Aging Western societies are facing an increasing prevalence of chronicautoimmune-mediated inflammatory disorders (AIMIDs) for which treatments that are safe and effective are scarce. One of themain reasons for this situation is the lack of animal models, which accurately replicateclinical and pathological aspects of the human diseases. One important AIMID is theneuroinflammatory disease multiple sclerosis (MS), for which the mouse experimentalautoimmune encephalomyelitis (EAE) model has been frequently used in preclinicalresearch. Despite some successes, there is a long list of experimental treatments thathave failed to reproduce promising effects observed in murine EAE models when they weretested in the clinic. This frustrating situation indicates a wide validity gap betweenmouse EAE and MS. This monography describes the development of an EAE model in nonhumanprimates, which may help to bridge the gap. Luca Massacesi, who pioneered themodel in the laboratory of Prof. Steve Hauser at UCSF . During my research at the Biomedical Primate Research Centre  I had already some experience with an EAE model in rhesus monkeys , but was rather unhappy with the anacute clinical course of the model and the destructive neuropathology, which more closelyresembled acute postinfectious demyelinating disease, such as acute disseminatedencephalomyelitis (ADEM) than multiple sclerosis . Thedescription of the new model in marmosets looked much better than our rhesus monkey EAEmodel and this became clearer once we started collecting our own data.An important success factor for the model has been our choice to focus ontranslational research into the pathogenesis as well as the treatment of MS.Our research thus stood on two legs, an exploratory leg where we unraveled(immuno)pathogenic mechanisms and an applied leg where the efficacy andsafety of new therapies were tested . The underlying thoughtwas that via this strategy we could use information from the applied leg tovalidate new pathogenic concepts developed in the exploratory leg.At the start of our MS research in the marmoset EAE model, our thinking was stronglyinfluenced by concepts developed in well-established EAE models in immunologicallynaïve rodents . Indeed, we assumed that just like in the rodentmodels, autoreactive T cells in marmosets are naïve and require strong stimulationwith danger signals for escaping regulatory mechanisms that keep them inactive. It took us many years to realize that this rather ignorant view wasfundamentally wrong and that the immune systems of conventionally reared marmosets andSPF-bred mice or rats are for a large part incomparable. The same is true for the humanimmune system, as we can learn from the seminal work of Mark Davis and colleagues atStanford University  . There is now alsomounting evidence that environmental factors have a profound influence on theimmunocompetence of animal disease models. As an example, cohousing of SPF-bredlaboratory mice with mice from pet shops or the wild creates a more human-like immunesystem . As stated by the authors of this hallmark publication, it isindeed ironic that an immunologically inexperienced 10–12-week-old mouse has become derigueur for studies on the complex human immune system in health and disease .We made the exciting discovery that EAE in marmosets is not driven by a single pathwaybut by two fundamentally different autoimmune pathways, one of which is more or lessidentical to the one in mouse EAE, while the other is completely different. Theobservation that the T cells that drive disease progression in the marmoset EAE modelmight be recruited from a repertoire of antigen-experienced T cells, which are absent ininexperienced SPF-bred rodents, underlies the concept that autoimmunity in MS might notbe elicited by an infection (response-to-infection paradigm), but by primary injuryinside the central nervous system  .The Laboratory Primate . That work brought us a nicemultidisciplinary publication, in which MRI, neuropathology and immunology wereintegrated . Figure 2 shows a compilation of figures from thispublication and illustrates the strategy that we followed for obtaining histologicalinformation on MRI-detectable abnormalities in the EAE-affected marmoset brain.Particularly stimulating was the support from MS neuropathologists, who appreciated theremarkable neuropathological similarity of the model with MS. In retrospect, it isregrettable that we restricted our neuropathology examination in the first paper to thewhite matter. If we would have performed immunostaining for proteolipid protein (PLP), we might have been the first to report on the dramaticdemyelination of cortical grey matter, a now very important pathological feature of MSthat was then unknown .Our first experiments addressed basic questions on the diagnosis of disease symptoms andthe visualization of CNS pathology in living animals. We designed a panel of MRIsequences that could be used for visualization and quantification of brain pathology insedated EAE marmosets. A picture of the equipment can be found in the book Which immunopathogenic mechanisms are operational in the periphery andwithin the lesions?What is the fine specificity of the T cells and antibodies that mediate theautoimmune attack on the CNS?Can the EAE induction protocol be refined by eliminating the role ofnonspecifically acting factors, such as components of the adjuvant?Can methods be developed for longitudinal monitoring of the(immuno)pathogenic process?Can the MRI protocol be refined to get more information on lesiondevelopment in the intact animal?The only research groups performing immunological research on marmosets were Prof. ClaudeGenain's group at UCSF (who stopped in 2007) and our group at the Biomedical PrimateResearch Centre (BPRC). Hence, we needed to work hard on the filling of our research toolbox withreagents  that could beused for immune profiling of our marmoset EAE model. We also had to develop assayssuitable for the availability of only 3 mL venous blood per month. An overview of thecurrently used tools has been published . The stream ofpublications in the peer-reviewed literature summarized in this monography shows that wehave achieved most of our goals.The questions for subsequent experiments unfolded more or less logically:The question of whether there is a future for the marmoset EAE model has to be asked inthese days, where biomedical research using live animals, primates in particular, isheavily debated in Europe and beyond. Scientists using nonhuman primates for the study ofserious human diseases need to deal with increasing political and societal pressure aswell as shortage of funding, both creating huge challenges. Our only arguments in thefight are the results of our research, which cannot be obtained in models in lowerspecies, nor in patients, nor in cell or organ cultures.Our research in the marmoset EAE model has provided highly relevant information forbetter understanding of MS as a basis for more effective therapies. In the past 5 yearswe have even entered the terra incognita of progressive MS, for which there is no validalternative animal model and no effective treatment. I sincerely hope that thismonography will enhance the appreciation of the translational relevance of nonhumanprimate disease models among our diverse stakeholders. In addition, it might provideuseful information in the dialogue with sectors of the human population criticizingnonhuman primate usage in biomedical research.2Due to their close phylogenetic relationship with humans, nonhuman primates (NHPs)provide translationally relevant models of a variety of diseases that threaten the aginghuman population . The highdegree of biological similarity of NHPs and humans is reflected in the immune system,which according to most experts is a central driver of the autoimmune neurologicaldisease multiple sclerosis (MS). The translational relevance of NHP disease models shouldalways be weighed against the high costs and justified ethical concerns, i.e., whetherthe same information can be obtained in a lower-ranked model or without animals. On theother side of the scale is the interest of the MS patients, who are desperately waitingfor an effective treatment for their disease. I posit here that MS models in NHPs areindispensable in preclinical MS research as they can provide novel insights intomechanisms operating in the initiation and especially the progression of the disease thatcannot be obtained in any other MS animal model, nor in the patient. Obviously, NHPs willnever replace corresponding experimental models in adolescent (8–12 weeks old)inbred/SPF laboratory mice, but they definitely add complementary translational relevanceto these models.Callithrix jacchus). In this publication onlythe highlights can be discussed. For the underlying hard data, the reader isreferred to the original publications given in the reference list.This monography reviews insights that we have gained during 25 years(1995–2020) of research in a model of experimental autoimmuneencephalomyelitis (EAE) in the common marmoset , for which no effective treatmentsexist, such as MS and type I diabetes. Despite significantly increased investment in thedevelopment of innovative therapies, the rate of drug candidates that reproduce promisingeffects observed in animal models when they are tested in the clinic, remainsdisappointingly low. The reasons for the low efficiency rate in therapy development forAIMID are unforeseen toxicity and a lack of efficacy, indicating that the predictivevalue of animal models for the clinical success of a candidate treatment is insufficient. However, funding for research aiming at understanding thereasons why treatments developed via forward translation (from the laboratory to theclinic) fail is limited. This is an unfortunate situation, as much can be learned fromreverse translation research (from the clinic back to the laboratory) and the newinsights can be used to improve currently used animal models .The subject of this monography is the EAE model, an experimentally induced autoimmunedisease in  susceptible laboratory animals that is projected on MS. EAE isnot only the most frequently used animal model in preclinical therapy research for MS buthas also often been used by immunologists as a generic model for testing basic conceptsof immune tolerance and autoimmunity .However, mounting evidence indicates a wide immunological gap between frequently used EAEmodels in adolescent (8–12 weeks of age) mice or rats, which are bred and housed undervery clean (SPF) conditions, and the MS patient. The important influence of theenvironment is elegantly illustrated by cohousing of SPF-bred mice with dirty mice, e.g.,those purchased from the pet shop, which gives the SPF mice a more human-like immunesystem . The decisive influence of gut microbiota on EAEsusceptibility is also illustrated in studies by Berer et al., who showed that EAE-pronetransgenic mice, which do not develop evident disease under germ-free conditions, becomespontaneously sick after administration of normal commensal gut microbiota . These and other studies underline the strong influence that gut microbiota canhave on the competence of the murine immune system.The fact that nonhuman primates are bred and raised under conventional conditions impliesthat, similar to the