Trafficking of immune cells is controlled by directed migration of relevant cells toward chemotactic signals. Actin cytoskeleton undergoes continuous remodeling and serves as machinery for cell migration. The mDia family of formins and the Wiskott-Aldrich syndrome protein (WASP)–Arp2/3 system are two major actin nucleating–polymerizing systems in mammalian cells, with the former producing long straight actin filaments and the latter producing branched actin meshwork. Although much is known about the latter, the physiological functions of mDia proteins are unclear. We generated mice deficient in one mDia isoform, mDia1. Although mDia1−/− mice were born and developed without apparent abnormality, mDia1−/− T lymphocytes exhibited impaired trafficking to secondary lymphoid organs in vivo and showed reduced chemotaxis, little actin filament formation, and impaired polarity in response to chemotactic stimuli in vitro. Similarly, mDia1−/− thymocytes showed reduced chemotaxis and impaired egression from the thymus. These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.
Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by recurrent eczematous legions and intense itch. Itch can be induced by various chemical mediators. Among them, much attention has been paid to interleukin 31 (IL-31) as an AD-associated itch mediator since the discovery of the pruritogenic action of IL-31 in mice. IL-31 is mainly produced by CD4+ T cells and transmits the signals via a heterodimeric receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR), both of which are expressed in dorsal root ganglion (DRG) neurons. However, the neuronal mechanism underlying IL-31–induced itch sensation is poorly understood. By analyzing a mouse model for atopic dermatitis, we found that the expression of Tac2, which encodes neurokinin B (NKB), markedly increased in the DRG neurons in response to IL-31. While NKB-deficient mice lost IL-31–induced itch response, scratching behaviors induced by other pruritogens such as histamine, chloroquine and protease-activated receptor 2 (PAR2) agonist were unaffected in the absence of NKB. NKB transmits the signal through neurokinin 3 receptor (NK3R), a G protein-coupled tachykinin receptor. When wild-type mice were pre-treated with NK3R antagonists, IL-31–induced scratching was significantly attenuated, without affecting itch responses induced by other pruritogens. These results indicate that NKB-NK3R axis could be a novel therapeutic target controlling IL-31–induced itch in AD patients.
UV radiation induces systemic immunosuppression. Because nonsteroidal anti-inflammatory drugs suppress UV-induced immunosuppression, prostanoids have been suspected as a crucial mediator of this UV effect. However, the identity of the prostanoid involved and its mechanism of action remain unclear. Here, we addressed this issue by subjecting mice deficient in each prostanoid receptor individually or mice treated with a subtype-specific antagonist to UV irradiation. Mice treated with an antagonist for prostaglandin E receptor subtype 4 (EP4), but not those deficient in other prostanoid receptors, show impaired UV-induced immunosuppression, whereas administration of an EP4 agonist rescues the impairment of the UV-induced immunosuppression in indomethacin-treated mice. The EP4 antagonist treatment suppresses an increase in the number of CD4(+)/forkhead box P3-positive (Foxp3(+)) regulatory T cells (Treg cells) in the peripheral lymph nodes (LNs) and dendritic cells expressing DEC205 in the LNs and the skin after UV irradiation. Furthermore, the EP4 antagonist treatment down-regulates UV-induced expression of receptor activator of NF-κB ligand (RANKL) in skin keratinocytes. Finally, administration of anti-RANKL antibody abolishes the restoration of UV-induced immunosuppression by EP4 agonism in indomethacin-treated mice. Thus, prostaglandin E(2) (PGE(2))-EP4 signaling mediates UV-induced immunosuppression by elevating the number of Treg cells through regulation of RANKL expression in the epidermis.
