Gain-of-function Pyrin mutations induce NLRP3 protein-independent interleukin-1ß activation and severe autoinflammation in mice.

Missense mutations in the C-terminal B30.2 domain of pyrin cause familial Mediterranean fever (FMF), the most common Mendelian autoinflammatory disease. However, it remains controversial as to whether FMF is due to the loss of an inhibitor of inflammation or to the activity of a proinflammatory molecule. We generated both pyrin-deficient mice and "knockin" mice harboring mutant human B30.2 domains. Homozygous knockin, but not pyrin-deficient, mice exhibited spontaneous bone marrow-dependent inflammation similar to but more severe than human FMF. Caspase-1 was constitutively activated in knockin macrophages and active IL-1ß was secreted when stimulated with lipopolysaccharide alone, which is also observed in FMF patients. The inflammatory phenotype of knockin mice was completely ablated by crossing with IL-1 receptor-deficient or adaptor molecule ASC-deficient mice, but not NLRP3-deficient mice. Thus, our data provide evidence for an ASC-dependent NLRP3-independent inflammasome in which gain-of-function pyrin mutations cause autoinflammatory disease.

Histone deacetylase 6 inhibition impairs effector CD8 T-cell functions during skin inflammation.

BACKGROUND: Broad-spectrum histone deacetylase (HDAC) inhibitors are useful in the treatment of allergic and autoimmune diseases and malignancy. However, use of more specific HDAC inhibitors might limit the toxicities caused by HDAC inhibition. HDAC6, a member of the HDAC family, is highly expressed on CD8 T cells and has been shown to regulate immune responses through interactions between T cells and antigen-presenting cells. However, the mechanism by which HDAC6 inhibition affects the activation and functions of CD8 T cells is unclear. OBJECTIVES: We investigated the role or roles of HDAC6 in CD8 T-cell activation and functions during skin inflammation in vitro and in vivo and examined the mechanism by which HDAC6 inhibition modifies T-cell receptor signaling in vitro. METHODS: We assessed the clinical and biological effects of ACY-1215, an HDAC6-specific inhibitor, by using murine CD8 T cell-related skin disease models, including contact hypersensitivity (CHS) and experimental graft-versus-host disease (GVHD)-like disease. RESULTS: ACY-1215, an HDAC6 inhibitor, prevented the development of CHS and GVHD-like disease in vivo by modulating CD8 T-cell activation and functions; abrogated the induction of effector T cells from naive CD8 T cells by means of anti-CD3/CD28 antibody- or antigen-specific stimulation in vitro; and enhanced the binding of acetylated heat shock protein 90 to lymphocyte-specific protein tyrosine kinase in vitro, disrupting lymphocyte-specific protein tyrosine kinase phosphorylation and leading to impairment of the mitogen-activated protein kinase pathway. CONCLUSION: HDAC6, a key modifier of T-cell receptor signaling, might represent a novel target for the treatment of CD8 T cell-related skin diseases, including CHS and GVHD.

Interferon regulatory factor 8 integrates T-cell receptor and cytokine-signaling pathways and drives effector differentiation of CD8 T cells.

We recently demonstrated that differentiation of cytotoxic T cells requires cooperation between T-cell receptor (TCR)/costimulation and γc-cytokines. Here we demonstrate that the transcription factor IFN regulatory factor 8 (IRF8) is expressed in CD8 T cells by the combination of these two signals. More importantly, depletion of IRF8 in these cells abrogated the differentiation of naive CD8 T cells into effector cells in an experimental graft-vs.-host disease mouse model. We also show that IRF8 seems to not operate upstream of other critical factors such as T-bet and eomesodermin, which have been implicated in effector maturation. Collectively, our work shows that IRF8 integrates the TCR/costimulation and γc-cytokine-signaling pathways and mediates the transition of naive CD8 T cells to effector cells, thus identifying IRF8 as one of the molecular regulators of CD8 T-cell differentiation.

Programmed cell death 1 (PD-1) regulates the effector function of CD8 T cells via PD-L1 expressed on target keratinocytes.