situation in humans, their immune system has been trained from earlylife onwards by genetic diversity and the exposure to environmental pathogens, gutmicrobiota as well as to chronic latent infection with herpes- and polyomavirus. Of noteis that the gut microbiota of marmosets is highly enriched with bifidobacteria, whichmost likely is an adaptation to their specific dietary habits  ; it most closely resembles the microbiome of human neonates (own unpublishedobservation). The fact that marmosets are naturally infected with In summary, the fact that in nonhuman primates, autoimmune reactions develop within apathogen-trained immune context marks an important difference with the situation inimmunologically naïve inbred/SPF mice.4MS is a chronic progressive neurological disease that specifically affects the centralnervous system (CNS), which comprises the brain and spinal cord. Clinically, MS ischaracterized by increasing defects of sensory, motoric and/or cognitive functions. Forquantification of the disease severity, symptoms are ranked on an expanded disabilityscoring scale (EDSS) . The EDSS is commonly used in clinical trials forquantifying the effect of a new treatment on the disease .Conceptually, the disease course, as seen in the majority of MS patients  pathological processes. We identified aclassical combination of humoral and cellular autoimmune reactions, which induce episodicloss of neurological functions (relapses). In addition, we identified a novel cytopathicmechanism that seems to amplify the normal age-associated degeneration ofmyelin/oligodendrocyte complexes .The exact nature of the events occurring at the transition from pre-symptomatic torelapsing–remitting MS (transition 1) and from relapsing–remitting to secondaryprogressive MS (transition 2) is debated. The simplest, albeit not necessarily correct,explanation may be as proposed in Fig. 4b, namely that transition 1 occurs when themounting inflammatory pathology caused by the autoimmune attack exceeds a clinicalthreshold, whereas transition 2 occurs when the pathogenic influence of the immune systemdeclines and progressive loss of function by irreversible degeneration of neuro-axonaland oligodendrocyte–myelin complexes becomes dominant. It is also proposed that in MSthe age-related degeneration of oligodendrocyte–myelin complexes is enhanced by the pathological process. As was already mentioned and will befurther discussed later, data from the marmoset EAE model revealed that this amplifieddegeneration may be mediated by a newly discovered pathogenic mechanism resemblingneurodegenerative diseases  .The pathological hallmark of MS and the most likely cause of the accumulatingneurological deficits is the lesion, a usually well-defined area in the CNS white matter(WM), where the insulating myelin layers around axons are destroyed, but myelin-formingoligodendrocytes are spared. Depending on their age, activity and localization, lesionscontain variable degrees of inflammation, repair (remyelination), astrogliosis (scarformation) and neuro-axonal pathology . The presence of immunecells and molecules  in inflammatory active lesions, which areusually characterized by enhanced permeability of the blood–brain barrier (BBB),indicates involvement of the immune system in lesion formation. While MS was initiallyconsidered to be a typical autoimmune disease of the WM, it is now clear that the greymatter (GM) is affected as well, maybe even to a greater extent than WM . In progressive MS the extent of cortical GM (cGM) demyelination, whichcan be focal in leukocortical and intracortical lesions or widespread in subpial lesions,often exceeds the load of WM lesions. Lesions in the cGM are often paucicellular, exceptin the very beginning of the disease , with only a rim ofactivated microglia cells. It is therefore suspected that demyelination of white and greymatter is caused by different pathogenic mechanisms. This view is supported by data fromthe marmoset EAE model, as will be discussed later in this monography.In debates on the start of MS the terms cause and trigger are often used as synonyms, butI believe that these terms indicate clearly different entities. I like to use the FirstWorld War (WW1) as a metaphor to explain the difference. The cause of WW1 was a complexset of factors, including mounting economic problems in European countries and theincreasing militarization of Imperial Germany. The trigger of WW1, however, was a singlefactor, namely the assassination of Archduke Franz Ferdinand of Austria and his wifeSophie by the Bosnian-Serb Gavrilo Princip. Whether the assassination would have sparkedsuch a tragedy if the economic and social situation in Europe were more stable remains anopen question.Mutatis mutandis, the same may hold true for MS. While an undisputed trigger of MS hasnot been identified thus far, the disease risk profile indicates that there is a varietyof causal factors that influence MS susceptibility; these can be genetic or environmental. Genomewide association studies (GWASs) reveal that the vastmajority of genetic risk factors have a function in the immune system, although theindividual contribution of each gene to the pathogenic process is often not clear.Environmental risk factors are diverse and include exposure to infectious (withEpstein–Barr virus) as well as noninfectious factors . In the course of our research thequestion of whether MS might be triggered by an endogenous factor or process emerged.Our work in the marmoset EAE model has been mainly focused on investigatingthe immunobiology of these MS risk factors, as potential causes of MS. Theunderlying thought was that via the analysis of MS risk factors insightcould be gained into critical steps in the disease process.5In the literature, two essentially complementary etiological concepts for MS arepresented  . According to an outside-in paradigm, autoimmunityin MS is triggered in genetically predisposed individuals by infection with an exogenouspathogen; various mechanisms have been proposed, such as molecular mimicry and bystanderactivation . An inside-out paradigm proposes that MS starts as adegenerative process inside CNS myelin, indicated here as a primary lesion. Conceptually,the immune system reacts against antigens released from this primary lesion. It must beemphasized here that both concepts are based on dynamic interactions of genetic andenvironmental factors. However, while in the outside-in paradigm pathogens are the directtrigger of the autoimmune process, in the inside-out concept gene–pathogen interactionsare a cause of MS, but not a trigger. Instead, they set the immune system in ahyper-responsive state to injury, which is triggered by another event . We will discuss later that data obtained in the marmoset EAE model are stronglysupportive for the inside-out paradigm.autoimmunity is not itself an entity, but a physiological response tosustained excess antigen turnover in diseased tissues (the primary lesion).those who develop clinical disease are viewed as high responders tocritical antigens.The first proposition implicates a mechanism that amplifies the normal age-associateddegeneration of myelin-oligodendrocyte and neuro-axonal complexes . As will be discussed later, such a mechanism has been found in the marmoset EAEmodel. The second proposition implies a hyper-immune response of MS patients againstmyelin components compared to healthy individuals. This has been shown by several authors. As will be discussed later, wefound a dominant antigenic role of the quantitatively minor constituent myelinoligodendrocyte glycoprotein (MOG) in the marmoset EAE model.The inside-out paradigm harmonizes nicely with a much older “primary lesion” theory ofTerence Wilkin . This theory essentially implies thatMacaca fuscata). The disease could beattributed to infection with a new It is heavily debated whether studies in an EAE model can provide relevant insights intothe mechanisms that trigger and/or perpetuate MS . The essential difference between MSand EAE is that MS develops spontaneously in  predisposed individuals, whileEAE is experimentally induced in otherwise healthy animals by means of rather artificialprocedures . Historical data indicate, however, that there may be nosharp separation between the two diseases. Humans were found to be susceptible to EAE. Infact, the EAE model stems from the observation by Pasteur that humans vaccinated withinactivated rabies virus grown on rabbit brains, developed brain pathology reminiscent ofan acute demyelinating disease in humans, called acute disseminated encephalomyelitis(ADEM) . Subsequent work by Rivers et al. (1933), Rivers andSchwenkter (1935) and Kabat et al. (1947) in rhesus macaques clarified that thisneurological disease was not caused by the virus, but induced by components from therabbit brain that contaminated the vaccine. Collectively, the published data imply thatsensitization of humans and nonhuman primates  against antigenspresent in the rabbit brain elicits similar ADEM-like pathology and disease. On the otherhand, there is a report from the Oregon National Primate Research Center describing cases of spontaneous MS-like diseasein a colony of Japanese snow monkeys (To our knowledge there is no clear evidence that 6Rivers and Schwenkter (1935) reported that macaquesgiven repeated (60 or more) intramuscular injections of alcohol-ether extracts fromnormal rabbit brain developed similar ADEM-like pathology as observed in earlier studies. Subsequently, Kabat et al. (1947) observed that ADEM-like disease could also be induced with only a fewinjections  wasinitiated by Margreet Jonker, who set up the model in rhesus monkeys . Observations by Rose et al. (1994) could be replicated, namely thatintracutaneous injection with myelin or with the quantitatively majormyelin basic protein (MBP) formulated with CFA elicited an acuteneurological disease with serious inflammatory/necrotic lesions. Disease development inthis model was associated with increased numbers of neutrophilic granulocytes in theblood as well as in the lesions. Interestingly, the same formulations elicited EAE withonly mild inflammatory lesions in marmosets . The beneficial effect ofanti-CD4 monoclonal antibody in the rhesus monkey EAE model indicates that there must bean underlying adaptive autoimmune process , which maybe aggravated by innate immune reactions against components of the adjuvant that causethe serious damage. Recent work by Dunham et al. (2017b) shows that the combination of intra-CNS oxyradical production by infiltratedneutrophils and redistribution of iron generates toxic oxygen , we believe that the EAE model in marmosets is more relevant for MS.77.1Marmosets are small-bodied nonprotected primates, weighing 350–400 g at adult age,which have their natural habitat in the Amazon forest. Figure 6a shows the features andsize of these animals. As a detailed description of the biology of marmosets and theirusage in preclinical research is beyond the scope of this monography, we refer the readerto reviews published elsewhere . To name a few important aspects,marmosets breed well in captivity, where they can give birth to two nonidentical twinsper year. Due to the sharing of the placental bloodstream, the immune systems offraternal siblings are educated in the same thymic compartment and are therefore