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Central T cell tolerance operates intrathymically in order to eliminate self-reactive T cells and depends on the cooperation between thymic epithelial cells and hematopoietic myeloid cells.Lymphocytes with low to intermediate affinity to self antigens, or those receiving insufficient antigenic stimulation, can escape central tolerance and populate secondary lymphoid organs consequently, autoreactive T cells require control by additional mechanisms of peripheral tolerance.Foxp3+ regulatory T cells (Tregs) critically provide one of these mechanisms by suppressing autoreactive CD4+ T cells through dominant peripheral tolerance. The importance of Tregs is illustrated by rare mutations of the human Foxp3 gene, resulting in the life-threatening Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome [3]. Affected newborn boys suffer from severe diarrhea, type-1 diabetes, skin inflammation, haemolytic anemia and other autoimmune manifestations, typically causing death within the first years of life if left untreated [4,5]. Immunosuppressive therapy with glucocorticoids, cyclosporine, tacrolimus or rapamycin can only partially suppress autoimmunity, and the combination of two or several of these drugs is often necessary [4]. Drug type and dosing may require frequent modulation due to the lack of a sufficient clinical response or because of side effects. So far, bone marrow transplantation is the only cure for the IPEX syndrome [4]. A defective balance between Foxp3+ Tregs and autoreactive T cells has also been implied in the pathogenesis of various other autoimmune diseases [6]. The function of Foxp3 is highly conserved among mammal species. Foxp3 mutant scurfy mice develop a lethal autoimmune disease which closely resembles the IPEX syndrome [3,7]. Moreover, the ablation of Foxp3+ Tregs in genetically modified mice induces a scurfy-like disease and unleashes immunity against self/ tumour antigens [8e10]. This is caused by the specific ablation of Foxp3+ Tregs, yet a minor population of CD8+Foxp3+ T cells also exists in mice [11e14]. Consequently, these mouse models are valuable for studying novel immunosuppressive therapies with relevance to the human IPEX syndrome and other autoimmune diseases. Besides dominant tolerance mediated by Foxp3+ Tregs, intrinsic/recessive mechanisms of peripheral tolerance may also contribute to the control of autoreactive cells, e.g. through induction of clonal anergy or clonal deletion [2]. One important mechanism counter-regulating recessive tolerance is co-stimulation. The co-stimulatory TNF receptor family members OX40 and CD30 belong to the group of TNF receptor-associated factor (TRAF) binding receptors that recruit TRAF proteins and induce NF-kB and stress kinase signaling [15]. OX40 and CD30 signals promote the differentiation and survival of CD4+ T cells which are known to initiate autoimmunity in sf mice [16,17]. Indeed, OX40 and CD30 have been shown to play an essential role in the disease pathogenesis of sf mice, and OX40 was implicated in various other autoimmune conditions [18,19]. Thus, a complex interplay of multiple layers shapes self tolerance [20]. Novel therapeutic options promoting tolerance e particularly in scenarios of impaired Foxp3+ Treg function e are urgently needed. Non-depleting monoclonal anti-CD4 antibodies including the clone YTS177.9 were shown to suppress or even prevent CD4+ T cell-mediated pathologies via dominant infectious tolerance [21e 24]. Surprisingly, the exact mode of action and the cellular targets involved have remained incompletely resolved. Because anti-CD4 antibodies are currently entering various clinical trials [25], insights into their mechanism of action are of strong relevance. Interestingly, Foxp3+ Tregs have recently been suggested to be the central underlying mechanism of anti-CD4-induced tolerance in both mice and humans [25e27]. By using Foxp3DTR knock-in mice and natural scurfy (sf) mutant mice, either conditionally or constitutively ablated of functional Foxp3+ Tregs [7,10], we discovered a previously unknown mechanism of anti-CD4-induced tolerance that is Foxp3+ Treg-independent but directly acts on CD4+Foxp3 autoreactive T cells. The characteristics of Foxp3-independent tolerance are distinct from classical dominant infectious tolerance and are powerful enough to inhibit autoimmunity. Our results highlight recessive peripheral tolerance as a powerful pathway to control clinically relevant autoimmune diseases.

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Central tolerance1. Introductioneliminate self-reactive T cells Lymphocytes with low affinity to self antigensPeripheral tolerance(thymic epithelial cells and hematopoietic myeloid cells)Foxp3+ TregsClonal anergy or clonal deletionCentral T cell tolerance operates intrathymically in order to eliminate self-reactive T cells and depends on the cooperation between thymic epithelial cells and hematopoietic myeloid cells.Lymphocytes with low to intermediate affinity to self antigens, or those receiving insufficient antigenic stimulation, can escape central tolerance and populate secondary lymphoid organs consequently, autoreactive T cells require control by additional mechanisms of peripheral tolerance.

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IPEXOX40 and CD30 Disease pathogenesis Autoimmune conditionsPlays an essential role in the development and function of D4+CD25+ Regulatory T cells.

FOXP3 act as a transcriptional repressor of cytokine promotors.

The function of Foxp3 is highly conserved among mammal species.

Foxp3 mutant scurfy miceImmunosuppressive therapy Bone marrow transplantation For studying novel immunosuppressive therapies with relevance to the human IPEX syndrome and other autoimmune diseasesone mechanism counter-regulating recessive tolerance is co-stimulation.Foxp3+ regulatory T cells (Tregs) critically provide one of these mechanisms by suppressing autoreactive CD4+ T cells through dominant peripheral tolerance. Affected newborn boys suffer from severe diarrhea, type-1 diabetes, skin inflammation, haemolytic anemia and other autoimmune manifestations, typically causing death within the first years of life if left untreated. Immunosuppressive therapy with glucocorticoids, cyclosporine, tacrolimus or rapamycin can only partially suppress autoimmunity, and the combination of two or several of these drugs is often necessary. Bone marrow transplantation is the only cure for the IPEX syndrome

One important mechanism counter-regulating recessive tolerance is co-stimulation. OX40 and CD30 have been shown to play an essential role in the disease pathogenesis of sf mice, and OX40 was implicated in various other autoimmune conditions.

