somatic mutation of the PIG-A gene that is essential for the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI). The clinical phenotype of PNH consists of hemolysis, thrombophilia and bone marrow failure (BMF). While hemolysis and thrombosis are largely due to deficiency of the GPI-linked complement inhibitors, the pathogenesis of BMF remains unknown.Previous studies have indicated that CD1d-restricted, GPI-specific T cells are present in PNH patients and might underlie BMF. Interestingly, these cells display a heavily biased T cell receptor repertoire with enrichment for T cell receptor (TR) α chains encoded by the TRAV21/TRAJ31-1 genes and frequently carrying a distinctive complementarity-determining region 3 (CDR3: AVNNNARLM) combined by TR β chains encoded by the TRBV19 gene, often with restricted CDR3 as well, strongly alluding to selection by common (auto)antigen(s).
Aims
Prompted by these findings, here we sought to obtain more evidence about the nature of the implicated immune response through detailed analysis of the repertoire of GPI-specific T cell populations.
Methods
Our study included samples from 11 patients and 9 normal donors. TRAV21 gene rearrangements were RT-PCR amplified on RNA from T cell subsets sorted from peripheral blood mononuclear cells on the basis of: (i) expression of CD48, [positive in GPI+ cells] and vice versa; and, (ii) expression of TR β chains encoded by the TRBV19 gene versus TRBV19 negative. Both criteria applied to PNH patients, whereas the sole criterion used for normal donors was TRBV19 expression. PCR products were sequenced on 454 GS Junior (Roche) platform. Sequences were submitted to IMGT/HighV-QUEST, and metadata was processed by an in-house bioinformatics pipeline. Since all studied rearrangements utilized the same TRAV gene, we defined as clonotypes TRA rearrangements with identical TRA joining (TRAJ) gene usage and amino acid CDR3 sequence.
Results
Overall, significant differences (p<0.05) were identified in PNH patients vs normal controls for 13 of 50 functional TRAJ genes, the most pronounced concerning the TRAJ39, TRAJ57 and TRAJ58 genes. Similarly, significant differences (p<0.05) emerged when comparing the TRAJ gene repertoire between T cell subsets in PNH patients. In particular, (i) the TRAJ15 and TRAJ4 genes were over-represented amongst CD48+ cells; while the TRAJ24 and TRAJ33 were over-represented amongst CD48- cells, (ii) the “GPI-specific” CDR3 sequence (AVNNNARLM) was identified with higher frequency in CD48+ versus CD48- cells, however it was also present, with statistically significant lower frequency (p<0.05) amongst healthy donors ; (iii) the frequency of the “GPI-specific” CDR3 sequence was identified in GPI- positive up to 73.8% and in GPI-negative T – cell fractions up to 25.6%. When comparing the CDR3 length in i) patients versus healthy individuals, and ii) GPI-positive versus GPI-negative T cells, we noted a significant (p<0.05) bias to longer CDR3s in patients vs healthy donors; and, CD48- vs CD48+ cells. Cluster analysis of the CDR3 sequences of all PNH cases identified 21 different clonotypes that were shared by PNH patients but not by healthy individuals.
Conclusion
The present study documents noteworthy restrictions in the TR repertoire in PNH, indicative of antigen selection. The existence of clonotypes shared between different patients suggests that common antigen driven immune responses may be implicated in the pathogenesis of BMF.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
Keyword(s): Bone marrow failure, Paroxysmal nocturnal hemoglobinuria (PNH), T cell repertoire
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Room H5
Background
Paroxysmal nocturnal hemoglubinuria (PNH) is an acquired clonal disease of hematopoietic stem cells caused by somatic mutation of the PIG-A gene that is essential for the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI). The clinical phenotype of PNH consists of hemolysis, thrombophilia and bone marrow failure (BMF). While hemolysis and thrombosis are largely due to deficiency of the GPI-linked complement inhibitors, the pathogenesis of BMF remains unknown.Previous studies have indicated that CD1d-restricted, GPI-specific T cells are present in PNH patients and might underlie BMF. Interestingly, these cells display a heavily biased T cell receptor repertoire with enrichment for T cell receptor (TR) α chains encoded by the TRAV21/TRAJ31-1 genes and frequently carrying a distinctive complementarity-determining region 3 (CDR3: AVNNNARLM) combined by TR β chains encoded by the TRBV19 gene, often with restricted CDR3 as well, strongly alluding to selection by common (auto)antigen(s).
Aims
Prompted by these findings, here we sought to obtain more evidence about the nature of the implicated immune response through detailed analysis of the repertoire of GPI-specific T cell populations.