verycomparable. This creates an attractive experimental setting for two-leg studies comparingthe effect of an experimental variable, a new therapy for example, with a controlpreparation.The BPRC houses a large self-sustaining colony of marmosets, comprising between 100 and150 animals. The monkeys are kept in family groups that are housed in spacious cages withindoor and outdoor enclosures. This means that our marmosets are exposed from birth toenvironmental microbes as well as to intestinal microbiota and chronic latent infections carried by family members. Evidence shows that thesemicroorganisms have an important effect on the immune status; in other words marmosetshave a pathogen-educated immune system. Mounting evidence indicates that this dailycontact with bacteria and viruses from in- and outside the body generates immunefunctions that are complementary to the classical ones found in the inbred/SPF laboratorymice used in most immunological research.7.2MHC. Genomewide association studies in MS and results obtained in rodentEAE models show that the highly polymorphic major histocompatibility complex (MHC) classII region exerts the strongest genetic influence on disease susceptibility . The main biological function of MHC class II molecules is to presentantigens to CD4Callithrix jacchus); Caja-DP genes are absent . ThreeCaja-DR loci have been identified: two produce functional transcripts, namely themonomorphic Caja-DRB*W1201 and the polymorphic Caja-DRB*W16 loci, while the Caja-DRB1*03locus contains only pseudogenes. Two hybrid Caja-DRB transcripts were found, which arecomposed of exon 1 and 3 of Caja-DRB*W16 alleles with exon 2 of Caja-DRB1*03 alleles.Furthermore, oligomorphic Caja-DQA1 , -DQB1  and -DQB2 genes were found. The invariant Caja-DRB*W1201 product is particularly relevant for theEAE model as it restricts the activation of CD4The MHC class II region of marmosets comprises the equivalents of HLA-DR and -DQ, whichare respectively indicated with the acronyms Caja-DR and -DQ . Sequences comparable to the classical HLA-A, -B and -C genes have not beenfound. Evidence was found for functional transcripts of the oligomorphic Caja-E locus . Caja-E has an important role in the marmoset EAE model as it restricts theactivation of autoaggressive cytotoxic T cells (CTL) specific for rhMOG epitope 40–48 . The classicalantigen presentation function to CD8TCR. The repertoire of T cell antigen receptors (TCRs) has been unraveled (inpart) . Overall a high similarity of marmoset andhuman TCRBV-D-J-C sequences was observed, illustrating the closephylogenetic relationship between the species.BCR. The repertoire of B cell antigen receptors (BCR) has been partlyunraveled as well . Also at the level of the immunoglobulinheavy variable (IGHV) gene repertoire a high similarity was found between marmosets andhumans.Other relevant immune molecules. The initiation and perpetuation of(auto)immune reactions involves interaction of various cell types via multiple cell-boundor secreted molecules. Some insight into the degree of relatedness between thosemolecules from humans and marmosets can be gained by testing whether monoclonalantibodies (mAbs) raised against human surface-expressed CD molecules cross-react withmarmoset cells. It is obviously a major effort to do this for all immune molecules, butfor several the data have been published and the list is periodically updated .In conclusion, the relatively close evolutionary distance of marmosets and humans, whichhas been estimated at about 30 million years , is reflected by a high degree ofimmunological similarity. This makes the marmoset an exquisitely attractive animal modelof human AIMIDs.7.3Mycobacterium tuberculosis. In addition, they gaveintravenous injections of 10Bordetella pertussis particlesat the day of immunization and 2 d later. This resulted in a relapsing–remitting (RR)disease course with inflammatory–demyelinating pathology. The authors also showed thatEAE could be transferred within (bone marrow chimeric) twins from an EAE-affected monkeyto its fraternal sibling using MBP-specific T cell lines. In later studies by this group,which were led by the neurologist Dr. Claude Genain, several important findings weremade, such as the detailed unraveling of the repertoire of immune reactions against MOG,the crucial role of anti-MOG antibodies for demyelination, the presence of anti-MOGantibodies inside lesions and the unexpected late exacerbation of neurological diseaseoccurring after tolerization against MOG .The first report on successful EAE induction in marmosets was published in 1995. The authors used a rather harsh immunization protocol,involving human brain homogenate formulated with CFA containing3 mg mLThe harsh experimental procedures used by Genain and Hauser (2001) are a clear reflection of the prevailing SPF rodent-based conceptson EAE and MS in the 1990s, without taking into account that the immune systems from SPFrodents and non-SPF primates might differ fundamentally. Our group at the BPRC choseanother approach, namely a stepwise refinement of the marmoset EAE model to the minimalcomponents needed for the induction of MS-like pathology and disease. These efforts aresummarized in the following.Inspired by the seminal publication of Massacesi et al. (1995), we used the same EAEinduction protocol in marmosets from our own purpose-bred colony .The only modification was that we used myelin isolated from an MS brain, which wepurchased from the Netherlands Brain Bank . Similar to the original study, weobtained relapsing–remitting (RR) EAE with severe inflammatory pathology. However, wenoticed that clinically evident EAE, albeit with a more protracted course and lessdestructive pathology, could be obtained when we used a commercially available CFApreparation (Difco) containing only 1 mg mLThe observation by McFarland et al. (1999), that demyelination in marmosets immunized with a chimericMBP/PLP protein in the adjuvant Titermax was always associated with spreading of theimmune response to MOG, underscores the pathogenic relevance of this quantitatively minorCNS myelin component. Data summarized in Box 2 indicate that MOG is a remarkable moleculewith a Dr. Jekyll/Mr. Hyde-type of role in the brain, which depends on its glycosylationstate. We tested whether immunity to MOG is indeed essential for EAE development. Ourapproach was to immunize fraternal twin siblings with myelin from B6 mice, which wereeither wild-type or MOG-deficient mice. MOG-deficient mice had been developed byDelarasse et al. (2003). We observed that in one twin with acute onset EAE the absence or presence of MOGin the myelin inoculum made no difference for the EAE presentation, while in the othertwin, development of chronic EAE was impairedwhen MOG was absent. At the pathological level, demyelination was reduced albeit notabsent (surprisingly) in the sibling immunized with MOG-deficient myelin !Escherichia coli bacteriathat represents the extracellular domain (residues 1–125). Possible epitopes located inthe transmembrane and intracellular parts of the molecule, which were identified for miceand rats, were excluded from our studies . The protein wasformulated with CFA and the emulsion was injected once at day 0. The clinical coursepattern in a representative cohort of The previous findings show that autoimmunity against MOG is indispensable for thedevelopment of chronic EAE in marmosets. To further investigate the anticipated complexpatterns of cellular and humoral autoimmunity against MOG in the genetically outbredmarmoset EAE model, we immunized marmosets with a nonglycosylated recombinant proteinexpressed in The immunological analysis of the rhMOG/CFA model revealed two distinct pathogenicmechanisms . We found that the 100 % EAE incidence maps to a commonautoimmune mechanism, namely the Caja-DRB*W1201-restricted activation of Th1 cellsspecific for epitope MOG24-36 . By contrast, we found that thevariable EAE course was related to the degree of diversity of the T-cell response againstMOG peptides . Counterintuitively, monkeys that progressed relativelyfast to clinically definitive EAE were characterized by a broader T-cell reactivity withrhMOG peptides than slow progressor cases. This is opposite to mouse models, where EAEprogression is associated with progressive diversification of autoreactive T-cellreactivity, a phenomenon known as “epitope spreading” . Ourinterpretation were that while fishing in the pool of MOG breakdown products, certain MHCalleles present in the repertoire of fast progressor monkey bind peptides that canactivate T cells with the capacity to accelerate EAE development. Later studies showed arole of IL-7 in the pathogenic function of these accelerator T cells . Candidate epitopes emerging from T-cell reactivity scans were MOG4-26, MOG34-56and MOG64-86/74-96 . The encephalitogenic activityof MOG4-26 was not (yet) tested. For the other two, we observed potent encephalitogenicactivity for MOG34-56, while MOG74-86/74-96 was inactive . Subsequentstudies revealed that the core pathogenic epitope is MOG40-48 and that this epitope canbe presented to (antigen experienced) cytotoxic T cells (CTLs) via Caja-E molecules. Besides the MHC-E restriction of epitope recognition, theseCTLs express the natural killer (NK) cell marker CD56 and theMOG40-48 epitope shares almost sequence similarity with a peptide derived from the majorcapsid protein of cytomegalovirus  we tentatively identified theseCTLs as being potentially related to or similar to CMV-induced effector memory T cells.In summary, we showed two independent pathogenic mechanisms in the rhMOG/CFA marmoset EAEmodel: a mouse EAE-like pathway operating early in the disease and a novel pathwaystarting late in the disease and mediating EAE progression.Based on findings discussed above we immunized marmosets with MOG34-56 or MOG74-96, eachformulated with CFA. Of these two, MOG34-56emerged as the superior encephalitogenic peptide . Intriguingly, weobserved 100 % EAE incidence in marmosets sensitized against MOG34-56 together withwidespread demyelination in the white and grey matter of brain and spinal cord ,despite the absence of detectable levels of myelin-binding antibodies in the serum. Inaddition, we observed that CFA could even be replaced by IFA, which is only the mineraloil without mycobacteria . A comparable mechanism has not beenfound in any other EAE model, indicating that this might represent a novel pathogenicmechanism that may be confined to the pathogen-educated primate immune system. Indeed,characterization of T-cell responses in this model revealed that they phenotypicallyresemble IL-17AA serendipitous finding revealed which of the two pathways described in Box 3 can beassociated with GM pathology. We observed in an atypical marmoset EAE model induced withrecombinant human (rh) MOG formulated with incomplete Freund's adjuvant (IFA) theactivation of T cells against MOG24-36 together with antimyelin antibodies. However, inthese animals T- and B-cell reactivity with the MOG34-56 peptide was lacking . Histological analysis of the brain showed profound demyelination of whitematter , while cortical grey matter demyelination was minimal(own unpublished results). This suggests acausal relation between the absence of autoimmunity against MOG34-56 and the absence ofgrey matter pathology. Indeed, sensitization