------------------------Key features and differences between nTREGcells and iTREGcells. Both subsets express the transcription factor FOXP3 and a variety of other cell surface factors, but they are strikingly different in origin, induction, TCR repertoire and in antigen specificity. Abbreviations: iTREGcell, induced regulatory T cell; nTREGcell, natural regulatory T cell; TREGcell, regulatory T cell; TCR, T-cell receptor3

OX40 activates PI3k/PKB, NF-B1, and NFAT pathways.Suppressive events central memory cells (Tcm) effector memory cells (Tem)These signaling pathways include upregulating genes that control T cell division and survival, and promoting transcription of cytokine genes, as well as expression of cytokine receptors. CD30/TNFRSF8OX40Is a cell membrane protein TNF-R family Expressed by activated T and B cells. Interact with TRAF2 and TRAF5 (NF-kappa-B)Play a role in the regulation of cellular growth and transformation of activated lymphoblasts.

Three models for effector and memory T cell generation are depicted. A) Upon activation, nave CD4 T cells develop into either central memory cells (Tcm) or effector memory cells (Tem). The Tcm and Tem fate decision occurs early during priming, perhaps determined by antigen access and/or dose, before OX40 is ligated on recently activated naive T cells. OX40 signals promote clonal expansion and survival of Tem precursors that differentiate in a step-wise manner into true primary effector cells and then into resting Tem after antigen is cleared. Tcm precursors expand and enter into the central memory pool in an OX40 independent fashion. OX40 is critical to CD4 T cell memory because in general Tem predominate. B) Following activation of nave CD8 T cells, they expand and develop into effector cells that may contain both Tcm and Tem precursors. In this context, OX40 signals promote clonal expansion and support survival of most effector CD8 T cells that enter into the memory pool regardless of their lineage potential, hence OX40 is critical to CD8 T cell memory generation. This situation might apply to responses against tumors, auto or allo-antigen, or select infectious agents. C) Activation of nave CD8 T cells under other inflammatory conditions results in the daughter cells developing into SLEC (short-lived effector cells) and MPEC (memory precursor effector cells). During priming, OX40 promotes expansion and survival of MPEC and further inhibits conversion of MPEC into SLEC. At later times, most SLEC die, but multipotent MPEC survive and give rise to transitional Tem that progressively differentiate into long-lived Tcm. Furthermore, OX40 signals to MPEC provided during the primary effector phase impart signals to maintain the later self-renewing capacity of Tcm in the absence of antigen. This situation might apply to responses against select infectious agents, and again OX40 becomes critical for memory generation because of the control of MPEC.-------------------------OX40L binding to OX40 results in recruitment of TRAF2 and the formation of a signaling complex containing IKK and IKK, as well as PI3k and PKB (Akt). This complex, upon translocation into lipid rafts, is sufficient to activate NF-B1 in an antigen independent manner, via phosphorylation and degradation of IB, leading to entry of p50 and RelA into the nucleus. In contrast, OX40 ligation does not effectively lead to phosphorylation of PKB, but OX40 co-operates with TCR signals brought about by antigen recognition, to augment PKB activation, possibly reflective of a requirement to recruit and activate PDK1. OX40 also synergizes with TCR signals to augment intracellular Ca2+, through an unknown mechanism, that leads to enhanced nuclear import of NFAT. The downstream targets of these signaling pathways include upregulating genes that control T cell division and survival, and promoting transcription of cytokine genes, as well as expression of cytokine receptors. Suppressive events brought about by OX40 signaling include downregulation of CTLA-4 and Foxp3.

--------------------------OX40 (CD134) and its binding partner, OX40L (CD252), are members of the tumor necrosis factor receptor/tumor necrosis factor superfamily and are expressed on activated CD4(+) and CD8(+) T cells as well as on a number of other lymphoid and non-lymphoid cells. Costimulatory signals from OX40 to a conventional T cell promote division and survival, augmenting the clonal expansion of effector and memory populations as they are being generated to antigen. OX40 additionally suppresses the differentiation and activity of T-regulatory cells, further amplifying this process. OX40 and OX40L also regulate cytokine production from T cells, antigen-presenting cells, natural killer cells, and natural killer T cells, and modulate cytokine receptor signaling. In line with these important modulatory functions, OX40-OX40L interactions have been found to play a central role in the development of multiple inflammatory and autoimmune diseases, making them attractive candidates for intervention in the clinic. Conversely, stimulating OX40 has shown it to be a candidate for therapeutic immunization strategies for cancer and infectious disease. This review provides a broad overview of the biology of OX40 including the intracellular signals from OX40 that impact many aspects of immune function and have promoted OX40 as one of the most prominent costimulatory molecules known to control T cells.---------------------------------Human CD30, also known as TNFRSF8, is a cell membrane protein of the tumor necrosis factor receptor family and tumor marker. TNFRSF-8 is expressed by activated, but not by resting, T and B cells. Also, CD30 is expressed on classical Hodgkin Lymphoma cells together with CD15. CD30 is the receptor for TNFSF8/CD30L. CD30 can interact with TRAF2 and TRAF5, and mediate the signal transduction that leads to the activation of NF-kappa-B. TNFRSF8 may play a role in the regulation of cellular growth and transformation of activated lymphoblasts. TNFRSF8 is a positive regulator of apoptosis, and also has been shown to limit the proliferative potential of autoreactive CD8 effector T cells and protect the body against autoimmunity.

