Characterization of human gamma delta T cells in allogeneic hematopoietic stem cell transplantation

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.