Characterization of human gamma delta T cells in allogeneic hematopoietic stem cell transplantation
Author: Gaballa, Ahmed
Date: 2020-08-28
Location: Sal 4X, Alfred Nobel alle 8, plan 4, Karolinska Institutet, Flemingsberg
Time: 09.00
Department: Inst för klinisk vetenskap, intervention och teknik / Dept of Clinical Science, Intervention and Technology
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Thesis (2.749Mb)
Abstract
Over the last five decades, allogeneic hematopoietic stem cell transplantation (HSCT) has evolved rapidly, continuing to offer a cure for several hematological diseases. Nevertheless, associated life-threatening complications remain an obstacle against exploiting its full therapeutic benefit. Among these complications, infection, relapse, and graft-versus-host disease (GVHD) represent not only the most common but also the most serious ones. Though commonly regarded as distinct clinical events, their underlying pathophysiology is firmly related from an immunological perspective. T lymphocytes are key players in HSCT complications and their proper reconstitution following allogeneic HSCT is central for beneficial clinical outcome. The last two decades have witnessed a growing interest in a subset of T cells known as gamma delta (γδ) T cells. The immunological capabilities of these unconventional cells have been intensively explored. However, more efforts aimed at unraveling the immunobiological features of different γδ subsets are warranted to effectively exploit their full immunotherapeutic potential.
In the present work, I tried to tackle several immune-related aspects that directly influence allogeneic HSCT outcome with a special focus on γδ T cells. In paper I, the main objective was to address the impact of different GVHD prophylaxis regimens on de novo generation of T and B lymphocytes. Using PCR methods, T cell receptor recombination excision circle (TREC), kappa deleting recombination excision circle (KREC), and telomere length (TL) were quantified in the peripheral blood (PB) of transplanted patients at several time intervals. Although there was no significant difference between the two GVHD prophylaxis groups, we identified other transplant related factors that were associated with reduced TREC and/or KREC levels after HSCT. Furthermore, we showed that high levels of these excision circles correlated with favorable outcome post HSCT.
In paper II-IV, more attention was paid to explore the role of γδ T cells in donor grafts. Using multicolor flow cytometry together with other molecular and functional assays, we found a significant association between graft frequencies of CD8+γδ T cells and acute GVHD (aGVHD) grade II-III in Paper II. Additionally, we showed that higher frequencies of CD27+ γδ T cells in the stem cell grafts were correlated with both less relapse and CMV incidences. The results from paper II highlighting a potential role of CD8+γδ T cells in donor grafts raised our interest to further investigate this subset to elucidate their immunological characteristics. In paper III we thoroughly analysed γδ T cells in BM grafts using multicolor flow cytometry and TCR repertoire analysis using next generation sequencing (NGS). We showed that grafts from CMV+ donors contained higher proportions of CD8+γδ T that preferentially expressed Vγ9- and differentiated towards terminal effector memory phenotype. Additionally, analysis of TCRγ chain revealed a clonally focused repertoire in CMV+ donor grafts. We also showed that CD8+γδ T cells differentially respond to TCR stimuli suggesting adaptive-like phenotype. In paper IV, we sought to address whether allogeneic HSCT outcome is influenced by γδ TCR repertoire composition in donor grafts. Immunosequencing of TCRγ chain by NGS revealed a more public repertoire and increased presence of long sequence clonotypes in graft given to non-relapsed patients. Further analysis of the amino acid sequences identified 12 public and 4 private sequences that were exclusively found in high frequencies in grafts given to nonrelapsed patients.
Finally, in paper V we aimed to optimize a protocol for efficient in-vitro expansion of Vγ9Vδ2 T cells from umbilical cord blood (UCB). Phenotypical and functional characterization of expanded cells was comparable to PB and suggests that UCB can be a reliable source for Vγ9Vδ2 T cell expansion.
In the present work, I tried to tackle several immune-related aspects that directly influence allogeneic HSCT outcome with a special focus on γδ T cells. In paper I, the main objective was to address the impact of different GVHD prophylaxis regimens on de novo generation of T and B lymphocytes. Using PCR methods, T cell receptor recombination excision circle (TREC), kappa deleting recombination excision circle (KREC), and telomere length (TL) were quantified in the peripheral blood (PB) of transplanted patients at several time intervals. Although there was no significant difference between the two GVHD prophylaxis groups, we identified other transplant related factors that were associated with reduced TREC and/or KREC levels after HSCT. Furthermore, we showed that high levels of these excision circles correlated with favorable outcome post HSCT.