Methods
Our study included samples from 11 patients and 9 normal donors. TRAV21 gene rearrangements were RT-PCR amplified on RNA from T cell subsets sorted from peripheral blood mononuclear cells on the basis of: (i) expression of CD48, [positive in GPI+ cells] and vice versa; and, (ii) expression of TR β chains encoded by the TRBV19 gene versus TRBV19 negative. Both criteria applied to PNH patients, whereas the sole criterion used for normal donors was TRBV19 expression. PCR products were sequenced on 454 GS Junior (Roche) platform. Sequences were submitted to IMGT/HighV-QUEST, and metadata was processed by an in-house bioinformatics pipeline. Since all studied rearrangements utilized the same TRAV gene, we defined as clonotypes TRA rearrangements with identical TRA joining (TRAJ) gene usage and amino acid CDR3 sequence.
Results
Overall, significant differences (p<0.05) were identified in PNH patients vs normal controls for 13 of 50 functional TRAJ genes, the most pronounced concerning the TRAJ39, TRAJ57 and TRAJ58 genes. Similarly, significant differences (p<0.05) emerged when comparing the TRAJ gene repertoire between T cell subsets in PNH patients. In particular, (i) the TRAJ15 and TRAJ4 genes were over-represented amongst CD48+ cells; while the TRAJ24 and TRAJ33 were over-represented amongst CD48- cells, (ii) the “GPI-specific” CDR3 sequence (AVNNNARLM) was identified with higher frequency in CD48+ versus CD48- cells, however it was also present, with statistically significant lower frequency (p<0.05) amongst healthy donors ; (iii) the frequency of the “GPI-specific” CDR3 sequence was identified in GPI- positive up to 73.8% and in GPI-negative T – cell fractions up to 25.6%. When comparing the CDR3 length in i) patients versus healthy individuals, and ii) GPI-positive versus GPI-negative T cells, we noted a significant (p<0.05) bias to longer CDR3s in patients vs healthy donors; and, CD48- vs CD48+ cells. Cluster analysis of the CDR3 sequences of all PNH cases identified 21 different clonotypes that were shared by PNH patients but not by healthy individuals.
Conclusion
The present study documents noteworthy restrictions in the TR repertoire in PNH, indicative of antigen selection. The existence of clonotypes shared between different patients suggests that common antigen driven immune responses may be implicated in the pathogenesis of BMF.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
Keyword(s): Bone marrow failure, Paroxysmal nocturnal hemoglobinuria (PNH), T cell repertoire
Abstract: S473
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Room H5
Background
Paroxysmal nocturnal hemoglubinuria (PNH) is an acquired clonal disease of hematopoietic stem cells caused by somatic mutation of the PIG-A gene that is essential for the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI). The clinical phenotype of PNH consists of hemolysis, thrombophilia and bone marrow failure (BMF). While hemolysis and thrombosis are largely due to deficiency of the GPI-linked complement inhibitors, the pathogenesis of BMF remains unknown.Previous studies have indicated that CD1d-restricted, GPI-specific T cells are present in PNH patients and might underlie BMF. Interestingly, these cells display a heavily biased T cell receptor repertoire with enrichment for T cell receptor (TR) α chains encoded by the TRAV21/TRAJ31-1 genes and frequently carrying a distinctive complementarity-determining region 3 (CDR3: AVNNNARLM) combined by TR β chains encoded by the TRBV19 gene, often with restricted CDR3 as well, strongly alluding to selection by common (auto)antigen(s).
Aims
Prompted by these findings, here we sought to obtain more evidence about the nature of the implicated immune response through detailed analysis of the repertoire of GPI-specific T cell populations.
Methods
Our study included samples from 11 patients and 9 normal donors. TRAV21 gene rearrangements were RT-PCR amplified on RNA from T cell subsets sorted from peripheral blood mononuclear cells on the basis of: (i) expression of CD48, [positive in GPI+ cells] and vice versa; and, (ii) expression of TR β chains encoded by the TRBV19 gene versus TRBV19 negative. Both criteria applied to PNH patients, whereas the sole criterion used for normal donors was TRBV19 expression. PCR products were sequenced on 454 GS Junior (Roche) platform. Sequences were submitted to IMGT/HighV-QUEST, and metadata was processed by an in-house bioinformatics pipeline. Since all studied rearrangements utilized the same TRAV gene, we defined as clonotypes TRA rearrangements with identical TRA joining (TRAJ) gene usage and amino acid CDR3 sequence.
