Abstract:
The invention is in the field of molecular immunology, more in particular in the field of medical treatment of animals such as humans suffering from unwanted immune reactions. The invention relates to methods for the treatment of unwanted immune reactions and provides means and methods for suppressing an immune response. The present invention relates in particular to regulatory T cells and methods of long-term, culture-expanding, activating and using same in immunotherapy and for the suppression of autoimmune responses, allergies and inflammatory diseases. The invention provides a sia alpha 2,3-conjugated antigen for use in the suppression of an immune response in a patient in need of such a treatment.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention is in the field of molecular immunology, more in particular in the field of medical treatment of patients suffering from unwanted immune reactions. The invention relates to methods for the treatment of unwanted immune reactions and provides means and methods for suppressing an immune response. The present invention relates in particular to regulatory T cells and methods of long-term, culture-expanding, activating and using same in immunotherapy and for the suppression of autoimmune responses, allergies and inflammatory diseases. 
       BACKGROUND OF THE INVENTION 
       [0002]    It has long been thought that suppressor cells play a role in the progression of cancer (Dye et al., J. Exp. Med. 154:1033-1042 (1981)). In fact, active suppression by T regulatory cells plays an important role in the down-regulation of T cell responses to foreign and self-antigens. 
         [0003]    T cells are a class of lymphocytes, having specific T cell receptors (TCRs) that are produced as a result of gene rearrangement. T cells have diverse roles, which are accomplished by the differentiation of distinct subsets of T cells, recognizable by discrete patterns of gene expression. Several major T cell subsets are recognized based on receptor expression, such as TCR-[alpha]/[beta], and TCR [gamma]/[delta] and invariant natural killer cells. Other T cell subsets are defined by the surface molecules and cytokines secreted therefrom. 
         [0004]    For example, T helper cells (CD4 cells) secrete cytokines, and help B cells and cytotoxic T cells to survive and carry out effector functions. Cytotoxic T cells (CTLs) are generally CD8 cells, and they are specialized to kill target cells, such as infected cells or tumor cells. Natural killer (NK) cells are related to T cells, but do not have TCRs, and have a shorter lifespan, although they do share some functions with T cells and are able to secrete cytokines and kill some kinds of target cells. 
         [0005]    Human and mouse peripheral blood contains a small population of T cell lymphocytes that express the T regulatory phenotype (“Treg”), i.e., positive for both CD4 and CD25 antigens (i.e., those CD4-positive T cells that are also distinctly positive for CD25). First characterized in mice, where they constitute 6-10% of lymph node and splenic CD4-positive T cell populations, this population of CD4-positive CD25-positive cells represents approximately only 5-10% of human peripheral blood mononuclear cells (PBMC), or 2-7% of CD4-positive T cells, although some donors exhibit a more distinct population of CD4-positive and CD25-positive cells. About 1-2% of human peripheral blood PBMCs are both CD4 positive (CD4-positive) and CD25 brightly positive (CD25-positive) cells. 
         [0006]    There are several subsets of Treg cells (Bluestone et al., Nature Rev. Immunol. 3:253 (2003)). One subset of regulatory cells develops in the thymus. Thymic derived Treg cells function by a cytokine-independent mechanism, which involves cell to cell contact (Shevach, Nature Rev. Immunol 2:389 (2002)). They are essential for the induction and maintenance of self-tolerance and for the prevention of autoimmunity (Shevach, Annu. Rev. Immunol. 18:423-449 (2000); Stephens et al., 2001; Turns et al., 2001; Thornton et al., 1998; Salomon et al., Immunity 12:431-440 (2000); Sakaguchi et al., Immunol. Rev. 182:18-32 (2001)). 
         [0007]    These professional regulatory cells prevent the activation and proliferation of autoreactive T cells that have escaped thymic deletion or recognize extrathymic antigens, thus they are critical for homeostasis and immune regulation, as well as for protecting the host against the development of autoimmunity (Suri-Payer et al., J. Immunol. 157:1799-1805 (1996); Asano et al., J. Exp. Med. 184:387-396 (1996); Bonomo et al., J. Immunol. 154:6602-6611 (1995); Willerford et al., Immunity 3:521-530 (1995); Takahashi et al., Int. Immunol. 10:1969-1980 (1998); Salomon et al., Immunity 12:431-440 (2000); Read et al., J. Exp. Med. 192:295-302 (2000). Thus, immune regulatory CD4-positive CD25-positive T cells are often referred to as “professional suppressor cells.” 
         [0008]    However, Treg cells can also be generated by the activation of mature, peripheral CD4-positive T cells. Studies have indicated that peripherally derived Treg cells mediate their inhibitory activities by producing immunosuppressive cytokines, such as transforming growth factor-beta (TGF-[beta]) and IL-10 (Kingsley et al., J. Immunol. 168:1080 (2002); Nakamura et al., J. Exp. Med. 194:629-644 (2001)). After antigen-specific activation, these Treg cells can non-specifically suppress proliferation of either CD4-positive or CD25-positive T cells (demonstrated by FACS sorting in low dose immobilized anti-CD3 mAb-based co-culture suppressor assays by Baecher-Allan et al., J. Immunol. 167(3):1245-1253 (2001)). 
         [0009]    Studies have shown that CD4-positive CD25-positive cells are able to inhibit anti-CD3 stimulation of T cells when co-cultured with autologous antigen presenting cells (APC), but only through direct contact (Stephens et al., Eur. J. Immunol. 31:1247-1254 (2001); Taams et al., Eur. J. Immunol. 31:1122-1131 (2001); Thornton et al., J. Exp. Med. 188:287-296 (1998)). However, in mice this inhibitory effect was not able to overcome direct T cell stimulation with immobilized anti-CD3 or with anti-CD3/CD28 (Thornton et al., 1998). In previous reports, human CD4-positive CD25-positive T cells isolated from peripheral blood required pre-activation in order to reveal their suppressive properties, as direct culture of the regulatory cells was generally insufficient to mediate suppressive effects (Dieckmann et al., J. Exp. Med. 193:1303-1310 (2001)). 
         [0010]    Others have also found that the inhibitory properties of human CD4-positive CD25-positive T cells are activation-dependent, but antigen-nonspecific (Jonuleit et al., J. Exp. Med. 193:1285-1294 (2001); Levings et al., J. Exp. Med. 193(11):1295-1302 (2001); Yamagiwa et al., J. Immunol. 166:7282-7289 (2001)), and have demonstrated constitutive expression of intracellular stores of cytotoxic T lymphocyte antigen-4 (CTLA-4) (Jonuleit et al., 2001; Read et al., J. Exp. Med. 192:295-302 (2000); Yamagiwa et al., 2001; Takahashi et al., J. Exp. Med. 192:303-310 (2000)). Moreover, after T-cell receptor (TCR)-mediated stimulation, CD4-positive CD25-positive T cells suppress the activation of naive CD4-positive CD25-negative T cells activated by alloantigens and mitogens (Jonuleit et al., 2001). 
         [0011]    Both mouse and human Treg cells express CTLA-4, however the role of CTLA-4 in tolerance induction and its capacity to impart inhibitory function to regulatory CD4-positive CD25-positive T cells is controversial. CTLA-4 (also known as CD152) is a homolog of CD28 and is a receptor for the CD80 and CD86 ligands. CTLA-4 inhibits T cell responses in an antigen and TCR-dependent manner. T cells that have impaired CTLA-4 function have enhanced T cell proliferation and cytokine production. In contrast, enhanced CTLA-4 function leads to inhibited cytokine secretion and impaired cell cycle progression both in vitro and in vivo. In the mouse, CTLA-4 is not required for suppressive function of the Treg cells, as opposed to its requirement in humans. 
         [0012]    A recent study has shown that Treg cells grow extensively in vivo (Tang, J. Immunol. 171:3348 (2003)), while others have suggested that the efficacy of therapeutic cancer vaccination in mice can be enhanced by removing CD4-positive CD25-positive T cells (Sutmuller et al., J. Exp. Med. 194:823-832 (2001)). Studies have also indicated that depletion of regulatory cells led to increased tumor-specific immune responses and eradication of tumors in otherwise non-responding animals (Onizuka et al., Cancer Res. 59:3128-3133 (1999); Shimizu et al., J. Immunol. 163:5211-5218 (1999)). Susceptible mouse strains that were made CD4-positive CD25-positive deficient by neonatal thymectomy were shown to develop a wide spectrum of organ-specific autoimmunities that could be prevented by an infusion of CD4-positive CD25-positive T cells by 10-14 days of age (Suri-Payer et al., J. Immunol. 160:1212-1218 (1998)). That study also found that CD4-positive CD25-positive T cells could inhibit autoimmunity induced by autoantigen-specific T cell clones. The transfer of CD4-positive CD25-negative T cells into nude mice also reportedly led to the development of autoimmune disorders which could be prevented by the co-transfer of CD4-positive CD25-positive T cells using lymphocytes first depleted of CD25-positive cells (Sakaguchi et al., J. Immunol. 155:1151-1164 (1995)). 
         [0013]    Hereafter, the transcription factor Forkhead box P3 (FoxP3) was related to the generation and fuction of naturally occurring Treg. Mice in which FoxP3 protein was deleted due to a mutation in the FoxP3 gene, developed severe autoimmune syndroms and wasting diseases (socalled “scurfy” mice; Brunkow et al., Nat Genet. 27:68-73, 2001). This seminal discovery enabled to attribute the cause of the X-linked IPEX syndrome (Immunodysregulation, Polyendocrinopathy, and Enteropathy, X-linked) in humans to a mutation in the FoxP3 gene (Bennett et al. Nat Genet. 27: 20-21; 2001). Later studies also demonstrated the presence of FoxP3 in some adaptive Treg subsets. 
         [0014]    However, data also indicate that the role of CD4-positive CD25-positive cells is not limited to self-tolerance and the prevention of autoimmunity. While few studies have addressed the role of CD4-positive CD25-positive T cells in alloresponses or in transplantation, CD4-positive CD25-positive T cells have been reported to prevent allograft rejection, both in vitro and in vivo (Hara et al., J. Immunol. 166:3789-3796 (2001); Taylor et al., J. Exp. Med. 193:1311-1318 (2001)). Allogeneic stimulation of human T cell proliferation is also blocked by CD4-positive CD25-positive T cells (Yamagiwa et al., 2001), whereas Wood&#39;s laboratory has shown that CD4-positive CD25-positive T cells suppress mixed lymphocyte responses (MLR), but only when the alloantigen was presented by the indirect, and not the direct, pathway of allorecognition (Hara et al., 2001). It is likely that direct antigen presentation occurs between the regulatory T cells and the anti-CD3/28 stimulated responder T cells, as the sorted CD4-positive 25-positive cells are highly depleted of professional APC. 
         [0015]    The absence of Tregs or depletion of Tregs is shown to result in the development of auto-immunity, such as Type 1 Diabetes, Inflammatory bowel disease (IBD), thyroididites, Multiple Sclerosis and Systemic lupus erythematosus (SLE). Moreover the disease can be reversed by the adoptive transfer of CD4+CD25+Treg cells. Besides a deficiency in Treg number, T cell regulation in autoimmunity has also been shown to fail due to a deficiency in the function of Treg to inhibit effector T cells. It is clear that defects in Treg cell number and function can contribute to disease and therapies directed at these defects have the potential to prevent and also cure these diseases. Animal studies suggest that an increase in Treg cell number at the site of inflammation is likely to be therapeutic in autoimmunity. This can be achieved by adoptive transfer of in-vitro expanded autologous Tregs or by the use of agents that promote Treg cell proliferation, survival and induction. The identity of factors that influence cell number and function of Tregs are not clearly identified at the moment, and may be crucial for the application of autoimmune diseases. 
         [0016]    Antigen Presenting cells such as DC are known for their capacity to differentiate naive CD4 T cells into different lineage of T cells, such as Th1, Th2, Th17 and Treg. Recent studies demonstrate that a population of gut DC, particularly lamina propria CD103+ DCs, can promote the conversion of naive CD4+ T cells into FoxP3+ iTregs through the secretion of retinoic acid (RA) in conjunction with TGF-β. DC express various receptors such as CD80/86 that can be bound by CTLA-4 on Tregs that triggers the induction of the enzyme indoleamine 2,3 dioxygenase (IDO) in DC. IDO converts tryptophan into pro-apoptotic metabolites that suppress effector T cells. On the other hand engagement of MHC class II on DC by LAG3 on Tregs suppresses APC maturation and reduces their ability to activate T cells. These findings demonstrate that DC may differentiate CD4 T cells into Tregs. However, little is still known on the mechanism and signals that reach DC to instruct CD4 naïve T cells to differentiate into Tregs. 
         [0017]    Patients suffering from autoimmune diseases or inflammatory diseases would greatly benefit from treatments wherein the Treg numbers or function are improved. 
         [0018]    Applicants have established that the uptake of specific glycosylated antigens by DCs regulates the number and function of Tregs. This opens new opportunities for the treatment of unwanted immune reactions and leads to new methods and means for the treatment of autoimmune diseases and inflammatory diseases. 
       SUMMARY OF THE INVENTION 
       [0019]    We found that sialic acids on self and non-self antigens play an important role in the induction of tolerance. As a model system, applicants investigated the well-known food allergy against ovalbumin (OVA). Ovalbumin is the major allergen in chicken egg. In humans, CD4 T-cell responses against OVA have been detected (Heine et al, Currebt Allergy and Asthma reports 6, 145-152, 2006). To study responses in mice, T-cell receptor transgenic mice have been generated that express a OVA-specific TCR on all CD4 T-cells (OT-II transgenic mice). These mice are widely used.) 
         [0020]    We therefore set out to modify the model antigen OVA with Neu5Aca2-3Galβ1-4Glc, creating sia-alpha 2,3-OVA and assessed the functional consequences on CD4 +  T-cell activation and differentiation upon co-culture with sia-2,3-OVA-loaded DC. 
         [0021]    It was established that such a sia alpha 2,3-conjugated antigen was capable of suppressing an immune response and could therefore advantageously be used in the suppression of an immune response in a patient allergic to ovalbumin. 
         [0022]    In a more general concept, the invention therefore relates to a sia alpha 2,3-conjugated antigen for use in the suppression of an immune response in a patient in need of such a treatment. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0023]    Sialic acids are the most prevalent terminal monosaccharide on the surface of mammalian cells. The most common mammalia sialic acids are N-acetylneuraminic acid (Neu5Ac) and N-glycolyneuraminic acid (Neu5Gc). Humans are unable to synthesize Neu5Gc due to an irreversible mutation in the gene encoding the enzyme responsible for conversion of Neu5Gc from Neu5Ac. Sialic acids may be α2,3-, α2,6- or α2,8-linked to the underlying glycan. Sialic acids are often found at the outer ends of surface exposed oligosaccharide chains, attached to proteins and lipids. In this terminal position, they serve as ligands for lectins such as Sialic acid binding Ig-like lectins (Siglecs). 
         [0024]    We started by assessing whether conjugation of alpha-sia-2,3 to OVA (hereafter referred to as OVA-sia-2,3) essentially affected OVA-specific CD4+ T-cell responses in-vitro. Hereto, naive CD4+CD62Lhi CD25− T-cells were isolated from OT-II mice and co-cultured with BMDC that had been loaded with OVA-sia-2,3 or native OVA for 4 h. Six days later, CD4 T helper differentiation was analysed by staining for FoxP3 or intracellular IFNγ. We observed that naive CD4+ T-cells were converted into FoxP3+ T-cells when primed by DC loaded with OVA-sia-2,3 ( FIG. 1A , upper panels). 
         [0025]    DC loaded with native OVA did not prime T-cells to differentiate into Treg. By contrast, these T-cells were converted into effector T-cells as shown by IFNγ staining ( FIG. 1A , lower panels). This was confirmed when examining the supernatant of these cultures ( FIG. 1B , upper panel). In addition high levels of the T-cell effector cytokines TNFα and IL-6 were detected in these cultures. By contrast, these were virtually absent in cultures of T-cells primed by OVA-sia-2,3 DC. Since the generation of FoxP3+ Treg is closely related to Th17 generation, we assessed the presence of Th17 cells by analysing IL17A in the supernatant. No significant amounts of IL17A were detectable in the supernatant of OVA-sia2,3 DC-T-cell co-cultures. 
         [0026]    Moreover, we observed that the amount of Th1 effector cytokines IFNγ and TNFα was still significantly lower when naïve T-cells were primed by OVA-sia-2,3-DC than by OVA-DC in the presence of Th1- or Th17-promoting stimuli ( FIG. 1C ). Only in the presence of the Th17-promoting agent prostaglandin (PGN) high levels of IL17A were detected in cultures with OVA-sia-2,3-DC. No significant amounts of IL10 were detected in the DC-T-cell co-cultures (data not shown). 
         [0027]    Together, these data show that priming of naive CD4 T-cells by OVA-sia2,3 loaded DC promotes de novo generation of FoxP3+ T-cells. Furthermore, the generation of effector T-cells is prevented, even in a Th1- or Th17-promoting environment. However, more IL17A secreting T-cells are present when naïve CD4+ T-cells are primed by OVA-sia-2,3 loaded DC in a Th17-skewing milieu. 
         [0028]    As a control, the following experiment was performed. The generation of FoxP3+ T cells in the absence of effector T-cells upon priming of naive CD4 T-cells with OVA-sia-2,3 loaded DC could theoretically be the result of low amounts of antigen presented by the DC in MHC class II molecules. To address this, we incubated BM DC with fluorescent labeled OVA-sia-2,3 and assessed both binding as well as uptake at various time points. It is clear from  FIGS. 2A  and B that modification of OVA with sia-2,3 results in significant better binding and uptake by BMDC compared to nonmodified OVA. Thus, the uptake of OVA-sia-2,3 by BMDC is increased. 
         [0029]    Despite this increased uptake, it is possible that OVA-sia-2,3 is rapidly degraded upon internalization. To rule out this possibility, we subsequently used these antigen-loaded DC in an antigen-presentation assay with purified OVA-specific CD4+ T-cells. Native OVA is well presented in MHC class II molecules as shown by significant T-cell proliferation ( FIG. 2C ). The proliferation of the CD4+ T-cells was only significantly different when a high dose of sialic acid-conjugated OVA was used. At lower doses no significant difference in CD4 T-cell proliferation induced by DC loaded with native OVA or sia-2,3-OVA was detected. These data may indicate that OVA-sia-2,3 enters a similar processing and presentation pathway as native OVA. 
         [0030]    In view of the data on antigen uptake and presentation, we hypothesized that uptake of OVA-sia-2,3 triggers a signaling cascade, resulting in modulation of the DC phenotype. Therefore we examined the expression of costimulatory molecule transcripts in BMDC upon 6h incubation with OVA-sia-2,3 and compared it with expression in BMDC incubated with native OVA or BMDC incubated in medium only. From  FIG. 3A  it is clear that the expression of CD80 and CD86 is lower on BMDC incubated with OVA-sia-2,3 than with native OVA, albeit not significantly. Similar data were obtained for CD40 and MHC-class II (data not shown). Furthermore, molecules associated with tolerance were not distinctively or higher expressed by the OVA-sia-2,3-loaded BMDC. Moreover, the expression of PD-L2 seems to be decreased on OVA-sia-2,3-loaded BMDC. 
         [0031]    Analysis of cytokine mRNA expression revealed a significant lower expression of IL1β levels in OVA-sia-2,3-loaded DC ( FIG. 3B ). By contrast, the expression of IL23p19, which can associate with the p40 subunit of IL12 to form IL23, was significantly elevated in OVA-sia-2,3-loaded DC. Furthermore examination of mRNA encoding the anti-inflammatory cytokines IL10 or TGFb revealed no significant difference ( FIG. 3B ). 
         [0032]    Together, these data indicate that uptake of OVA-sia-2,3 in the absence of additional stimuli does not result in expression of well known tolerogenic markers. We have therefore demonstrated that a sia-2,3 modified antigen taken up by DC modifies the differentiation of naive CD4 T cells into Tregs. Our data demonstrate that this is not the result of low dose of antigen presentation, as high antigen dose were taken up, similar as un-modified antigen or sia-2,6 modified antigens. We have analysed whether the uptake of sia-2,3 may modify the tolerogenic phenotype of DC, but we did not see any major alterations in the expression of CD80/CD86, CD40 or MHC class II. We observed that the expression of the co-stimulatory molecule PDL-2 was lower on DC that had taken up OVA-sia-2,3 compared to native OVA. Upon analysis of the cytokine production by DC we observed that the inflammatory cytokine profile IFNy, IL-6 and TNFa) was reduced by DC upon uptake of OVA-sia-2,3, illustrating a potentiation towards an anti-inflammatory signature. When analysing the anti-inflammatory cytokine profile we observed little enhanced production of TGFb and no alterations in IL-10 and IL17A. 
         [0033]    Our finding that alpha-2,3 sialylation of antigen enhances the differentiation of antigen specific FoxP3+ regulatory T cells, sheds new light on how on the mechanism and signals that reach DC to instruct CD4 naive T cells to differentiate into Tregs. It also enables a whole new area of treatment for autoimmune diseases and inflammatory diseases. The invention therefore relates to a sia alpha 2,3-conjugated antigen for use in the suppression of an immune response in a patient in need of such a treatment. 
         [0034]    The term sia-alpha 2,3 conjugated antigen refers to an antigen such as a protein, polypeptide, lipid or otherwise, covalently attached to the sialic acid Neu5Acα2-3Galβ1-4Glc, creating sia-alpha 2,3-conjugated antigen. 
         [0035]    Such an antigen may effectively be used for the treatment of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, diabetes type 1, gastritis and inflammatory bowel disease. It may also be used for the treatment of inflammatory diseases, such as psoriasis, allergy, Alzheimer&#39;s disease, Parkinson&#39;s disease and transplantation. 
         [0036]    In another embodiment, the invention relates to a method for suppressing an immune response in a patient in need of such a treatment wherein a sial alpha 2,3 modified antigen is administered to said patient. 
         [0037]    It may be envisaged that the immune response is even better suppressed when disease-specific antigens are sialylated and administered to patients. The invention therefore also relates to a sia alpha 2,3-conjugated antigen for use in the suppression of an immune response in a patient in need of such a treatment wherein the antigen is disease-specific. Several examples of disease specific antigens that work well in the methods according to the invention are listed in table 1. 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Disease 
                 Disease-specific antigens 
               
