Multiple genetic paths including massive gene amplification allow Mycobacterium tuberculosis to overcome loss of ESX-3 secretion system substrates.

Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.

Standardising personalised diabetes care across European health settings: A person-centred outcome set agreed in a multinational Delphi study.

OBJECTIVE: Standardised person-reported outcomes (PRO) data can contextualise clinical outcomes enabling precision diabetes monitoring and care. Comprehensive outcome sets can guide this process, but their implementation in routine diabetes care has remained challenging and unsuccessful at international level. We aimed to address this by developing a person-centred outcome set for Type 1 and Type 2 diabetes, using a methodology with prospects for increased implementability and sustainability in international health settings. METHODS: We used a three-round questionnaire-based Delphi study to reach consensus on the outcome set. We invited key stakeholders from 19 countries via purposive snowball sampling, namely people with diabetes (N = 94), healthcare professionals (N = 65), industry (N = 22) and health authorities (N = 3), to vote on the relevance and measurement frequency of 64 previously identified clinical and person-reported outcomes. Subsequent consensus meetings concluded the study. RESULTS: The list of preliminary outcomes was shortlisted via the consensus process to 46 outcomes (27 clinical outcomes and 19 PROs). Two main collection times were recommended: (1) linked to a medical visit (e.g. diabetes-specific well-being, symptoms and psychological health) and (2) annually (e.g. clinical data, general well-being and diabetes self management-related outcomes). CONCLUSIONS: PROs are often considered in a non-standardised way in routine diabetes care. We propose a person-centred outcome set for diabetes, specifically considering psychosocial and behavioural aspects, which was agreed by four international key stakeholder groups. It guides standardised collection of meaningful outcomes at scale, supporting individual and population level healthcare decision making. It will be implemented and tested in Europe as part of the H2O project.

The Hidden Genomics of Chlamydia trachomatis.

The application of whole-genome sequencing has moved us on from sequencing single genomes to defining unravelling population structures in different niches, and at the -species, -serotype or even -genus level, and in local, national and global settings. This has been instrumental in cataloguing and revealing a huge a range of diversity in this bacterium, when at first we thought there was little. Genomics has challenged assumptions, added insight, as well as confusion and glimpses of truths. What is clear is that at a time when we start to realise the extent and nature of the diversity contained within a genus or a species like this, the huge depth of knowledge communities have developed, through cell biology, as well as the new found molecular approaches will be more precious than ever to link genotype to phenotype. Here we detail the technological developments and insights we have seen during the relatively short time since we began to see the hidden genome of Chlamydia trachomatis.

Canadian healthcare capacity gaps for disease-modifying treatment in Huntington's disease: a survey of current practice and modelling of future needs.

OBJECTIVES: Disease-modifying therapies in development for Huntington's disease (HD) may require specialised administration and additional resource capacity. We sought to understand current and future capacity for HD management in Canada considering the possible introduction of an intrathecal (IT) disease-modifying treatment (DMT). DESIGN, SETTING AND PARTICIPANTS: Using a case study, mixed methods framework, online surveys followed by semistructured interviews were conducted in late 2020 and early 2021. Neurologists from Canadian HD (n=16) and community (n=11) centres and social workers (n=16) were invited to complete online surveys assessing current HD management and potential capacity to support administration of an IT DMT. OUTCOME MEASURES: Survey responses, anticipated demand and assumed resource requirements were modelled to reveal capacity to treat (ie, % of eligible patients) by centre. Resource bottlenecks and incremental support required (full-time equivalent, FTE) were also determined. RESULTS: Neurologists from 15/16 HD centres and 5/11 community centres, plus 16/16 social workers participated. HD centres manage 94% of patients with HD currently seeking care in Canada, however, only 20% of IT DMT-eligible patients are currently seen by neurologists. One-third of centres have no access to nursing support. The average national incremental nursing, room, neurologist and social worker support required to provide IT DMT to all eligible patients is 0.73, 0.36, 0.30 and 0.21 FTE per HD centre, respectively. At peak demand, current capacity would support the treatment of 6% of IT DMT-eligible patients. If frequency of administration is halved, capacity for IT-DMT administration only increases to 11%. CONCLUSIONS: In Canada, there is little to no capacity to support the administration of an IT DMT for HD. Current inequitable and inadequate resourcing will require solutions that consider regional gaps and patient needs.