Programmed cell death 1 (PD-1) is an inhibitory molecule expressed by activated T cells. Its ligands (PD-L1 and -L2; PD-Ls) are expressed not only by a variety of leukocytes but also by stromal cells. To assess the role of PD-1 in CD8 T cell-mediated diseases, we used PD-1-knockout (KO) OVA-specific T cell-receptor transgenic (Tg) CD8 T cells (OT-I cells) in a murine model of mucocutaneous graft-versus-host disease (GVHD). We found that mice expressing OVA on epidermal keratinocytes (K14-mOVA mice) developed markedly enhanced GVHD-like disease after transfer of PD-1-KO OT-I cells as compared to those mice transferred with wild-type OT-I cells. In addition, K14-mOVA × OT-I double Tg (DTg) mice do not develop GVHD-like disease after adoptive transfer of OT-I cells, while transfer of PD-1-KO OT-I cells caused GVHD-like disease in a Fas/Fas-L independent manner. These results suggest that PD-1/PD-Ls-interactions have stronger inhibitory effects on pathogenic CD8 T cells than does Fas/Fas-L-interactions. Keratinocytes from K14-mOVA mice with GVHD-like skin lesions express PD-L1, while those from mice without the disease do not. These findings reflect the fact that primary keratinocytes express PD-L1 when stimulated by interferon-γ in vitro. When co-cultured with K14-mOVA keratinocytes for 2 days, PD-1-KO OT-I cells exhibited enhanced proliferation and activation compared to wild-type OT-I cells. In addition, knockdown of 50% PD-L1 expression on the keratinocytes with transfection of PD-L1-siRNA enhanced OT-I cell proliferation. In aggregate, our data strongly suggest that PD-L1, expressed on activated target keratinocytes presenting autoantigens, regulates autoaggressive CD8 T cells, and inhibits the development of mucocutaneous autoimmune diseases.

Self-peptides prolong survival in murine autoimmunity via reduced IL-2/IL-7-mediated STAT5 signaling, CD8 coreceptor, and V alpha 2 down-regulation.

Although the pathogenic role of B cells and CD4 T cells has been studied extensively, less is known about the role of CD8 T cells in autoimmunity and self-tolerance. To evaluate the role of CD8 T cells in autoimmunity and its modulation using self-peptides, we used mice expressing soluble OVA (sOVA) under control of the keratin-14 promoter. Spontaneous autoimmunity occurred when sOVA mice were crossed with OT-I mice, whose CD8 T cells carry a Valpha2/Vbeta5-transgenic TCR with specificity for the OVA(257-264) peptide. Eighty-three percent of OVA/OT-I mice died during the first 2 wk of life due to multiple organ inflammation. In contrast, preventive or therapeutic OVA(257-264) peptide injections induced a dose-dependent increase in survival. Healthy survivors exhibited reductions in peripheral CD8 T cells, CD8 coreceptor, and Valpha2 expression. Furthermore, CD8 T cells from healthy mice were anergic and could not be activated by exogenous IL-2. A block in IL-2/IL-7 signaling via the STAT5 pathway provided the basis for low surface expression of the CD8 coreceptor and failure of IL-2 to break CD8 T cell anergy. Thus, the soluble TCR ligand triggered multiple tolerance mechanisms in these sOVA/OT-I mice, making this treatment approach a potential paradigm for modulating human autoimmune diseases.

The use of mouse models to better understand mechanisms of autoimmunity and tolerance.

A major emphasis of our studies has been on developing a better understanding of how and why the skin serves as a target for immune reactions as well as how the skin evades becoming a target for destruction. For these studies we developed transgenic mice that express a membrane-tethered form of a model self antigen, chicken ovalbumin (mOVA), under the control of a keratin 14 (K14) promoter. K14-mOVA transgenic mice that express OVA mRNA and protein in the epithelia have been assessed for their immune responsiveness to OVA and are being used as targets for T cells obtained from OT-1 transgenic mice whose CD8+ T cells carry a Vα2/Vß5-transgenic T cell receptor with specificity for the OVA(257-264)-peptides (OVAp) in association with class I MHC antigens. Some of the K14-mOVA transgenic mice develop a graft-versus-host-like disease (GvHD) when the OT-1 cells are injected while others appear to be tolerant to the OT-1 cells. We found that γc cytokines, especially IL-15, determine whether autoimmunity or tolerance ensues in K14-mOVA Tg mice. We also developed transgenic mice that express soluble OVA under the control of a K14 promoter (K14-sOVA) that die within 5-8 days after adoptive transfer of OT-1 cells and identified these mice as a model for more acute GvHD-like reactions. Spontaneous autoimmunity occurs when these K14-sOVA mice are crossed with the OT-I mice. In contrast, we found that preventive or therapeutic OVAp injections induced a dose-dependent increase in survival. In this review the characterization of 5 strains of K14-OVATg mice and underlying mechanisms involved in autoimmune reactions in these Tg mice are discussed. We also describe a strategy to break tolerance and describe how the autoimmunity can be obviated using OVAp. Finally, a historical overview of using transgenic mice to assess the mechanisms of tolerance is also provided.