               
                   
               
             
             
               
                 Multiple Sclerosis 
                 Myelin, MOG 
               
               
                 Rheumatoid Arthritis 
                 citrullinated proteins; human cartilage 
               
               
                   
                 gp39; HSP70, HSP60; type II collagen 
               
               
                 Type 1 Diabetes 
                 preproinsulin; GAD65; IGRP; IA-2; 
               
               
                   
                 preproIAPP; Zinctransporter 8 
               
               
                 Allergies 
                 Animal products Fel d 1 (cat allergy) fur 
               
               
                   
                 and dander; cockroach calyx; wool; dust 
               
               
                   
                 mite excretion. penicillin; sulfonamides; 
               
               
                   
                 salicylates local anaesthetics, celery and 
               
               
                   
                 celeriac; corn or maize; eggs, fruit; 
               
               
                   
                 pumpkin, beans; peas; peanuts; 
               
               
                   
                 soybeans; milk; seafood; sesame; soy; 
               
               
                   
                 tree nuts; pecans; almonds; wheat. bee 
               
               
                   
                 sting venom; wasp sting venom; mosquito 
               
               
                   
                 stings, latex, metal, Plant pollens (hay 
               
               
                   
                 fever) grass, ryegrass, timothy-grass 
               
               
                   
                 weeds, ragweed, plantago, nettle, 
               
               
                   
                 artemisia vulgaris, chenopodium album, 
               
               
                   
                 sorrel trees, birch, alder, hazel, 
               
               
                   
                 hornbeam, aesculus, willow, poplar, 
               
               
                   
                 platanus, tilia, olea, Ashe juniper. 
               
