Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies, and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly diagnosed diabetes. Multidimensional cluster analysis showed two equal-sized patient agglomerations characterized by proinflammatory (interferon-γ–positive, multiautoantibody-positive) and partially regulated (interleukin-10–positive, pauci-autoantibody–positive) responses. Multiautoantibody-positive nondiabetic siblings at high risk of disease progression showed similar clustering. Additionally, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesions distinguishable by the degree of cellular infiltrate and presence of B cells that we termed “hyper-immune CD20Hi” and “pauci-immune CD20Lo.” Of note, subjects had only one infiltration phenotype and were partitioned by this into two equal-sized groups that differed significantly by age at diagnosis, with hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment and prevention strategies.

Type 1 diabetes results from T cell–mediated β-cell destruction. The HLA-A*24 class I gene confers significant risk of disease and early onset. We tested the hypothesis that HLA-A24 molecules on islet cells present preproinsulin (PPI) peptide epitopes to CD8 cytotoxic T cells (CTLs). Surrogate β-cell lines secreting proinsulin and expressing HLA-A24 were generated and their peptide ligandome examined by mass spectrometry to discover naturally processed and HLA-A24–presented PPI epitopes. A novel PPI epitope was identified and used to generate HLA-A24 tetramers and examine the frequency of PPI-specific T cells in new-onset HLA-A*24+ patients and control subjects. We identified a novel naturally processed and HLA-A24–presented PPI signal peptide epitope (PPI3–11; LWMRLLPLL). HLA-A24 tetramer analysis reveals a significant expansion of PPI3–11-specific CD8 T cells in the blood of HLA-A*24+ recent-onset patients compared with HLA-matched control subjects. Moreover, a patient-derived PPI3–11-specific CD8 T-cell clone shows a proinflammatory phenotype and kills surrogate β-cells and human HLA-A*24+ islet cells in vitro. These results indicate that the type 1 diabetes susceptibility molecule HLA-A24 presents a naturally processed PPI signal peptide epitope. PPI-specific, HLA-A24–restricted CD8 T cells are expanded in patients with recent-onset disease. Human islet cells process and present PPI3–11, rendering themselves targets for CTL-mediated killing.

! 1! Generation) of) human) islet0specific) regulatory) T) cells) by) TCR) gene) transfer.) )Caroline)M.)Hull 1,5) *,)Lauren)E.)Nickolay 1,5 ,)Megan)Estorninho 1,5 ,)Max)W.) Richardson 2 ,)James)L.)Riley 2 ,)Mark)Peakman 1,3,4,5 ,)John)Maher 5,6,7 ,)Timothy) I.M.)Tree 1,5) *) ) 1 Department)of)Immunobiology,)Faculty)of)Life)Sciences)&)Medicine,)King's) College)London,)London)SE1)9RT,)UK) ) 2 Department)of)Microbiology,)University)of)Pennsylvania)School)of)Medicine,) Philadelphia,)Pennsylvania,)USA) ) 3 Peak)Therapeutics,)London)SE24)9LG,)UK ) 4 Division)of)Diabetes)and)Nutrition,)King's)College)London)SE1)1UL,)UK) ) 5 NIHR)Biomedical)Research)Centre,)Guy's)and)St)Thomas')NHS)Foundation) Trust)and)King's)College)London,)London)SE1)9RT,)UK) ) 6) CAR)Mechanics)Group,)Division)of)Cancer)Studies,)Guy's)Hospital)Campus,) King's)College)London)School)of)Medicine,)London)SE1)1UL,)UK) ) 7 Department)of)Allergy)and)Immunology,)King's)College)Hospital)National) Health)Service)Foundation)Trust,)London)SE5)9RS,)UK) ) ) Short)Running)Title:)Generation)of)human)islet[specific)Tregs.) ) ) ) *Corresponding)authors) ) caroline.hull@kcl.ac.uk) timothy.tree@kcl.ac.uk) ) ! 2! Abstract) )Based)on)the)success)in)animal)models)of)type)1)diabetes)(T1D),)clinical)trials) of) adoptive) regulatory) T) cell) (Treg)) therapy) are) underway) using) ex# vivo) expanded)polyclonal)Tregs.)However,)pre[clinical)data)also)demonstrate)that)islet[specific) Tregs) are) more) potent) than) polyclonal) Tregs) at) reversing) T1D.)Translation) of) this) approach) into) man) will) require) methods) to) generate) large) populations) of) islet[specific) Tregs) which,) to) date,) has) proved) to) be) a) major)hurdle.)Here)we)demonstrate)the)feasibility)of)lentiviral[mediated)T)cell)receptor) (TCR))gene)transfer)to)confer)antigen)specificity)on)polyclonal)human)Tregs.)Targeting) has) been) achieved) using) TCRs) isolated) from) human) islet [specific) and) viral[specific) CD4+) T) cell) clones.) Engineered) T) cells) demonstrated) expression)of)ectopically[delivered)TCRs,)resulting)in)endowment)of)cognate)antigen[specific) responses.) This) enabled) antigen[specific) suppression) at) increased)potency)compared)to)polyclonal)Tregs.)However,)cells)transduced) with) islet[specific) TCRs) were) less) responsive) to) cognate) antigen) than) viral[ specific) TCRs,) and) in) some) cases,) required) additional) methods) to) isolate) functional)antigen[specific)Tregs.)This)study)demonstrates)the)potential)of)TCR) gene)transfer)to)develop)islet[specific)Treg)therapies)for)effective)treatment)of) T1D,)but)also)highlights)that)additional)optimisation)may)be)required)to)achieve)Populations) of) Tregs) found) in) the) periphery,) including) those) expressing) the)with)T1D.)This)abnormality)is)evident)before)clinical)diagnosis,)at)the)time)of) diagnosis) and) many) years) following) onset) of) T1D [4[7].) Since) defective) Treg) function) appears) to) be) central) to) the) pathogenesis) of) T1D,) it) is) logical) to) hypothesise) that) correction) of) this) imbalance) may...

Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis