A very rapid and simple assay based on trogocytosis to detect and measure specific T and B cell reactivity by flow cytometry

Abstract

Detection, quantification, separation and characterization of T and B cells reactive to specific antigens are important tasks in both basic and clinical immunology. Here, we describe an approach allowing the performance of all four tasks on a functional basis by flow cytometry. The assay is based on the property of lymphocytes to capture membrane components from the cells they interact with, in a process we call trogocytosis. Working with CD8 + CTL and target cells labeled with membrane markers, we describe the conditions allowing reactive lymphocytes to be detected rapidly and inexpensively within mixed populations. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this method to the detection of antigen-specific CD4 + T and B cells. While our method is, for the time being, not as sensitive as staining of CTL with MHC class I multimers, it allows the simultaneous quantitative identification of reactive CD8 + , CD4 + and B cells. Altogether, our method offers a simple and general alternative to other methods previously described to detect and quantify lymphocyte reactivity, and it can also be used in combination with those.

Intercellular Trogocytosis Plays an Important Role in Modulation of Immune Responses

“…The peptide-MHC complexes transferred from APCs to T cells are the best studied examples of protein transfer that occurs via trogocytosis. Here, T cells can acquire both MHC class I and class II proteins from APCs (19,23,24,(53)(54)(55). Reports of intercellular transfer of membrane fragments from APCs to T cells (24,54) and from target cells to NK cells (31), and even through homotypic interactions between cells like Daudi cells (56), are consistent with the membrane transfer mechanism that involves the transfer of membrane fragments derived from the intercellular contact or IS (57).…”

Suitability of various membrane lipophilic probes for the detection of trogocytosis by flow cytometry

“…Several important physiological and pathological consequences of trogocytosis in the immune system have been suggested or, in some cases demonstrated (1,2,4). The occurrence of trogocytosis can be evidenced by labeling the APCs' plasma membrane either with fluorescent lipophilic probes such as DiO C18, DiI C18, PKH26, and PKH67, via biotinylation of the proteins, or modified carbohydrate precursors and detecting the presence of these probes onto lymphocytes after their co-culture with APC expressing the antigen (5)(6)(7).…”

“…For example, the use of fluorescent probes staining either the plasma membrane or the cytosol of APCs revealed that trogocytosis concerns plasma membrane but not cytosolic components (7). In addition, biotinylation of APC surface proteins indicated that only a selected set of these proteins was transferred onto lymphocytes via trogocytosis (6,8).…”

“…In the past, we and others have shown that the transfer of labels from APC to the lymphocytes during trogocytosis makes it possible to use trogocytosis analysis protocol (TRAP) assays to identify and enumerate those lymphocytes that react specifically with the antigen-loaded APCs (5,7,(9)(10)(11)(12). Among the many advantages of TRAP assays over previous assays (10), the most significant are their simplicity, the fact that they can work for all types and subtypes of lymphocytes concomitantly and that it is not necessary to know the precise nature of the antigen recognized.…”

B‐ and T‐cell memory elicited by a seasonal live attenuated reassortant influenza vaccine: assessment of local antibody avidity and virus‐specific memory T‐cells using trogocytosis‐based method

“…In our study, we modified the [T‐cell recognition of antigen‐presenting cells (TRAP) by protein capture] assay that measures antigen‐activated cells involved in trogocytosis, i.e., membrane fragments interchange between T cells and APCs during antigen presentation 13 . The TRAP method has been previously used to study trogocytosis in vitro 14 and to determine in vivo T cells specific for herpes virus, 15 lymphocytic choriomeningitis virus, 16 and ovalbumin 14 …”