               
                   
               
             
          
         
       
     
         [0038]    In this way one would modify specific antigens, related to the disease, as proteins, or peptides, or as nanoparticules or encapsulated particules, and use the alpha 2,3 sialic acid as the glycan structure that can ex-vivo or in-vivo instruct APC such as DC to start an anti-inflammatory program and enhance the induction of Tregs that dampen the inflammations and will recover the disease. 
     
    
     
       LEGEND TO THE FIGURES  
         [0039]      FIG. 1 : Priming of naive CD4 T-cells with OVA-sia-2,3-loaded DC results in de novo generation of FoxP3+ T-cells and prevents effector T-cell formation. 
           [0040]    A. Immature BMDC were incubated with 50 ug/ml OVA or OVA-sia-2,3 for 4 hours. After extensive washing, naive OVA-specific CD4 T-cells were added at a 1:10 ratio. On day 6 of culture, cells were harvested, fixed and permeabilised and stained for CD4 and FoxP3 (upper panel) or, after 6 h stimulation with PMA/ionomycin/BrefeldinA, for IFNγ (lower panel). Results are representative of five independent experiments. B, supernatants of these cultures were examined for the presence of effector T-cell cytokines (IFNγ, TNFα, 1L6, and II17A) as well as the anti-inflammatory cytokine IL10. C. The amount of effector T-cell cytokines IFNγ and TNFα is also reduced when CpG or PGN were added to co-cultures containing OVA-sia-2,3-loaded DC. Only in the presence PGN, OVA-sia-2,3-loaded DC promote Th17 differentiation. Depicted results are representative of four independent experiments. 
           [0041]      FIG. 2 : No enhanced MHC class II presentation of OVA-sia-2,3 despite increased binding and uptake by DC. 
           [0042]    The OVA-neo-glycoconjugate OVA-sia-2,3 was fluorescently labeled to assess binding and uptake by BMDC. A, To assess binding of the neo-glycoprotein, 10 5  BMDC were incubated with 50 μg/m1 of antigen for 30 min at 4 C. Binding was compared with native OVA. CTRL indicates cells incubated with medium only, which were used to as negative control. Binding was assesed by flow cytometry. Representative facs plots are shown. B, In addition, uptake was determined by incubating BMDC with 50 μg/ml of antigen at 37 C. Uptake of antigen was determined at indicated time points using flow cytometry and represented as MFI. One representative experiment out of three is shown. C. To examine whether increased uptake of OVA-sia-2,3 also increased presentation in MHC class II, we co-cultured 2.5×10 4  CD11c +  BMDC, pulsed with indicated concentrations of OVA-sia-2,3 or native OVA, with purified OVA-specific CD4 +  T-cells. Proliferation was determined by addition of 3H-Thymidine during the last 16 h of a three day culture period. 
           [0043]      FIG. 3 : No induction of a tolerogenic signature in BMDC after incubation with OVA-sia-2,3. 
           [0044]    To examine whether incubation of BMDC with OVA-sia-2,3 induced a tolerogenic phenotype in BMDC, we incubated 10 5  BMDC with 50 μg/ml of antigen. This was compared with the phenotype induced by incubation of BMDC with native OVA. Six hours later, RNA was isolated and expression of A. co-stimulatory markers and B. cytokines was examined using RT-PCR. One representative experiment out of three is shown. P-value &lt;0.05 was considered significantly different from responses to native OVA. 
           [0045]      FIG. 4 : Priming of naive CD4 T-cells with Sia-OVA-loaded ex-vivo isolated splenic DC results in de novo generation of FoxP3+ T-cells with suppressive properties. 
           [0046]    Ex-vivo isolated CD11c +  splenic DC were incubated with 50 ug/ml Sia-OVA or native OVA for 4 hours. After extensive washing, naïve OVA-specific CD62L hi CD4 +  T-cells were added at a 1:10 ratio. On day 6 of culture, cells were harvested, fixed and permeabilised and stained for CD4 and FoxP3 (A) or, after 6 h stimulation with PMA/ionomycin/BrefeldinA, for IFNγ (B). The supernatants of the cultures were examined for the presence of effector cytokines (IFNγ, TNFα, IL6) as well as the anti-inflammatory cytokine IL10 (D). In addition, by adding these T-cells to co-cultures of naive CFSE-labeled OT-II T-cells and OVA-loaded DC at a 1:1 ratio, potential suppressive properties could be evaluated. The proliferation of responder T-cells was analysed 4 days later using flow cytometry (C). Results are representative of two independent experiments. 
           [0047]      FIG. 5 : Uptake of sialylated antigen results in tolerogenic DC even in the presence of a pro-inflammatory stimultus. 
           [0048]    Ex-vivo isolated splenic DC were incubated with 50 ug/ml Sia-OVA in the presence of 100 ng/ml LPS. Four hours later, cells were extensively washed and naïve OVA-specific CD62L hi  CD4 +  T-cells were added at a 1:10 ratio. On day 6 of culture, cells were harvested, fixed and permeabilised and stained for CD4 and FoxP3 (A, upper panel) or, after 6 h stimulation with PMA/ionomycin/BrefeldinA, for the effector cytokine IFNγ (A, lower panel). In addition, the culture supernatants were analysed for the presence of effector cytokines (IFNγ, TNFα, IL6) (B). 
           [0049]      FIG. 6 : De novo induction of FoxP3 +  T cells upon intravenous injection of Sia-OVA. 
           [0050]      FIG. 6A : C57BL/6 mice transferred with CFSE-labeled OT-II T-cells and one day later injected with PBS; OVA or Sia-OVA intravenously. Analysis of OT-II T cells (identified based on Tg T-cell receptor) for dilution of CFSE in spleen (left) and lymph nodes (right)  FIG. 6B : C57BL/6 mice transferred with CFSE-labeled OT-II T-cells one day later injected with PBS; OVA or Sia-OVA subcutaneously Analysis of spleens (left) and lymph nodes (right). To examine whether Sia-OVA also has tolerogenic properties in-vivo we injected C57BL/6 mice that were adoptively transferred with CFSE labeled CD4 +  CD25 −  OT-II cells with 100 ug Sia-OVA i.v. (A) or s.c. (B). This was compared with injection of 100 ug OVA. Control mice received PBS. Four days later, the spleen and axillary and inguinal lymph nodes were isolated and single cell suspensions were stained for Tg TCR (Valfa2, Valfa5), CD4 and CFSE dilution of the Tg CD4 T cells was analysed. Additionally, cells were co-stained for FoxP3 (after fix and permeabilisation) and the amount of FoxP3+ CFSE+ TCR Tg T-cells was determined after i.v. injection of antigen (C). The adoptively transferred CD4+ T-cell population contained 99% CD25− T cells, indicating that no naturally occurring CD4+ CD25+ Treg was transferred (D). One representative experiment out of two is shown. P-value &lt;0.05 was considered significantly different from responses to native OVA. 
           [0051]      FIG. 7 : Injection of Sia-OVA prevents the generation of effector cells in-vivo. To examine the strength of Sia-OVA induced tolerance in-vivo, C57BL/6 mice were injected with 100 ug Sia-OVA i.v. Control mice were injected with 100 ug native OVA. One week later, mice were sensitized by injection with 200 ug OVA/25 ug antiCD40 and 50 ug poly I:C. Another week later, mice were sacrificed, spleens were isolated and evaluated for the presence of FoxP3 +  T cells, either after fixating, permeabilisation and staining for CD4 and FoxP3 (A. Left panel) or by RT-PCR after RNA isolation (A, right panel). In addition, splenocyted were restimulated for 5 h with OVA 257-264 in the presence of BrefeldinA, cells were harvested, fixed and permeabilised and stained for CD4 and IFNγ (B, left panel). In addition, the presence of IFNγ in culture supernatants was analysed by ELISA (B, right panel). Additionally, spleen cells were restimulated for 24 h with OVA 265-279; BrefeldinA was present during the last 6 hours. Cells were harvested, fixed and permeabilised and stained for CD4 and IFNγ (C), or IL10 (D, left panel). The presence of IL10 in culture supernatants was also analysed by ELISA in cultures that didnot contain BrefeldinA (D, right panel). One representative experiment out of three is shown. Responses were compared with non-treated naive mice. P-value &lt;0.05 was considered significantly different from responses to native OVA. 
           [0052]      FIG. 8 : Low CD40 expression on DC loaded with Sia-OVA. BMDC were incubated with Sia-OVA or native OVA in the absence or presence of LPS. Control DC were incubated with medium or LPS. 24 h later, cells were stained with anti-CD40 and CD11c antibodies and expression of CD40 on CD11c+ DC was analysed using flow cytometry. 
       
    
    