CD30 signals through TRAFs: upon stimulation by CD30L, CD30 recruits TRAF2 via interaction with its TRAF binding motif, resulting in TRAF2 degradation. TNFR1, on the other hand, signals through TRADD leading to activation of caspases and ultimately apoptosis or activation of the classical NF-B pathway and ultimately survival and proliferation. Signaling through CD30 depletes intracellular TRAF2 diverting signaling through TNFR1 to apoptosis [Miret al.2000]. Not all TRAF2 undergoes degradation upon CD30 stimulation. In certain ALCL cells, TRAF2 translocates to a detergent-insoluble cellular compartment, and this translocation has been associated with NF-B pathway activation [Wrightet al.2007]. Activation of the classical NF-B pathway involves activation of the IKK complex and proteasomal degradation of the IB proteins. The proteasomal degradation of TRAF2, TRAF3, and c-IAP-1/2 which hold NIK upon recruitment by a subset of TNF receptor superfamily proteins, notably CD40, leads to the activation of the alternative NF-B pathway via liberation and stabilization of NIK [Staudt, 2010]. Activation of the alternative NF-B pathway involves activation of IKK1 and proteolytic processing of the p100 precursor protein to the p52 subunit of NF- B. (Illustration courtesy of Alessandro Baliani. Copyright 2012.) CD30, cluster of differentiation 30; CD40, cluster of differentiation 40; CD30L, CD30 ligand; c-IAP1/2, cellular inhibitor of apoptosis protein; IB, inhibitor of kappa B; IKK1/2, IB kinase; NF-B, nuclear factor kappa B; NIK, NF-B inducible kinase; P, phosphate; p50, p52, p65 (RelA), RelB, p50, p52, p65 (RelA), RelB subunits of NF-B; p100, p52 precursor subunit of NF-B; TNF, tumor necrosis factor; TNFR1, TNF receptor 1; TRADD, TNF receptor-associated death domain; TRAF2 and TRAF3, TNF receptor-associated factor 2 and 3, respectively; Ub, ubiquitin.

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X-linked recessive inheritance Lethality at 21-25 days Wasting syndrome Exfoliative dermatitis Small, thickened earsWild typeScurfy

B6.Cg-Foxp3sf/J (sf)C57BL/62. Material and methodsB6.Cg-Foxp3sf/J (sf)

sf Thy1.1 mice

Rag1/ mice

Foxp3DTR mice Severe autoimmune disorder Hepatosplenomegaly Enlarged lymph nodes Multi-organ lymphocytic infiltrates Elevated cytokines (GM-CSF, IL-2, -4, -5,-6,-7,-10, IFN-g, TNFa)Wild typeScurfyScurfy mice have a deletion in the forkhead domain of Foxp3, fail to develop thymic-derived Foxp3+regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation.

RAG1 and RAG2 are two proteins necessary for immunoglobulin and T-cell receptor gene recombination. RAG stands for recombination-activating gene. RAG1 and RAG2 work closely together in receptor gene recombination. Therefore, immunologists often refer to the two proteins together as RAG protein. RAG1 and RAG2 make up the lymphoid-specific parts of V(D)J recombinase, a complex of enzymes that work together to join gene segments of B-cell and T-cell receptor genes (Janewayet al., 2005). All species that carry out V(D)J recombination have RAG1 and RAG2, and the expression levels of RAG1 and RAG2 correlate directly with V(D)J recombination activity--------------------------------------B6.Cg-Foxp3sf/JScurfy mice have a deletion in the forkhead domain of Foxp3, fail to develop thymic-derived Foxp3+regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation.

Scurfy mice have defective T cell tolerance leading to an X-linked lymphoproliferative disease that parallels the X-linked autoimmunity-allergic disregulation syndrome (XLAAD) in humans. Hemizygous males are characterized by runting, scaly, crusty skin on the eyelids, ears and tails, dermal thickening, squinted eyes, cachexia, reddening and swelling of the genital papilla, and small testicles that are retained in the abdominal cavity. Homozygous scurfy females develop the same disease phenotype seen in hemizygous males, but they have a normal reproductive tract.

DescriptionScurfy mice develop an X-linked lymphoproliferative disease resulting from defective T cell tolerance. Phenotypes associated with these mice include runting, scaly, crusty skin on the eyelids, ears and tails, dermal thickening, squinted eyes, cachexia, reddening and swelling of the genital papilla, and small testicles that are retained in the abdominal cavity. This disorder, which parallels X-linked autoimmunity-allergic disregulation syndrome (XLAAD) in humans, results in Coombs' test-positive anemia, hypergammaglobulinemia, a small, thin thymus, and lymphohistiocytic proliferation in the skin and lymphoid organs, with splenomegaly, lymphadenomegaly, and hepatomegaly.Foxp3sf/Y males generally die by 16-25 days of age. Transgenic expression ofFoxp3prevents scurfy disease inFoxp3sf/Y mice.Neonatal thymectomy of scurfy males ameliorates disease and increases lifespan; athymic nude (Foxn1nu/Foxn1nu)Foxp3sf/Y mice do not develop scurfy. WhileCd4+peripheral T cells from scurfy mice can transfer the scurfy disease phenotype to wild type, histocompatibleFoxn1nu/Foxn1nuorPrkdcscid/Prkdcscidhosts, bone marrow transplantation from scurfy homozygotes fails to transfer disease. Also, neither neonatal inoculation with wild type bone marrow, nor thymic lobe transplants from wild type donors into carrier males prevents disease. Northern blot analysis of skin, lymph nodes and spleen revealed over-expression ofIl2, Il4, Il5, Il10, Il6, IFNg,andTNFa; over-expression of these last three is especially high. PeripheralCd4+T cells from scurfy mice are hyper-responsive to antigen, have an activated phenotype (Cd44+,Cd69+,Cd25+,Cd80+,Cd86+), a decreased requirement forCd28co-stimulation, and a decreased sensitivity to tyrosine kinase inhibitors and cyclosporin A. Prenatal or neonatal injection with anti-Cd4antibodies can delay the onset of disease, as can the targeted disruption ofCd4.Cd8+cells do not transfer disease, and targeted disruption of B2m does not alter disease onset. Activation of peripheral T cells is necessary to initiate the scurfy pathology;Foxp3sf/Y mice carrying a transgene for an ovalbumin-specific TCR and a targeted mutation ofRag1fail to develop the scurfy disease phenotype until challenged with ovalbumin.Foxp3sfhomozygous females can not be generated through traditional breeding because carrier males die by 25 days of age. By breeding nudeFoxp3sf/Y males withFoxp3sf/+ females, however, homozygous scurfy females can be generated that are heterozygous for the recessiveFoxn1numutation. These homozygous scurfy females develop the same disease phenotype seen in hemizygous males, but they have a normal reproductive tract.DevelopmentThe scurfy mutation arose spontaneously at the Oak Ridge National Laboratory in 1949 in the partially inbred MR stock. This strain was a multiple recessive stock of seven mutations, primarily coat color mutations. Scurfy was maintained either by backcross onto 129/Rl-p Tyrch/p Tyrcor by breeding heterozygous females to (C3H/Rl x 101/Rl)F1 or (101/Rl x C3H/Rl)F1 males at each generation to keep it on a non-inbred background. Means et al. obtained scurfy mice from Yvonne Boyd at Harwell where they were maintained by breeding to (C3H/Rl x 101/Rl)F1. Means et al. backcrossedFoxp3sf/+ females to C57BL/6NTac males. In 2001 The Jackson Laboratory received N8 mice and backcrossed to C57BL/6J. (Russell et al., 1959; Godfrey et al., 1991; Means et al., 2000.)