of marmosets against the MOG34-56 peptide inIFA induced fulminant cortical grey matter demyelination, with a crucial role ofEBV-infected B cells .What is the nature of the primary lesion that starts the release of myelinantigens?What is the nature of the antigen-presenting cells (APCs) that capture andprocess these antigens, with a special focus on MOG, and present them to encephalitogenicT cell (response-to-injury paradigm)?The consideration that MS is obviously not elicited by injection of anantigen/adjuvant formulation but develops spontaneously raises at least twoimportant questions:8The review of immune responses against CNS myelin injury will be briefly interrupted herefor a brief summary of our current insights into the primary lesion. Theresponse-to-damage/inside-out paradigm is based on the assumption that some pathologicalevent inside the CNS precedes the activation of autoreactive T and B cells that may bemore responsive to the damage due to prior antigenic experience. In the EAE model theantigenic experience comes from active or passive immunization; in MS this might ratherbe infection with a pathogen sharing molecular mimicry with a CNS antigen.Already in 1990, Terence Wilkin postulated in his primary lesion theory that“autoimmunity is a genetically predisposed immune hyper-reaction to the excess ofautoantigen released from a primary lesion” . Shedding of fragments fromaging myelin sheaths seems to be a normal physiological process .Our own data show that this may not inevitably lead to autoimmunity, as binding tomyeloid APCs is mediated by normally glycosylated MOG. It was observed that MOG isolatedfrom a nonpathological brain binds to the C-type lectin receptor DC-SIGN , which is expressed within the human CNS on microglia and within primatecervical lymph nodes (CLNs) on the phagocytes that have captured the myelin. DC-SIGN is best known for its role in antigen capture andpresentation by myeloid dendritic cells (mDCs). Ligand activation of DC-SIGN relaysinhibitory signals to the dendritic cells (DCs), which keeps themin an immature, tolerogenic state  and prevents the assembly ofinflammasomes, which are needed for the production of bioactive IL-1The inflammasome response of cells is regulated by autophagy; more specifically,inflammasomes can be removed from cells via autophagy . Micedefective in autophagy genes display intracellular accumulation of protein aggregates inthe cytosol of neurons and hepatocytes. This raises the question of whether MS patientsmay be defective in autophagy. Although this subject has not been intensively studied, apublication by Igci et al. (2016) reported altered expression levels for several genesencoding autophagy-related proteins in MS versus healthy controls.These (and other) findings led to the concept that myelin binding to phagocytes indraining lymph nodes is mediated via MOG, which via its N-linked glycan attached to Asn31bind the C-type lectin receptor DC-SIGN. The response of the APC to myelin debristherefore depends on whether MOG exposed on the myelin particles is normally orabnormally glycosylated . This concept may narrowthe problem of autoimmunity initiation in MS to the question of what causes thedisturbance of normal MOG glycosylation. Intriguingly, we observed that myelin producedunder inflammatory conditions  a.k.a. microglia nodules  a.k.a. newly forming lesions, but likely represent the same pathological entity . Interestingly, similar aggregates of microglia were found in the EAE modelsin marmosets  and in rhesus monkeys . In themarmoset EAE model the non-demyelinating inflammatory microglia nodules correlated withsmall-sized prelesional MRI-detectable blood–brain-barrier leakage and were“contaminated” with some immune cells  . Itis currently unclear whether they are completely comparable to the pure microglia nodulesin MS. The observation by Singh et al. (2013), that microglia nodules are formed around adegenerating axon, draws the interest towards the axo-myelinic synapse, a new functionalconcept for the axon–myelin unit . A graphic representation of the unit isdepicted in Box 4. As discussed elsewhere, an intact unit is essential for adequatetrophic support to high nutrient demanding axons . We posit that thesefindings underlie the inside-out concept that the primary cause of MS may be instabilityof the axon–myelin unit . One can only speculate why this happens. Itcould have an internal cause, such as a genetic or metabolic defect and subtle molecularchanges , or an external cause, which could be associated witha Western lifestyle  (see Box 4), an infection or a combination of allfeatures. It is pertinent to emphasize, however, that our choice for the inside-outparadigm as a working concept does not negate the possibility that MS is triggered by anexternal factor.99.1Conceptually, the cells that capture, process and present antigens to the immune systemhave a decisive influence on the way the immune system responds to injury. The originalthought that the CNS lacks lymphatic vessels via which CSF and interstitial fluids can bedrained to the lymphoid system needed to be adjusted after the discovery of suchstructures in the meninges that surround the brain and spinal cord . Note, similar structures were found in the marmoset brain. Via these lymph vessels, fluids draining from the brain andspinal cord respectively pass through the cervical and lumbar lymph nodes (CLNs andLLNs), where antigens might be captured by local APCs. Via in situ immunochemistry wecould demonstrate the localization of myelin antigens within DC-SIGN9.2For many years, translational therapy research in MS was mainly focused on the modulationor suppression of autoreactive CD4An alternative method for B cell depletion is by capturing cytokines that B cells needfor survival and differentiation, i.e., BlyS/BAFF or APRIL . Toachieve this, the chimeric protein atacicept has been developed by joining TACI, thejoint receptor for BlyS and APRIL on B cells, with an Fc fragment of human immunoglobulin G. Unexpectedly, atacicept showed norelevant clinical effect in RRMS and even worsened lesion activity, necessitating thetermination of two clinical trials .These paradoxical clinical findings in MS sparked our interest in comparing the effect ofmAbs against CD20, BlyS and APRIL in the marmoset EAE model; we used the models inducedwith rhMOG/CFA or with MOG34-56/IFA. As rituximab failed to bind marmoset B cells, weselected a clonal variant of ofatumumab (HuMab7D8), which cross-reacted with marmoset Bcells. We observed that late-stage treatment  with the anti-CD20 mAb abrogated EAE development in marmosets immunized withrhMOG/CFA , whereas the anti-BlyS and anti-APRIL mAbs only moderatelydelayed EAE onset . As depletion of B cells from the circulationwas observed in both types of treatment, we focused our analysis on the secondarylymphoid organs (SLOs), i.e., lymph nodes and spleen. Intriguingly, we observed that inthe monkeys treated with anti-CD20 mAb, but not in those treated with anti-BlyS oranti-APRIL mAbs, T cells within SLOs remained strongly positive for CCR7, indicating thattheir release into the circulation might have been impaired .Furthermore, we observed that in the monkeys treated with anti-CD20 mAb, but not in thosetreated with anti-BlyS or anti-APRIL mAb, the copy numbers of CalHV3 DNA, being alymphocryptovirus of marmosets, was strongly reduced, indicating the depletion ofCalHV3-infected B cells .that B cells act as requisite antigen-presenting cells for the T cells thatinitiate and perpetuate EAE in marmosets,that this role may be primarily executed by the CalHV3-infected B cellfraction.This concept was further tested in the MOG34-56/IFA EAE model in which lesion developmentin WM and cGM occurs independent of anti-MOG antibodies . Asexpected, we also observed that the development of clinical signs and CNS pathology inthis model was substantially reduced by the treatment with anti-CD20mAb, hinting at adirect role of B cells in the activation of pathogenic T cells .Collectively, these data underlie the following concepts:9.3We then asked why LCV/CalHV3 infection of B cells is important for their role as APCs inthe marmoset EAE model, in particular for presentation of the vulnerable MOG34-56 peptideto pathogenic T cells. Inspired by a seminal publication from Manoury et al. (2002), whofirst presented the concept of destructive processing of autoantigen (MBP) in thymic APCsas an explanation for why certain autoreactive T cells escape thymic selection, weproposed that this might also be the case for MOG . Our hypothesiswas supported by recent studies in mice, indicating that autoreactive T cells specificfor the MOG40-48 epitope escape negative thymic selection because the epitope isdestroyed by a thymus-specific serine protease (TSSP), which is expressed in thymic APCs.TSSP cleaves at SP/PP residues within the MOG40-48 epitope (see Box 3). We proposed thatperipheral activation of these escaped autoreactive T cells is prohibited by B cellsexpressing the lysosomal serine protease cathepsin G (catG). CatG cleaves the epitope atthe ArgMalignant transformation. Transformed B cells are constitutively in anactivated state and are relatively resistant to apoptosis. Moreover, they have thecapacity to migrate across the BBB into the CNS . We indeed observed thatinfusion of marmosets with autologous BLC, which were prepulsed in vitro with MOG34-56,evoked mononuclear cell infiltrates in meninges and small-sized perivascular inflammatorylesions .Immune evasion. One of the immune escape mechanisms employed by LCVs is toavoid T-cell recognition of viral antigens by cytotoxic T cells through mitigation ofantigen presentation via MHC class Ia  and MHC class II (MHC-DR) molecules. The absence of MHC class I molecules evokes an attack by NK cellson the B cells, which can be repelled by the upregulation of MHC-E molecules. To achievethis, MHC-E must be occupied by epitopes expressing a so-called QdM motif, which directsthe interaction of MHC-E towards inhibitory CD94-NKG2A heterodimeric NK receptors. Binding of peptides lacking a QdM binding motif directs MHC-Ebinding to activating CD94/NKG2C heterodimeric NK receptors .Interestingly, the MOG40-48 epitope was found to bind with high affinity to HLA-E andCaja-E molecules expressed by transfectant K562 cells .Antigen cross-presentation. LCV-infected B cells have the capacity tocross-present peptide antigens via MHC class Ib/Caja-E molecules to CD8Citrullination. The upregulation of peptidylarginine deïminase (PAD)enzymes in LCV-infected B cells enables the conversion of arginine residuesinto citrullines .Autophagy. The autophagy pathway seems to be constitutively activated inLCV-infected B cells. Evidence from EBV-infected B cells suggests that constitutiveactivation is induced by the viral antigen LMP2A, which drives B cell function andsurvival . LMP2A replaces the B-cell receptor as inducer of asignaling cascade also involving Bruton's tyrosine kinase (Btk) .Each of the above-listed capacities of LCV-infected B cells can contribute to the stillelusive association of MS with EBV infection, especially around adolescent age. However,evidence was found that the combination of antigen citrullination and autophagyactivation underlies the presentation of the potent encephalitogenic MOG34-56 peptide