     EXAMPLES  
     Example 1 
     Mice 
       [0053]    C57BL/6 mice were purchased from Charles River Laboratories and used at 8-12 weeks of age. OT-1 and OT-11 TCR transgenic mice were bred and kept in our animal facility under specific pathogen-free conditions. All experiments were approved by the Animal Experiments Committee of the VUmc. 
       Example 2 
     Bone Marrow-Derived DC 
       [0054]    BMDC were cultured as previously described by Lutz et. al. J.I. Methods 223, 77-92,1999) with minor modifications. Femur and tibia of mice were removed, both ends were cut and the marrow was flushed with Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM; Gibco, CA, USA). The resulting marrow suspension was passed over 100 μm gauze to obtain a single cell suspension. After washing, 2×106 cells were seeded per 100 mm dish (Greiner Bio-One, Alphen aan de Rijn, The Netherlands) in 10 ml IMDM, supplemented with 10% FCS; 2 mM L-glutamine, 50 U/ml penicillin, 50 ug/ml streptomycin (BioWhittaker, Walkersville, Md.) and 50 μM β-mercaptoethanol (Merck, Damstadt, Germany) (=IMDMc) and containing 30 ng/ml recombinant murine GM-CSF (rmGM-CSF). At day 2, 10 ml medium containing 30 ng/ml rmGM-CSF was added. At day 5 another 30 ng/ml rmGM-CSF was added to each plate. From day 6 onwards, the non-adherent DC were harvested and used for subsequent experiments. 
       Example 3 
     Antibodies 
       [0055]    Unconjugated mouse anti-chicken egg albumin (OVA) antibody (OVA-14) was purchased from Sigma Aldrich. FITC-labeled antibodies used were anti-CD11c (clone N418) and anti-CD4 (clone GK1.5). 
         [0056]    PE-labeled antibodies were anti-IL-4 (clone 11B11), anti-IL-17 (clone eBioTC11-18H10.1), anti-CD40 (clone MR1), anti-CD80 (clone 16-10-A1), anti-CD86 (clone GL-1), anti-MHC class-II (clone ?,-. APC-labeled antibodies used were anti-CD11c (clone N418), anti-IFNγ (clone XMG1.2) and anti-FoxP3 (clone FJK-16s). All antibodies were purchased from e-Bioscience (Belgium) or BD Biosciences (Belgium)). 
         [0057]    Secondary antibodies used in this study were peroxidase-labeled goat anti-human IgG and goat anti-mouse IgG (Jackson, West grove, Pa., USA). 
       Example 4 
     Generation of sia-2,3-OVA 
       [0058]    3′-Sialyllactose (Neu5Acα2-3Galβ1-4Glc; Dextra labs, UK) was conjugated to Ovalbumin (Calbiochem, Darmstadt, Germany) creating OVA-sia-2,3 using a bifunctional cross linker (4-N-Maleimidophenyl butyric acid hydrazide; MPBH; Pierce, Rockford, USA). In short, via reductive amination, the hydrazide moiety of the linker is covalently linked to the reducing end of the carbohydrate. Hereto, the mixtures were incubated for 2 h at 70°°C. After cooling down to RT, 1 ml ice-cold isopropanol (HPLC grade; Riedel de Haan, Seelze, Germany) was added and the mixture was further incubated at −20° C. for 1 h. Subsequently, the precipitated derivatised carbohydrates were pelleted and dissolved in 1 mM HCl. Ovalbumin was added to derivatised carbohydrates at a 1:10 molar ratio (OVA:carbohydrate) and conjugation was performed o/n at 4° C. The neo-glycoconjugate was separated from reaction-reductants using a PD-10 desalting column (Pierce, Rockford, USA). The concentration of OVA was determined using the bicinchoninic acid assay (Pierce, Rockford, Ill.). Potential endotoxin contamination was determined using a chromogenic LAL endotoxin assay kit (fabrikant). Both OVA-sia2,3 and native OVA were devoid of any endotoxin (Supplemental  FIG. 1A ). 
         [0059]    Additionally, a Dylight 549-N-hydroxysuccimide (NHS) label (Thermo Scientific, Rockford, USA) was covalently coupled to OVA or OVA-sia-2,3 (Dylight-549-OVA). Free label was removed using a PD-10 column (Pierce). 
         [0060]    Presence of sia-2,3 on OVA was measured by ELISA. In brief, OVA-sia-2,3 was coated directly onto ELISA plates (NUNC Maxisorb, Roskilde, Denmark) and binding of the plant lectin Maackia amurensis (MAA, Vector Laboratories Inc) was determined as described {Singh, 2010 90/id}, data are shown in Supplemental  FIG. 1B . 
       Example 5 
     Binding/Uptake Assays 
       [0061]    5×10 4  BMDC were plated in 96 well round-bottom plates and Dylight 549-labeled antigen (30 μg/ml) was added. Cells were incubated with antigen for 30 min at 4° C. to determine binding, or 1, 2 and 4 h at 37° C. to determine binding/uptake. 
       MHC Class I and Class II-Restricted Antigen-Presentation Assay 
       [0062]    BMDC (2.5×10 4 /well) were incubated with indicated concentrations of antigen in 96-well round bottom plates for four hours. After washing, either 5×10 4  purified OVA-specific CD4+ or CD8+ T-cells were added to each well. OVA-specific CD4+ and CD8+ T-cells were isolated from lymphoid tissue of OT-I or OT-II mice, respectively. In brief, lymph nodes and spleen were collected and single cell suspensions were obtained by straining the spleens and lymph nodes through a 100 μm gauze. Erythrocytes were depleted by incubation in ACK-lysis buffer and CD4+ or CD8+ T-cells were isolated from the single cell suspensions using the Dynal mouse CD4 or CD8 negative isolation kit (Invitrogen, CA, USA) according to the manufacturer&#39;s protocol. Proliferation was assessed by [3H]-thymidine incorporation. [3H]-thymidine (1 μC/well; Amersham Biosciences, NJ, USA) was added for the last 16 h of a 3 day culture. Cells were harvested onto filters and [3H]-thymidine incorporation was assessed using a Wallac microbeta counter (Perkin-Elmer, USA). 
       Example 6 
     In-vitro CD4+Thelper Differentiation Assay 
       [0063]    10 4  BMDC were incubated with 30 μg/ml neo-glycoconjugate or native OVA for 4 h in 96-wells round bottom plates. After washing, 5×104 purified naive CD4+CD62LhiCD25− T-cells isolated from OT-II mice were added to each well. On day 2, 10 IU rmlL-2 was added. On day 7, expression of FoxP3 was analysed using the FoxP3 staining kit (e-Bioscience). Addionally, the frequency of IFNg+, IL4+ or IL17A+ T-cells was determined by intracellular staining. Hereto, T-cells were activated with PMA and ionomycin (100 ng/ml and 1 μg/ml; Sigma) for 6h in the presence of Brefeldin A (Sigma). Cells were co-stained for CD4 and analyzed using a FACScalibur. 
       Example 7 
     cDNA Synthesis and Real Time PCR 
       [0064]    mRNA was isolated by capturing poly(A+)RNA in streptavidin-coated tubes using a mRNA Capture kit (Roche, Basel, Switzerland). cDNA was synthesized using the Reverse Transcription System kit (Promega, WI, USA) following manufacturers guidelines. Real time PCR reactions were performed using the SYBR Green method in an ABI 7900HT sequence detection system (Applied Biosystems). 
       Example 8 
     In-vitro Analysis of Treq Induction 
       [0065]    Loading of ex-vivo isolated splenic DC with Sia-OVA in-vitro results in generation of tolerogenic DC that induce naïve CD4 +  Thelper differentiation towards Treg lineage 
         [0066]    10 4  BMDC were incubated with 30 μg/ml Sia-OVA or native OVA for 4 h in 96-wells round bottom plates. After washing, 5×104 purified naive CD4 + CD62L hi CD25 −  T-cells isolated from secondary lymphoid tissue of OT-Il Tg mice were added to each well. On day 2, 10 IU rmlL-2 was added. On day 7, expression of FoxP3 was analyzed using a FoxP3 staining kit (e-Bioscience). Additionally, the frequency of IFNγ + , IL4 +  and IL17A +  T-cells was determined by intracellular staining. Hereto, T-cells were activated with PMA and ionomycin (100 ng/ml and 1 μg/ml; Sigma) for 6 h in the presence of Brefeldin A (Sigma). Cells were co-stained for CD4 and analyzed using a FACScalibur. 
         [0067]    We observed that also incubation of naïve OVA-specific CD4 +  T-cells with ex-vivo isolated and Sia-OVA loaded splenic DC results in generation of increased numbers of FoxP3+ CD4+ T-cells compared to native OVA-loaded DC ( FIG. 4A ). Hardly any IFNγ-producing T-cells were detected ( FIG. 4B ). Neither IL4- nor IL17-producing T-cells were detected in T-cells primed by SIA-OVA or native OVA-loaded DC (not shown). 
         [0068]    The induced FoxP3 +  T cells were tested for their suppressive capacities. Hereto, they were added to co-cultures of naïve CD4+ OT-II responder T-cells and OVA-loaded DC. By labeling the responder T cells with CFSE, their proliferation can be analyzed via flow cytometry. Only T-cells primed by Sia-OVA-loaded DC suppressed the proliferation of responder T cells ( FIG. 4C ). T-cells primed by OVA-loaded DC or naive T cells did not affect the proliferation of the responder T cells. 
         [0069]    To assess the strength of DC modulation by SIA-OVA uptake (and thus the applicability of administration of sialylated antigens in patients with ongoing immune responses), we loaded ex-vivo isolated splenic DC with Sia-OVA in the presence of LPS (100 ng/ml). Even in this setting, FoxP3 +  T-cell generation was detected. Moreover, whereas OVA-LPS loaded DC induced IFNγ production in OVA-reactive T cells, this was not observed in cultures with Sia-OVA-LPS loaded DC ( FIG. 5A ). Analysis of culture supernatants showed reduced TNFα, IFNγ and IL6 concentrations than culture supernatants from T cells and DC-OVA-LPS ( FIG. 5B ). 
       Example 9 
     In vivo Experiments 
       [0070]    The potency of sialylated antigens to induce tolerance in-vivo was analyzed in different models. 
         [0071]    C57BL/6 mice were adoptively transferred with CFSE-labeled CD4+ OT-II T-cells. One day later, mice were injected with 100 μg OVA-SIA or native OVA i.v. 
         [0072]    or s.c. and three days later, lymphoid tissues were analyzed for the proliferation of the transferred OVA-specific CD4 T-cells. Control mice received PBS, which did not lead to proliferation of the transferred CD4 T-cells ( FIG. 6A ). We observed that injection of OVA induced massive proliferation ( FIG. 6A ), irrespective of site used for injection (i.v. or s.c.). However, i.v. injection of Sia-OVA resulted in reduced proliferation of the transferred OT-II T cells. The reduction in proliferation was observed systemically (spleen and lymph nodes). Injection of Sia-OVA s.c. did not show prominent effects on OT-II T cell proliferation in the draining lymph nodes compared to OVA ( FIG. 6B ). When analyzing the phenotype of the transferred OT-II T cells we observed that only in the Sia-OVA injected mice, the T cells were positive for FoxP3 ( FIG. 6C ). Since the injected OT-II T-cells were CD25 − CD4 +  T cells, thus devoid of CD25 + CD4 +  naturally occurring Treg, these data show that injection of Sia-OVA results in de novo induction of FoxP3+ Treg ( FIG. 6D ). 
         [0073]    Furthermore, these data suggest that the receptor for Sia is mostly present on antigen presenting cells, in particular on DC in the spleen. 
         [0074]    Since i.v. injection of Sia-OVA had such prominent effects on FoxP3 +  T cell generation in-vivo, we assessed whether these cells could prevent the generation of effector T cells. Hereto, C57BL/6 mice were treated with Sia-OVA before immunization. This group was compared with mice treated with OVA. Mice were immunized one week later by i.v. injection of 100 μg OVA mixed with 25 μg aCD40 and poly I:C. One week after immunization, spleens were collected and the frequency of FoxP3 +  CD4 +  T cells was analyzed by flow cytometry. Compared to naïve control mice, there was a significant increase in the percentage of FoxP3 +  T-cells detected in the spleens of Sia-OVA but not native OVA treated mice. This was also significantly higher than the percentage detected in spleens of native OVA treated mice ( FIG. 7A  left panel), which was confirmed by RT-PCR on total splenocytes ( FIG. 7A  right panel). 
         [0075]    In addition, the presence of CD8 and CD4 effector T cells was determined upon in-vitro re-stimulation with OVA peptides (OVA 257-264  and OVA 265-279 , respectively) and intracellular cytokine staining. The percentage of IFNγ-producing CD8 T-cells was significantly reduced in Sia-OVA treated mice compared to OVA treated mice ( FIG. 7B , left). This was confirmed when measuring IFNγ levels in the supernatant of parallel cultures ( FIG. 7B , right). Analysis of IFNg production by CD4 T cells did not show significant differences ( FIG. 7C ). This may be due to the fact that induced Treg have been shown to produce IFNγ as well (e.g. Tr1 cells). Hereto, simultaneous analysis for IL10 should be performed in future to discriminate these IL10 and IFNγ-producing Treg from IFNγ-producing effector T cells. 
         [0076]    Analysis of IL10-producing CD4 T-cells showed that there was a significantly increased percentage of IL10-secreting T cells in the spleens of SIA-OVA treated mice. However, this was not significantly different from the percentage that was found in spleens of native OVA treated mice ( FIG. 7D , left). These data were confirmed when analyzing the supernatants of splenocytes after o/n culture ( FIG. 7D , right). 
         [0077]    Furthermore, our experiments clearly showed that when we injected DC in vitro loaded with SIA-OVA into C57BL/6 mice, followed by a challenge with OVA+CpG, we observed a strong induction of FoxP3+Treg and a decrease of effector CD4 T-cell induction. This clearly shows that induction of tolerance in vivo is mediated by DC. 
       Example 10 
     Modulation of DC 
       [0078]    We have analysed the phenotype of DC after taking up Sia-OVA and compared it with the phenotype of DC that ingested native OVA. This was done in both the absence and presence of LPS. It was shown that CD40 is consistently lower on Sia-OVA loaded DC when compared to OVA loaded DC. 
         [0079]    To get more insight in the underlying mechanism of tolerance induction by Sia-OVA loaded DC, we performed a micro-array analysis. Hereto, DC were incubated with 50 μg/ml Sia-OVA or native OVA and 1 and 6 h later, DC were harvested and RNA was extracted using the nucleospin kit. Genomic DNA was removed using DNAse treatment. RNA quality and integrity was checked by Service XS (Leiden). Based on good quality, RNA was amplified, labeled and hybridized on BeadChip Arrays (MouseWG-6 v2, Illumina). We have compared the normalized gene expression of Sia-OVA DC with OVA-DC and all samples that show more than 10-fold differences (higher or lower) are in Table 2. Most interesting genes seem AIRE (higher in Sia-OVA DC) and the switching on of a type I IFN pathway. Both have been related to tolerance and also seem to be connected with each other. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                 OVA- 
                   