---------------------------------------B6.129S7-Rag1tm1Mom/JMice homozygous for theRag1tm1Mommutation produce no mature T cells or B cells. Their phenotype can be described as a "non-leaky" immune deficiency.DescriptionMice homozygous for theRag1tm1Mommutation produce no mature T cells or B cells. WhereasPrkdcscidhomozygotes produce some B cells and IgM (i.e., are "leaky"),Rag1tm1Momhomozygotes lack all mature lymphocytes (i.e., are "non-leaky"). Rag1 null mice have no CD3+or T cell receptor (TCR) alpha-beta positive cells. The thymus of the mutant mice contains 15 to 130 times fewer cells than heterozygous or wildtype siblings. The thymocytes are CD8-CD4-and most are IL2 receptor-positive. Neither the spleen nor the bone marrow contain any IgM or IgD staining cells, indicating an absence of mature B cells. These and other data suggest that B cell and T cell development has been arrested at an early stage. Macroscopically, the mutants are indistinguishable from heterozygotes or normal wildtype siblings.DevelopmentTheRag1tm1Mommutant strain was developed by Dr. Peter Mombaerts in the laboratory of Dr. Susumu Tonegawa at the Center for Cancer Research, Massachusetts Institute of Technology. A replacement targeting vector with thePgk-neomarker was used. Homologous recombination of the targeting vector resulted in a 1356 bp deletion in the 5' end of the coding sequence. The 129S7/SvEvBrd-Hprt+-derived AB1 embryonic stem cell line was used. The C57BL/6J strain was generated by backcrossing mice carrying theRag1tm1Mommutation ten times to C57BL/6J inbred mice.

--------------------------B6.Cg-Rag1tm1MomThy1aTg(Tcra2C,Tcrb2C)1Dlo/JThese compound mutant mice on aThy1a(Thy1.1) C57BL/6J congenic background carry a targeted knockout of theRag1(recombination activating gene 1) gene and express the T cell receptor (TCR) from cytotoxic T-lymphocyte (CTL) clone 2C derived fromH2bBALB.B mice (2C TCR). This strain provides a tool to track and examine T cell selection, homeostasis, and lymphomagenesis and acts as a TCR model with known peptide specificity.DescriptionThese compound mutant mice on aThy1a(Thy1.1) C57BL/6J congenic background carry a targeted knockout of theRag1(recombination activating gene 1) gene and express the T cell receptor (TCR) from cytotoxic T-lymphocyte (CTL) clone 2C derived fromH2bBALB.B mice (2C TCR). Clone 2C was originally isolated as an allospecific T cell that recognizedH2-Ldon mastocytoma P815. In addition to peptide p2C, 2C TCR binds peptide SIYRYYGYL (in the context ofH2-Kb) and many other V8-specific superantigens. This strain provides a tool to track and examine T cell selection, homeostasis, and lymphomagenesis and acts as a TCR model with known peptide specificity.Mice homozygous for theRag1knockout and hemizygous for the TCR transgene exhibit altered thymic maturation. Fewer thymocytes mature into CD4-CD8-peripheral T cells in the compound mutant TCR transgenic/RAG1 knockout mice (5%) than in the TCR transgenic mice (45%). In contrast, there are proportionally twice as many 2C TCR clonotypic antibody 1B2+CD4+CD8+thymocytes in the TCR transgenic/RAG knockout thymus as in the TCR transgenic thymus. On theRag1knockout background, no endogenously rearranged TCRs (Thy1b-positive/1B2-negative) are reportedly detected on splenic T cells. T cells derived from TCR transgenic/RAG1 knockout mice can be activated to a high levelin vivoby administration of a self-MHC-restricted antigenic peptide. Mice have mainly CD8 single positive T cells, as well as some CD4 single positive and double negative 2C positive cells in the periphery. Animals have a risk of thymoma as they age.DevelopmentConstructions of functionally-rearranged 2C-specificTcraandTcrbmouse genes were assembled from several phage and cosmid libraries. Linearized vectors were co-injected into fertilized (C57L x SJL)F2 embryos which lack the V8 gene family. The transgenic animals were then intercrossed withRag1tm1Mommutant mice (e.g. Stock No.002216) to create a compound mutant strain. TheThy1a(Thy1.1) allele was introduced through crosses with a C57BL/6 backgroundThy1acongenic strain (see Stock No.000406). This strain was backcrossed to C57BL/6J for more than 12 generations by the donating laboratory.