The current status of investigative dermatology II.

Major advances in skin biology and skin diseases are heralding new and more specific forms of treatment that are based on better characterization of pathological mechanisms involved in the individual diseases. The advances that we have seen are being made by dermatologists, skin biologists, and others who have come to appreciate the skin as an organ that is reflective of many important systemic mechanisms. In this commentary, I identify some of what I feel are the most important advances that will be the basis for many future studies.

IL-15 serves as a costimulator in determining the activity of autoreactive CD8 T cells in an experimental mouse model of graft-versus-host-like disease.

To elucidate the mechanisms controlling peripheral tolerance, we established two transgenic (Tg) mouse strains expressing different levels of membrane-bound OVA (mOVA) as a skin-associated self-Ag. When we transferred autoreactive TCR-Tg CD8 T cells (OT-I cells), keratin 14 (K14)-mOVA(high) Tg mice developed autoreactive skin disease (graft-vs-host disease (GVHD)-like skin lesions) while K14-mOVA(low) Tg mice did not. OT-I cells in K14-mOVA(high) Tg mice were fully activated with full development of effector function. In contrast, OT-I cells in K14-mOVA(low) Tg mice proliferated but did not gain effector function. Exogenous IL-15 altered the functional status of OT-I cells and concomitantly induced disease in K14-mOVA(low) Tg mice. Conversely, neutralization of endogenous IL-15 activity in K14-mOVA(high) Tg mice attenuated GVHD-like skin lesions induced by OT-I cell transfer. Futhermore, K14-mOVA(high) Tg mice on IL-15 knockout or IL-15Ralpha knockout backgrounds did not develop skin lesions after adoptive transfer of OT-I cells. These results identify IL-15 as an indispensable costimulator that can determine the functional fate of autoreactive CD8 T cells and whether immunity or tolerance ensues, and they suggest that inhibition of IL-15 function may be efficacious in blocking expression of autoimmunity where a breach in peripheral tolerance is suspected.

Response to: "Rescuing the NIH before it is too late".

We, the directors of the 27 NIH institutes and centers, wanted to respond to the points made by Andrew Marks in his recent editorial. While we appreciate that the scientific community has concerns, the current initiatives and directions of the NIH have been developed through planning processes that reflect openness and continued constituency input, all aimed at assessing scientific opportunities and addressing public health needs.

Contact Hypersensitivity.

Contact hypersensitivity (CHS) is a simple in vivo assay of cell-mediated immune function in which exposure of epidermal and dermal cells to exogenous haptens results in a delayed-type hypersensitivity (DTH) reaction that can be measured and quantified. Epidermal Langerhans cells and dermal dendritic cells are the critical antigen-presenting cells in this reaction which initiate sensitization to haptens by presenting antigens to CD4- and CD8-bearing T lymphocytes which, in turn, secrete cytokines and recruit other cells to the site of the reaction. In the protocol described here, mice are shaved and the skin of their abdomens is exposed to a hapten. After 5 or 6 days (the afferent phase), the baseline ear thickness is measured prior to initiation of the efferent phase. Finally, the ear is treated epicutaneously with the hapten solution and ear thickness is measured in ∼24 hr. The magnitude of the ear swelling reaction after allergen treatment reflects the strength of the immune response.

Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells.