                   
               
               
                   
                   
                 OVA- 
                 sa2,3  
                 OVA- 
                 OVA- 
               
               
                   
                   
                 sa2,3 vs 
                 vs 
                 sa2,6 vs 
                 sa2,6 vs 
               
               
                   
                   
                 OVA 1 h 
                 OVA 6 h 
                 OVA 1 h 
                 OVA 6 h 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 ILMN_2659408 
                 Rel 
                 1,028345 
                 1,103712 
                 0,103688 
                 11,1404 
               
               
                 ILMN_1249750 
                 Reln 
                 0,101298 
                 0,101936 
                 0,999407 
                 102,914 
               
               
                 ILMN_2674533 
                 Renbp 
                 99,51158 
                 0,986405 
                 97,55804 
                 0,978967 
               
               
                 ILMN_2641270 
                 AA536717 
                 0,098526 
                 10,40813 
                 0,995059 
                 98,62595 
               
               
                 ILMN_2605630 
                 AA881470 
                 101,2935 
                 0,977818 
                 98,89889 
                 0,96501 
               
               
                 ILMN_2719139 
                 AB124611 
                 98,90014 
                 0,098043 
                 100,2287 
                 0,930972 
               
               
                 ILMN_1218537 
                 Abca15 
                 102,7413 
                 1,008956 
                 100,6122 
                 9,991803 
               
               
                 ILMN_2663015 
                 Abcb8 
                 1,024122 
                 0,100682 
                 0,099732 
                 100,361 
               
               
                 ILMN_2685157 
                 Abcc3 
                 999,247 
                 0,930306 
                 1001,941 
                 0,938589 
               
               
                 ILMN_1253491 
                 Abcc9 
                 1,006649 
                 0,959709 
                 1,002382 
                 102,7102 
               
               
                 ILMN_2687062 
                 Abr 
                 99,01085 
                 0,997722 
                 98,67048 
                 0,9895 
               
               
                 ILMN_2739219 
                 Acad10 
                 1020,163 
                 0,964181 
                 1012,151 
                 0,984681 
               
               
                 ILMN_1220016 
                 Acbd5 
                 0,984563 
                 0,991942 
                 0,099628 
                 98,83858 
               
               
                 ILMN_2770667 
                 Acin1 
                 0,979681 
                 0,097175 
                 0,946506 
                 99,56963 
               
               
                 ILMN_1216022 
                 Aclp7 
                 1,00318 
                 10,64468 
                 10,17214 
                 104,08 
               
               
                 ILMN_2745889 
                 Acot2 
                 0,980553 
                 9,743136 
                 0,977466 
                 95,41733 
               
               
                 ILMN_1213138 
                 Acy1 
                 97,356 
                 0,952368 
                 99,73741 
                 0,965828 
               
               
                 ILMN_3139103 
                 Adam15 
                 0,099838 
                 8,929052 
                 1,004738 
                 87,97479 
               
               
                 ILMN_1240629 
                 Adam15 
                 104,2488 
                 0,967151 
                 102,0592 
                 0,094326 
               
               
                 ILMN_3134632 
                 Adam22 
                 102,1393 
                 10,06842 
                 100,5282 
                 1,016651 
               
               
                 ILMN_3033533 
                 Add1 
                 1,00665 
                 9,572825 
                 1,012456 
                 93,49712 
               
               
                 ILMN_2738082 
                 Adipoq 
                 0,993226 
                 0,096595 
                 0,995041 
                 99,89807 
               
               
                 ILMN_1215394 
                 Adpgk 
                 10,10091 
                 99,35876 
                 9,956478 
                 0,991647 
               
               
                 ILMN_1215901 
                 Agpat2 
                 10,13781 
                 1,05875 
                 97,95148 
                 1,041015 
               
               
                 ILMN_2972521 
                 Agtr1a 
                 0,09689 
                 1,010311 
                 0,974424 
                 102,1489 
               
               
                 ILMN_2590950 
                 Agtrap 
                 9,612647 
                 103,6355 
                 0,009756 
                 0,102844 
               
               
                 ILMN_2916008 
                 Agxt2l2 
                 0,972468 
                 99,42319 
                 0,950237 
                 0,993325 
               
               
                 ILMN_1258578 
                 Ahnak 
                 1,038872 
                 9,531327 
                 0,105231 
                 95,0189 
               
               
                 ILMN_2684007 
                 Al844366 
                 0,997183 
                 1,013509 
                 1,013799 
                 99,31121 
               
               
                 ILMN_1216550 
                 Al851790 
                 0,993945 
                 0,998739 
                 0,989183 
                 100,3487 
               
               
                 ILMN_2673099 
                 A1987944 
                 0,98241 
                 1,018939 
                 0,996969 
                 101,1484 
               
               
                 ILMN_1213787 
                 Aire 
                 1,026076 
                 0,998319 
                 1,012772 
                 100,1469 
               
               
                 ILMN_1235909 
                 Ak2 
                 9,870461 
                 0,109321 
                 97,77247 
                 1,08545 
               
               
                 ILMN_1246068 
                 Akap12 
                 100,2487 
                 0,102842 
                 101,4649 
                 1,049984 
               
               
                 ILMN_3116504 
                 Akap2 
                 0,100643 
                 1,09025 
                 0,09927 
                 105,2205 
               
               
                 ILMN_2627299 
                 Akap9 
                 1,040274 
                 95,7044 
                 0,987936 
                 9,503727 
               
               
                 ILMN_2661287 
                 Akp2 
                 0,991037 
                 0,102454 
                 0,97956 
                 98,13207 
               
               
                 ILMN_2481458 
                 Akr1b3 
                 99,68519 
                 0,975054 
                 99,46308 
                 1034,701 
               
               
                 ILMN_1214358 
                 Akt1s1 
                 1,017785 
                 103,7958 
                 10,22891 
                 1,061528 
               
               
                 ILMN_3100276 
                 Aldh1l1 
                 10,07558 
                 0,980369 
                 100,8811 
                 0,984862 
               
               
                 ILMN_1224012 
                 Aldob 
                 9,665992 
                 0,985546 
                 97,35459 
                 0,100766 
               
               
                 ILMN_2660414 
                 Alg5 
                 99,73186 
                 1,018703 
                 101,2032 
                 0,098715 
               
               
                 ILMN_2892292 
                 Alg9 
                 1,011797 
                 1,011511 
                 1,016208 
                 102,1624 
               
               
                 ILMN_1235966 
                 Alox12b 
                 99,32697 
                 1,023599 
                 100,3686 
                 101,8012 
               
               
                 ILMN_2681123 
                 Als2cr2 
                 976,0651 
                 0,960205 
                 1005,91 
                 0,091872 
               
               
                 ILMN_2718293 
                 Amelx 
                 99,37019 
                 9,926122 
                 101,5065 
                 0,975022 
               
               
                 ILMN_2859778 
                 Anapc4 
                 0,971498 
                 0,977219 
                 1,006495 
                 100,3286 
               
               
                 ILMN_2568390 
                 Angptl3 
                 0,977892 
                 0,097669 
                 0,999894 
                 974,3339 
               
               
                 ILMN_1253761 
                 Ankrd39 
                 9,849003 
                 96,10191 
                 10,3044 
                 0,95381 
               
               
                 ILMN_2592358 
                 Ankrd49 
                 0,970275 
                 0,977431 
                 0,918654 
                 101,763 
               
               
                 ILMN_1217993 
                 Ankrd6 
                 1,012684 
                 99,42222 
                 0,974492 
                 0,992855 
               
               
                 ILMN_2665496 
                 Ankrd9 
                 102,6115 
                 1,01209 
                 100,6919 
                 1,022893 
               
               
                 ILMN_2735877 
                 Anks3 
                 103,0724 
                 0,910252 
                 103,0553 
                 8,992366 
               
               
                 ILMN_2685507 
                 Anp32a 
                 0,098381 
                 0,953522 
                 1,016315 
                 94,34225 
               
               
                 ILMN_1230010 
                 Anxa10 
                 9,946819 
                 101,7208 
                 9,982971 
                 1,031209 
               
               
                 ILMN_1219115 
                 Apc 
                 0,987325 
                 98,93451 
                 0,961718 
                 0,992878 
               
               
                 ILMN_2449193 
                 Apg4d 
                 97,58354 
                 0,099454 
                 97,66596 
                 0,986757 
               
               
                 ILMN_1232821 
                 Aph1a 
                 102,3822 
                 0,995923 
                 10,24009 
                 100,4748 
               
               
                 ILMN_2916782 
                 Apom 
                 0,988538 
                 0,893169 
                 0,97074 
                 86,82716 
               
               
                 ILMN_2724868 
                 Appbp2 
                 96,78724 
                 1,000733 
                 92,39066 
                 10,28014 
               
               
                 ILMN_1225901 
                 Aqp11 
                 0,998861 
                 0,995061 
                 1,005773 
                 96,73764 
               
               
                 ILMN_2943165 
                 Aqp7 
                 0,097988 
                 0,996017 
                 0,990391 
                 995,3483 
               
               
                 ILMN_1237241 
                 Araf 
                 10,37345 
                 96,13192 
                 10,44231 
                 0,096285 
               
               
                 ILMN_2649846 
                 Arcn1 
                 103,324 
                 0,963489 
                 10,08846 
                 0,098458 
               
               
                 ILMN_2743425 
                 Arfip1 
                 10,03356 
                 0,961122 
                 100,2037 
                 0,935154 
               
               
                 ILMN_2613531 
                 Arhgap21 
                 1,003831 
                 0,981488 
                 0,980804 
                 96,2602 
               
               
                 ILMN_2589999 
                 Arl10c 
                 0,985403 
                 89,7199 
                 0,975282 
                 8,821913 
               
               
                 ILMN_3066763 
                 Arl4a 
                 104,433 
                 1,050157 
                 103,2892 
                 1,022906 
               