In paper II-IV, more attention was paid to explore the role of γδ T cells in donor grafts. Using multicolor flow cytometry together with other molecular and functional assays, we found a significant association between graft frequencies of CD8+γδ T cells and acute GVHD (aGVHD) grade II-III in Paper II. Additionally, we showed that higher frequencies of CD27+ γδ T cells in the stem cell grafts were correlated with both less relapse and CMV incidences. The results from paper II highlighting a potential role of CD8+γδ T cells in donor grafts raised our interest to further investigate this subset to elucidate their immunological characteristics. In paper III we thoroughly analysed γδ T cells in BM grafts using multicolor flow cytometry and TCR repertoire analysis using next generation sequencing (NGS). We showed that grafts from CMV+ donors contained higher proportions of CD8+γδ T that preferentially expressed Vγ9- and differentiated towards terminal effector memory phenotype. Additionally, analysis of TCRγ chain revealed a clonally focused repertoire in CMV+ donor grafts. We also showed that CD8+γδ T cells differentially respond to TCR stimuli suggesting adaptive-like phenotype. In paper IV, we sought to address whether allogeneic HSCT outcome is influenced by γδ TCR repertoire composition in donor grafts. Immunosequencing of TCRγ chain by NGS revealed a more public repertoire and increased presence of long sequence clonotypes in graft given to non-relapsed patients. Further analysis of the amino acid sequences identified 12 public and 4 private sequences that were exclusively found in high frequencies in grafts given to nonrelapsed patients.
Finally, in paper V we aimed to optimize a protocol for efficient in-vitro expansion of Vγ9Vδ2 T cells from umbilical cord blood (UCB). Phenotypical and functional characterization of expanded cells was comparable to PB and suggests that UCB can be a reliable source for Vγ9Vδ2 T cell expansion.
List of papers:
I. Törlén J, Gaballa A, Remberger M, Mörk LM, Sundberg B, Mattsson J, Uhlin M. Effect of Graft-versus-Host Disease Prophylaxis Regimens on T and B Cell Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation. Biology of blood and marrow transplantation. 2019 25;6 1260-1268.
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II. Gaballa A, Stikvoort A, Önfelt B, Mattsson J, Sundin M, Watz E, Uhlin M. T-cell frequencies of CD8+ γδ and CD27+ γδ cells in the stem cell graft predict the outcome after allogeneic hematopoietic cell transplantation. Bone marrow transplantation. 2019 54;10 1562-1574.
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III. Gaballa A, Arruda LCM, Radestad E, Uhlin M. CD8(+)gamma delta T Cells Are More Frequent in CMV Seropositive Bone Marrow Grafts and Display Phenotype of an Adaptive Immune Response. Stem Cells International. 2019:6348060.
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IV. Arruda LCM, Gaballa A, Uhlin M. Graft γδ TCR Sequencing Identifies Public Clonotypes Associated with Hematopoietic Stem Cell Transplantation Efficacy in Acute Myeloid Leukemia Patients and Unravels Cytomegalovirus Impact on Repertoire Distribution. Journal of immunology. 2019 202;6 1859-1870.
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V. Berglund S, Gaballa A, Sawaisorn P, Sundberg B, Uhlin M. Expansion of Gammadelta T Cells from Cord Blood: A Therapeutical Possibility. Stem Cells International. 2018:8529104.
Fulltext (DOI)
Pubmed
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I. Törlén J, Gaballa A, Remberger M, Mörk LM, Sundberg B, Mattsson J, Uhlin M. Effect of Graft-versus-Host Disease Prophylaxis Regimens on T and B Cell Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation. Biology of blood and marrow transplantation. 2019 25;6 1260-1268.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Gaballa A, Stikvoort A, Önfelt B, Mattsson J, Sundin M, Watz E, Uhlin M. T-cell frequencies of CD8+ γδ and CD27+ γδ cells in the stem cell graft predict the outcome after allogeneic hematopoietic cell transplantation. Bone marrow transplantation. 2019 54;10 1562-1574.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Gaballa A, Arruda LCM, Radestad E, Uhlin M. CD8(+)gamma delta T Cells Are More Frequent in CMV Seropositive Bone Marrow Grafts and Display Phenotype of an Adaptive Immune Response. Stem Cells International. 2019:6348060.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Arruda LCM, Gaballa A, Uhlin M. Graft γδ TCR Sequencing Identifies Public Clonotypes Associated with Hematopoietic Stem Cell Transplantation Efficacy in Acute Myeloid Leukemia Patients and Unravels Cytomegalovirus Impact on Repertoire Distribution. Journal of immunology. 2019 202;6 1859-1870.
Fulltext (DOI)
Pubmed
View record in Web of Science®
V. Berglund S, Gaballa A, Sawaisorn P, Sundberg B, Uhlin M. Expansion of Gammadelta T Cells from Cord Blood: A Therapeutical Possibility. Stem Cells International. 2018:8529104.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Uhlin, Michael
Co-supervisor: Hassan, Moustapha; Mattsson, Jonas; Thunberg, Sarah
Issue date: 2020-08-07
Rights:
Publication year: 2020
ISBN: 978-91-7831-874-2
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