Results
Overall, significant differences (p<0.05) were identified in PNH patients vs normal controls for 13 of 50 functional TRAJ genes, the most pronounced concerning the TRAJ39, TRAJ57 and TRAJ58 genes. Similarly, significant differences (p<0.05) emerged when comparing the TRAJ gene repertoire between T cell subsets in PNH patients. In particular, (i) the TRAJ15 and TRAJ4 genes were over-represented amongst CD48+ cells; while the TRAJ24 and TRAJ33 were over-represented amongst CD48- cells, (ii) the “GPI-specific” CDR3 sequence (AVNNNARLM) was identified with higher frequency in CD48+ versus CD48- cells, however it was also present, with statistically significant lower frequency (p<0.05) amongst healthy donors ; (iii) the frequency of the “GPI-specific” CDR3 sequence was identified in GPI- positive up to 73.8% and in GPI-negative T – cell fractions up to 25.6%. When comparing the CDR3 length in i) patients versus healthy individuals, and ii) GPI-positive versus GPI-negative T cells, we noted a significant (p<0.05) bias to longer CDR3s in patients vs healthy donors; and, CD48- vs CD48+ cells. Cluster analysis of the CDR3 sequences of all PNH cases identified 21 different clonotypes that were shared by PNH patients but not by healthy individuals.
Conclusion
The present study documents noteworthy restrictions in the TR repertoire in PNH, indicative of antigen selection. The existence of clonotypes shared between different patients suggests that common antigen driven immune responses may be implicated in the pathogenesis of BMF.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
Keyword(s): Bone marrow failure, Paroxysmal nocturnal hemoglobinuria (PNH), T cell repertoire
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Room H5
Background
Paroxysmal nocturnal hemoglubinuria (PNH) is an acquired clonal disease of hematopoietic stem cells caused by somatic mutation of the PIG-A gene that is essential for the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI). The clinical phenotype of PNH consists of hemolysis, thrombophilia and bone marrow failure (BMF). While hemolysis and thrombosis are largely due to deficiency of the GPI-linked complement inhibitors, the pathogenesis of BMF remains unknown.Previous studies have indicated that CD1d-restricted, GPI-specific T cells are present in PNH patients and might underlie BMF. Interestingly, these cells display a heavily biased T cell receptor repertoire with enrichment for T cell receptor (TR) α chains encoded by the TRAV21/TRAJ31-1 genes and frequently carrying a distinctive complementarity-determining region 3 (CDR3: AVNNNARLM) combined by TR β chains encoded by the TRBV19 gene, often with restricted CDR3 as well, strongly alluding to selection by common (auto)antigen(s).
Aims
Prompted by these findings, here we sought to obtain more evidence about the nature of the implicated immune response through detailed analysis of the repertoire of GPI-specific T cell populations.
Methods
Our study included samples from 11 patients and 9 normal donors. TRAV21 gene rearrangements were RT-PCR amplified on RNA from T cell subsets sorted from peripheral blood mononuclear cells on the basis of: (i) expression of CD48, [positive in GPI+ cells] and vice versa; and, (ii) expression of TR β chains encoded by the TRBV19 gene versus TRBV19 negative. Both criteria applied to PNH patients, whereas the sole criterion used for normal donors was TRBV19 expression. PCR products were sequenced on 454 GS Junior (Roche) platform. Sequences were submitted to IMGT/HighV-QUEST, and metadata was processed by an in-house bioinformatics pipeline. Since all studied rearrangements utilized the same TRAV gene, we defined as clonotypes TRA rearrangements with identical TRA joining (TRAJ) gene usage and amino acid CDR3 sequence.
Results
Overall, significant differences (p<0.05) were identified in PNH patients vs normal controls for 13 of 50 functional TRAJ genes, the most pronounced concerning the TRAJ39, TRAJ57 and TRAJ58 genes. Similarly, significant differences (p<0.05) emerged when comparing the TRAJ gene repertoire between T cell subsets in PNH patients. In particular, (i) the TRAJ15 and TRAJ4 genes were over-represented amongst CD48+ cells; while the TRAJ24 and TRAJ33 were over-represented amongst CD48- cells, (ii) the “GPI-specific” CDR3 sequence (AVNNNARLM) was identified with higher frequency in CD48+ versus CD48- cells, however it was also present, with statistically significant lower frequency (p<0.05) amongst healthy donors ; (iii) the frequency of the “GPI-specific” CDR3 sequence was identified in GPI- positive up to 73.8% and in GPI-negative T – cell fractions up to 25.6%. When comparing the CDR3 length in i) patients versus healthy individuals, and ii) GPI-positive versus GPI-negative T cells, we noted a significant (p<0.05) bias to longer CDR3s in patients vs healthy donors; and, CD48- vs CD48+ cells. Cluster analysis of the CDR3 sequences of all PNH cases identified 21 different clonotypes that were shared by PNH patients but not by healthy individuals.
Conclusion
The present study documents noteworthy restrictions in the TR repertoire in PNH, indicative of antigen selection. The existence of clonotypes shared between different patients suggests that common antigen driven immune responses may be implicated in the pathogenesis of BMF.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
Keyword(s): Bone marrow failure, Paroxysmal nocturnal hemoglobinuria (PNH), T cell repertoire
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