In this study, we have demonstrated a novel procedure by which to isolate activated TNF-producing lymphocytes. Through the use of a TACE inhibitor, we take advantage of the unique mechanism of TNF production in order to prevent TNF release from activated T cells, thereby allowing their direct identification and isolation. We find that this procedure is highly reproducible, yields similar results to intracellular cytokine staining, and is of sufficient sensitivity to identify rare populations of antigen-specific CD8+ T cells. We demonstrate that this assay can be used to isolate viable TNF-producing antigen-specific T cells, allowing for subsequent studies requiring cell culture or RNA-based analyses.
Many different cell types of both lymphoid and non-lymphoid origin produce TNF; we show here that our assay is amenable for the identification of TNF-producing CD4+ and CD8+ T lymphocytes, as well as NK cells. It is important to note in particular that CD4+ T cells appear to exhibit substantially higher background TNF staining in the absence of stimulation compared to both CD8+ T cells and NK cells, as has been reported previously (Horton et al., 2007
). Although this would likely reduce the sensitivity of our assay for CD4+ T cell sorting, combining TNF labeling with another measure of CD4+ T cell activation, such as upregulation of CD40L (Chattopadhyay et al., 2005
) or CD69 (Waldrop et al., 1998
) will reduce sensitivity concerns and still allow CD4+ T cells to be sorted viably and specifically based on TNF production.
Viable isolation of functional antigen-specific T cells is of particular importance for both basic and translational immunological research. As we have shown, cell sorting via TACE-inhibition of TNF release permits the direct isolation of viable antigen-specific CD8+ T cells for clonotypic analysis of expressed TCRs. Our results demonstrate both the specificity and utility of the TNF-SS sorting procedure. Thus, compared to CD8+ T cells sorted on the basis of physical binding to cognate MHC class I tetramer, the TNF-SS sorting procedure identified the same dominant clonotypes at the molecular level. Interestingly, however, the TNF assay revealed that the dominant clonotypes identified by MHC class I tetramer binding were not equally capable of producing TNF, thereby indicating functional heterogeneity within the peptide-specific CD8+ T cell population at the clonotypic level.
From the standpoint of translational research, there is a need for procedures that permit the isolation and expansion of antigen-specific T cells for use in immunotherapeutic strategies. Commonly, MHC class I tetramers/multimers are used for such isolation procedures; however, as shown herein and elsewhere (Appay et al., 2000
; Oxenius et al., 2002
; Gu et al., 2007
; Rehr et al., 2008
), not all MHC class I tetramer-binding cells are fully functional. For instance, not all MHC class I tetramer-binding cells in a population produce IFN-γ after stimulation with cognate peptide (Goepfert et al., 2000
). Alternatively, surface mobilized CD107a can be used as a means to isolate viable CD8+ T cells. Again, however, not all antigen-specific CD107a+CD8+ T cells produce IFN-γ(Makedonas et al.; Betts et al., 2004
; Betts et al., 2006
). In addition to this, as T cells mature and become terminally differentiated, TNF production is lost (La Gruta et al., 2004
). Together, this demonstrates functional heterogeneity within the overall population of responding T cells to any given antigen. Thus, the ability to isolate cells based upon the ability to produce TNF is of particular use, as it will allow the isolation of CD8+ T cells with increased functional capacity (Gu et al., 2007
) at an earlier stage of differentiation. This may be of great importance, as T cell-based therapies typically require in vitro
expansion of the T cells to generate sufficient numbers for effective transfer.
In conclusion, we have demonstrated a novel procedure by which to identify and isolate antigen-specific T cells based upon their ability to produce TNF. This procedure is highly sensitive and specific, and can be used to isolate viable cells for mRNA-based analyses as well as subsequent cell culture and proliferation. Alone, or in combination with other functional sorting methods, this procedure will facilitate further investigations into the underlying biological properties of antigen-specific T cells. This procedure will also be valuable for T cell-based immunological therapies, as TNF-producing T cells can either be enriched or depleted from therapeutic products.