Supporting Trial Participation in People with the Huntington's Gene: A Patient-Centered, Theory-Guided Survey of Barriers and Enablers.

BACKGROUND: Under-recruitment regularly impedes clinical trials, leading to wasted resources and opportunity costs. Methods for designing trial participation strategies rarely consider behavior change theory. OBJECTIVE: Informed by the Theoretical Domains Framework, we identified factors important to participating in Huntington's disease research and provide examples of how such a theory-informed approach can make specific suggestions about how to design targeted recruitment strategies. METHODS: We identified a range of trial participation barriers and enablers based on interviews of key informants and implemented an online survey of members of the Huntington's disease community, asking them to rate the extent to which different factors would affect likelihood to participate in a generic Huntington's disease trial. RESULTS: From 4,195 members, we received 323 responses and 243 completed surveys (323/4,195 or 8% participation, 243/323 or 75% completion). Respondents endorsed 9 barriers and 23 enablers relevant to trial participation. Most frequently endorsed barriers were travel to the study site (69%), worry about unknown side effects (65%), trial documents being difficult to understand (64%), and participation affecting other activities (49%). Enablers included optimism about likelihood of trial participation leading to a cure (98%), helping others (98%), contributing to science (97%), and having helpful people available to help with the participation decision (89%). CONCLUSION: Our theory-informed survey to identify barriers to and enablers of Huntington's disease trial participation identified 32 factors, from 13 theoretical domains relevant to trial participation, and suggests effective approaches for improving trial participation and patient experience.

A patient-focused, theory-guided approach to survey design identified barriers to and drivers of clinical trial participation.

OBJECTIVES: Despite clear evidence showing that many clinical trials fail or are delayed because of poor patient recruitment, there is surprisingly little empirically supported guidance for trialists seeking to optimize their trial recruitment strategies. We propose that the challenges of recruitment can be better understood and addressed by thinking of research participation as one or more behaviors, subject to the same forces as other human behaviors. In this article, we describe an adaptable, behavioral theory-driven approach for designing pretrial surveys of the barriers and drivers relevant to trial participation. Instead of proposing a single survey instrument intended to be used uniformly across many situations, we propose that tailored surveys be informed by a common comprehensive, theory-guided development approach that ensures all domains potentially guiding participation are considered. STUDY DESIGN AND SETTING: We used the Theoretical Domains Framework (TDF), which organizes over 100 constructs known to be associated with behavior and behavior change into 14 domains that describe determinants of professional and patient health behaviors, to inform the development of tailored surveys about barriers to and drivers of clinical trial participation. After searching the literature for barriers and drivers to trial recruitment relevant to each of the TDF domains, we developed separate surveys for members of two national health charities (Canadian Breast Cancer Network, Huntington Society of Canada) to exemplify how the approach can be adapted across settings. We conducted think-aloud interviews with members of each group to maximize the clarity and usability of the surveys, elicited opinions about which barriers/drivers were relevant for each patient group, and identified additional barriers/drivers. Interviews proceeded iteratively with changes incorporated into subsequent interviews. Here, we describe our two target patient groups, as well as our process of modifying, adding, and deleting barrier/driver items for each group and across theoretical domains. RESULTS: We interviewed 8 women with a history of breast cancer from the Canadian Breast Cancer Network (48-65 year old) and 11 Huntington Disease community members (9 women) from the Huntington Society of Canada (26-70 year old). After the iterative development interviews, the breast cancer group had identified 38 barriers/drivers thought relevant to their participation in clinical trials across 12 TDF domains. The Huntington group identified 47 items across 13 TDF domains. CONCLUSION: Our patient-focused and theory-guided approach was able to identify a more comprehensive range of barriers to and drivers of trial participation than existing published tools. Our approach is also more broadly adaptable than such tools, in that it uses a theoretical framework and in-depth piloting to generate a set of items tailored to each specific clinical area, rather than a single set of items intended to be applicable to all situations. This theory-guided approach also enables more specific recruitment strategies to be developed once domain-specific barriers are known, potentially optimizing participation for a given trial and helping build a cumulative evidence of barriers/drivers and strategies for addressing them.

Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains.

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis, is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

From research to rapid response: mass COVID-19 testing by volunteers at the Centre for Genomic Regulation.

