The evolution of CD4+ T cell clonality in a murine model of inflammatory arthritis

Immunological tolerance is an important aspect of immunity preventing responses being mounted against self-peptides and other innocuous foreign antigens. A breach in self-tolerance can lead to the development of autoimmune diseases such as Rheumatoid arthritis (RA). RA is a chronic inflammatory autoimmune disease that is characterised by synovial inflammation and joint erosion. CD4+ T cells have been shown to play a key role in disease progression, and their ability to infiltrate joints is associated with perpetuation of local and systemic inflammatory responses. A diverse range of T cell receptor (TCR) usage has been demonstrated in RA patients, however how such diversity arises and is shaped remains unclear. Understanding the development of CD4+ T cell antigen specific responses will therefore be important for the development and application of antigen-specific therapeutic tolerance regimes. To investigate these antigen specific responses, CD4+ T cell clonality was examined using the OVA induced breach of tolerance model of experimental arthritis, allowing the assessment of developing antigen specific responses in the early stages of arthritis. The initial articular CD4+ T cell response was found to be oligoclonal in nature, with enrichment of several TCRVβ families in the inflamed joint. Moreover, the enrichment for some families is associated with joint derived antigens. Next-generation sequencing analysis of CDR3β sequences of CD4+ T cell clones revealed the dynamics of clonal responses between the inflamed joint and its associated draining lymph node and how these responses change with the progression of the disease. Inflamed joints displayed similar CD4+ T cell repertoire diversity at early and late stages of the disease, while inflamed lymph nodes displayed increased repertoire diversity with disease progression. Moreover, the number of CD4+ T cell clones shared between the inflamed joint and lymph node decreased with time. However, correlation analyses of highly abundant clones between inflamed joints and lymph nodes suggested continued migration of CD4+ T cell clones from inflamed lymph nodes to the joints. The decreased diversity in inflamed lymph nodes at the later time point may be a reflection of epitope spreading to the initial inciting antigen as well as development of new responses to neo antigens released as a result of continued joint damage. The hypothesis is that the reduced CD4+ T cell diversity in the inflamed lymph nodes will eventually be mirrored in the joint if the disease continues untreated. This research provides insight on the dynamics of the antigen specific response between the inflamed tissue and its draining lymph node with disease progression, highlighting important site specific and temporal differences in clonal diversity with disease development and also highlights the role autoreactive CD4+ T cell responses play in disease progression. By understating the evolution of CD4+ T cell responses in RA, more informed decisions can be made on how antigen-specific therapeutics should be applied and will help develop more effective regimes to reinstate self-tolerance, with the ultimate goal of moving towards drug free remission and a cure

TCRß sequencing reveals spatial and temporal evolution of clonal CD4 T cell responses in a breach of tolerance model of inflammatory arthritis

Effective tolerogenic intervention in Rheumatoid Arthritis (RA) will rely upon understanding the evolution of articular antigen specific CD4 T cell responses. TCR clonality of endogenous CD4 T cell infiltrates in early inflammatory arthritis was assessed to monitor evolution of the TCR repertoire in the inflamed joint and associated lymph node (LN). Mouse models of antigen-induced breach of self-tolerance and chronic polyarthritis were used to recapitulate early and late phases of RA. The infiltrating endogenous, antigen experienced CD4 T cells in inflamed joints and LNs were analysed using flow cytometry and TCRβ sequencing. TCR repertoires from inflamed late phase LNs displayed increased clonality and diversity compared to early phase LNs, while inflamed joints remained similar with time. Repertoires from late phase LNs accumulated clones with a diverse range of TRBV genes, while inflamed joints at both phases contained clones expressing similar TRBV genes. Repertoires from LNs and joints at the late phase displayed reduced CDR3β sequence overlap compared to the early disease phase, however the most abundant clones in LNs accumulate in the joint at the later phase. The results indicate CD4 T cell repertoire clonality and diversity broadens with progression of inflammatory arthritis and is first reflected in LNs before mirroring in the joint. These observations imply that antigen specific tolerogenic therapies could be more effective if targeted at earlier phases of disease when CD4 T cell clonality is least diverse

Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription Translation (TX-TL) Systems

RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo

Tolerance induction in memory CD4 T cells is partial and reversible

Memory T cells respond rapidly in part because they are less reliant on heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of re‐activating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggest they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re‐infection. These data indicate that antigen specific tolerogenic strategies must examine multiple parameters of T cell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells

Preclinical models of arthritis for studying immunotherapy and immune tolerance

Increasingly earlier identification of individuals at high risk of rheumatoid arthritis (RA) (eg, with autoantibodies and mild symptoms) improves the feasibility of preventing or curing disease. The use of antigen-specific immunotherapies to reinstate immunological self-tolerance represent a highly attractive strategy due to their potential to induce disease resolution, in contrast to existing approaches that require long-term treatment of underlying symptoms. Preclinical animal models have been used to understand disease mechanisms and to evaluate novel immunotherapeutic approaches. However, models are required to understand critical processes supporting disease development such as the breach of self-tolerance that triggers autoimmunity and the progression from asymptomatic autoimmunity to joint pain and bone loss. These models would also be useful in evaluating the response to treatment in the pre-RA period. This review proposes that focusing on immune processes contributing to initial disease induction rather than end-stage pathological consequences is essential to allow development and evaluation of novel immunotherapies for early intervention. We will describe and critique existing models in arthritis and the broader field of autoimmunity that may fulfil these criteria. We will also identify key gaps in our ability to study these processes in animal models, to highlight where further research should be targeted

Oocyte Developmental Competence Requires Follicle-Stimulating Hormone.

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Follicle-stimulating hormone is not required for oocyte growth or acquisition of meiotic competence in vivo.

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Role of FSH in oocyte competence and embryo development.

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Follicle-stimulating hormone accelerates mouse oocyte development in vivo.

During folliculogenesis, oocytes grow and acquire developmental competence in a mutually dependent relationship with their adjacent somatic cells. Follicle-stimulating hormone (FSH) plays an essential and well-established role in the differentiation of somatic follicular cells, but its function in the development of the oocyte has still not been elucidated. We report here that oocytes of Fshb(-/-) mice, which cannot produce FSH, grow at the same rate and reach the same size as those of wild-type mice. Consistent with this observation, the granulosa cells of Fshb(-/-) mice express the normal quantity of mRNA encoding Kit ligand, which has been implicated in oocyte growth. Oocytes of Fshb(-/-) mice also accumulate normal quantities of cyclin B1 and CDK1 proteins and mitochondrial DNA. Moreover, they acquire the ability to complete meiotic maturation in vitro and undergo transition from non-surrounded nucleolus to surrounded nucleolus. However, these events of late oocyte development are significantly delayed. Following in vitro maturation and fertilization, only a small number of embryos derived from oocytes of Fshb(-/-) mice reach the blastocyst stage. Administration of equine chorionic gonadotropin, which provides FSH activity, 48 h before in vitro maturation increases the number of blastocysts obtained subsequently. These results indicate that FSH is not absolutely required for oocyte development in vivo but that this process occurs more rapidly in its presence. We suggest that FSH may coordinate the development of the germline and somatic compartments of the follicle, ensuring that ovulation releases a developmentally competent egg.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Role of follicle stimulating hormone in the acquisition of developmental competence.

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