               
                 ILMN_1247625 
                 Arp3b-pending 
                 1,006938 
                 0,971162 
                 1,001264 
                 964,7989 
               
               
                 ILMN_2666279 
                 Arrdc3 
                 1,077088 
                 0,086627 
                 1,058399 
                 897,2452 
               
               
                 ILMN_2679609 
                 Art1 
                 0,101671 
                 9,850694 
                 1,014597 
                 101,1813 
               
               
                 ILMN_2629591 
                 Asah1 
                 105,1951 
                 0,118185 
                 10,55519 
                 1,141375 
               
               
                 ILMN_2663555 
                 Asb3 
                 101,6378 
                 1,13001 
                 9,793706 
                 1,121915 
               
               
                 ILMN_3075168 
                 Ash2l 
                 0,969681 
                 0,098741 
                 1,00595 
                 100,8362 
               
               
                 ILMN_3006123 
                 Asns 
                 96,1865 
                 1,016376 
                 98,12903 
                 0,968906 
               
               
                 ILMN_2776700 
                 Asph 
                 10,012 
                 101,4094 
                 10,22624 
                 0,999028 
               
               
                 ILMN_2594584 
                 Asph 
                 100,5377 
                 9,837391 
                 98,59214 
                 0,101903 
               
               
                 ILMN_2629103 
                 Atcay 
                 10,20191 
                 0,977784 
                 9,779767 
                 99,57801 
               
               
                 ILMN_2620574 
                 Atg16l1 
                 982,569 
                 9,468761 
                 101,9885 
                 0,967669 
               
               
                 ILMN_2606567 
                 Atic 
                 97,46479 
                 0,963219 
                 99,36086 
                 1,006792 
               
               
                 ILMN_1258206 
                 Atm 
                 99,02622 
                 0,977615 
                 9,747839 
                 1,012082 
               
               
                 ILMN_3038944 
                 Atp2b2 
                 1,032362 
                 1011,428 
                 0,980498 
                 0,999334 
               
               
                 ILMN_2973897 
                 Atp5l 
                 95,48242 
                 1,000159 
                 97,48098 
                 0,994841 
               
               
                 ILMN_2680440 
                 Atp6v1b2 
                 99,90241 
                 0,953126 
                 100,8208 
                 0,9605 
               
               
                 ILMN_2755322 
                 Atp6v1e2 
                 101,832 
                 0,994822 
                 99,59874 
                 1,0165 
               
               
                 ILMN_1255220 
                 Atp9a 
                 0,100331 
                 10,10136 
                 1,008405 
                 101,7454 
               
               
                 ILMN_1229377 
                 AU017455 
                 0,955379 
                 992,8015 
                 0,94251 
                 0,998757 
               
               
                 ILMN_2919343 
                 Aven 
                 97,77753 
                 1,005044 
                 99,18759 
                 10,18624 
               
               
                 ILMN_2755585 
                 Avpi1 
                 1,012887 
                 1,08022 
                 1,030473 
                 110,9546 
               
               
                 ILMN_1251934 
                 Azi2 
                 101,4731 
                 9,879583 
                 99,25178 
                 9,993418 
               
               
                 ILMN_1247168 
                 B130032G09Rik 
                 9,890892 
                 10,15862 
                 100,8566 
                 1,005667 
               
               
                 ILMN_1257672 
                 B230205M18 
                 1,005101 
                 0,999803 
                 1,014716 
                 97,46832 
               
               
                 ILMN_2565428 
                 B230325K09Rik 
                 9,968016 
                 0,983847 
                 9,758051 
                 995,26 
               
               
                 ILMN_1235144 
                 B230399H06Rik 
                 101,9651 
                 0,102365 
                 100,7778 
                 1,012485 
               
               
                 ILMN_2669708 
                 B3gat2 
                 1011,864 
                 0,98462 
                 1008,746 
                 1,014485 
               
               
                 ILMN_3149776 
                 B3gnt8 
                 10,06867 
                 90,3311 
                 103,1875 
                 0,91006 
               
               
                 ILMN_1216802 
                 Bad 
                 0,102461 
                 0,957779 
                 0,009748 
                 94,21028 
               
               
                 ILMN_2665609 
                 Baiap2l1 
                 988,7948 
                 1,024328 
                 976,3332 
                 0,999748 
               
               
                 ILMN_2749866 
                 Bap1 
                 9,676667 
                 0,959977 
                 0,973136 
                 95,05828 
               
               
                 ILMN_2652385 
                 Baz2a 
                 1,000717 
                 9,973698 
                 0,964416 
                 98,74386 
               
               
                 ILMN_2684272 
                 Bbs9 
                 102,191 
                 1,011412 
                 101,3924 
                 9,963482 
               
               
                 ILMN_3006534 
                 BC003885 
                 99,0739 
                 1,03535 
                 101,7832 
                 1,02412 
               
               
                 ILMN_3133238 
                 BC013491 
                 99,14106 
                 0,990603 
                 97,97291 
                 1,035456 
               
               
                 ILMN_2688176 
                 BC046418 
                 0,983674 
                 0,985768 
                 1,005455 
                 96,46881 
               
               
                 ILMN_2960128 
                 BC048502 
                 0,099621 
                 0,979413 
                 0,992124 
                 100,9488 
               
               
                 ILMN_2664291 
                 BC055111 
                 99,83961 
                 0,099096 
                 98,75682 
                 0,984474 
               
               
                 ILMN_2993962 
                 BC099439 
                 0,981124 
                 1033,503 
                 0,098784 
                 1,026491 
               
               
                 ILMN_2677422 
                 Bcl2l14 
                 100,8667 
                 0,98433 
                 102,2935 
                 0,959676 
               
               
                 ILMN_2713638 
                 Bcmo1 
                 0,997612 
                 0,993911 
                 0,992652 
                 98,05958 
               
               
                 ILMN_2639819 
                 Bet1l 
                 9,780487 
                 0,998096 
                 97,10562 
                 0,100206 
               
               
                 ILMN_2681241 
                 Birc5 
                 0,102187 
                 101,0209 
                 0,099758 
                 0,98774 
               
               
                 ILMN_2910258 
                 Bnc1 
                 1,050197 
                 0,978384 
                 1,004996 
                 103,3005 
               
               
                 ILMN_2846368 
                 Bola2 
                 98,00094 
                 0,937006 
                 97,37207 
                 0,930611 
               
               
                 ILMN_1253942 
                 Bop1 
                 93,89475 
                 10,0153 
                 94,85711 
                 1,036676 
               
               
                 ILMN_1243635 
                 Brunol4 
                 97,2682 
                 1,007737 
                 98,66806 
                 1,014305 
               
               
                 ILMN_1224958 
                 C030015H18 
                 98,21568 
                 0,09935 
                 100,2623 
                 0,989865 
               
               
                 ILMN_1259185 
                 C030048B08Rik 
                 101,1684 
                 0,995838 
                 102,8585 
                 10,23537 
               
               
                 ILMN_1233652 
                 C130015E15Rik 
                 103,0576 
                 1,014571 
                 99,38498 
                 0,985424 
               
               
                 ILMN_2753279 
                 C130023O10Rik 
                 96,23538 
                 10,02187 
                 98,53216 
                 1,002206 
               
               
                 ILMN_2754119 
                 C130039O16Rik 
                 0,976549 
                 1,000482 
                 0,997887 
                 986,9044 
               
               
                 ILMN_1223290 
                 C130046N05Rik 
                 1,038869 
                 0,100686 
                 1,016037 
                 10044,12 
               
               
                 ILMN_1228917 
                 C330023M02Rik 
                 0,956375 
                 1,032472 
                 0,945739 
                 1018,341 
               
               
                 ILMN_2702286 
                 Cacnb3 
                 1,020904 
                 1,150696 
                 1,025121 
                 114,2253 
               
               
                 ILMN_1241128 
                 Calcoco1 
                 996,5213 
                 0,089271 
                 100,732 
                 0,874231 
               
               
                 ILMN_1257323 
                 Car6 
                 1,012838 
                 0,978974 
                 0,989067 
                 1002,46 
               
               
                 ILMN_2866175 
                 Card14 
                 9,803762 
                 10,16014 
                 9,931693 
                 102,0377 
               
               
                 ILMN_1220811 
                 Caskin1 
                 1,014175 
                 0,996775 
                 1,009265 
                 98,46722 
               
               
                 ILMN_2865939 
                 Ccdc100 
                 9,960177 
                 9,40414 
                 10,06811 
                 92,64171 
               
               
                 ILMN_2745151 
                 Ccdc123 
                 0,098653 
                 0,972197 
                 0,961661 
                 96,12732 
               
               
                 ILMN_2756733 
                 Ccdc130 
                 100,0595 
                 0,959578 
                 99,43568 
                 0,951075 
               
               
                 ILMN_2671436 
                 Ccdc77 
                 101,2349 
                 0,964637 
                 96,68356 
                 0,988785 
               
               
                 ILMN_2752408 
                 Ccdc90b 
                 1,007825 
                 0,10722 
                 0,991932 
                 103,8052 
               
               
                 ILMN_2862179 
                 Ccl11 
                 98,9178 
                 0,982289 
                 97,0845 
                 0,983082 
               
               
                 ILMN_2771176 
                 Ccl7 
                 83,83454 
                 0,122139 
                 89,36513 
                 1,25809 
               
               
                 ILMN_2863768 
                 Ccnb3 
                 0,992992 
                 0,999488 
                 0,988422 
                 97,21907 
               
               
                 ILMN_2669793 
                 Ccnd1 
                 0,998637 
                 0,101775 
                 0,966321 
                 102,7623 
               
               
                 ILMN_3131063 
                 Ccnd3 
                 0,963618 
                 8,522801 
                 0,098333 
                 85,32755 
               
               
                 ILMN_2696291 
                 Cd209d 
                 100,111 
                 0,100882 
                 101,6694 
                 0,990543 
               
               
                 ILMN_2665757 
                 Cd209e 
                 0,977415 
                 0,100134 
                 0,982201 
                 9,986747 
               
               
                 ILMN_3117602 
                 Cd6 
                 1022,289 
                 9,172848 
                 1033,979 
                 0,091576 
               
               
                 ILMN_2586179 
                 Cd69 
                 0,969605 
                 1,010149 
                 9,560333 
                 103,03 
               
               
                 ILMN_2731282 
                 Cd8a 
                 10,24021 
                 1,014975 
                 10,22786 
                 1016,535 
               
               
                 ILMN_1244296 
                 Cdc14b 
                 0,101063 
                 1,001597 
                 1,007298 
                 98,53194 
               
               
                 ILMN_2612206 
                 Cdc20 
                 1006,605 
                 0,93934 
                 974,0858 
                 9,245129 
               
               
                 ILMN_1250900 
                 Cdk7 
                 101,045 
                 0,982047 
                 985,5148 
                 0,956287 
               
               
                 ILMN_2732437 
                 Chrna6 
                 1,018822 
                 1,05132 
                 1,008538 
                 995,2643 
               
               
                 ILMN_1235663 
                 Cnot8 
                 101,847 
                 1,01818 
                 102,0317 
                 9,862585 
               
               
                 ILMN_2589422 
                 Col6a1 
                 0,97806 
                 0,984973 
                 1,011739 
                 97,71503 
               
               
                 ILMN_2671689 
                 Cox7b 
                 100,9042 
                 1,056407 
                 100,3663 
                 1,022861 
               
               
                 ILMN_1236346 
                 Cpeb2 
                 1,0092 
                 1,006872 
                 1,004107 
                 101,0207 
               