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.

TCR-beta chains derived from peripheral gammadelta T cells can take part in alphabeta T-cell development.

Between 10 and 20% of the peripheral gammadelta T cells express cytoplasmic TCR-beta proteins, but whether such TCR-beta chains can partake in alphabeta T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3epsilon-deficient mice with Pax5-TCR-beta deficient proB cells expressing, via a retroviral vector, TCR-beta chains from either peripheral gammadelta or alphabeta T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (<15x10(6) cells), contained few gammadelta T but no alphabeta T cells. In contrast, thymi from mice receiving proB cells containing gammadelta or alphabeta T-cell-derived TCR-beta chains contained 80-130x10(6) cells, and showed a normal CD4, CD8 and alphabeta TCR expression pattern. However, regardless of the source of TCR-beta chain, reconstituted mice rapidly showed signs of autoimmunity dying 5-15 wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-beta chains from gammadelta T cells can efficiently take part in alphabeta T-cell development. The implications of these findings for gammadelta T-cell development will be discussed.

Whole genome sequence of Vibrio cholerae directly from dried spotted filter paper.

BACKGROUND: Global estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately contribute new information to shape public policy response on cholera control and elimination. METHODOLOGY/PRINCIPAL FINDINGS: Whole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/µL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor. CONCLUSIONS: WGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers. This provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas.

The Chlamydia muridarum plasmid revisited : new insights into growth kinetics.

Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence.  Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. Methods: We measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies. Results: We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication. Conclusions: Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes.  There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum.  We have proven that the differences described are solely due to the plasmid pNigg.

Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25- T cells.

CD4+CD25+ regulatory T (TR) cells have been described in both humans and mice. In mice, TR are thymically derived, and lack of TR leads to organ-specific autoimmunity. Recently, the forkhead/winged helix transcription factor, FoxP3, has been shown to be important for the function of TR cells in mice. In this study, human TR cells were examined and, in results similar to those of studies done in mice, expression of FoxP3 was found exclusively in CD4+CD25+ T cells and correlated with the suppressive activity of these cells. In contrast to the mouse studies, activation of human CD4+CD25- T cells led to expression of FoxP3. Expression of FoxP3 in activated human CD4+CD25+ cells also correlated with suppression of proliferation by these cells in freshly isolated CD4+CD25- T cells from the same donor. This suppression was cell-contact dependent and cytokine independent. Thus, in humans, during activation of CD4+CD25- T cells in an immune response, two populations of cells may arise, effector CD4+CD25+ and regulatory CD4+CD25+ T cells, with expression of FoxP3 correlated with regulatory activity. These data also raise the possibility that a failure to generate peripheral TR cells properly may contribute to autoimmune disease and suggest a possible therapeutic role for FoxP3 in the treatment of such diseases.

Mycobacterium ulcerans Mouse Model Refinement for Pre-Clinical Profiling of Vaccine Candidates.

Buruli Ulcer is a neglected tropical disease leading to extensive disabilities and morbidity in West Africa. In this paper we sought to characterize various strains of Mycobacterium ulcerans (M.ulcerans) with different origins and laboratory passage records while refining a mouse model for Buruli ulcer. We described, compared and followed the kinetics of the histo-pathological outcome of infection of a collection of strains at various anatomical sites of infection in order to find a suitable model for further immunization studies. Moreover we compared the outcome of infection in C57Bl/6 and Balbc/J mice. Specifically we described thoroughly one M. ulcerans strain characterized by slow growth rate and limited tissue necrosis, which presents close ressemblance with the infection kinetics in humans. This strain caused macrophages as well as T and B cells infiltration, correlating with mycobacterial proliferation at the site of infection as well as in the draining lymph nodes, making it a suitable strain to screen vaccine candidates efficacy.

Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer.

BACKGROUND: Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters. METHODOLOGY/PRINCIPAL FINDINGS: To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a TH1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model. CONCLUSIONS: Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci.

Herpes simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin and neural disease.