5306 Anti-tumor immune responses in tumor-bearing hosts are initiated by tumor-specific antigen uptake into dendritic cells (DC) and the subsequent migration of these cells into secondary lymphoid organs. Although prostaglandin E2 (PGE2), a major cyclooxygenase product, is overproduced in many tumors and plays important roles in inflammation and immune responses, its role in DC function in tumor-bearing hosts remains unclear. Here using prostaglandin EP2 and EP4 receptor deficient mice and an EP4 receptor specific antagonist, we investigated the roles of these receptors in regulating DC migration in tumor-bearing hosts. We found that the EP2 receptor but not the EP4 receptor mediates PGE2-induced inhibition of DC migration. DCs from EP2 receptor deficient tumor-bearing mice showed a significantly increased ability to home to secondary lymph organs compared with those from tumor-bearing wild type (wt) mice, whereas complete deletion of the EP4 receptor or blocking of EP4 receptor signaling using a specific antagonist does not restore defective DC migration. These observations are consistent with the reduced tumor growth in EP2 knockout (ko) mice and increased T cell numbers in draining lymph nodes as well as an increased T cell infiltration in tumors derived from EP2 ko animals. We also found that the expression of the EP2 receptor was dramatically increased in DCs derived from tumor-bearing hosts and PGE2-EP2 signaling significantly inhibited DC chemotaxis in response to CCL19 and CCL21 in vitro. Together, our data demonstrate PGE2-EP2 signaling negatively regulates dendritic cell migration in tumor-bearing hosts and thus selective blockade of this receptor may improve anti-tumor immune responses.
Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting 15% to 20% of the general population in developed countries.1Weidinger S. Beck L.A. Beiber T. Kabashima K. Irvine A.D. Atopic dermatitis.Nat Rev Dis Primers. 2018; 4: 1Crossref PubMed Scopus (127) Google Scholar It is characterized by recurrent eczematous legions and intense itch. Because the itch sensation induces scratching behavior, which exacerbates the skin inflammation and disturbs the quality of life of affected individuals, chronic itch is a challenging clinical problem in the treatment of AD. Itch can be induced by various chemical mediators. Among them, much attention has been paid to IL-31 as an AD-associated itch mediator since the discovery of the pruritogenic action of IL-31 in mice.2Dillon S.R. Sprecher C. Hammond A. Bilsborough J. Rosenfeld-Franklin M. Presnell S.R. et al.Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice.Nat Immunol. 2004; 5: 752-760Crossref PubMed Scopus (658) Google Scholar IL-31 is mainly produced by CD4+ T cells and transmits the signals via a heterodimeric receptor composed of IL-31 receptor A and oncostatin M receptor (OSMR), both of which are expressed in various cell types including dorsal root ganglion (DRG) neurons.3Cevikbas F. Wang X. Akiyama T. Kempkes C. Savinko T. Antal A. et al.A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.J Allergy Clin Immunol. 2014; 133: 448-460Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar A recent clinical study has demonstrated that blockade of IL-31 signals by a specific antibody for IL-31 receptor A alleviates pruritus in patients with AD.4Ruzicka T. Hanifin J.M. Furue M. Pulka G. Mlynarczyk I. Wollenberg A. et al.Anti-interleukin-31 receptor A antibody for atopic dermatitis.N Engl J Med. 2017; 376: 826-835Crossref PubMed Scopus (308) Google Scholar However, the neuronal mechanism underlying IL-31–induced itch sensation is poorly understood. Mutations of DOCK8 in humans cause a combined immunodeficiency characterized by AD. We have previously reported that DOCK8-deficient (Dock8–/–), but not Dock8+/–, mice spontaneously develop AD-like skin disease when crossed with transgenic mice expressing AND T-cell receptor (designated AND Tg mice). In this model, the disease manifestations were completely lost when either OSMR or IL-31 was deleted (see Fig E1 in this article's Online Repository at www.jacionline.org). To identify candidate molecules that mediate IL-31–induced itch sensation, we performed microarray analysis of the DRG neurons and found that 698 genes were expressed at higher levels in Dock8–/– AND Tg mice than in Dock8+/– AND Tg mice (see Table E1 in this article's Online Repository at www.jacionline.org), the second highest of