----------------------------------B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/JFoxp3DTRmutant mice express knocked-in human diphtheria toxin receptor and EGFP genes from theFoxp3locus--without disrupting expression of the endogenousFoxp3gene. These mice may be useful for regulatory T cell visualizing and ablation.DescriptionFoxp3DTRmutant mice contain an internal ribosome entry site (IRES), a human diphtheria toxin receptor (DTR), and an enhanced green fluorescent protein (EGFP) downstream of the internal stop codon of the forkhead box P3 (Foxp3) gene. FOXP3 is a transcription factor required for the development and function of regulatory T (Treg) cells, which are required for suppression of self-reactive T cells and prevention of some autoimmune diseases. In these mice, DTR-EGFP expression is evident in FOXP3+Tregcells. Diphtheria toxin (DT) administration results in ablation of Tregcells in thymus, lymph nodes, and spleen 2 days after injection, with cell numbers rebounding 10-15 days post-injection. Neonates injected with DT daily all die from lymphoproliferative disease, indicated by failure to thrive, lack of mobility, ventral skin lesions, hunched posture, and conjunctivitis, within 27 days of birth. With the same DT treatment, adults exhibit a more rapid development of autoimmune disease and die within 3 weeks. These mice show an increase in CD4+T cell activation, as well as an increase in the number of B cells, macrophages, granulocytes, natural killer, and dendritic cells in the spleen and lymph nodes. Homozygous females and hemizygous males are viable, fertile, and normal in size. These mice may be useful for visualizing and specifically eliminating regulatory T cells, and for studying autoimmunity and immune dysfunction.DevelopmentTheFoxp3DTRtargeting vector was designed with an internal ribosome entry site (IRES), a human diphtheria toxin receptor (DTR), and an enhanced green fluorescent protein (EGFP), followed by afrt-flanked neomycin (neo) resistance cassette inserted downstream of the internal stop codon of the X-linked forkhead box P3 (Foxp3) gene. This construct was injected into (129X1/SvJ x 129S1/Sv)F1-Kitl+-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The donating investigator reported that the resulting mice were backcrossed to C57BL/6NTac mice for at least 8 generations (see SNP note below). Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6NJ inbred mice (Stock No.005304) for at least one generation.A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6 genetic background.

52.2. Rescue and therapy experiments

IgG2a isotype control YTS177.9Day 3Every 4 days

IgG2a isotype control YTS177.9WTSf maleDay 17-19Day 15-21

IgG2a isotype control DTDay 34 times per weekIgG2a isotype control YTS177.9Foxp3DTRDay 50YTS177.9On days 28, 0Bled on days 7, 14, 21 and 28 for peripheral blood analysis.

Survival curves indicate the time to death.

euthanasia. (-25% of weight or mobility impairment)

Anti-CD4 antibody [YTS177.9]. Reported to block MHC class II T cell responses in vivo and in vitro and induces tolerance.Express a simian diphtheria toxin. DT administration results in ablation of Foxp3+CD4+T reg cells.2.2. Rescue and therapy experiments

For sf rescue experiments, wt and sf male pups were injected at the latest on day 3 after birth with 20 mg YTS177.9 (low endotoxin, AbD Serotec or BioXcell), 20 mg rat IgG2a isotype control (low endotoxin, AbD Serotec or BioXcell) or were left untreated. For sf therapy experiments, 17 e 19 days old wt and sf males were treated every 4 days with 10 mg/kg YTS177.9. Mice were killed after 15e21 days or if moribund for analyses. Foxp3DTR mice were treated 4 times per week with 20 mg/kg of DT i.p. for 50 days. In addition, mice were injected with 10 mg/kg YTS177.9 (anti-CD4) or isotype control antibody, on days 28 and 0. Foxp3DTR mice were bled on days 7, 14, 21 and 28 for peripheral blood analysis. Survival curves indicate the time to death, loss of 25% of body weight or the development of severe mobility impairment requiring euthanasia.

--------------------------------Anti-CD4 antibody [YTS177.9]. Reported to block MHC class II T cell responses in vivo and in vitro and induces tolerance.Isotype IgG2a

Foxp3DTR mice. These "depletion of regulatory T cell" (DEREG) BAC transgenic mice express a simian diphtheria toxin receptor-enhanced green fluorescent protein (DTR-eGFP) fusion protein under control of the endogenous forkhead box P3 promoter/enhancer regions on the BAC transgene. DTR-eGFP expression is observed in fully functional Foxp3+CD4+regulatory T cell populations allowing fluorescent detection or diphtheria toxin-induced ablation Foxp3+T reg cells.Diphtheria toxin (DT) administration results in ablation of Foxp3+CD4+T reg cells with no apparent affect on CD25+effector T cells. DT-induced depletion of Foxp3+CD4+T reg cells is associated with enhanced and prolonged delayed-type hypersensitivity (DTH) responses and neonatal development of scurfy-like symptoms.62.3. Adoptive T cell transfer

CD4+ T cells (spleens and lymph nodes )YTS177.9 or isotype sf Adoptive transfer of CD4 T Cells (i.v)sf mice Rag1-/- mice (age > 8 weeks). Untreated sf Thy1.1 CD4+ T cells monitored and sacrificed when reaching 20% weight loss.

strainmutation produce no mature T cells or B cellshave no CD3+or T cell receptor (TCR)thymocytes are CD8-CD4-LB IgM or IgD -/-

T lymphocyte specific Thy1a allele.costimulatory signaling through CD28 in mouse T cells (as a substitute activating signal for TCR signaling.2.3. Adoptive T cell transfer