Subunit vaccines comprised of glycoprotein D (gD-2) failed to prevent HSV-2 highlighting need for novel strategies. To test the hypothesis that deletion of gD-2 unmasks protective antigens, we evaluated the efficacy and safety of an HSV-2 virus deleted in gD-2 and complemented allowing a single round of replication on cells expressing HSV-1 gD (ΔgD(-/+gD-1)). Subcutaneous immunization of C57BL/6 or BALB/c mice with ΔgD(-/+gD1) provided 100% protection against lethal intravaginal or skin challenges and prevented latency. ΔgD(-/+gD1) elicited no disease in SCID mice, whereas 1000-fold lower doses of wild-type virus were lethal. HSV-specific antibodies were detected in serum (titer 1:800,000) following immunization and in vaginal washes after intravaginal challenge. The antibodies elicited cell-mediated cytotoxicity, but little neutralizing activity. Passive transfer of immune serum completely protected wild-type, but not Fcγ-receptor or neonatal Fc-receptor knock-out mice. These studies demonstrate that non-neutralizing Fc-mediated humoral responses confer protection and support advancement of this attenuated vaccine.

Auto-reconstitution of the T-cell compartment by radioresistant hematopoietic cells following lethal irradiation and bone marrow transplantation.

OBJECTIVE: In lethally irradiated bone marrow chimeras, part of the reconstituted T-cell compartment is derived from the irradiated host, but the detailed origin and functional activity of host-derived T cells has not been thoroughly analyzed. Herein, we determine the origin and function of radioresistant host-derived T cells. MATERIALS AND METHODS: Lethally irradiated thymectomized or nonthymectomized C57BL/6 host mice were reconstituted with syngeneic bone marrow, itself incapable of generating T cells. Using fetal thymic organ cultures, bulk and limiting dilution assays on OP9-DL1 stromal cells, unambiguous cohorts of thymus-derived and peripheral T-cell-derived T cells were phenotypically characterized by flow cytometry and functionally characterized by their ability to participate in a T-cell-dependent antibody response. RESULTS: Both thymus-derived and peripheral T-cell-derived host T cells are functional and can reconstitute 35% of the normal T-cell pool. By comparing thymectomized vs nonthymectomized hosts, host-derived T cells were shown to comprise a major (70%) subpopulation of de novo generated, thymus-derived, polyclonal, naïve cells, and a minor subpopulation of surviving, peripheral, oligoclonal, memory-like cells. Unlike euthymic recipients, mice whose T cells were derived from surviving peripheral T cells were frequently incapable of mounting a T-cell-dependent antibody response. Host-derived thymocytes regenerated in an interleukin-7-dependent fashion from conventional DN2 thymocytes and their differentiation recapitulated normal thymic ontogeny. CONCLUSION: We characterized, for the first time, functional radioresistant DN2-phenotype thymic T-cell precursors, the T-cell progeny of which might provide a first line of defense against infections during the lymphopenic phase post-bone marrow transplantation.

Serological evaluation of Mycobacterium ulcerans antigens identified by comparative genomics.

A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were able to discriminate between these two groups. Nevertheless, six of the seven proteins showed an ability to distinguish people living in an endemic area from those in a non-endemic area with an average sensitivity of 69% and specificity of 88%, suggesting exposure to M. ulcerans. Further validation of these six proteins is now underway to assess their suitability for use in Buruli ulcer seroepidemiological studies. Such studies are urgently needed to assist efforts to uncover environmental reservoirs and understand transmission pathways of the M. ulcerans.

Regulatory T cells control autoimmunity following syngeneic bone marrow transplantation.

Sublethally irradiated, immunodeficient, C57BL/6 RAG-2 gene-deleted recipient mice reconstituted with T cell-depleted bone marrow (BM) grafts frequently developed diarrhea, lost weight and showed signs of autoimmunity, dying between 4 and 7 weeks after reconstitution. Mice died despite evidence of efficient donor-derived hemato-lymphoid reconstitution, and disease was associated with the presence of IgG anti-nuclear antibodies. Autoimmunity was initiated by T cells, but could be prevented by transfer of naturally arising regulatory T cells. In contrast, lethally irradiated, BM-reconstituted immunocompetent, C57BL/6 mice survived without signs of autoimmunity. Survival of immunocompetent mice was shown to be due to the presence of residual, extra-thymically located, radio-resistant, functional regulatory T cells. The importance of regulatory T cells was further shown by the reduced survival of immunocompetent BM recipients whose CD25+ T cells had been depleted prior to bone marrow transplantation. The implications of these results in the context of syngeneic graft-versus-host disease following BM transplantation are discussed.