byEBV-infected B cells . Our specific interest for antigenprocessing and presentation in B cells was sparked by some remarkable observations.First, we observed that immunization with MOG34-56/CFA elicited severe clinical EAE inrhesus monkeys , while in monkeys immunized with MOG34-56/IFA neitherclinical signs nor T- or B-cell responses against the peptide could be measured (ownunpublished observation). However, when the peptide was administered via ex vivo-pulsedLCV-infected B lymphoblastoid cell lines, T-cell reactivity with the peptide could bemeasured ex vivo. Second, we observed that T- or B-cell reactivity with the MOG34-56peptide was conspicuously absent in marmosets immunized with rhMOG/IFA , while T- and B-cell immunity is clearly detectable in marmosets immunized withMOG34-56 peptide in IFA . Moreover, we observed that infusion withLCV-infected B lymphoblastoid cells pulsed ex vivo with the peptide elicited T- andB-cell reactivity in marmosets .We observed that infection of B cells with the EBV-related lymphocryptoviruses (LCVs)induced a variety of cell biological changes that potentiate their pathogenic role in theEAE model. These include the following:Collectively, these findings led to us to hypothesize that the MOG40-48 epitope might bedestroyed during normal processing of rhMOG in APCs and that LCV infection of B cellsmight cause the conversion of destructive processing of the MOG40-48 epitope intoproductive processing of the epitope from the immunizing MOG34-56 peptide andpresentation via MHC-E molecules (see Box 5 for details). This concept was tested inEBV-infected and CpG-stimulated human B cells . An importantdiscovery was that LCV infection of B cells induces activation of the autophagy pathway(see Box 6). In brief, we observed that the replacement of only the ArgOur interpretation of these data was that citrullination of ArgIn discussions about the causal association of EBV infection with MS the counter argumentis often raised that the EBV infection prevalence in the human population (90 %) ismuch higher than the prevalence of MS (9.4The observations discussed thus far imply that myelin antigens (MOG for example) arecaptured by myeloid APCs in CLNs or LLNs, while the B cell depletion studies reveal a keyfunction of LCV-infected B cells in the EAE model. This somewhat paradoxical situationcan be understood when MOG is in some way transferred from the myeloid APCs to theLCV-infected B cells for further processing. However, in the absence of direct evidence,this explanation can only be speculative. The fact that pathogenic anti-MOG antibodiesbind to a conformational epitope formed by three loops at the apical tip of the molecule, implies that the B cells should receive via their BCR conformationally intactMOG from the DCs. A publication by MacPherson  seems to beimportant in this context. The author reports that part of the protein antigens that aretaken up by DCs is not processed but transferred to B cells in the intact form .In the rhMOG/IFA model only CD4Regarding PAD, it was discussed that anincreasing proportion of the myelin antigens in the MS brain is citrullinated through theupregulation of PAD2 by inflammatory factors and an increase in [In the mouse EAE model, the Arg residues are contact sites for the T cell antigenreceptor. Hence, their post-translational modification (PTM) potentially reduces theamount of available antigen for the induction of relapses by pro-inflammatory T cells.This notion underlies the new concept that as a consequence of PTM the autoreactive T-and B-cell-driven inflammatory demyelination will gradually fade out and be replaced by apathogenic process inducing progressive disease.1010.1Data discussed thus far posit a central pathogenic role of LCV-infected B cells in theautoimmune pathway(s) leading to cortical grey matter (cGM) pathology . Their originally proposed role is the activation of effector memoryCTL specific for MOG40-48, which can kill oligodendrocytes .However, in contrast to observations in MS lesions , co-localizationof T cells with MHC-E expressing oligodendrocytes could not yet be detected in themarmoset model. In fact, T cells were rare, if present at all, in subpial cGM lesions,but they were frequently found in the adjacent meninges .https://www.youtube.com/watch?v=-3k1ubpq0Zo, last access:17 April 2019). This theory is in remarkableagreement with observations reported 40 years ago on the detection of a high moleculartransmissible factor in the MS brain, termed MS-associated agent (MAA), that is absent inthe healthy brain and that was found to induce depression of neutrophils in mice.We were inspired by the concept that the interaction of T and B cells in meningealinfiltrates might produce (a) cytopathic factor(s) capable of directly inducing cGMdemyelination . In the ratmodel this activity was found to be mediated by pro-inflammatory cytokines, IFNto test whether infusion of autologousLCV-infected B cells prepulsed in vitro with MOG34-56 peptide induces EAE pathology inrhesus monkeys and marmosetsto examine whether the interaction of LCV-infected B cells with orwithout MOG34-56 peptide with T cells from EAE marmosets in vitro elicits production ofcytotoxic factors that may be implicated in cGM demyelination .Concerning study 1, we observed the formation of T and B cells containing meningealinfiltrates (in both species), together with small-sized perivascular inflammatorylesions .These findings prompted the following experiments:Concerning study 2, we observed peptide-dependent production of IL-17A,while IFNThere is another argument against the induction of oligodendrocyte depletion byT-cell-derived cytokines. The necessity to citrullinate the Arg residues within theMOG40-48 epitope for protection of the immunizing MOG34-56 peptide against destructiveprocessing may affect T-cell recognition of the epitope. As was already mentioned, it hasbeen shown in B6 mice that the Arg residues at positions 41 and 46 contact the antigenreceptor of I-A10.2The observations discussed thus far seem to dispute the concept that the depletion ofoligodendrocytes in cGM lesions occurs by physical interaction with cytotoxic T cells orby T-cell-derived cytotoxic factors. Hence, we tested an alternative option, namelywhether the MOG34-56 peptide itself or a metabolite thereof might be the elusivecytopathic factor.Figure 10a depicts the 3-D configuration of monomeric human MOG as elucidated byBreithaupt et al. (2003). The structure is rich in We observed the following: (1) replacement of these residues by citrulline protects theMOG34-56 peptide against cleavage by catG and (2) that citrullination promotesspontaneous aggregation of the peptide in the following order:MOG34-56 < MOG34-56c41,46 These findings may shed an entirely new light on the role of LCV-infected B cells in cGMdemyelination. It has been well-established that EBV-infected B cells secreteantigen-containing vesicles for spreading of an immune response .The notion that the MOG34-56 peptide associates with autophagosomes via LC3 raises thequestion of whether LCV-infected B cells might secrete LC3The next question was whether the vesicles contain MOG34-56 peptide. This was testedusing synthetic peptides containing a bio-orthogonal group for attachment of afluorochrome to peptide inside (fixed and permeabilized) cells. The peptides wereunmodified (CA-1) or contained citrulline at the positions 41 and 46 (CA-2) or 46 and 52(CA-3). Titrating doses of the peptides were fed to human EBV-infected BLCs for 48 h inserum-free medium.Column (a) shows that BLC incubated without peptide express only low stainingof LC3.Column (b) shows the same incubation conditions of the peptides, but withoutBLC. In the absence of BLC no precipitate of CA-1 is detectable, whileaggregates of peptides CA-2 and CA-3 were spun down on the glass slides.Column (c) shows BLC incubated with two doses of CA-1, -2 and -3, specifically 6.2and 25 Also in the incubations with peptide CA-2 (citrullination at positions 41and 46), albeit at lower concentration than CA-1, globular structuresassociated with diffuse aggregates of CA-1 were detectable with only scarceLC3In the incubations of CA-3 (citrullination at positions 46 and 52) with the B cellsmainly large-sized aggregates were found surrounded by LC3-expressing BLCs.Figure 14 shows stainings of cytospin preparations from BLCs incubated with 6.2or 25 These data strongly suggest that the CA-1 peptide has successfully survived passagethrough the B cells, which according to previous insights  implies that position 46 has been citrullinated, possibly via secretedPAD. In the cultures containing the globular structures, reduction of LC3 staining wasobserved, which points at a possible toxic effect.We observed that immunization of marmosets with MOG34-56/IFA activates apathway that leads to demyelination of white and cortical grey matter of the brain. Theheart of this nonclassical pathogenic mechanism is formed by CD8We observed a special relevance of the citrullination of the Arg46 residue,which is located within a surface-exposed putative F-LIR motif that enables associationof the immunizing MOG34-56 peptide with autophagosomes, as shown in EBV-infected human Blymphoblastoid cells fed with the peptide. In this way the peptide is protected againstfast endolysosomal degradation, which enhances immunogenicity .The observations that treatment of the cells with rapamycine, a stimulator of autophagy,enhances protection of the peptide against degradation and treatment with 3MA, aninhibitor of autophagy, enhances degradation of the peptide support this concept.The replacement of arginine residues by citrullines impaired T-cell stimulation(proliferation). This finding argues against demyelination induction via cytotoxic Tcells.The paradoxical situation that citrullination of the ArgAnother line of research involved the serendipitous discovery thatcitrullinated MOG34-56 spontaneously aggregates to multimeric structures, which displayseeding behavior comparable to amyloid-In summary, we make the following four points:Based on these collective data I posit that EBV-infected B cells may function as a Trojanhorse in the progressive phase of MS, bringing MOG peptide oligomeric aggregates into theCNS and releasing them locally as packages within cytotoxic vesicles.11It was already recognized in the beginning of the model development that thehuman-like architecture of the marmoset brain and the MS-like histology ofthe EAE lesions might be relevant for studies on the monitoring of brainpathology development and treatment with magnetic resonance imaging (MRI).Our group at BPRC has developed a variety of sequences for visualizing and quantifyingMRI sequences that were subsequently used for testing the radiological and clinicaleffects of therapeutic antibodies against human IL-12p40 and CD40 . In addition, we used magnetic resonance (MR) spectroscopy of 24 h urine samples collected at 7 dintervals during the disease course. The demonstration that urine samples collectedbefore, shortly after and late after EAE onset have different “metabolite fingerprints”indicates the presence of urinary biomarkers for the different pathological processes . However, the primary focus of our work has been on the afferent armof the disease, being the initiation and perpetuation of the autoimmune mechanisms