To avoid any binding of anti-CD4 antibodies, CD4+ T cells were negatively selected from pooled spleens and lymph nodes of diseased sf mice using the Dynal Mouse CD4 Cell Isolation Kit (Invitrogen) according to the manufacturers instructions. The purity of enriched CD4+ T cells was typically >80%. CD4+ T cells were incubated for 1 h on ice with either 50 mg/ml YTS177.9 or isotype control followed by washing away all unbound antibodies. 6 105 cells were transferred i.v. into Rag1/ mice (age > 8 weeks). For mixed transfer experiments, YTS177.9- or isotype-treated sf CD4+ T cells were mixed with untreated sf Thy1.1 CD4+ T cells at an approximate 1:1 ratio and a total of 6 105 cells were transferred.Rag1/ mice were frequently monitored and sacrificed when reaching 20% weight loss. Disease was considered to be established when one of the following criteria was fulfilled: severe and progressing blepharitis/conjunctivitis over two consecutive time points of observation, exfoliative dermatitis at the tail or at any other body location, weight loss of 20%.

72.4. Flow cytometryFc receptors were blocked using CD16/32CD3CD8 CD30 CD44 CD62L CD69 CD90 Foxp3 OX40 CD4 Labeled:ethidium monoazide photolysispropidium iodide DAPI.

Antibodies2.4. Flow cytometryFc receptors were blocked using aCD16/32 (2.4G2, produced in house). The following antibodies and secondary reagents were purchased from eBioscience: aCD3 (145-2C11), aCD8a (53-6.7), aCD30 (mCD30.1), aCD44 (IM7), aCD62L (MEL-14), aCD69 (H1.2F3), aCD90.1/Thy1.1 (HIS51), aFoxp3 (FJK-16s), aOX40 (OX-86) and appropriate isotype controls. aCD4 (RmCD4-2)was kindly provided by Guenther Bernhard (Hanover Medical School) and Elisabeth Kremmer (Helmholtz Center, Munich). Intranuclear Foxp3 staining was carried out using the Foxp3 fixation/permeabilization kit (eBioscience). Dead cells were labeled by ethidium monoazide photolysis, propidium iodide or DAPI (all from Sigma). Cells were acquired using LSRII, FACSCanto (both BD) or Cyan (Beckman Coulter) and data were analyzed with FlowJo (Tristar). Live cells were gated and aggregates were routinely excluded by SSC pulse width.

CD90 is a 25-35 kD GPI-anchored protein, also known as Thy-1. It belongs to Ig superfamily. Human CD90 is expressed on neuronal cells, a subset of CD34+cells, a subset of fetal liver cells and fetal thymocytes, fibroblasts, activated endothelial cells, and some leukemia cell lines. CD34+CD90+cells are primitive hematopoietic stem cells. It has been reported that Thy-1 binds with 2 and 3 integrins and plays bimodal roles in the regulation of cell adhesion and neurite outgrowth, and inhibits hematopoietic stem cells proliferation and differentiation.8

2.5. qPCRTnfrsf45-gcttggagttgactgtgttcc-35- gggtctgctttccagataagg-3 Tnfrsf8 5-tggagaggaggttgtcaagtc-35-gaggaaggcagctcacagat-3

On negatively isolated splenic CD4+ T cells 2.5. qPCRQuantitative real-time PCR on negatively isolated splenic CD4+ T cells was performed as described using the following additional primer pairs: Tnfrsf4 (50-gcttggagttgactgtgttcc-30; 50- gggtctgctttccagataagg-30); Tnfrsf8 (50-tggagaggaggttgtcaagtc-30; 50-gaggaaggcagctcacagat-30). Hprt and wt CD4+ T cells were used as references.

92.6. Histological analysis and scoring

Analysed using the program AutMess

Sirius Red staining collagen fibres in liver sectionsLiverLungPancraetic SkinThe severity of autoimmunity:Inflammatory cell InfiltrationNecrosis2.6. Histological analysis and scoringH&E stainings and microscopy of paraffin-embedded organ sections were performed as described [8] using AxioImager Z1, Axiovision software and AxioCam MRm (all Zeiss). The severity of autoimmunity affecting the different organs was scored across specimen in a blinded fashion according to the following definitions.The liver score is the sum of individual scores for inflammatory cell infiltration in portal tracts, parenchyma, and necrosis. Individual scores were given as follows. Portal inflammation: 0, no inflammatory infiltrate; 1, low level of inflammatory cell infiltration; 2, moderate level of inflammatory cell infiltration; 3, severe inflammation. Lobular inflammation: 0, no inflammatory infiltrate; 1, low level of inflammatory cell infiltration; 2, moderate level of inflammatory cell infiltration; 3, severe inflammation (>50% of parenchyma). Necrosis: 0, none; 1, small necroses; 2, large necrotic areas; 3, bridging necroses. Lung inflammation: 0, normal; 1, minor perivascular inflammation; 2, increased perivascular and peribronchial inflammation; 3, severe perivascular, peribronchial and interstitial inflammation. Pancraetic inflammation: 0, no inflammation; 1, mild perivascular inflammation; 2, moderate perivascular and intralobular inflammation; 3, severe inflammation; 4, severe inflammation and acinar necrosis. Skin inflammation: 0, normal; 1, minimal inflammation: few scattered lymphocytes plasma cells multifocally in the pinnae; 2, mild inflammation: increased numbers of lymphocytes, plasma cells neutrophils multifocally in the pinnae; 3, moderate inflammation: diffuse infiltration of lymphocytes, plasma cells and neutrophils associated with multifocal abrasion or ulcerations in the pinnae; 4, severe inflammation: diffuse, coalescing infiltrates of neutrophils, lymphocytes and plasma cells, multifocal abrasions, extensive ulcerations (covered by a serocellular crust in the pinnae).Histochemical staining of collagen fibres in liver sections was carried out by Sirius Red staining [28]. For the quantification of collagen, pictures of three high power fields (high power field 0.287 mm2) per liver section were taken and analysed using the program AutMess (Carl Zeiss Microscopy GmbH). Values were averaged for each section.102.7. ELISAAMA titers antimitochondrial antibodies

Autoantibodies

anti-mouse IgG OptEIA TMB

Optical density 450 nm vs 570 nm.90% of patients with autoimmune primary biliary cirrhosis develop high levels of AMAThe presence of serum antimitochondrial antibodies (AMA) is a highly specific indication ofprimary biliary cirrhosis(PBC).The reference range of antimitochondrial antibody immunoassay is a titer less than 1:40 on enzyme immunoassay (EIA) and negative on indirect immunofluorescence (IF).