We describe a novel culture system for generating large numbers of murine skin-associated mast cells and distinguish their characteristics from bone marrow-derived cultured mast cells. Culture of day 16 fetal skin single cell suspensions in the presence of interleukin-3 and stem cell factor allowed expansion and maturation of mast cells in the presence of stromal cells. The average yield of mast cells after 2 wk was 7.3 million cells per fetus at a purity of 96%. These fetal skin-derived cultured mast cells increased their histamine content in a time-dependent manner to 3.6 pg per cell after 2 wk and 6.7 pg per cell after 4 wk. Phenotypic analyses revealed much greater expression of CD49b and CD81 and lesser expression of CD77 and CD102 on fetal skin-derived cultured mast cells as compared with bone marrow-derived cultured mast cells. These findings suggest a close similarity between fetal skin-derived cultured mast cells and freshly isolated cutaneous mast cells. Connective tissue mast cell characteristics of fetal skin-derived cultured mast cells were evidenced by: (1) their greater histamine content than bone marrow-derived cultured mast cells; (2) the presence of heparin; and (3) their degranulation in response to compound 48/80 and substance P. Importantly, fetal skin-derived cultured mast cells secreted greater amounts of interleukin-13 but much less MIP-1beta and interleukin-6 than bone marrow-derived cultured mast cells in response to ionomycin. Thus fetal skin-derived cultured mast cells have many characteristics distinct from bone marrow-derived cultured mast cells and can be used as a model of cutaneous mast cells to discern their functions.

Induction of GVHD-like skin disease by passively transferred CD8(+) T-cell receptor transgenic T cells into keratin 14-ovalbumin transgenic mice.

To understand the mechanisms involved in immunological tolerance to skin-associated antigens, we have developed transgenic (Tg) mice that express a model self-antigen, membrane-bound chicken ovalbumin (OVA), under the control of a keratin 14 (K14) promoter. K14-mOVA Tg mice express OVA mRNA in the epidermis, and appear normal. K14-mOVA Tg mice failed to mount T cell and delayed type hypersensitivity reactions to OVA, suggesting that the Tg mice were tolerant to OVA. Skin dendritic cells, including Langerhans cells, may contribute to the tolerance induction because migratory skin DC derived from K14-mOVA efficiently activated CD8(+) T cells from OVA-specific T-cell receptor (Va2/Vb5) Tg (OT-I) mice. OT-I cells expanded and accumulated in skin-draining lymph nodes after intravenous injected into K14-mOVA mice and exhibited activation markers. Graft-versus-host disease-like skin lesions appeared in K14-mOVA mice by day 7 after injection of OT-I cells. These studies demonstrate that K14-mOVA Tg mice are susceptible to an autoimmunelike skin disease induced by passively transferred naïve CD8(+) OVA T-cell receptor Tg T cells, and serve as a good model for understanding self-tolerance and for the investigation of the pathogenesis, treatment and potential prevention of cell-mediated autoimmune reactions in skin.

Fluorine-18 labeled mouse bone marrow-derived dendritic cells can be detected in vivo by high resolution projection imaging.

Immunization with ex vivo generated dendritic cells has become a focus for many clinical applications. The optimal site of injection and the migration pattern of these cells remain to be elucidated. We therefore developed a novel method for labeling mouse bone marrow-derived dendritic cells (BMDC) with the positron emitting radioisotope F-18 using N-succinimidyl-4-[F-18]fluorobenzoate, which covalently binds to the lysine residues of cell surface proteins. When we determined the stability of F-18 labeled BMDC, we found that at 4 h only 44+/-10% of the initial cell-bound activity was retained at 37 degrees C, whereas considerably more (91+/-3%) was retained at 4 degrees C. Labeled cells did not exhibit any significant alteration in cell viability or phenotype as determined by trypan blue exclusion and FACS analysis 24 h after radiolabeling. Furthermore, F-18-labeled BMDC stimulated allogeneic T cells in a mixed leukocyte reaction as potently as did sham-treated BMDC and migrated towards secondary lymphoid tissue chemokine (SLC) in a chemotaxis assay in vitro with the same efficiency as sham-treated BMDC. Migration of F-18-labeled BMDC was studied after footpad injection by (1) ex vivo counting of dissected tissues using a gamma counter and (2) in vivo by imaging mice with PiPET, a 2-mm resolution positron projection imager. After 4 h, the ratio between measured activity in draining vs. contralateral (D/C) lymph nodes (LN) was 166+/-96 (n=7) in the case of live cell injections, whereas if we injected heat-killed F-18-labeled BMDC or F-18-labeled macrophages the D/C ratios were 17+/-2 (n=2) and 14+/-4 (n=2), respectively. Injection of cell-free activity in the form of F-18-labeled 4-fluorobenzoic acid resulted in a D/C ratio of 7+/-2 (n=3), suggesting that the activity measured in the draining lymph node was associated with migrated F-18-labeled BMDC. When F-18-labeled live cells were injected into the footpad, 0.18+/-0.04% (n=7) of footpad activity was found in the draining LN within 4 h, whereas none was found in the contralateral LN. Quantitative assessment of cell migration by PET projection imaging of mice confirmed the ex-vivo counting results. These studies indicate that PET imaging offers a new approach for in vivo studies of dendritic cell biodistribution and migration.

