Interleukin-8 and RANTES are signature cytokines made by HOZOT, a new type of regulatory T cells.
Distinct cytokine production profiles define the effector functions of both helper and regulatory T cells. Recently, we established novel cytotoxic regulatory T (Treg) cell lines, HOZOT, which have been characterized as IL-10-producing T cells. In this study, we further characterized HOZOT by performing comprehensive analyses of cytokines produced by HOZOTs in order to identify a signature cytokine profile. Using DNA microarrays, we compared the gene expression profiles of HOZOT-4, a representative HOZOT cell line, under three different conditions. Seven genes, including IL-8, IL-10, IL-13, MIP-1alpha, and MIP-1beta, were identified as inducible cytokines when stimulated with stromal cells or anti-CD3/CD28 antibodies. Twelve genes, including IL-2, IL-3, IL-4, IL-22, CCL1, and lymphotactin, were categorized as antibody stimulation-responsive but stromal cell-non-responsive. Three genes, IL-32, RANTES, and CCL23, were constitutively expressed irrespective of stimulation condition. Among these cytokines, we focused on two chemokines, IL-8 and RANTES for further studies, and found that only HOZOT produced both of them at considerable levels whereas other T cell subsets, including Tregs and helper T cells, did not. Kinetic and inhibition experiments revealed contrasting properties for the two chemokines. IL-8 was induced only after stimulation, whereas RANTES mRNA and protein accumulated to high levels even before stimulation. Interestingly, IL-8 mRNA was induced by cycloheximide treatment and RANTES showed reduced mRNA but increased protein expression by antibody stimulation. As a whole, the unique cytokine signature profile consisting of Th1, Th2, and cytolytic T cell cytokines as well as Treg cytokines reflect the multifunctional nature of HOZOT. In particular, the dual production of IL-8 and RANTES by distinct mechanisms is a hallmark of HOZOT.
Harashima
Akira
A
Cell Biology Institute, Research Center, Hayashibara Biochemical Laboratories Inc, Okayama, Japan. aharashi@hayashibara.co.jp
Toraya
Terumasa
T
Okochi
Ayumi
A
Yamamoto
Mayuko
M
Suzuki
Motoyuki
M
Otani
Takeshi
T
Inoue
Toshiya
T
Tsuji-Takayama
Kazue
K
Sugimoto
Akira
A
Takeuchi
Makoto
M
Yamasaki
Fumiyuki
F
Nakamura
Shuji
S
Kibata
Masayoshi
M
D002460:Cell Line; D018946:Chemokine CCL5 / Q000096:biosynthesis* / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis / Q000276:immunology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000276:immunology; D020411:Oligonucleotide Array Sequence Analysis; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology / Q000378:metabolism*
C483024:CCL5 protein, human; C508606:CXCL8 protein, human; D018946:Chemokine CCL5; D016207:Cytokines; D016209:Interleukin-8