The TMB substrate reagent set is designed for use in BD OptEIA ELISA sets. Substrate Reagent A, which is clear and colorless, contains hydrogen peroxide. Substrate Reagent B is a colorless to a very light amber solution containing 3,3', 5,5' tetramethylbenzidine (TMB) in an organic solvent.

2.7. ELISAAMA titers were determined as described with the exception that autoantibodies were revealed using HRP-conjugated goat anti-mouse IgG (Jackson Immunoresearch). HRP was revealed using the OptEIA TMB substrate reagent set (BD). After acidification, the optical density was measured at 450 nm against 570 nm as reference.

112.8. Cytometric bead assay (CBA)Serum levels of: IL-5 IFN-Mouse Th1/Th2 Multiplex Kit BMS FlowCytomix Software

2.8. Cytometric bead assay (CBA)Serum levels of IL-5 and IFN-g were determined using the Mouse Th1/Th2 Multiplex Kit (eBioscience) and BMS FlowCytomix Software (BenderMed Systems) according to the manufacturers instructions.

12Fig. 1. Anti-CD4 treatment rescues sf mice devoid of functional Foxp3+ Tregs.

Developed typical autoimmune disease (exfoliative dermatitis of the tail, ears, growth retardation and lethargy) 2- 3 wRescue at 21 days of ageLacked signs autoimmune desease.

To examine whether anti-CD4 treatment also reduced the autoimmunedamage of these organs,Inflammation scores of liver, lung and pancreas were all clearly reduced in YTS177.9-treatedA single injection of anti-CD4 inhibits systemic autoimmunity independent of Foxp3 Tregs in vivo.

Fig. 2. CD4 blockade tolerizes autoreactive sf CD4+ T cells. Isotype- and YTS177.9- treated mice were sacrificed at 8-9 days of age.

For explored if anti-CD4-induced tolerance on a cellular level.polyclonal CD4 T cell activation (62L/69)

P.447 JANEWAY TABLA PROTEINAS16

Sf IFN- and IL-5

Tx IFN- and IL-5Analyzed AMA as an indirect activity of autoantigen-specific CD4 T cells.PDC-E2-specifc CD4 T cells were tolerized by the non-depleting anti-CD4 antibody in vivo.Fig. 3. Anti-CD4 treatment is required concurrent to regulatory T cell depletion in order to prevent autoreactive T cell activation and autoimmunity.

Efficient depletion ofFoxp3 Tregs in all DT-treatedFoxp3DTRReduced CD4 T cell activation 7 days after the first DTDT 0 dayIsotype 0,28 danti-CD4 antibody 28 danti-CD4 antibody on day 0.Peripheral bloodCD44+CD62Llow T cells

anti-CD4 antibody can only induce tolerance if autoreactive T cells are stimulated simultaneouslyno inhibition of T cell activationmedian life expectancy of foxp3DTR mice in contrast to Prior treatment and isotype treatmentaugmented the Survival1Fig. 4. YTS177.9 directly tolerizes scurfy CD4+ T cells in a recessive and non-infectious fashion

investigated the characteristicsof anti-CD4-induced Foxp3-independent tolerance in detail.The disease-free survival improved RAG -/- The adoptive transfer of sf CD4 T cells into Rag1-/- mice is sufficient to transfer fulminant autoimmunity 2 To distinguish between recessive T cell-intrinsic tolerization and classical dominant / infectious tolerance1YTS177.9 / CD4 T cells + untx sf Th1.1 CD4 T cells

developed diseaseIsotype / sf CD4 T cells + untx sf Th1.1 CD4 T cells

sf(YTS177.9) sf(Iso)

55 days after mixed T cell transferthe frequencies of sf CD4 T cells among the mixture had not changedMixtures of sfCD4 T cells were analyzed prior to injectionThy1.1 vs. CD4 expression of CD4+CD3+ T cellsCx. YTS177.9-induced tolerance is transient, T cell-intrinsic and non infectious in the absence of Foxp3 Tregs.sf(YTS177.9) have no survival- or proliferation defect compared to sf(Iso)Fig. 5. Anti-CD4 therapy ameliorates established autoimmunity in sf mice

every 4 days anti-CD4 treatment could ameliorate pre-established autoimmune disease?TX. were more mobile but they had to be sacrificed due to progressing skin inflammation (ears)REST was not altered by anti-CD4 therapyliver pathology was clearlyreduced following anti-CD4 therapy123

(AMA) associated with autoimmune liver diseases were reduced.serum**

Fig. 6. Reduced liver damage in sf mice following YTS177.9 therapy.

Chronic liver inflammation resulting in fibrosis and eventually liver cirrhosis Evaluated collagen deposition in liver sections from sf mice12 fibrosisreduced Sirius Red stainingCX. anti-CD4 therapy not only reduced inflammation but alsosecondary fibrosis and tissue damage.Fig. 7. CD4 blockade impairs CD30 and OX40 expression by scurfy CD4+ T cells.