thatcause the pathology. We mainly used the information from the therapy trials to assess therelevance of an immunological mechanism for MS and to adjust the model where needed .The effector arm of the disease has been studied in great detail by others. In studies byAbsinta et al. (2016) and Maggi etal. (2017) a myelin-induced marmoset EAE model was examinedwith high-contrast MR images recorded at 7 T to monitor the initiation and development of lesions in the whitematter. An extensive discussion of results obtained by these authors is beyond the scopeof this monography. Hence, they will be briefly summarized. For more detail theinterested reader is referred to publications from this group .As discussed above, it is highly difficult to assess whether inflammation precedes orfollows lesion onset in the MS brain. In brief, analysis of the temporal development ofbrain white matter lesions in a myelin-induced marmoset EAE model show that lesions startas a small-sized focal enhancement of blood–brain barrier permeability developing intoan acute inflammatory lesion (< 1 week old), which is cuffed around a centralparenchymal vein and is composed of Iba-1The main message from these studies is that perivenular inflammation, i.e., lymphocytecuffs and microglia activation, precedes the formation of lesions. Lesions develop bycentrifugal expansion from non-demyelinated nodules of lymphoid and myeloid cells arounda leaky blood vessel via which they have infiltrated the CNS parenchyma. Although theseobservations clearly support a primary inflammatory ontogeny of MS lesions, they do notanswer the question of where autoimmunity is induced.12Research discussed thus far has led to the conclusion that the heart of the marmoset EAEmodel is formed by pathogenic functions that B cells acquire via LCV infection:protection of the vulnerable MOG40-48 epitope against fast degradation,cross-presentation of the epitope via Caja-E to effector memory CTLs and secretion ofpost-translationally modified MOG34-56 as toxic aggregates, either packaged or notpackaged in vesicles. This concept may provide a plausible mechanistic explanation forthe association of EBV infection with MS susceptibility.The clear core pathogenic role of LCV-infected B cells raises the question of whetherother environmental factors that influence the disease expression in the model may exerttheir effect via LCV-infected B cells. A serendipitous finding showed that this may bethe case for (a) component(s) of a new dietary supplement that was introduced into themarmoset colony at BPRC in January 2014. Recent literature is flooded with publicationsabout the role of a gut–immune–brain axis in the initiation and course of EAE . At this point the marmoset EAEmodel has also yielded some intriguing new insights, which are summarized in thefollowing paragraphs.A direct consequence of the high level of refinement of the marmoset EAE model has beenthe loss of robustness of the model, which is probably due to higher sensitivity for thevariable influence of genetic and environmental factors. This has led to increasedvariation in the response to the immunizing antigen or to an experimental treatment, including nonresponders, and the potential loss of statisticalpower . In the course of 4 years (2014–2018) we were facing anunexplained sudden decrement in the disease incidence of the atypical EAE models inducedwith MOG34-56/IFA (from 90 % to 65 %) or rhMOG/IFA (from 100 % to 60 %). The reduced EAE susceptibility coincided with a sharp decrease in the numberof marmosets in the BPRC colony testing positive for CalHV3 . It has taken usconsiderable time to find out that the sudden drop of EAE susceptibility coincided withthe modification of a dietary supplement, which was introduced in January 2014. Themodification was rather modest, namely from a water-based to a yogurt-based formulation,which also contained the double dose of B vitamins. Nevertheless, the effects on themodel were rather dramatic .These enigmatic observations prompted a controlled study in marmoset twins,which had been fed the new dietary supplement from weaning onwards. For theexperiment, one sibling of each twin was reverted to the old water-basedsupplement (WBS), starting 8 weeks prior to EAE induction, while the othersibling remained on the new yogurt-based supplement (YBS). In brief, the EAEincidence in the old WBS and new YBS groups differed, albeit notsignificantly . Thisis exactly what we found in the twin study . We observed a positiveeffect of the YBS on the proportion of bifidobacteria in the marmoset stool. According toanother theory, yogurt is a dietary source of tryptophan, which can be metabolized by themarmoset gut microbiota, the bifidobacteria in particular. Tryptophan can be converted byprobiotic gut microbiota into high-affinity ligands of the aryl-hydrocarbon receptor(AHR), such as kynurenine . Ligand-activated AHR counteracts the increased expression ofgenes relevant for B-cell growth and function, such as early B-cell factor 1 (EBF1), inEBV-infected B cells . Upregulation of EBF1 induced by EBNA1 isassociated with EBNA1's role in maintaining the viability of infected B cells and keepingthe EBV genome intact during latent infection . This suggests thatthe yogurt intake in combination with higher levels of bifidobacteria in the gut may leadto the production of factors mediating AHR activation, which might have caused thereduced survival of CalHV3-infected B cells.The translational relevance of these findings for the MS patient may be thatthe pathogen-educated immune system of adult outbred marmosets issusceptible to dietary modification in such a way that immune responsesagainst MOG released from a primary lesion in the CNS are reduced. Furtherwork should reveal which components in the YBS diet are responsible for thiseffect.13One of the strongest arguments for developing nonhuman primate EAE models of MS is thatthey provide relevant systems for translational research into the pathogenesis andtreatment of the disease. Relevant features in this line of research are that manybiologicals developed for immunosuppressive or immunomodulatory therapy in MS cross-reactbetween marmosets and humans and that marmosets and humans share a high level ofimmunological similarity. Indeed, reports from others  and us  documented the remarkable similarities in the afferent and effectorarms between marmoset EAE and MS.The diagram in Fig. 1 shows that the integration of exploratory research of pathogenicmechanisms and applied research of new therapies creates a powerful research strategy.The integration of forward and reverse translation creates an iterative scientificprocess that potentially provides deep insight into the critical steps in the pathogenicprocess and delivers better therapies. We have used this iterative strategy in themarmoset EAE model. More specifically, we tested therapies that worked or did not work inthe clinic for eliminating pathogenic mechanisms in the EAE model that are irrelevant forthe human disease. The ultimate aim of this strategy was to create the most relevantpreclinical model for MS .It is believed that the immunopathogenic process within the CNS starts withthe infiltration of peripherally activated CD4T cells that have achieved transmigration of the blood–brain barrier need toengage in cognate interaction with local antigen-presenting cells (APCs) for unfoldingtheir complete pathogenic activity. This interaction comprises three signals: signal 1,the formation of a trimolecular complex composed of a peptide presented by MHC moleculesand the antigen receptor of T cells; signal 2, the interaction of co-stimulatorymolecules expressed by T cells and APCs, such as CD28 with CD80/86 or CD40 ligand withCD40; and signal 3, cytokines that steer functional polarization of the activated Tcells, such as IL12, IL-23 and IL-7. A frequently tested approach in the broad spectrumof immune-based inflammatory disorders is the functional ablation of inflammatory T cellsby blocking signal 2.The infiltration of CD4Mounting evidence indicates that MS patients have an inherently deficientrepair capacity for myelin damage by recruitment of oligodendrocyte precursor cells (c)into lesions, where they are supposed to differentiate into mature oligodendrocytes whichare capable of producing new myelin sheaths.The demyelination process focusses on the oligodendrocyte/myelin–axon units via twomechanisms, namely cytotoxic damage to myelin sheaths via complement (CDC) or macrophages(ADCC) and T-cell-mediated cytotoxicity towards oligodendrocytes.Below follows an overview of the types of therapy that were tested in themarmoset EAE model.Figure 16 gives a graphical presentation of relevant factors in the pathogenicprocess inside the CNS, each of which could be a target for therapeuticintervention. Several of these targets have been validated in the marmosetEAE model:Category 1: therapies targeting T cells. Rodent EAE models show a centralpathogenic role of two types of pro-inflammatory CD4IL-12/IL-23. The CD4In conclusion, the two experiments show that the mAb had a strong beneficial effect whentreatment was started early in the disease, while the treatment was less effective oncethe disease was ongoing. Moreover, we observed a dissociation between the strong effectof the mAb on white matter pathology and the modest, albeit statistically significant,effect on the clinical signs. An explanation for this clinical–pathological paradox maybe the existence of two immunopathogenic pathways, an initiation pathway driven byIFNIFNIn conclusion, these observations argue against an important pathogenic roleof Th1 cells in the marmoset EAE model.IL-17A. In a third experiment, we tested the effect of a humanized IgG4In conclusion, we found that treatment with anti-IL-17A antibody induced amoderate delay of clinical EAE in marmosets, but EAE development was notcompletely abrogated. This finding hints at a pathogenic role for IL-17A inthe marmoset EAE model and maybe in MS, but IL-17A may not be the only keypathogenic cytokine.IL-7 receptor/CD127. IL-7 is a cytokine produced by stroma cells that has abroad activity range. In the bone marrow, IL-7 stimulates the differentiation ofhemopoietic stem cells into lymphoid progenitor cells. Moreover, the cytokine stimulatesproliferation and differentiation of T cells, B cells and NK cells. We have tested achimeric IgG mAb raised against marmoset IL-7 in the MOG34-56/IFA model . Treatment of seven twins with mAb or PBS intravenously administered was started at21 d after the first immunization. Although the mAb blocked CD127 function in allmonkeys, as assessed by IL-7-induced STAT5 phosphorylation, we observed a beneficialeffect on the disease course only in twins with fast-progressing disease. This remarkabledichotomous effect indicates that even in the highly refined MOG34-56/IFA model diseaseheterogeneity can be observed.In conclusion, The IL-7 pathway seems a relevant target of therapy for a subset of MSpatients.Overall conclusion of this part. These experiments seem to confirm observationsin MS clinical trials, namely that therapies targeting only CD4Therapies targeting B cells. The remarkable results of the anti-CD20 mAbrituximab in RRMS  created a paradigm shift in MS. While B cellswere for a long time viewed only as producers of myelin opsonizing