Refine
Has Fulltext
- yes (2)
Is part of the Bibliography
- yes (2)
Document Type
- Doctoral Thesis (2)
Language
- English (2) (remove)
Keywords
- T-Lymphozyt (2) (remove)
Institute
Human Vγ9Vδ2 T cells are the main γδ T cell subset in the circulation, accounting for up to 5% of the total peripheral blood lymphocyte population. They have been suggested to be important in response to tumors and infections. Their immune mechanisms encompass cell killing via cytotoxicity and secretion of pro-inflammatory cytokines such as IFNγ and tumor necrosis factor (TNF). The main stimulators of Vγ9Vδ2 T cells are isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), denominated phosphoantigens (PAg).
A major advance in the understanding of PAg detection and Vγ9Vδ2 T cell activation has been the identification of the butyrophlin 3A (BTN3A) proteins as key mediators in these processes. In humans, three isoforms constitute the BTN3A family: BTN3A1, BTN3A2, and BTN3A3; and their genes are localized on the short arm of chromosome 6. The role of BTN3A1 has been highlighted by BTN3A-specific monoclonal antibody 20.1 (mAb 20.1), which has an agonist effect and causes proliferation, expansion, and activation of primary human Vγ9Vδ2 T cells. On the other hand, BTN3A-specific monoclonal antibody 103.2 (mAb 103.2) is antagonistic, inhibiting the Vγ9Vδ2 T cell response. The actual mechanism underlying both PAg- and mAb 20.1-mediated activation is not completely elucidated, but the importance of BTN3A1 is clear.
The main objective of this dissertation was to characterize the role of BTN3A1 in the PAg-dependent and PAg-independent Vγ9Vδ2 T cell activation and to evaluate its contribution in the response to influeza A virus infected cells. This research work demonstrated, by using Vγ9Vδ2 TCR MOP-transduced murine cells (reporter cells), that human chromosome 6 (Chr6) is mandatory for PAg-induced stimulation, but not for stimulation with mAb 20.1. The reporter cells responded to mAb 20.1 in cultures with BTN3A1-transduced Chinese hamster ovary cells (CHO BTN3A1) as antigen presenting cells. Nevertheless, for PAg-dependent activation the presence of Chr6 in CHO BTN3A1 was mandatory.
Although reporter cells expressing clonotypically different Vγ9Vδ2 TCRs showed similar PAg response, they clearly differed in the mAb 20.1 response. The reporter cell line transduced with Vγ9Vδ2 TCR D1C55 demonstrated essentially no response to mAb 20.1 compared to Vγ9Vδ2 TCR MOP cells. These findings were further supported by experiments performed with human PBMCs-derived Vγ9Vδ2 T cell clones. The results indicate heterogeneity in the PAg- and 20.1-dependent responses, in terms of CD25 and CD69 expression, among three different Vγ9Vδ2 T cells clones.
Co-cultures of reporter cells with Raji RT1BI and PAg plus mAb 20.1 or single chain antibody 20.1 (sc 20.1) revealed no additive or synergistic activating effects. In contrast, mAb 20.1 or sc 20.1 inhibited the PAg-mediated activation of the reporter cells.
The comparison of the relative contribution of the isoforms BTN3A2 and BTN3A3, in the activation of Vγ9Vδ2 T cells, was undertaken by overexpression of these isoforms in CHO cells. The results showed that BTN3A2 contributes to both PAg- and mAb-induced Vγ9Vδ2 T cell activation. On the contrary, BTN3A3 does not support PAg-mediated γδ T cell response.
Additionally, mutations in the proposed PAg- and mAb 20.1-binding sites of the extracellular BTN3A1 domain were generated by means of site-directed mutagenesis. These mutations revoked the mAb 20.1-induced Vγ9Vδ2 T cell activation, but not that induced by PAg.
