CXCR5+PD-1++ CD4+ T cells colonize infant intestines early in life and promote B cell maturation.

Gastrointestinal infections are a major cause for serious clinical complications in infants. The induction of antibody responses by B cells is critical for protective immunity against infections and requires CXCR5+PD-1++ CD4+ T cells (TFH cells). We investigated the ontogeny of CXCR5+PD-1++ CD4+ T cells in human intestines. While CXCR5+PD-1++ CD4+ T cells were absent in fetal intestines, CXCR5+PD-1++ CD4+ T cells increased after birth and were abundant in infant intestines, resulting in significant higher numbers compared to adults. These findings were supported by scRNAseq analyses, showing increased frequencies of CD4+ T cells with a TFH gene signature in infant intestines compared to blood. Co-cultures of autologous infant intestinal CXCR5+PD-1+/-CD4+ T cells with B cells further demonstrated that infant intestinal TFH cells were able to effectively promote class switching and antibody production by B cells. Taken together, we demonstrate that functional TFH cells are numerous in infant intestines, making them a promising target for oral pediatric vaccine strategies.

Do Relapses Follow ANCA Rises? A Systematic Review and Meta-Analysis on the Value of Serial ANCA Level Evaluation.

Objectives: ANCA-vasculitis (AAV) patients frequently suffer from relapses and risk subsequent organ damage. There is much debate on the value of serial ANCA level evaluation to monitor disease activity. We aimed to evaluate the association between ANCA rises and disease relapses at (I) moment of the rise, (II) within 6 months or (III) within a year from the rise. Methods: 3 databases (MEDLINE, EMBASE, COCHRANE) were searched from 1993 through September 2021. We included studies that reported relapse incidence within 12 months after an ANCA rise measured by antigen-specific immunoassays in peripheral blood of AAV patients in remission. Quality assessment was performed using QUADAS-2. Finally, a meta-analysis was carried out to estimate average OR using a random effects model. Results: Twenty unique studies were included. The methodological quality was limited due to risk of selection bias. An ANCA rise often preceded a disease relapse within 6 months (OR 3.65, 95% CI 1.66-8.03) and less often within 12 months (OR 2.88, 95% CI 1.21-6.88), while it was not indicative of a concurrent relapse (OR 0.13, 95% CI 0.03-0.53). Once a relapse is diagnosed, ANCA is significantly more often present than not (OR 10.80, 95% CI 3.82-30.55). As expected based on clinical, technical and methodological variability between studies, there was substantial heterogeneity across studies in all analyses (I2 = 70-87%). Conclusion: In previously ANCA-positive patients, the ANCA test is often positive upon clinical suspicion of a disease relapse. Patients with a rise in ANCA are at risk of encountering disease relapses in the upcoming 6 or 12 months.

The Germinal Center Milieu in Rheumatoid Arthritis: The Immunological Drummer or Dancer?

Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation, affecting approximately 1% of the general population. To alleviate symptoms and ameliorate joint damage, chronic use of immunosuppressives is needed. However, these treatments are only partially effective and may lead to unwanted side effects. Therefore, a more profound understanding of the pathophysiology might lead to more effective therapies, or better still, a cure. The presence of autoantibodies in RA indicates that B cells might have a pivotal role in the disease. This concept is further supported by the fact that a diverse antibody response to various arthritis-related epitopes is associated with arthritis development. In this context, attention has focused in recent years on the role of Germinal Centers (GCs) in RA. Since GCs act as the main anatomic location of somatic hypermutations, and, thus, contributing to the diversity and specificity of (auto) antibodies, it has been speculated that defects in germinal center reactions might be crucial in the initiation and maintenance of auto-immune events. In this paper, we discuss current evidence that various processes within GCs can result in the aberrant production of B cells that possess autoreactive properties and might result in the production of RA related autoantibodies. Secondly, we discuss various (pre-)clinical studies that have targeted various GC processes as novel therapies for RA treatment.