antibodies, whichinduce demyelination via complement- (CDC) or macrophage-dependent (ADCC) cytotoxicityreactions, they have now gained a central position in the MS pathogenic process . These findings prompted us to test the second generationanti-CD20 mAb HuMab7D8, which is a clonal variant of the human mAb ofatumumab withconfirmed cross-reactivity with marmoset B cells in the EAE model. We made a number ofnoticeable observations.CD20. We tested the efficacy of HuMab7D8 in the rhMOG/CFA model, starting weeklytreatment at 21 d postimmunization. The antibody induced almost complete and persistentdepletion of CD20One of the presumed roles of B cells in MS is antigen presentation toautoaggressive T cells . This was tested in the MOG34-56/IFA model, in which pathology anddisease is induced by the action of autoaggressive T cells, without thesupport of myelin-opsonizing autoantibodies. Also in this model, we observedprofound suppression of EAE symptoms and pathology, as well as suppressionof humoral and cellular autoimmune parameters .BlyS and APRIL. An alternative method for B cell depletion is the capture ofcytokines that B cells need for survival and differentiation, i.e., BlyS/BAFF or APRIL. This prompted us to test two mAbs in the rhMOG/CFA model, namelyanti-human BlyS mAb (belimumab) and ananti-human APRIL mAb. Treatment was again started at 21 d after the immunization. Bothantibodies caused only a moderate, albeit statistically significant, delay of the EAEonset . Note, examination of the secondary lymphoid organs didnot reveal retention of activated autoaggressive T cells .CD40. The treatments with anti-BlyS/APRIL and anti-CD127 mAbs hinted at aspecial role of B cells expressing high levels of CD40 in the EAE model (see above). Thisconclusion is supported by data from earlier studies in which we tested murine, chimericand humanized variants of the human CD40 blocking mAb 5D12 in the EAE model. In brief, weobserved in a marmoset EAE model induced with human myelin/CFA that treatment with amouse anti-human CD40 mAb (5D12) delays the onset of clinical signs .In the same study, we observed that the mAb gains access to inflammatory active lesions.To assess whether treatment efficacy might be limited by the formation of antidrugantibodies we gave a single intravenous injection of the murine mAb and tested serumantibody levels at several time points thereafter. Indeed, free test substance levelswere substantially lower in mAb-treatment cases than in a naïve monkey.Based on these encouraging findings, a chimeric mouse–human IgG4 mAb was developed(ch5D12), which was tested in the EAE model induced with rhMOG/CFA .Also in this experiment, we observed profound suppression of EAE clinical signs andpartial suppression of brain and spinal cord pathology. As was expected, the induction ofanti-rhMOG IgG antibodies was suppressed as well.Overall conclusion. These immunotherapy findings support a core pathogenic roleof CD20Cell-based regenerative therapies. One of the great challenges in MS research isto develop treatments with which CNS damage can be repaired once the (immuno)pathogenicprocess has been stopped. Much is expected from treatment with stem cells (SCs), whichare now available in different flavors, such as hemopoietic SCs, neural stem/precursorcells (NPCs), mesenchymal SCs or induced pluripotent stem cells (iPSs) . We have tested two types of stem cells in the marmoset EAEmodel, namely human NPC, grown as homogenous cell line, and iPS cells derived fromreprogrammed human skin fibroblasts, differentiated into oligodendrocyte precursor cells(OPCs).NPC therapy. This form of therapy is based on the administration ofself-renewing, multipotent cells isolated from the CNS. For our study, we used humaneGFP-transduced NPC, grown as a homogenous cell line, to enable the detection of injectedcells in postmortem brain tissue . The cells were administered torhMOG/CFA-immunized monkeys either intra-CSF via injection into the cisterna magna orinto the blood stream via the tail vein. We injected a low dose of cyclosporine A . We haveused this exciting technology to generate OPCs from reprogrammed human skin fibroblasts. The potency of these (GFP-labeled) OPCs to remyelinateaxons that were denuded by autoimmune demyelination was tested in the MOG34-56/IFAmarmoset EAE model, The OPC graft was injected into the brain at some distance from anMRI-detectable (T2) lesion. Like in the previous experiment, low dose CsA the immunological similarity between marmosets and humans and (2) the neuroanatomicalsimilarity of marmoset and humans.1414.1replacement, reduction andrefinement . Strict adherence to these principles isnow common practice, especially in research with sentient animals, such as NHPs. However,it is rather remarkable that an obviously important fourth R is lacking, namely the relevance of an animal model. In the following, these four R's will be brieflydiscussed as well as why difficulties encountered when harmonizing them have made thegeneration of a clinically relevant EAE model an acrobatic balancing act .The leading principles in preclinical research with living animals have been formulatedby Russell and Burch as the “3 R's”: Brachydanio rerio), Drosophila flies andCaenorhabditis elegans worms. However,although important insights into novel disease mechanisms can be obtained from thesemodels, it is felt that model systems more closely related to the patient are needed forthe integration of all information into a coherent pathogenic concept that can betranslated into new therapies.The replacement principle encourages scientists to use methods in their research thatavoid or replace the use of living animals. Although in vitro models with cells ortissues and in silico models of human pathology are increasingly used in preclinicalresearch, it is generally felt that the complexity of a pathological process in relationwith other physiological body systems is only displayed in living animals. In that casethe lowest animal species from which relevant information can be obtained should beselected. This consideration underlies the increasing use of evolutionary distant modelsystems, such as zebrafish  imaging techniques , but compliance with the formercondition appeared to be more problematic. Reviewers of projects and publications oftenlimit the interpretation of the reduction principle only to the minimal number of animalsthat should be used for a “usable” result. Power calculations are applied to determinethe minimal size of experimental and control groups, so that the effect of anexperimental variable can be statistically tested. Critical variables in powercalculations are the disease incidence, the anticipated treatment effect and the expectedvariation in read-out parameters, such as the time to clinically evident disease or thedisease severity. The outbred nature of NHPs and their conventional housing conditionsimply a variable influence of genetic background and environmental factors, ironicallybeing dominant autoimmune disease risk factors in the human population. The inevitableconsequence of variation is that experimental groups may contain clinically lowresponders or nonresponders, which reduces disease incidence and thus requires largergroup size for statistical power. In rodent disease models this dilemma is bypassed byusing well-established genetically homogeneous (inbred) SPF-bred strains and the usage ofpotent adjuvants to obtain high disease incidence and a synchronous disease course.However, a recent study raised questions on the relevance of immunologically immature SPFmice as models of complex human diseases . Moreover, the usage ofstrong bacterial adjuvants clearly conflicts with the refinement and relevance principles(see below).M. tuberculosis or butyricum) in mineral oil.The mineral oil, also known as incomplete Freund's adjuvant (IFA), forms a depot for thefinely dispersed antigen solution in aqueous buffer; the mycobacteria provide dangersignals needed for “awakening” of the tolerized T and B cells. However, CFA isnotorious for its seriously detrimental side effects, in particular the induction ofsevere ulcerative skin lesions at the injection sites, which are clearly caused by themycobacteria. Usage of CFA in nonhuman primates is therefore discouraged. As discussedabove, in the marmoset EAE model IFA can be used, which implies a major reduction ofdiscomfort to immunized animals compared to animals immunized with CFA.The refinement principle encourages scientists to employ research methods that minimizethe discomfort from experimental procedures to the animals. A major concern with respectto the EAE model is that for reproducible disease induction strong adjuvants need to beused to pepper the immunogenic potency of self-antigens by disrupting the regulatorymechanisms that keep autoaggressive T and B cells under control . Themost frequently used is complete Freund's adjuvant (CFA), which is an emulsion ofheat-killed mycobacteria  can also be found in MS lesions .Compliance of the marmoset EAE model with the fourth R has been achieved via an iterativestrategy depicted in Fig. 13. In brief, research in the exploratory arm aimed at maximumrefinement of the original EAE model, which was induced by immunization with myelinisolated from the brain of an MS patient formulated with the bacterial adjuvant CFA . The stepwise refinement of this complex model towards the minimallyneeded components yielded a highly refined model that displays essential pathologicalaspects of MS  . In the applied arm, the consecutive stepsof the refinement process were validated with clinically relevant therapeutic mAbs. TheThe discomfort to the animals in experiment was reduced,The immunogenicity of administered biological therapeutics, which limitedtheir activity window, was reduced,The dogma that danger signals are absolutely needed for autoimmunityinduction may need to be adjusted,A new pathogenic mechanism was discovered.Concerning the third issue a caveat may be needed as the injection of antigen/IFAemulsion will certainly cause damage and induce the release of damage-associatedmolecular patterns, which can relay danger signals to APCs through DAMP receptors . The released DAMPs cause visible skin irritation. However, these signals appear tooweak for the elicitation of EAE in genetically susceptible, but immunologically immature,SPF mice .The replacement of CFA for IFA in the translationally relevant atypical EAEmodels had various important consequences:14.2Compliance of the primate EAE model with the refinement and relevance principlesintroduced an unforeseen conflict with the reduction principle. It was observed that thereplacement of CFA with IFA produced less robust EAE models, which were more prone tovariation in the response against immunization as well as in the response to treatment. Alikely explanation is that these refined disease models are more sensitive to thevariable influences of genetic and environmental factors. An experiment in which weencountered the consequences was reported recently . In brief, wetested the efficacy of a mAb raised against the human IL-7 receptor CD127 in a poweredtwo-leg study in marmoset twins significance and  relevance for the highly refined EAE models. One solutioncould be that reviewers of grants and publications on proof of concept studies choose notto apply the normal sample size dogmas developed for homogeneous models, such as those ininbred/SPF mice, to studies in more complex and more heterogeneous disease models innonhuman primates . The central argument is that the valueof a study does not increase proportionally with the number of animals added to a testgroup, while the burden from discomfort on study participants does incrementproportionally .15A final relevant question is whether insights that we gained through studiesof the marmoset EAE model may help explain the disease course depicted inFig. 