CD4⁺CD8β⁺ double-positive T cells in skin-draining lymph nodes respond to inflammatory signals from the skin

CD4+CD8+ double-positive (DP), mature, peripheral T cells are readily detectable in a variety of species and tissues. Despite a common association with autoimmune and malignant skin disorders, however, little is understood about their role or function. Herein, we show that DP T cells are readily detectable in the blood, spleen, and peripheral lymph nodes of naïve C57BL/6 mice.

DP T cells were also present in Jα-/- and CD1d-/- mice, indicating that these cells are not NK-T cells. After skin administration of CASAC adjuvant, but not Quil A adjuvant, both total DP T cells and skin-infiltrating DP T cells increased in number. We explored the possibility that DP T cells could represent aggregates between CD4+ and CD8+ single-positive T cells and found strong evidence that a large proportion of apparent DP T cells were indeed aggregates.

However, the existence of true CD4+CD8+ DP T cells was confirmed by Amnis Image-Stream (Millipore Sigma, Billerica, MA, USA) imaging. Multiple rounds of FACS sorting separated true DP cells from aggregates and indicated that conventional analyses may lead to ~10-fold overestimation of DP T cell numbers.

The high degree of aggregate contamination and overestimation of DP abundance using conventional analysis techniques may explain discrepancies reported in the literature for DP T cell origin, phenotype, and function.