CXCR3-mediated skin homing of autoreactive CD8 T cells is a key determinant in murine graft-versus-host disease.

The pathomechanisms underlying the development of cutaneous graft-versus-host disease (GVHD) are incompletely defined. We previously reported that K14-mOVA mice expressing membrane ovalbumin (mOVA), driven by the keratin 14 (K14) promoter, developed GVHD-like mucocutaneous disease and weight loss following transfer of OVA-specific, CD8(+) OT-I T cells. In this study, we demonstrate that early in the course of disease, the kinetics of epidermal expression of C-X-C motif chemokine ligand 9 (CXCL9) and CXCL10, interferon-γ-inducible chemokines that bind the C-X-C motif chemokine receptor 3 (CXCR3) receptor, coincides with CXCR3 expression by OT-I cells in secondary lymphoid organs. Recruitment of OT-I cells into the skin began by day 5 with progressive accumulation through day 13 post transfer. Transfer of CXCR3-knockout (CXCR3KO) OT-I cells into K14-mOVA mice resulted in strikingly attenuated skin disease. CXCR3KO OT-I cells retained full activation and effector function, but preferentially accumulated in the spleen, in contrast to wild-type (WT) OT-I cells that accumulated in skin-draining lymph nodes. Moreover, OT-I cells accounted for a significantly reduced percentage of skin-infiltrating lymphocytes in mice receiving CXCR3KO OT-I cells compared with WT OT-I cells. These results identify CXCR3 as being critical to the skin-selective effector T-cell recruitment underlying autoreactive GVHD, suggesting CXCR3 as a potential target in the treatment of GVHD and related skin diseases.

Reversal of CD8 T-cell-mediated mucocutaneous graft-versus-host-like disease by the JAK inhibitor tofacitinib.

The utility of allogeneic hematopoietic stem cell transplantation is limited by graft-versus-host disease (GVHD), a significant cause of morbidity and mortality. Patients with GVHD exhibit cutaneous manifestations with histological features of interface dermatitis followed by scleroderma-like changes. JAK inhibitors represent a class of immunomodulatory drugs that inhibit signaling by multiple cytokines. Herein we report the effects of tofacitinib in a murine model of GVHD. Oral administration of tofacitinib prevented GVHD-like disease manifested by weight loss and mucocutaneous lesions. More importantly, tofacitinib was also effective in reversing established disease. Tofacitinib diminished the expansion and activation of murine CD8 T cells in this model, and had similar effects on IL-2-stimulated human CD8 T cells. Tofacitinib also inhibited the expression of IFN-γ-inducible chemoattractants by keratinocytes, and IFN-γ-inducible cell death of keratinocytes. Tofacitinib may be an effective drug for treatment against CD8 T-cell-mediated mucocutaneous diseases in patients with GVHD.

Identification of CD3+CD4-CD8- T cells as potential regulatory cells in an experimental murine model of graft-versus-host skin disease (GVHD).

We have developed K14-mOVA transgenic (Tg) mice that express membrane-associated ovalbumin (mOVA) under the control of a K14 promoter, as well as double Tg mice, by crossing them with OT-I mice that have a TCR recognizing the OVA peptide. When injected with CD8(+) OT-I cells, K14-mOVA Tg mice develop graft-versus-host disease (GVHD), whereas double Tg mice are protected. This suggests that, in double Tg mice, regulatory mechanisms may prevent infused OT-I cells from inducing GVHD. We demonstrated that, after adoptive transfer, TCRαß(+)CD3(+)CD4(-)CD8(-)NK1.1(-) double-negative (DN) T cells are increased in the peripheral lymphoid organs and skin of double Tg mice and exhibit a Vα2(+)Vß5(+)TCR that has the same TCR specificity as OT-I cells. These DN T cells isolated from tolerant double Tg mice proliferated in response to OVA peptide and produced IFN-γ in the presence of IL-2. These cells could also suppress the proliferation of OT-I cells and were able to specifically kill activated OT-I cells through Fas/Fas ligand interaction. These findings suggest that DN T cells that accumulate in double Tg mice have regulatory functions and may have a role in the maintenance of peripheral tolerance in vivo.