               
                 ILMN_2877900 
                 Cpne5 
                 0,99407 
                 1,018435 
                 1,014962 
                 102,8263 
               
               
                 ILMN_2913078 
                 Cps1 
                 9,861924 
                 0,100572 
                 9,963818 
                 101,7166 
               
               
                 ILMN_1213549 
                 Creb3l4 
                 0,95467 
                 9995,844 
                 9,819902 
                 0,983614 
               
               
                 ILMN_1216758 
                 Crem 
                 101,0591 
                 0,992101 
                 100,802 
                 0,970744 
               
               
                 ILMN_1233069 
                 Crh 
                 97,64674 
                 0,999484 
                 1010,487 
                 0,986246 
               
               
                 ILMN_2907964 
                 Crim2 
                 0,989478 
                 0,929252 
                 0,99025 
                 93,03724 
               
               
                 ILMN_2987844 
                 Crk 
                 101,1075 
                 1,011071 
                 101,1205 
                 0,993613 
               
               
                 ILMN_2668253 
                 Crkrs 
                 0,965559 
                 1,010495 
                 0,099876 
                 99,35456 
               
               
                 ILMN_2728094 
                 Cryba1 
                 100,1304 
                 0,986998 
                 100,073 
                 1,024256 
               
               
                 ILMN_2613659 
                 Ctdp1 
                 94,92608 
                 9,832516 
                 97,15404 
                 0,958597 
               
               
                 ILMN_2858769 
                 Ctps2 
                 1,009855 
                 0,980505 
                 0,99267 
                 98,51342 
               
               
                 ILMN_1253235 
                 Cugbp2 
                 98,95966 
                 0,932621 
                 97,68206 
                 0,992364 
               
               
                 ILMN_2760019 
                 Cxcl13 
                 98,46601 
                 1,022856 
                 98,65985 
                 1,014522 
               
               
                 ILMN_2659426 
                 Cxcl14 
                 1,001054 
                 0,953478 
                 0,997075 
                 982,147 
               
               
                 ILMN_3078306 
                 Cyb561d1 
                 100,5749 
                 0,952014 
                 101,5275 
                 0,945079 
               
               
                 ILMN_1241818 
                 Cyp2c54 
                 10,05082 
                 0,994231 
                 100,6704 
                 1,033107 
               
               
                 ILMN_2525402 
                 D10Bwg1379e 
                 9968,751 
                 0,098971 
                 9781,422 
                 0,995327 
               
               
                 ILMN_2691157 
                 Dctn1 
                 0,986458 
                 0,104052 
                 0,095007 
                 108,3556 
               
               
                 ILMN_2446727 
                 Ddhd1 
                 105,4707 
                 0,900495 
                 103,8381 
                 0,09623 
               
               
                 ILMN_1259277 
                 Ddx28 
                 0,097655 
                 9,727763 
                 0,962635 
                 953,9783 
               
               
                 ILMN_2692412 
                 Defb2 
                 96,91346 
                 1,010442 
                 97,91363 
                 0,996252 
               
               
                 ILMN_1229247 
                 Defb41 
                 0,978942 
                 1,001083 
                 1,014259 
                 100,6946 
               
               
                 ILMN_2658961 
                 Dgka 
                 0,100532 
                 1,006368 
                 0,1036 
                 996,6944 
               
               
                 ILMN_3101919 
                 Dgkh 
                 0,995508 
                 1,000368 
                 1,027322 
                 96,16117 
               
               
                 ILMN_2462151 
                 Dgkq 
                 99,91771 
                 1,008908 
                 100,2425 
                 0,984353 
               
               
                 ILMN_2915059 
                 Dgkz 
                 94,59734 
                 8,56831 
                 9,67789 
                 8,70217 
               
               
                 ILMN_1222841 
                 Dgl1-pending 
                 97,55169 
                 0,989549 
                 99,01643 
                 1,003833 
               
               
                 ILMN_1233008 
                 Dhx30 
                 98,64991 
                 0,967717 
                 100,4042 
                 0,009669 
               
               
                 ILMN_2611098 
                 Dip2b 
                 100,9192 
                 9,737461 
                 100,2285 
                 0,98252 
               
               
                 ILMN_2746556 
                 Dkk3 
                 99,38057 
                 0,965588 
                 100,6638 
                 10,00113 
               
               
                 ILMN_2627081 
                 Dkkl1 
                 102,0234 
                 0,981478 
                 100,266 
                 1,000026 
               
               
                 ILMN_2914010 
                 Dmwd 
                 98,03993 
                 9,084114 
                 100,2711 
                 0,89593 
               
               
                 ILMN_2725428 
                 Dnajb10 
                 103,5862 
                 1,077698 
                 103,9828 
                 1,09103 
               
               
                 ILMN_2751925 
                 Dpp3 
                 95,03934 
                 0,957978 
                 95,86784 
                 0,936473 
               
               
                 ILMN_2677494 
                 Drg2 
                 1,001753 
                 1,032778 
                 0,998074 
                 99,72893 
               
               
                 ILMN_2775813 
                 Dusp12 
                 99,66745 
                 1,009156 
                 95,62582 
                 0,978808 
               
               
                 ILMN_3053158 
                 Dyrk1b 
                 103,1909 
                 0,937689 
                 106,0038 
                 0,927979 
               
               
                 ILMN_2572643 
                 E330034F13Rik 
                 0,100319 
                 10,24731 
                 1,010142 
                 1041,036 
               
               
                 ILMN_2702508 
                 Ebna1bp2 
                 9,687253 
                 0,099876 
                 97,12832 
                 10,03834 
               
               
                 ILMN_2861879 
                 Edar 
                 1,011165 
                 1,00212 
                 0,099657 
                 95,22979 
               
               
                 ILMN_2643355 
                 Edaradd 
                 1,00308 
                 1,01157 
                 0,098737 
                 100,847 
               
               
                 ILMN_2765015 
                 Eed 
                 100,1992 
                 0,999645 
                 99,85057 
                 1,023327 
               
               
                 ILMN_3061673 
                 Eef1d 
                 997,3336 
                 1,015619 
                 968,0081 
                 0,988045 
               
               
                 ILMN_2846821 
                 EG328280 
                 97,56242 
                 9,888982 
                 97,48851 
                 1,009572 
               
               
                 ILMN_2493668 
                 EG330031 
                 99,10159 
                 10,03361 
                 102,1472 
                 1,017641 
               
               
                 ILMN_1242669 
                 Egflam 
                 99,08995 
                 0,988674 
                 98,23838 
                 0,09474 
               
               
                 ILMN_2653543 
                 Egr3 
                 98,40926 
                 0,995354 
                 100,3774 
                 0,970772 
               
               
                 ILMN_2789601 
                 Eif3i 
                 0,993122 
                 0,977201 
                 0,099271 
                 99,0758 
               
               
                 ILMN_1243394 
                 Eif4b 
                 99,77838 
                 0,978956 
                 102,0176 
                 0,968495 
               
               
                 ILMN_1254206 
                 Eif4e1b 
                 99,26624 
                 1,009229 
                 98,58994 
                 1,023777 
               
               
                 ILMN_2697304 
                 Eln 
                 0,998608 
                 0,09873 
                 0,100388 
                 98,20773 
               
               
                 ILMN_2614752 
                 Elovl6 
                 97,7495 
                 0,939998 
                 103,1668 
                 0,092278 
               
               
                 ILMN_2757062 
                 ENSMUSG00000033219 
                 103,4994 
                 9,774745 
                 101,3423 
                 1,041206 
               
               
                 ILMN_1258722 
                 ENSMUSG00000042857 
                 101,8688 
                 9,759741 
                 100,9402 
                 1,000307 
               
               
                 ILMN_3129160 
                 Epas1 
                 99,12285 
                 0,098759 
                 99,41097 
                 0,097499 
               
               
                 ILMN_2686924 
                 Epha1 
                 98,41166 
                 0,99898 
                 98,07619 
                 10,22057 
               
               
                 ILMN_2679830 
                 Epsti1 
                 9,980848 
                 1,009307 
                 9,849766 
                 98,49 
               
               
                 ILMN_1250597 
                 Erbb3 
                 101,7993 
                 1,007096 
                 100,7874 
                 1,036176 
               
               
                 ILMN_2772035 
                 Erc1 
                 0,947979 
                 1,00777 
                 0,973254 
                 100,2977 
               
               
                 ILMN_2992541 
                 Ergic3 
                 10,17137 
                 0,097703 
                 10,27961 
                 95,0453 
               
               
                 ILMN_1213296 
                 Evi5l 
                 0,098612 
                 1,008619 
                 0,998532 
                 98,02823 
               
               
                 ILMN_1229242 
                 F830016N17Rik 
                 992,1156 
                 0,099309 
                 985,5693 
                 0,099541 
               
               
                 ILMN_2826304 
                 Fabp6 
                 103,1885 
                 1,00344 
                 102,212 
                 1,034919 
               
               
                 ILMN_3066293 
                 Fancc 
                 1006,906 
                 0,990837 
                 97,96588 
                 1,010898 
               
               
                 ILMN_2847136 
                 Fastk 
                 99,51694 
                 0,982594 
                 100,8734 
                 97,22678 
               
               
                 ILMN_1226274 
                 Fat4 
                 99,91808 
                 9,778576 
                 102,6167 
                 0,991788 
               
               
                 ILMN_3038394 
                 Fbxl10 
                 1,000968 
                 10,34846 
                 1,003316 
                 101,5194 
               
               
                 ILMN_2633301 
                 Fbxl7 
                 0,996523 
                 9,886947 
                 1,002087 
                 101,1812 
               
               
                 ILMN_2451855 
                 Fbxo45 
                 0,097758 
                 9,846192 
                 0,977467 
                 97,43176 
               
               
                 ILMN_2582084 
                 Fermt2 
                 100,7731 
                 0,997861 
                 103,4975 
                 1,018472 
               
               
                 ILMN_1229698 
                 Fgd4 
                 0,968829 
                 1,003967 
                 0,994546 
                 98,88624 
               
               
                 ILMN_2707356 
                 Fgf13 
                 98,5557 
                 0,979335 
                 101,3539 
                 0,989269 
               
               
                 ILMN_2832105 
                 Fgg 
                 976,712 
                 1,022724 
                 1000,745 
                 0,982923 
               
               
                 ILMN_2748680 
                 Fhit 
                 1,027531 
                 1,002197 
                 0,992864 
                 9753,363 
               
               
                 ILMN_2674132 
                 Fibp 
                 1,005565 
                 1,001045 
                 1,017715 
                 99,94015 
               
               
                 ILMN_1260135 
                 Flnc 
                 0,970813 
                 0,985748 
                 0,101869 
                 98,35351 
               
               
                 ILMN_2702464 
                 Flot1 
                 100,5289 
                 1,008288 
                 98,87429 
                 0,098905 
               
               
                 ILMN_2926842 
                 Flrt2 
                 100,8743 
                 0,99468 
                 98,35319 
                 0,95436 
               
               
                 ILMN_1248190 
                 Flvcr2 
                 101,0148 
                 0,866904 
                 100,9189 
                 0,886543 
               
               
                 ILMN_1240846 
                 Fndc1 
                 100,1766 
                 1,004825 
                 104,85 
                 0,099585 
               
               
                 ILMN_2670517 
                 Fntb 
                 1,004059 
                 0,961594 
                 0,983029 
                 97,38121 
               
               
                 ILMN_1252110 
                 Foxj2 
                 1,037745 
                 0,09719 
                 1,031479 
                 93,83965 
               
