Distinctive features of classic and nonclassic (Th17 derived) human Th1 cells

Abstract

T helper17 (Th17) lymphocytes represent a third arm of the CD4+ T-cell effector responses, in addition to Th1 and Th2 cells. Th17 cells have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in inflammatory sites. In humans, Keywords: CD161 r IL4I1 r RORC r Th17 Th1 cells derived from Th17 cells express CD161, whereas classic Th1 cells do not; these Th17-derived Th1 cells have been termed nonclassic Th1 cells. In this study, we examined similarities and differences between classic and nonclassic human Th1 cells by assessing a panel of T-cell clones, as well as CD161Introduction CD4 + T helper (Th) cells can be classified into lineages on the basis of cytokine production, the expression of specific transcription factors, and the immunological function they mediate: Th1 cells, which express the transcription factor T-box expressed in T cells (T-bet) and secrete IFN-γ, protect the host against intracellular infections; Th2 cells, which express GATA-3 and secrete IL-4, IL-5, and IL-13, mediate host defense against helminths [1,2]. Recently, additional subsets that preferentially produce distinct cytokines Correspondence: Prof. Francesco Annunziato e-mail: f.annunziato@dmi.unifi.it have been described. The most studied subset includes cells that selectively produce IL-17A (Th17 cells), express the transcription factor RAR-related orphan receptor (ROR)γt, and protect the host against infection with extracellular pathogens [3,4]. Human Th17 cells are, at least partially, different from murine Th17 cells [5,6]. Indeed, human Th17 cells express not only distinctive Th17 molecules, such as IL-23 receptor (IL-23R) and RORC, but also those typical of the Th1 phenotype such as IL-12Rβ2 and T-bet [6]. Moreover, we have previously discovered a subset of human Th17 cells that are also able to produce IFN-γ, named Th17/Th1 * These authors contributed equally to this work. To investigate the main features of these two different Th1 subsets, CD4+ CD161 + and CD4 + CD161 − T-cell populations were purified from peripheral blood (PB) of healthy human subjects and then cloned under limiting dilution conditions. As expected, clones derived from the CD4 + CD161 + T-cell fraction exhibited a Th17, a Th17/Th1, or a Th1 phenotype, whereas virtually all clones generated from the CD4 + CD161 − T-cell subset had a Th1 phenotype and none of them was able to produce IL-17A ( Fig. 1A and B). CD161 − Th1 clones were considered as classic Th1 cells, whereas CD161 + Th1 cells were considered as nonclassic (Th17-derived) cells. Transcription factorsThe expression of the transcription factors T-bet and RORC by classic and nonclassic Th1, as well as by Th17 or Th17/Th1, cells were compared by assessing 20 randomly selected T-cell clones from each phenotype with quantitative RT-PCR. In agreement with previously published data [15,16], the Th1-related transcription factor T-bet was found to be expressed by all four types of T-cell clones analyzed, reaching the highest mRNA levels in both classic and nonclassic-Th1 c...

IL‐4‐induced gene 1 maintains high Tob1 expression that contributes to TCR unresponsiveness in human T helper 17 cells

“…PB CD4 + CD161 + and CD4 + CD161 − T-cell populations were obtained by magnetic cell sorting [10] (Miltenyi Biotec, Bergisch Gladbach), further subdivided in CCR6 + and CCR6 − cell fraction by FACSAria (BD Biosciences) and then cultured under limiting dilution in the presence of allogeneic-irradiated feeder cells, PHA (2 μg/mL) and IL-2 (50 IU/mL). All clones were analyzed by flow cytometry for their ability to produce IL-17 and IFN-γ and pure Th17 and Th1 clones selected to perform further experiments [24].…”

“…CD4 + CD161 + and CD4 + CD161 − cells from PB of three healthy donors were stimulated with PMA-ionomycin, stained with IFN-γ and IL-17 catch reagents (Miltenyi Biotec), following manufacturer instructions and then stained with anti-CD3, -CD161, -IL-17, and -IFN-γ, and sorted by FACSAria into CD3 + CD161 + IL-17 + IFN-γ − and CD3 + CD161 − IL-17 − IFN-γ + cells [10].…”

“…We have also shown that virtually all human memory Th17 cells are contained within the CD161 + fraction of both circulating and tissue-infiltrating CD4 + T cells, and originate from CD161 + precursors present in umbilical cord blood and newborn thymus [9]. In humans, Th1 cells that derive from the shifting of Th17 cells have been defined as nonclassic Th1 cells [8,10], since they can be distinguished from classic Th1 cells by the expression of CD161, as expressed by Th17 cells [9][10][11].…”

“…We have also shown that virtually all human memory Th17 cells are contained within the CD161 + fraction of both circulating and tissue-infiltrating CD4 + T cells, and originate from CD161 + precursors present in umbilical cord blood and newborn thymus [9]. In humans, Th1 cells that derive from the shifting of Th17 cells have been defined as nonclassic Th1 cells [8,10], since they can be distinguished from classic Th1 cells by the expression of CD161, as expressed by Th17 cells [9][10][11].More recently, we have demonstrated that Th17 cells, unlike classic Th1 cells, do not proliferate in response to anti-CD3 plus anti-CD28 mAb stimulation; this unresponsiveness is due mainly to the inability of human Th17 cells to produce IL-2, which could be related to their reduced c-Fos, c-Jun, and NFAT activity [12]. Moreover, we found that the reduced activity of these transcription factors is associated with high expression of IL-4-induced gene 1 (IL4I1) mRNA, which encodes an L-phenylalanine oxidase able to downregulate the expression of CD3 chains in T cells, thus resulting in a poor ability to expand in response to TCR stimulation [12].…”

“…Accordingly, confocal microscopy analysis demonstrated both increased expression and nuclear localization of Tob1 in Th17 cells when compared to Th1-cell clones (Fig. 1B).In order to exclude the possibility that these differences were due to an artifact resulting from the use of long term in vitro cultured T cells (T-cell clones), Tob1 mRNA levels were also measured in ex vivo derived IL-17-or IFN-γ-producing T cells, which had been purified from the sorted CD161 + (Th17) and CD161 − (classic Th1) circulating CD4 + T-cell fractions by cytokine secretion assay, as already reported [10]. In agreement with data obtained using T-cell clones, in absence of any in vitro stimulation, ex vivo-derived Th17 cells expressed higher levels of Tob1 mRNA than classic Th1 cells (Fig.…”