which was Tac2 encoding neurokinin B. To confirm the microarray data, we performed real-time PCR analyses. Although the expression of Il31ra was comparable between Dock8–/– AND Tg and Dock8+/– AND Tg littermates, the expression of Tac2 in the DRG neurons increased 23-fold in Dock8–/– AND Tg mice, which was also canceled by deleting OSMR or IL-31 (Fig 1, A; see Fig E2 in this article's Online Repository at www.jacionline.org). Immunohistochemical analyses of Dock8–/– AND Tg mice revealed that neurokinin B was expressed by the IL-31 receptor A+ DRG neurons (Fig 1, B). Neurotransmitters are stored in synaptic vesicles and released on stimulation. Indeed, neurokinin B was released in vitro from primary DRG neurons of wild-type (WT; C57BL/6) mice in response to IL-31 (Fig 1, C). However, the release of neurokinin B was completely abolished when primary DRG neurons lack OSMR expression (Fig 1, C). Thus, neurokinin B is induced in and released from DRG neurons in response to IL-31. To examine the physiological significance of neurokinin B in IL-31–induced itch sensation, we developed 2 lines of Tac2-deficient (Δ4 and Δ15) mice by using the CRISPR/Cas9 nuclease system (see Fig E3 in this article's Online Repository at www.jacionline.org). Intradermal injection of histamine, chloroquine, and protease-activated receptor 2 agonist (SLIGRL-NH2) comparably induced scratching in both Tac2–/– (Δ4) and Tac2+/– mice (Fig 1, D), as was previously reported.5Mar L. Yang F.C. Ma Q. Genetic marking and characterization of Tac2-expressing neurons in the central and peripheral nervous system.Mol Brain. 2012; 5: 3Crossref PubMed Scopus (24) Google Scholar However, although IL-31 administration induced scratching in Tac2+/– mice, Tac2–/– (Δ4) mice were significantly less sensitive to IL-31 stimulation (Fig 1, D). Similar results were obtained when Tac2–/– mice (Δ15) were analyzed (see Fig E4 in this article's Online Repository at www.jacionline.org). Interestingly, Tac2 deficiency markedly improved the skin inflammation and scratching behavior in Dock8–/– AND Tg mice without affecting serum IL-31 levels (Fig 1, E-G). Thus, neurokinin B is selectively required for transmission of IL-31–induced itch sensation. Many pruritogens require natriuretic polypeptide b (Nppb) and gastrin-releasing peptide (GRP) to transmit itch sensation in the spinal cord.6Bautista D.M. Wilson S.R. Hoon M.A. Why we scratch an itch: the molecules, cells and circuits of itch.Nat Neurosci. 2014; 17: 175-182Crossref PubMed Scopus (205) Google Scholar To examine whether IL-31 uses GRP or Nppb to transmit itch sensation in the spinal cord, we specifically ablated neurons expressing GRP receptor or Nppb receptor by intrathecally injecting toxin (saporin)-conjugated GRP or Nppb, respectively. Although Nppb has been implicated in IL-31–mediated skin inflammation in the periphery,7Meng J. Moriyama M. Feld M. Buddenkotte J. Buhl T. Szöllösi A. et al.New mechanism underlying IL-31–induced atopic dermatitis.J Allergy Clin Immunol. 2018; 141: 1677-1689Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar IL-31–induced scratching was unaffected by intrathecal injection of Nppb-saporin (Fig 2, A). In contrast, treatment with GRP-saporin reduced IL-31–induced scratching (Fig 2, A). The Grp expression in the DRG neurons increased 4.5-fold in Dock8–/– AND Tg mice as compared with that in Dock8+/– AND littermates (see Fig E2). Although GRP was released in vitro from WT DRG neurons in response to IL-31, GRP release was hardly detected when primary DRG neurons from Tac2–/– (Δ4) mice were similarly stimulated (Fig 2, B). In addition, itch response induced by intrathecal injection of neurokinin B was lost when neurons expressing GRP receptor were ablated beforehand by toxin treatment (Fig 2, C). Neurokinin B transmits the signal through neurokinin 3 receptor (NK3R), a G protein–coupled tachykinin receptor. Immunohistochemical analyses of Dock8–/– AND Tg mice revealed that GRP was expressed by the NK3R+ DRG neurons and neurokinin B+ DRG neurons (Fig 2, D). These results indicate that neurokinin B acts upstream of GRP to transmit IL-31–induced itch sensation (Fig 2, E). So far, several selective antagonists for NK3R such as osanetant and fezolinetant have been developed.8Spooren W. Riemer C. Meltzer H. NK3 receptor antagonists: the next generation of antipsychotics?.Nat Rev Drug Discov. 