               
                 ILMN_1224018 
                 Foxk1 
                 0,955487 
                 0,985807 
                 0,995219 
                 98,28413 
               
               
                 ILMN_2656498 
                 Foxo1 
                 1,037234 
                 1,00469 
                 1,014846 
                 100,0742 
               
               
                 ILMN_1251126 
                 Foxp3 
                 9,77979 
                 9,4417 
                 9,726849 
                 91,25276 
               
               
                 ILMN_2659663 
                 Foxp2 
                 9,659801 
                 1,0488 
                 9,851438 
                 1,012678 
               
               
                 ILMN_2429551 
                 Frmd4a 
                 0,102648 
                 0,925808 
                 1,019704 
                 96,04416 
               
               
                 ILMN_2958016 
                 Fundc1 
                 1,073253 
                 1,094891 
                 1,055057 
                 109,6586 
               
               
                 ILMN_2674979 
                 Fus 
                 9,371913 
                 1,012155 
                 96,29235 
                 1,005847 
               
               
                 ILMN_2939666 
                 Fzd2 
                 992,6725 
                 9,804943 
                 1041,343 
                 0,992843 
               
               
                 ILMN_2774825 
                 G3bp1 
                 97,62113 
                 1,012218 
                 99,58618 
                 1,000295 
               
               
                 ILMN_2646380 
                 Gabpb1 
                 0,099944 
                 1,112443 
                 1,01149 
                 1118,014 
               
               
                 ILMN_3106849 
                 Gal3st3 
                 1,042885 
                 100,5308 
                 1,040509 
                 99,52122 
               
               
                 ILMN_2881155 
                 Gal3st4 
                 10,02421 
                 0,998784 
                 9,969722 
                 99,38768 
               
               
                 ILMN_2860649 
                 Gbp6 
                 100,0182 
                 0,99548 
                 95,27171 
                 0,974757 
               
               
                 ILMN_2875336 
                 Gcat 
                 100,5076 
                 0,958457 
                 101,5154 
                 9,983476 
               
               
                 ILMN_1228316 
                 Gdi1 
                 0,102815 
                 1,027637 
                 1,006134 
                 102,9727 
               
               
                 ILMN_1214319 
                 Gemin6 
                 1,011646 
                 0,992227 
                 1,009633 
                 97,57332 
               
               
                 ILMN_1236845 
                 Gfod2 
                 0,09598 
                 0,097142 
                 0,98029 
                 97,09654 
               
               
                 ILMN_2631363 
                 Gif 
                 0,983927 
                 0,97706 
                 0,965454 
                 97,4734 
               
               
                 ILMN_2721734 
                 Gjd2 
                 0,102873 
                 1,01547 
                 0,101304 
                 966,5576 
               
               
                 ILMN_2685506 
                 Gje1 
                 1,009132 
                 0,990791 
                 0,974513 
                 1011,543 
               
               
                 ILMN_2838605 
                 Glis3 
                 990,414 
                 0,009946 
                 990,063 
                 0,996183 
               
               
                 ILMN_2729364 
                 Glra2 
                 1,000599 
                 0,994161 
                 99,3303 
                 9,891735 
               
               
                 ILMN_1217767 
                 Glrx5 
                 0,094499 
                 9,159094 
                 0,955313 
                 91,33761 
               
               
                 ILMN_1248467 
                 Gm1027 
                 99,22358 
                 0,100963 
                 100,0698 
                 0,102133 
               
               
                 ILMN_2539428 
                 Gm1070 
                 0,959547 
                 99,624 
                 0,994394 
                 1,012881 
               
               
                 ILMN_3029489 
                 Gm129 
                 0,982231 
                 1,020812 
                 1,007998 
                 96,58471 
               
               
                 ILMN_1232057 
                 Gm26 
                 0,100459 
                 0,099178 
                 1,03716 
                 101,3112 
               
               
                 ILMN_1240736 
                 Gm318 
                 1,014537 
                 0,986561 
                 1,024794 
                 9945,749 
               
               
                 ILMN_2598594 
                 Gm443 
                 10,13529 
                 0,994511 
                 99,82462 
                 0,980946 
               
               
                 ILMN_2803319 
                 Gm606 
                 101,3642 
                 0,099711 
                 100,6399 
                 1,037138 
               
               
                 ILMN_3022025 
                 Gm732 
                 0,958279 
                 9,656418 
                 0,958082 
                 98,94724 
               
               
                 ILMN_1229324 
                 Gm757 
                 1,001783 
                 1,018478 
                 1,008558 
                 102,8928 
               
               
                 ILMN_2908855 
                 Gnai2 
                 9,832485 
                 0,094289 
                 9,895567 
                 95,49658 
               
               
                 ILMN_2733433 
                 Gnai3 
                 1020,027 
                 0,103899 
                 1028,753 
                 0,987284 
               
               
                 ILMN_2661635 
                 Gyg 
                 104,6152 
                 1,006181 
                 106,0327 
                 1,023016 
               
               
                 ILMN_2742160 
                 H13 
                 96,85991 
                 0,967374 
                 100,4352 
                 0,944166 
               
               
                 ILMN_2685581 
                 H2-Q5 
                 1,010285 
                 1,046776 
                 10,058 
                 106,6858 
               
               
                 ILMN_1230323 
                 Hbp1 
                 9750,761 
                 0,975145 
                 9891,527 
                 0,992563 
               
               
                 ILMN_2637982 
                 Herc1 
                 0,991905 
                 1,005105 
                 0,009839 
                 104,9426 
               
               
                 ILMN_2723631 
                 Hint1 
                 99,59557 
                 0,991124 
                 98,94354 
                 0,954837 
               
               
                 ILMN_1252995 
                 Hist1h2be 
                 9,861481 
                 9,457645 
                 9,881257 
                 93,87584 
               
               
                 ILMN_2677408 
                 Hrmt1l2 
                 0,967371 
                 0,098508 
                 0,975685 
                 1013,349 
               
               
                 ILMN_2658501 
                 Ifitm3 
                 1,029882 
                 0,104393 
                 1,038346 
                 104,5469 
               
               
                 ILMN_2658633 
                 Ifna7 
                 1,001601 
                 10,32717 
                 1,025378 
                 100,0743 
               
               
                 ILMN_1260493 
                 Ift140 
                 102,3462 
                 0,999771 
                 102,3426 
                 0,096394 
               
               
                 ILMN_2671767 
                 Ift20 
                 1,003421 
                 1,043429 
                 1,001482 
                 102,5302 
               
               
                 ILMN_2788283 
                 Ift52 
                 1,015203 
                 9,422482 
                 0,993875 
                 92,56936 
               
               
                 ILMN_2590585 
                 II1rapl2 
                 9,950188 
                 0,099529 
                 9,807601 
                 99,61221 
               
               
                 ILMN_3155812 
                 II20rb 
                 0,099269 
                 0,985523 
                 0,999115 
                 98,3642 
               
               
                 ILMN_1243066 
                 II1a 
                 0,10371 
                 0,164311 
                 1,108696 
                 0,016672 
               
               
                 ILMN_3155812 
                 II20rb 
                 0,099269 
                 0,985523 
                 0,999115 
                 98,3642 
               
               
                 ILMN_2590585 
                 II1rapl2 
                 9,950188 
                 0,099529 
                 9,807601 
                 99,61221 
               
               
                 ILMN_2695883 
                 Irf6 
                 98,07738 
                 1,018216 
                 99,05107 
                 0,098528 
               
               
                 ILMN_2623699 
                 Irf4 
                 10,11834 
                 1,043845 
                 10,07184 
                 10,42105 
               
               
                 ILMN_2727022 
                 Itgb1bp3 
                 0,099752 
                 1,0138 
                 10,24586 
                 0,009991 
               
               
                 ILMN_2658633 
                 Ifna7 
                 1,001601 
                 10,32717 
                 1,025378 
                 100,0743 
               
               
                 ILMN_2711910 
                 Ifnb1 
                 97,5166 
                 1,045995 
                 98,0811 
                 1,078197 
               
               
                 ILMN_3046362 
                 Traf5 
                 99,48784 
                 1,130779 
                 102,7819 
                 1,093667 
               
               
                 ILMN_3087518 
                 Dido1 
                 9,812321 
                 1,018148 
                 97,99077 
                 1,019697 
               
               
                 ILMN_1228448 
                 Cd19 
                 0,980663 
                 0,009872 
                 0,979249 
                 9,960054 
               
               
                 ILMN_2977690 
                 Tm9sf4 
                 0,992089 
                 10,24256 
                 0,980575 
                 105,3711 
               
               
                 ILMN_2505970 
                 Tmc5 
                 98,36548 
                 0,986988 
                 99,20843 
                 0,958709 
               
               
                 ILMN_2732649 
                 Tmem107 
                 99,31399 
                 0,98021 
                 101,2245 
                 0,986103 
               
               
                 ILMN_2645662 
                 Tmem86a 
                 0,985481 
                 8,866486 
                 0,975585 
                 878,4668 
               
               
                 ILMN_2441635 
                 Tomm34 
                 101,2956 
                 1,024865 
                 1022,77 
                 0,102703 
               
               
                 ILMN_1227012 
                 Ndufb4 
                 0,985371 
                 0,00103 
                 0,09931 
                 103,154 
               
               
                 ILMN_2419998 
                 Soat1 
                 1,003797 
                 8,423494 
                 0,097272 
                 83,96302 
               
               
                 ILMN_2607612 
                 Sp2 
                 100,5908 
                 1,00328 
                 102,9451 
                 0,103512 
               
               
                 ILMN_1221425 
                 Spaca5 
                 0,973755 
                 0,997833 
                 9,798517 
                 100,9814 
               
               
                 ILMN_1248179 
                 Spag11 
                 98,6397 
                 0,098584 
                 96,21745 
                 1,017883 
               
               
                 ILMN_1227250 
                 Specc1l 
                 0,963339 
                 1,036285 
                 0,930749 
                 101,1408 
               
               
                 ILMN_1227250 
                 Specc1l 
                 0,963339 
                 1,036285 
                 0,930749 
                 101,1408 
               
               
                 ILMN_2639777 
                 Sphk2 
                 10,1154 
                 1,011834 
                 10,1043 
                 100,6965 
               
               
                 ILMN_2818294 
                 Srpx2 
                 100,8231 
                 0,100387 
                 98,63012 
                 1,016853 
               
               
                 ILMN_3023573 
                 Ssbp1 
                 100,3159 
                 1,022076 
                 98,92587 
                 0,998213 
               
               
                 ILMN_2783117 
                 Tas2r140 
                 98,39451 
                 1,015511 
                 97,5502 
                 0,963632 
               
               
                 ILMN_2463080 
                 Tbx13 
                 10,10685 
                 10,24209 
                 98,87766 
                 101,2983 
               
               
                 ILMN_3072487 
                 Tcfap2b 
                 0,985614 
                 9,853532 
                 0,993813 
                 1061,242 
               
               
                 ILMN_2650280 
                 Sod2 
                 9,524808 
                 1,131742 
                 9,612056 
                 0,115816 
               
               
                 ILMN_1227889 
                 Pias3 
                 1,022192 
                 1,025845 
                 10,27761 
                 1040,663 
               
               
                 ILMN_2631014 
                 Pias3 
                 0,999235 
                 1,004021 
                 0,994658 
                 98,45135 
               
               
                 ILMN_2770667 
                 Acin1 
                 0,979681 
                 0,097175 
                 0,946506 
                 99,56963 
               
               
                 ILMN_1216022 
                 Aclp7 
                 1,00318 
                 10,64468 
                 10,17214 
                 104,08