4b. I posit the concept that the course of EAE in marmosets is driven bypost-translational modifications of the immunodominant myelin component MOG.From tolerance to autoimmunity. We showed that MOG in the healthy brainmaintains homeostasis via the interaction of its glycan epitope attached at theasparagine (Asn/N) 31 residue with the C-type lectin receptor DC-SIGN, which is expressedon microglia and dendritic cells in the draining  lymph nodes.Modification of this glycan, which may either have a genetic or a pathological cause(inflammation for example) uncovers the pathogenic role of MOG . Studies in the marmoset EAE model show that nonglycosylated rhMOG protein or MOGpeptide are strongly immunogenic and encephalitogenic. It is therefore proposed thatautoimmunity against MOG is induced when the protein loses its tolerogenic glycanepitope.From autoimmunity to relapsing disease. We identified two epitopes, MOG24-36 enzymes, which exist in five isoforms ; PAD2 is the mostprevalent in the CNS . Full activity of PADs depends on local[Data obtained in the rhMOG/IFA model show that the immune response against thenoncitrullinated protein is restricted to pro-inflammatory CD4In silico 3-D modeling of human MOG  shows that the sequence 34–56 is exposedat the surface of the molecule. The thee Arg residues are highlighted, showing that theArgFrom relapsing to progressive disease. Literature data suggest that with theprogression of time (and disease severity) the proportion of myelin protein that iscitrullinated increases . By the citrullination, T-cellrecognition of the antigen is disturbed, while the tendency to spontaneously formamyloid-type aggregates increases. Citrullination of the ArgOpen questions. The marmoset EAE model is now regarded by many as thetranslationally most relevant animal model of MS. Important aspects of the model are theclear pathogenic role of B cells infected with the lymphocryptovirus CalHV3. However, itis still an open question of whether the mechanisms uncovered in the model are relevantfor the human disease.The EAE model in marmosets is pathologically characterized by lesions in the white andgrey matter of the brain and the spinal cord. The model also revealed that lesions inwhite and grey matter are induced via distinct pathogenic mechanisms. Also, the questionof whether the mechanisms that we have explored are translatable to MS is still open.An intriguing new finding is that toxic amyloid-like aggregates are formed in theinteraction between citrullinated MOG34-56 peptide with EBV-infected B cells. It is stillan open question as to whether these aggregates are secreted by the B cells, in whichform this occurs and whether the aggregates are toxic towards neurons and glia cells.


Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aqueous environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solutions to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named ‘elevator-type’. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it ‘drags” the transported substrate along. Analysis of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Molecular dynamics simulations and experimental data help to understand how lipid bilayer properties may affect elevator movements and vice versa. Structural studies of membrane transporters from diverse protein families have revealed that alternating access may by achieved in many ways (reviewed recently ). The soThe elevator-type transport mechanism offers an alternative solution to achieve alternating access . ProteinPh [The first elevator-type mechanism was described in 2009 for the aspartate transporter GltPh , a membePh . In recePh . In thisPh .z-direction if the membrane plane is defined as the xy plane. In many cases, the domain movement is more complex than a simple translation, and the total distance over which the substrate is displaced is larger than the vertical distance , and the human neutral amino acid exchanger ASCT2 (Alanine Serine Cysteine Transporter 2)  family of solute transporters is structurally well-characterized with 39 available structures of four different family members: the prokaryotic sodium-dependent aspartate transporters Gltoccluded , inward-occluded  and inwaoccluded . We willLike all members of the SLC1 family, neutral amino acid transporter ASCT2 is a homotrimer. Each monomer consists of 8 transmembrane segments (TMs) that form a scaffold domain  and a transport domain . The transport domain additionally contains two helical hairpins (HP1 and HP2). In the outward-facing states the substrate-binding site is close to the extracellular side of the membrane, and the only difference between open and closed conformations is the position of HP2, which works as a gate to provide access to the binding site from the extracellular aqueous environment  Figure . When thTk [Ph [The fixed barrier elevator mechanism with one gate is likely conserved among the SLC1 family, as evidenced by recent single particle cryo-EM structures of GltTk , and molTk [Ph . Fixed bTk [Ph ) have chTk [Ph ,33 and CTk [Ph  indicateATP-binding Cassette (ABC) transporters do not use elevator-type mechanisms of transport, with the exception of the non-canonical subfamily of ECF (energy-coupling factor) transporters. ECF transporters are involved in uptake of vitamins or other micronutrients (reviewed in ). Two suThe concentrative nucleoside transporter CNT (a member of the SLC28 family) is a homotrimer , with ea+/succinate transporter VcINDY [+/citrate transporter SeCitS [It is possible that the location of the binding site between two domains in CNT necessitates the use of two gates, whereas an occluded binding site within the transport domain, as found in SLC1 transporters, may allow the use of a single gate. Most of the transporters with proposed elevator-like transport mechanisms have substrate-binding sites positioned at the interface of two domains . Transpor VcINDY  , and bir SeCitS  of the Sr SeCitS , but addThe bile acid transporter ASBT, and structurally related sodium-proton antiporters have 10 and 13 transmembrane helices respectively, with a transport domain  consisting of TM3–5, TM8–10 in ASBT , and a scaffold domain . Despite the movement of the substrate-binding site across the membrane during sliding of the transport domain relative to the scaffold , ASBT does not have a fixed barrier . Thus, tASBT is exceptional among elevator-type transporters because it is a monomeric protein. Another monomeric transporter, for which an elevator mechanism has been postulated, is CcdA . CcdA isPh show different extents of membrane deformation depending on the position of the transport domain [Ph in the outward-facing state induce very little local membrane curvature [It has been noticed that the TMs of the scaffold domains of many elevator-type transporters are shorter than those in transport domains, and often highly tilted . As a cot domain  , than in liposomes composed of Phosphatidylcholine (PC) [In vivo, allosteric modulation by lipid molecules has been observed in Xenopus oocytes expressing EAAT4 that displayed increased glutamate-induced currents when arachidonic acid was added [In only very few cases have the effects of the lipid environment been studied experimentally. In Gltine (PC) . This efine (PC) , but againe (PC) . In vivoas added . The preas added .The above examples show that lipids may affect protein function directly via interactions with amino acid residues, which could accelerate or slow down transport domain movements or stabilize the scaffold domain in the membrane. In addition, colligative bilayer properties are likely to affect the functioning of elevator-type transporters, because the lipid–protein interface must rearrange substantially during transport. Finally, also the domain structure of the proteins may affect the bilayer morphology, and consequently elevator dynamics.Importance of the field. Since the first description of an elevator-type transport mechanism for GltPh over a decade ago [cade ago , a varieA summary of the current thinking. In elevator-type transport mechanisms, one protein domain brings the substrate-binding site from one side of the membrane to the other by sliding through the lipid bilayer. The extent of the elevator movement, ranging from 21 Å in GltTk to 7.5 Å in ASBT, and number of gating elements (one or two) vary between different proteins  willulations .


One of the most common usages of radiation in current medical diagnosis is computed tomography (CT) using X-rays. The potential health risk of CT scans has been discussed in various studies to determine whether low-dose radiation from CT could enhance the chromosome aberration yields in pediatric patients and increase their risk of carcinogenesis. For this reason, it is of great interest to study the effects of low-dose radiation. The induction of DNA damage by a CT scan examination has been demonstrated in several reports by the γ-H2AX assay, the micronuclei assay and dicentrics measurements. However, the results of most studies showed limitations. On the other hand, epidemiological studies give contradictory results for post-natal radiation exposure in the low-dose range, so it is still difficult to draw conclusions about the effects of CT examinations and risk of carcinogenesis. This article provides an overview of previously published data and summarizes the current state of knowledge. However, the growing use of CT procedures on children raises concern over the long-term risk of cancer development associated with medical radiologic diagnostics and crosslinks assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. This study has several limitations that need to be emphasized, such as the small number of samples and the absence of any control subjects. Another study of Vandevoorde et al.(n = 51), at a low dose (blood doses in the range of 0.15–8.85 mGy). The low-dose hypersensitivity observed in the gamma-H2AX foci dose-response of this study indicates that risk estimates based on the LNT (linear-no-threshold) model may potentially underestimate the risks of pediatric CT imaging. The new in vitro CT study describes that age-related radiation sensitivity in children was not detected by applying the gamma-H2AX assay or DNA-dependent protein kinase spreads up to several megabase pairs around the damaged sites and related foci appear within a minute((Patient-specific factors such as diseases associated with changes to lymphocytes (e.g. infection and lymphoma) or various therapies (e.g. radiotherapy and chemotherapy) can also falsify measurable values. Such patients have been excluded in the most cited studiesin vitro and in vivo ((Several studies have reported the increase of chromosome aberrations in pediatric patients after CT examination  in vivo .. Nevert