2005; 4: 967-975Crossref PubMed Scopus (104) Google Scholar, 9Fraser G.L. Ramael S. Hoveyda H.R. Gheyle L. Combalbert J. The NK3 receptor antagonist ESN364 suppresses sex hormones in men and women.J Clin Endocrinol Metab. 2016; 101: 417-426Crossref PubMed Scopus (43) Google Scholar When osanetant was intraperitoneally injected into WT mice, IL-31–induced scratching was significantly attenuated (Fig 2, F). Similar results were obtained when fezolinetant was administered orally (see Fig E5 in this article's Online Repository at www.jacionline.org). In contrast, treatment with NK3R antagonists failed to suppress itch response induced by histamine, chloroquine, and SLIGRL-NH2 (Fig 2, F; see Fig E5). Thus, pharmacological inhibition of NK3R selectively attenuates IL-31–induced itch sensation. Although NK3R antagonists suppress sex hormones by modulating gonadotropin secretion, its effect is transient and reversible, with no major side effects reported as yet.8Spooren W. Riemer C. Meltzer H. NK3 receptor antagonists: the next generation of antipsychotics?.Nat Rev Drug Discov. 2005; 4: 967-975Crossref PubMed Scopus (104) Google Scholar, 9Fraser G.L. Ramael S. Hoveyda H.R. Gheyle L. Combalbert J. The NK3 receptor antagonist ESN364 suppresses sex hormones in men and women.J Clin Endocrinol Metab. 2016; 101: 417-426Crossref PubMed Scopus (43) Google Scholar Therefore, NK3R antagonists may be another option for treating AD-associated itch particularly in adults. Tac2–/– mice were developed by using the CRISPR/Cas9 genome editing system. A targeting site within the exon 4 of mouse Tac2 was selected using the CHOPCHOP web design tool (https://chopchop.rc.fas.harvard.edu/). Two complementary oligonucleotides (5′-CACCGAGTGCTGAGCAAGGCTAGCG-3′ and 5′-AAACCGCTAGCCTTGCTCAGCACTC-3′) containing the guide sequence (underlined) and Bbs I ligation adaptors were synthesized, annealed, and ligated into the Bbs I–digested px330 vector for coexpression of single guide RNA and Cas9 protein. The pX330 vector (5 ng/μL in Dulbecco's PBS) was injected into the pronuclei of in vitro fertilized eggs of C57BL/6 mice in M2 medium (Sigma-Aldrich, St Louis, Mo). The injected zygotes were cultured in CZB medium at 37oC, 5% CO2 until 2-cell stage embryos develop. Then, 24 to 36 embryos were transferred into the oviducts of pseudopregnant ICR female mice. Genotype of offspring mice was identified by genomic PCR using primers (5′-CTCTCCCCTACAAGGACTCTGA-3′ and 5′-CCAATCTAATCTTCAGAACGCC-3′) followed by TA cloning of the products and sequencing. The offspring mice carrying desired mutation (Δ4 and Δ15) were crossed with C57BL/6 mice or Dock8–/– AND Tg mice.E1Yamamura K. Uruno T. Shiraishi A. Tanaka Y. Ushijima M. Nakahara T. et al.The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction.Nat Commun. 2017; 8: 13946Crossref PubMed Scopus (39) Google Scholar Dock8+/– AND Tg and Dock8–/– AND Tg mice with or without OSMR expression have been described previously.E1Yamamura K. Uruno T. Shiraishi A. Tanaka Y. Ushijima M. Nakahara T. et al.The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction.Nat Commun. 2017; 8: 13946Crossref PubMed Scopus (39) Google Scholar For generation of Il31–/– mice, a targeting vector was designed on the basis of pNT1.1 vector to insert the gene encoding–enhanced green fluorescent protein and a flippase recognition target–flanked neomycin-resistant cassette (neo) immediately after the initiation codon and was introduced into embryonic stem cells by electroporation. Correctly targeted embryonic stem clones were microinjected into C57BL/6 blastocysts, and the male chimeras obtained were crossed with C57BL/6 female mice. Heterozygous mutant mice were crossed with CAG-FLPe transgenic mice (RBRC01843) to remove neo, and neo-deleted mutant mice were backcrossed with C57BL/6 mice for more than 5 generations before crossing with Dock8+/– AND Tg and Dock8–/– AND Tg mice. All mice were maintained under specific-pathogen-free conditions in the animal facility of Kyushu University, and age- and sex-matched littermate mice were used as controls. Dock8+/– AND Tg and Dock8–/– AND Tg mice with or without Osmr, Il31, and Tac2 were analyzed at age 12 to 18 weeks. Tac2–/– mice, Tac2+/– mice, and WT mice were analyzed at age 7 to 10 weeks. All animal experiments were conducted according to relevant national and international guideline contained in the “Act on Welfare and Management of Animals” (Ministry of Environment of Japan) and the “Regulation of Laboratory Animals” (Kyushu University). The