One surprise to come out of this work concerns the deduced prevalence of virus-specific T cells. ed with their affinity for the TCR and the level of TCR expression. Antigenspecific T cells were detectable in TCR transgenic mice. HLA DR4 tetramers were also made (J. Kappler, personal communication). We have prepared HLA DR1 coupled to an HIV gag peptide in this way (A. Kelleher, unpublished results). A drawback to this approach is that the peptide has to be engineered into the construct, which is cumbersome, but in this issue of the JCI, Novak et al.(16) describe production of HLA DR4 tetramers that contain an influenza virus epitope. DR4 was expressed in empty form in insect cells, with the 2 chains linked at their carboxy termini by a leucine zipper. Empty DR4 was purified and then soaked in the required epitope peptide to give a stable molecule that could be biotinylated and used to stain influenza virus–specific CD4+ T cells. This staining confirms the specificity of the reagent and affords Novak and colleagues the first glimpse of a human antiviral CD4+ T-cell response. They could detect no staining of T cells in blood from immune humans, but stimulation in vitro with influenza antigen induced CD4+ T cells that stained with the tetramer. By costaining these cultures with 5-carboxyfluorescein diacetate succinimidyl ester, which dilutes exponentially as the cells divide, Novak et al. could calculate the number of divisions that the cells completed—up to 10—within 7 days of antigen stimulation. These data allow the number of precursors in the original blood sample to be determined, avoiding the considerable difficulties associated with the LDA. Precursor frequency determined by these calculations agrees with estimates from the LDA (< 1 in 104).

The number of virus-specific CD4+ T cells appears to be lower than that of the CD8+ T cells cited above, but the latter were measured during persisting infections. Influenza virus is eliminated after each attack; specific CD8+ T cells can be found in the blood of healthy adults but at similar low levels (1 in 104). It remains to be seen whether the CD4+ T-cell response to a persisting or acute virus is smaller than the massive CD8+ T-cell response. Many questions about the human CD4+ T cell response to natural antigens can now be explored in detail.