A rarely challenged dogma in cell-mediated immune (CMI) assays is the incubation temperature, 37°C. Fever augments proinflammatory immune responses in vivo, and the aim of this study was to explore whether incubation at fever-range temperature could increase antigen-specific biomarker responses. We compared CMI responses following incubation of whole blood at 37°C and 39°C. Whole blood was obtained from (i) 34 healthy subjects whose blood was incubated with TB10. Cell-mediated immune (CMI) assays are important research tools used to monitor T cell-dependent immune responses toward antigens from, e.g., pathogens or vaccines.CMI assays rely on generation of an in vitro immune response and quantification of the proinflammatory immune response (26). In clinical medicine, CMI assays are used for the diagnosis of infections that cannot be detected with direct methods (28). An example is the diagnosis of latent infection with Mycobacterium tuberculosis, where either purified peripheral blood mononuclear cells (PBMCs) or whole blood is incubated with M. tuberculosis-specific antigens, followed by quantification of the gamma interferon (IFN-␥) response, hence the name IFN-␥ release assays (IGRAs) (3).A rarely challenged dogma in CMI assays-both in research and in diagnostic medicine-is that the incubation temperature is optimal at 37°C. Fever is a part of the innate immune response in vivo and can aid the immune defense by impairing the viability of pathogens with strict temperature preferences or by enhancing immunological reactions (reviewed in reference 38). Previous studies indicate that fever-range temperature in vivo and in vitro augments antigen presentation and coreceptor expression and skews antigen-presenting cells (APCs) toward activation, in concert with augmented T cell responsiveness and Th1 bias (5,6,17,33).One of the major challenges of CMI assays-and IGRAs, in particular-is achieving a consistent and strong response (i.e., high sensitivity) without generating nonspecific immune activation (i.e., compromising specificity).We and others have demonstrated that, compared to T cell responses, the APCs interacting with the specific T cells generate stronger chemokine and cytokine signals that could enable simpler or more sensitive readouts of CMI assays (reviewed in reference 24). In several studies, the chemokine IFN-␥-induced protein 10 (IP-10) has shown promise as a novel marker for immunodiagnosis of infection with M. tuberculosis in both children and adults (1,13,15,(21)(22)(23)30).In an attempt to further improve CMI assays, we hypothesized that incubation at fever-range temperatures in vitro could lead to an increase in proinflammatory immune responses and potentially an increase in the sensitivity of IGRA and CMI assays. Interleukin-7 (IL-7) is a cytokine that promotes survival of T cells (8, 9). IL-10 is an anti-inflammatory cytokine released by T cells and monocytes/macrophages with immune activation (25). Both IL-7 and blocking of IL-10 have previously been shown to augment IFN-␥ responses to M. tub...