protocol of animal experiments was approved by the Committee of Ethics on Animal Experiments of Kyushu University. Ablation of spinal cord neurons expressing GRP receptor or Nppb receptor was performed by intrathecal (segment L3/4) injection of GRP-saporin or Nppb-saporin, respectively (2 μg in 5 μL each, Advanced Targeting Systems, San Diego, Calif). As a control, nontargeted saporin (designated Blank) was also injected. Mice were used for experiments 2 weeks after toxin injection. Before experiments, mice were put into an acrylic cage (11 × 14 × 20 cm) for at least 1 hour for acclimation. Then, the itch-inducing substances dissolved in sterile saline were injected intradermally into the shoulder of the mice at a volume of 50 μL, and their behaviors were video-recorded using HDR-CX390 (Sony, Tokyo, Japan). Playback of the video was used for determination of the total number of scratching bouts per the specified times. When mice scratch, they stretch their hind paw toward the itchy spot, lean the head toward the hind paw, rapidly move the paw several times, and then lower it back to the floor. A series of these movements was counted as 1 bout of scratching. The following itch-inducing substances were used for intradermal injection in this study: IL-31 (1 μg in 50 μL; Peprotech, Rocky Hill, NJ), SLIGRL-NH2 (100 μg in 50 μL; BACHEM, Bubendorf, Switzerland), chloroquine (100 μg in 50 μL; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and histamine (100 μg in 50 μL; FUJIFILM Wako Pure Chemical Corporation). In some experiments, the NK3R antagonist osanetant (Axon Medchem, Groningen, The Netherlands) or fezolinetant (Haoyuan ChemExpress, Shanghai, China) was administered intraperitoneally or orally at concentration of 5 mg/kg or 10 mg/kg, respectively, 45 minutes before intradermal injection of itch-inducing substances. Skin tissues were fixed in 4% (w/v) paraformaldehyde and embedded in paraffin blocks. Sections (3 μm thick) were stained with hematoxylin and eosin, and examined by light microscopy. For immunofluorescence analyses of the DRG, mice were euthanized with isoflurane and subjected to perfusion with 4% paraformaldehyde in PBS. The DRG tissues were collected, postfixed overnight at 4°C, and cryoprotected overnight in 30% sucrose-PBS. Then, tissue samples were embedded in OCT compound (Sakura Finetech, Tokyo, Japan) and frozen on dry ice. After cryostat sections (10 μm thick) were prepared, they were blocked with G-Block (GenoStaff, Tokyo, Japan; GB-01) for 30 minutes at room temperature and incubated overnight at 4°C with primary antibodies. The staining was detected and visualized with fluorophore-conjugated secondary antibodies (Thermo Fisher, Waltham, Mass). 4′-6-Diamidino-2-phenylindole, dihydrochloride (DOJINDO, 1:5000) was used for nucleus staining. All images were obtained with a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan). The following primary antibodies were used in this study: rabbit antibody for neurokinin B (Novus Biologicals, Centennial, Colo; NB300-201, 1:500), goat antibody for IL-31 receptor A (R&D Systems, Minneapolis, Minn; AF2107, 1:100), rabbit antibody for NK3R (Novus Biologicals; NB300-102, 1:50), and goat antibody for GRP (Santa Cruz Biotechnology, Santa Cruz, Calif; sc-7788, 1:100). Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan), and cRNA was amplified and labeled using a Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, Calif). The cRNA was then hybridized to a 44 K 60-mer oligomicroarray (Whole Mouse Genome oligo DNA Microarray Kit version 2.0; Agilent Technologies). The hybridized microarray slides were scanned using an Agilent scanner. The relative hybridization intensities and background hybridization values were calculated using Feature Extraction Software version 9.5.1.1 (Agilent Technologies). Raw signal intensities and flags for each probe were calculated from the hybridization intensities and spot information, according to the procedures recommended by Agilent Technologies. To identify up- or downregulated genes in experimental samples, we calculated Z scores and ratios from the normalized signal intensities of each probe (upregulated genes, Z score > 2.0 and ratio > 1.5-fold; downregulated genes, Z score < −2.0 and ratio < 0.66-fold). The microarray data that support the finding of this study are available in Gene Omnibus with the primary accession code GSE118986. Total RNA from each tissue was isolated using