Hypothesized: anti-CD4 could attenuate signals from one or both receptors.

3-4 weeks oldOX40was strongly expressedsplenic CD4 T cellsCD4 T cells1No TxTx anti-CD4 treatment reduces OX40 and CD30 expression in vivo2?expression of both OX40- and CD30 mRNASf YTS177.9

Cx. anti-CD4-mediated tolerance acts in part via dampening pathogenic OX40 and CD30signalsYTS177.9 reduced OX40 protein expression on CD4 T cells when compared to isotype-treated sf miceAnti-CD4 antibodies can inhibit autoimmunity in mice completely devoid of functional Foxp3+ Tregs (therapy could not control all manifestations of autoimmunity).investigate whether the combination of anti-CD4 therapy with additional immunosuppressive therapies can result in enhanced control of autoimmunity.Represent a novel therapeutic option for IPEX patients or to other autoimmune diseases with Treg dysfunction.4. DiscussionIn contrast to the dogma of a Foxp3+ Treg-dependent mechanism, our study proves, for the first time, that anti-CD4 antibodies tolerize CD4+ T cells in a Foxp3+ Treg-independent manner in vivo.These results are unexpected yet do not contradict previous data [27]. In fact, we propose that both Treg-dependent- and independent mechanisms operate simultaneously in Foxp3-sufficient settings. By the use of constitutive Treg-deficient sf mice which develop autoimmunity without exogenous manipulation, we were able to examine Foxp3+ Treg-independent anti-CD4-induced tolerance in isolation. Our results are in line with a recent study showing that anti-CD4 induces tolerance to factor VIII in a Foxp3-independent fashion [40]. However, whereas tolerance to factorVIII was dependent on IL-10 [40], we found no increased IL-10 expression by tolerized CD4+ T cells and actually reduced IL-10 levels in the serum of tolerized sf mice. Supporting our findings, we report that Treg-indpendent tolerance is recessive and noninfectious.This contrasts with classical dominant infectious tolerance elicited by CD4 blockade which was indeed recently suggested to require Foxp3+ Tregs [24,27,41]. The non-infectious and recessive nature of Treg-independent tolerance could explain its transient nature because thymic output in sf mice likely replenishes pathogenic autoreactive CD4+ T cells once the anti-CD4 antibody is cleared. In line with this theory, a significant proportion of Rag1/ mice transferred with YTS177.9-treated sf CD4+ T cells were protected long-term in the absence of thymic output. However, since sf mice exhibit abnormal thymopoiesis secondary to autoimmunity [12,42], this aspect remains to be proven. Alternatively, the lack of humoral immunity in Rag1/ mice may prevent the full precipitation of autoimmunity after the transfer of anti-CD4-treated CD4+ sf T cells. Indeed, sf mice are known to develop autoantibodies [29,43], yet the contribution of B cells/autoantibodies to the sf pathology has not been examined in detail till date. However, B cells are implied in various human autoimmune diseases and B celldepleting agents are available as an immunosuppressive treatment [44,45].Whether recessive sf CD4+ T cell tolerance is based on anergy or deletion of autoreactive T cells remains to be explored in detail. Anergy of antigen-specific CD4+ T cells was previously reported following YTS177.9 exposure [22] and we found reduced expression of OX40 and CD30 on tolerized CD4+ T cells. Given that signals via these two receptors were shown to be crucial for the survival of antigen-experienced CD4+ T cells [17], it is possible that both scenarios are overlapping.Despite the advanced degree of autoimmunity in 17e19 days old sf mice, we demonstrated that continuous anti-CD4 monotherapy reduced liver inflammation, fibrosis and anti-mitochondrial autoantibodies. However, anti-CD4 therapy could not control all manifestations of autoimmunity. This may have different reasons. It has for example been shown that primed T cells are more resistant to tolerance induction than nave T cells [46]. CD4+ T cells of sf mice suffering from autoimmunity are expected to be chronically activated by self-antigens and/or inflammatory cytokines. Additionally, once CD4+ T cell help has initiated inflammatory reactions involving multiple effector immune cell types, autoimmunity may be sustained independent of CD4+ T cells and thus partially resist anti-CD4 tolerization. Insufficient access of the antibody to certain anatomic locations could be another factor. This may also extend to the availability of self-antigens which are required for tolerance induction. Furthermore, thymic T cell selection may be perturbed by chronic anti-CD4 therapy. Indeed, we found that CD4 expression is reduced on thymocytes following anti-CD4 treatment (data not shown) and suboptimal TCR signals due to ZAP-70 mutations were shown to result in defective negative selection and autoimmunity [47]. Nevertheless, the therapeutic benefits of anti-CD4 monotherapy in sf mice are remarkable. It will be interesting to investigate whether the combination of anti-CD4 therapy with additional immunosuppressive therapies can result in enhanced control of autoimmunity in both sf mice and Foxp3-sufficient settings of autoimmunity.

5. Conclusion

Non-depleting anti-CD4 antibodies are believed to induce tolerance via a Foxp3+ Treg-dependent mechanism. Thus, it was unclear whether anti-CD4 antibodies are suitable therapies for human autoimmune diseases characterized by Treg dysfunctions. In this study, we show that anti-CD4 antibodies can inhibit autoimmunity in mice completely devoid of functional Foxp3+ Tregs. This previously undefined tolerance mechanism is T cell-instrinsic and non-infectious. Thus, anti-CD4 therapy could unexpectedly represent a novel therapeutic option for IPEX patients suffering from autoimmunity due to Foxp3 mutations. This may also apply to other autoimmune diseases which are believed to arise from Treg dysfunction. These results have important implications for the clinical development of anti-CD4 antibodies and predict synergies with Treg-dependent therapies. The characteristics of anti-CD4 antibodies which trigger Treg-dependent versus Treg-independent tolerance programs in CD4+ T cells warrant further investigation.

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