Trogocytosis in multiple myeloma

Publication Type:
Thesis
Issue Date:
2012
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The term trogocytosis is used to describe the fast, cell-to-cell contact-dependent transfer of membrane proteins between cells, with T and B-lymphocytes, natural killer (NK) cells, antigen presenting cells (APC) and tumour cells being the most widely studied. This study aimed to: (1) identify the extent of trogocytosis in patients with multiple myeloma (MM), a malignancy of bone marrow plasma cells, compared with the other B cell malignancies, (2) to identify some of the molecules involved with trogocytosis in these patients and then (3) determine if cells that had acquired molecules had altered function. An in vitro model of trogocytosis was established in which plasma cell lines and flowsorted bone marrow plasma cells (CD38++) of patients with MM (n=11) or the malignant B cells from patients with chronic lymphocytic leukaemia (CLL) (CD5+CD19+) (n=4) and Waldenstrom macroglobulinaemia (WM) (CD19+) were biotinylated and then cultured with either patient or normal mononuclear cells. The acquisition of biotinylated membrane proteins was determined by flow cytometry and confocal microscopy. Screening for potential molecules involved suggested that CD86 and HLA-G were likely candidates for trogocytosis. CD86 is a co-stimulatory molecule at the immune synapse and HLA-G is a non-classical MHC class I molecule which prevents antigen-specific cytolysis by cytotoxic T lymphocytes (CTLs), inhibits the function of circulating NK cells and prevents proliferation of allogeneic CD4+ T cells. These observations have lead to the hypothesis that expression of HLAG may aid in the escape of tumours from immune surveillance. T cells acquired significantly more biotinylated proteins (mean=13.55%) than B cells (mean=2.43%; t=2.80; p<0.05) or NK cells (mean=3.15%; t=2.57; p<0.05). There was no significant difference between levels of biotin transferred to T cells from either plasma cell lines or primary plasma cells and acquisition was the same with autologous and allogeneic T cells. Significantly more trogocytosis was observed in myeloma patients than other B cell malignancies (n=5) as <1% T cells acquired membrane fragments when cultured with malignant B cells from patients with CLL or WM (t =3.86; p<0.05). Upon culture with biotinylated CD3+ flow-sorted normal T cells, approximately 2% of CD38++ plasma cells acquired membrane fragments, suggesting that in patients with myeloma, trogocytosis was predominantly unidirectional. Although HLA-G expression was found on 0.02 – 0.56% of normal T cells (mean =0.23%), 20% of MM patients (11 of 56) demonstrated a level of HLA-G+ CD3+ T cells above the normal range. Addition of flow-sorted CD3+ HLA-Gpos T cells led to a reduction in the proliferation of carboxyfluorescein succinimidyl ester (CFSE)- labelled CD3+ HLA-Gneg T cells stimulated with anti-CD3/CD28 beads and this inhibition was greater than the inhibition due to CD38++ HLA-Gpos plasma cells (t=2.64; p=0.046). The CD3+ HLA-Gpos T cells acquired inhibitory function but were not natural T regulatory cells as they were CD25neg. Overall survival was significantly worse for the 11/46 patients with HLA-Gpos plasma cells ( 2=12.4; p<0.0004). Flow cytometric analysis of CD38++ bone marrow plasma cells from MM patients showed varied HLA-G expression ranging from 0.2% to 96% (n=46). The clinical relevance of HLA-Gpos plasma cells was demonstrated by a significant reduction in overall survival (n= 46; χ2= 12.4; p<0.004). CD86 expression on T cells of myeloma patients (n=98) ranged from 0 – 30% (normals = 0 – 2.7%; n=10). T cells from myeloma patients (n=7), when co-cultured with CD86 expressing plasma cells, were found to acquire significantly (p<0.0001) higher levels of CD86. This study reports several new findings. It has shown that trogocytosis is more common in multiple myeloma than other B cell malignancies, is primarily unidirectional, HLA independent and T cells are more likely to be involved than other lymphocytes. T cells which acquire tumour antigens may have altered function and it has been demonstrated that HLA-Gpos T cells form a new subset of acquired regulatory T cells that inhibit the proliferation of HLA-Gneg T cells and therefore protect MM cells against the host’s immune defences.
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