ISOGEN (Nippon Gene). After treatment with RNase-free DNase I (Life Technologies), RNA samples were reverse-transcribed with oligo (dT) primers (Life Technologies) and SuperScript III reverse transcriptase (Life Technologies) for amplification by PCR. The following primers were used for real-time PCR: for Tac2, 5′-TCGTGAAAGTGCTGAGCAAG-3′ and 5′-GTGTCTGGTTGGCTGTTCCT-3′; for Grp, 5′-CACGGTCCTGGCTAAGATGT-3′ and 5′-ATACAGGGACGGGGATTCAT-3′; for Il31ra, 5′-TCCTGAGGATCCCAGATGTC-3′ and 5′-GGAGCCACTCCACTATCCAA-3′; and for Hprt, 5′-CTGGTGAAAAGGACCTCTCG-3′ and 5′-TGAAGTACTCATTATAGTCAAGGGCA-3′. Real-time PCR was performed on CFX Connect Thermal Cycler (BIO-RAD, Hercules, Calif) using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif). The expressions of mouse target genes were normalized to expression of Hprt gene. CFX Manager software (version 3.1) supplied with the instrument was used for analyses. Data are expressed as relative values to that of a sample from Dock8+/–Osmr+/+Il31+/+ AND Tg mice (Fig 1, A) or a sample from Dock8+/– AND Tg mice (Fig E2). DRG neurons were prepared from the specified mouse lines and stimulated in vitro with IL-31 (10 μg/mL; Peprotech). The culture supernatants were recovered 30 minutes after stimulation, and the concentrations of neurokinin B and GRP were measured with ELISA kits (MyBiosource, San Diego, Calif; MBS744693 for neurokinin B and RayBiotech, Norcross, Ga; EIAM-GRP for GRP), according to the manufacturers' instructions. Statistical analyses were performed using GraphPad Prism7 (GraphPad Software, La Jolla, Calif). The data were initially tested with a Kolmogorov-Smirnov test for normal distribution. Parametric data were analyzed using a 2-tailed unpaired Student t test when 2 groups were compared. Nonparametric data were analyzed with a Mann-Whitney test when 2 groups were compared. Data are expressed as mean ± SD, and P values of less than .05 were considered significant . The expression data on Tac2 gene encoding neurokinin B are indicated in boldface.
Intestinal microfold cells (M cells) in Peyer's patches are a special subset of epithelial cells that initiate mucosal immune responses through uptake of luminal antigens. Although the cytokine receptor activator of nuclear factor-κB ligand (RANKL) expressed on mesenchymal cells triggers differentiation into M cells, other environmental cues remain unknown. Here, we show that the metastasis-promoting protein S100A4 is required for development of mature M cells. S100A4-producing cells are a heterogenous cell population including lysozyme-expressing dendritic cells and group 3 innate lymphoid cells. We found that in the absence of DOCK8, a Cdc42 activator critical for interstitial leukocyte migration, S100A4-producing cells are reduced in the subepithelial dome, resulting in a maturation defect of M cells. While S100A4 promotes differentiation into mature M cells in organoid culture, genetic inactivation of S100a4 prevents the development of mature M cells in mice. Thus, S100A4 is a key environmental cue that regulates M cell differentiation in collaboration with RANKL.
we analysed B cells (immature, naı ¨ve, marginal-zone-like, IgM only and switched memories B cells) in non-treated patients presenting with a first demyelinating episode (CIS) and relapsing-remitting (RR)-MS.Immature B cells appeared to be dramatically reduced in the circulating compartment of both patients groups ( 5.5-and 3.7-fold respectively).We postulated a pathogenic tissue-specific recruitment of these cells.Thus, we analysed the a4, b1 and b7 integrins expression by different B-cell subsets.In both basal and pathological conditions, immature B cells expressed highly a4 and b7.Interestingly, immature B cells from CIS and RR-MS groups up-regulated b1.Patients treated with an anti-4 (Natalizumab), which block leukocytes recruitment to the CNS, showed normal immature B-cell proportions.Therefore, immature B cells might be involved in disease development by their ability to cross the inflamed blood-brain-barrier.Immature B cells are not primed; therefore, antigen-dependent activation of B cells might also occur inside the CNS.
Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3β-hydroxy-5-cholenoic acid (3β-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3β-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3β-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3β-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion.