Finally, co-cultures of Vγ9Vδ2 TCR MOP-transduced murine reporter cells with influenza A/PR/8/34-infected cells, or infection of PBMCs with this virus strain indicated that BTN3A1 might be dispensable for the Vγ9Vδ2 T cell response against influenza A.
The data of this research work points out that: i) in addition to BTN3A1, other Chr6-encoded genes are necessary for Vγ9Vδ2 T cell activation with PAg; ii) clonotypical (CDR3) differences influence the PAg- and mAb 20.1-mediated Vγ9Vδ2 T cell activation; iii) the PAg- and mAb 20.1-induced responses are not synergistic and interfere with each other; iv) BTN3A2 and BTN3A3 isoforms differ in the ability to support PAg- or mAb 20.1-dependent Vγ9Vδ2 T cell activation; v) the importance of the intracellular B30.2 domain of BTN3A1, in the Vγ9Vδ2 T cell activation, might be higher than that of the extracellular domain; and vi) in spite of the importance of BTN3A1 in the activation of Vγ9Vδ2 T cells, it is possible that many molecules with redundant functions are involved in the elimination of influenza virus infection by these cells.
In summary, it is possible to hypothesize a model in which BTN3A1 detects prenyl pyrophosphates in the cytoplasm via its B30.2 domain and in association with another protein(s). The binding of PAg to this domain induces a multimerization of BTN3A1 or a conformational change of its extracellular domain (mimicked by mAb 20.1). These modifications might be recognized by the Vγ9Vδ2 TCR or by an associated T cell protein. In the case that the TCR directly recognizes BTN3A1, the intensity of the response will depend on the Vγ9Vδ2 TCR clonotype. Future research will allow to gain a better understanding of BTN3A1, its interaction with other proteins, its actual role in the activation of Vγ9Vδ2 T cells, and its importance in specific models of cancer or infection. This knowledge will be necessary to transform these cells into effective tools in the clinic.
Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunologic research and for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However the large majority of human T-cells resides in tissues, especially in lymphatic organs, while only 1 % of the body’s T-cells circulate in the blood stream. Previous work in mice and humans had indicated that CD4 T-cells transiently lose antigen sensitivity when cellular contacts are lost, e.g. by leaving lymphoid organs such as lymph nodes (LNs) and entering the circulation. In this study, these findings were extended to CD8 T-cells. Thus, CD8 T-cell responses of the human tonsil show a significant drop in sensitivity to viral antigens if tissue-exit was simulated by keeping cells in dispersed culture at body temperature for two hours.
Conversely, tissue-like functionality in blood-derived CD8 T-cells was restored by applying the simple and robust RESTORE protocol. Indeed, application of the RESTORE protocol, i.e. pre-culturing PBMCs for two days at a high cell density before initiation of antigenic stimulation, demonstrated that CD8 T-cell responses to a broad range of viral and to tumor-associated antigens are greatly underestimated, and sometimes even remain undetected if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 (WT1)-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. My studies on the mechanism of the RESTORE protocol show that HD preculture of PBMCs does not involve antigen-or cytokine-driven clonal expansion of T-cells. Moreover, the gain in antigen sensitivity cannot be explained by a decreased activity of regulatory T-cells during the preculture step. The increased antigen sensitivity of CD8 T-cells from HD precultures of PBMCs is associated with tonic T-cell receptor signaling as indicated by enhanced tyrosine phosphorylation of the CD3 ζ chains and the tyrosine kinase Lck, thereby preparing T-cells for full responses. The upregulation of genes involved in aerobic glycolysis in “restored” CD8 memory T-cells relative to fresh cells might be an essential requirement for increased T-cell functionality including the regulation of IFN-γ production. Taken together, the RESTORE protocol, which was initially described for the CD4 T-cell response to the antibody TGN1412 permits a more meaningful monitoring of CD8 T-cell responses to viral infections and tumors. Furthermore, when generating T-cell lines for adoptive T-cell therapy, the RESTORE protocol allows the generation of CD8 T-cell lines with an improved representation of clones responding to low antigen concentrations.