Reply to Cohen

The problem identified by Cohen is real – the definition of Th1 and Th2 responses is difficult and various definitions are used by different researchers. Cohen points out that both the ratio and amount of cytokines are important, and suggests standardizing against a cytokine expressed at constant levels by all T cells.Unfortunately, interleukin 3 (IL-3) is not a good marker for general T-cell activation, as the level of IL-3 synthesized by various T cells can vary widely, and higher levels of IL-3 can be made in Th2 responses than in Th1 responses. In fact, I do not know of any cytokine that is produced at the same level by all T cells and until one is identified, there is no universal normalizing cytokine.The problem is actually very similar for the normalization of mRNA levels. Although Cohen correctly points out that actin is often used as a standard, this is problematic because actin is not expressed at constant levels – for example, in T cells, actin expression is increased very strongly by concanavalin A (ConA) activation. Other ‘housekeeping’ genes have also been used, but the problem is similar to the difficulty with cytokines – there may not be a gene that is expressed at constant levels in all cells.A related problem is that the cytokine patterns of an overall response do not always fit the basic cytokine patterns defined by Th2 or Th1 clones. Interferon γ (IFN-γ) and IL-4 are the most commonly used defining cytokines, as mentioned by Cohen, but this is probably due to a combination of historical and practical considerations. Lymphotoxin is another strong marker for Th1 responses, but is more difficult to assay and to distinguish from tumour necrosis factor α (TNF-α), which is produced by both cell types. IL-5 is a good marker for Th2 responses, although it does not share IL-4’s ability to induce differentiation into Th2 cells. Although these cytokines are recognizably Th1 or Th2, their levels do not always correlate with those of IFN-γ or IL-4, raising the question: which of these is a more valid description of type 1 or type 2 responses? Thus IFN-γ and IL-4 are useful markers for Th1 versus Th2 responses, but do not give definitive descriptions of the responses.The problem is exaggerated for mixed cell populations because of the different levels of cytokines made by CD4+ or CD8+ T-cell subsets, and the presence of other phenotypes such as Th3 or Treg1 (regulatory type 1 T cell)1xRegulatory T cell clones induced by oral tolerance (suppression of autoimmune encephalomyelitis) . Chen, Y. et al. Science. 1994; 265: 1237–1240Crossref | PubMedSee all References, 2xA CD4+ T cell subset inhibits antigen-specific T-cell responses and prevents colitis. Groux, H. et al. Nature. 1997; 389: 737–742Crossref | PubMed | Scopus (2752)See all References. IL-5 does not always correlate with IL-4, and IFN-γ often does not correlate with IL-2, at least partly because of the secretion of IL-2 but not IFN-γ by naive cells and uncommitted precursor memory cells3xSingle IL-2-secreting precursor CD4 T cell can develop into either Th1 or Th2 cytokine secretion phenotype. Sad, S. and Mosmann, T.R. J. Immunol. 1994; 153: 3514–3522PubMedSee all References, 4xTwo subsets of memory T lymphocytes with distinct homing potentials and effector functions. Sallusto, F. et al. Nature. 1999; 401: 708–712Crossref | PubMed | Scopus (3344)See all References.Overall, I think that the safest way to describe cytokine response patterns is to include information on both ratios and absolute amounts of cytokines – as mentioned by Cohen, both of these parameters are important. It is also useful to identify when a particular response fits the overall type 1 or type 2 pattern, and when there are differences such as independent variation of IL-4 and IL-5 levels.