T cell receptor repertoire characteristics both before and following immunotherapy correlate with clinical response in mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly lethal malignancy in need for new treatment options. Although immunotherapies have been shown to boost a tumor-specific immune response, not all patients respond and prognostic biomarkers are scarce. In this study, we determined the peripheral blood T cell receptor ß (TCRß) chain repertoire of nine MPM patients before and 5 weeks after the start of dendritic cell (DC)-based immunotherapy. MATERIALS AND METHODS: We separately profiled PD1+ and PD1-CD4+ and CD8+ T cells, as well as Tregs and analyzed 70 000 TCRß sequences per patient. RESULTS: Strikingly, limited TCRß repertoire diversity and high average clone sizes in total CD3+ T cells before the start of immunotherapy were associated with a better clinical response. To explore the differences in TCRß repertoire prior-DC-therapy and post-DC-therapy, for each patient the TCRß clones present in the total CD3+ T cell fractions were classified into five categories, based on therapy-associated frequency changes: expanding, decreasing, stable, newly appearing and disappearing clones. Subsequently, the presence of these five groups of clones was analyzed in the individual sorted T cell fractions. DC-therapy primarily induced TCRß repertoire changes in the PD1+CD4+ and PD1+CD8+ T cell fractions. In particular, in the PD1+CD8+ T cell subpopulation we found high frequencies of expanding, decreasing and newly appearing clones. Conversion from a PD1- to a PD1+ phenotype was significantly more frequent in CD8+ T cells than in CD4+ T cells. Hereby, the number of expanding PD1+CD8+ T cell clones-and not expanding PD1+CD4+ T cell clones following immunotherapy positively correlated with overall survival, progression-free survival and reduction of tumor volume. CONCLUSION: We conclude that the clinical response to DC-mediated immunotherapy is dependent on both the pre-existing TCRß repertoire of total CD3+ T cells and on therapy-induced changes, in particular expanding PD1+CD8+ T cell clones. Therefore, TCRß repertoire profiling in sorted T cell subsets could serve as predictive biomarker for the selection of MPM patients that benefit from immunotherapy. TRIAL REGISTRATION NUMBER: NCT02395679.

Eomes broadens the scope of CD8 T-cell memory by inhibiting apoptosis in cells of low affinity.

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.

Non-response to rituximab therapy in rheumatoid arthritis is associated with incomplete disruption of the B cell receptor repertoire.

OBJECTIVE: To gain more insight into the dynamics of lymphocyte depletion and develop new predictors of clinical response to rituximab in rheumatoid arthritis (RA). METHODS: RNA-based next-generation sequencing was used to analyse the B cell receptor (BCR) repertoire in peripheral blood and synovial tissue samples collected from 24 seropositive patients with RA treated with rituximab. Clonal expansion, mutation load and clonal overlap were assessed in samples collected before, at week 4 and at week 16 or 24 after treatment and correlated to the patients' clinical response. RESULTS: After 4 weeks of rituximab-induced B cell depletion, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones. No significant changes in the synovial tissue BCR repertoire were detected until week 16 post-treatment, when a reduced clonal overlap with baseline and an increased mutation load were observed. In patients who were non-responders at month 3 (n=5) using the European League Against Rheumatism response criteria, peripheral blood samples taken at week 4 after rituximab treatment showed more dominant clones compared with moderate responders (n=9) (median (IQR): 36 (27-52) vs 18 (16-26); p<0.01) and more clonal overlap with the baseline (median (IQR): 5% (2%-20%) vs 0% (0%-0%); p≤0.01). CONCLUSION: Significant changes in BCR clonality are observed in peripheral blood of patients 4 weeks after rituximab treatment, while changes in synovial tissue were observed at later time points. Incomplete depletion of the dominant baseline peripheral blood BCR repertoire in the first month of treatment might predict clinical non-response at 3 months.

Human Fetal TNF-α-Cytokine-Producing CD4+ Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life.

Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α)+CD4+CD69+ T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4+ T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4+ T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4+ Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4+ T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth.

In Rheumatoid Arthritis, Synovitis at Different Inflammatory Sites Is Dominated by Shared but Patient-Specific T Cell Clones.

Genetic and immunological evidence clearly points to a role for T cells in the pathogenesis of rheumatoid arthritis (RA). Selective targeting of such disease-associated T cell clones might be highly effective while having few side effects. However, such selective targeting may only be feasible if the same T cell clones dominate the immune response at different sites of inflammation. We leveraged high-throughput technology to quantitatively assess whether different T cell clones dominate the inflammatory infiltrate at various sites of inflammation in this prototypic autoimmune disease. In 13 RA patients, we performed quantitative next-generation sequencing-based human TCRß repertoire analysis in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, and from synovial fluid (SF) and peripheral blood (PB). Identical TCRß clones dominate inflammatory responses in ST samples taken from different locations within a single joint and when sampled in different joints. Although overall ST-SF overlap was comparable to higher ST-ST values, the overlap in dominant TCRß clones in ST-SF comparisons was much lower than ST-ST and comparable to the low ST-PB overlap. In individual RA patients, a limited number of TCRß clones dominate the immune response in the inflamed ST regardless of the location within a joint and which joint undergoes biopsy; in contrast, there is limited overlap of ST with SF or PB TCR repertoires. This limited breadth of the T cell response in ST of the individual RA patient indicates that development of immunotherapies that selectively modulate dominant T cell responses might be feasible.

Dominant B cell receptor clones in peripheral blood predict onset of arthritis in individuals at risk for rheumatoid arthritis.

BACKGROUND: The onset of seropositive rheumatoid arthritis (RA) is preceded by the presence of specific autoantibodies in the absence of synovial inflammation. Only a subset of these at-risk individuals will develop clinical disease. This impedes efforts to implement early interventions that may prevent onset of clinically manifest disease. Here we analyse whether clonal changes in the B cell receptor (BCR) repertoire can reliably predict onset of signs and symptoms. METHODS: In a prospective cohort study in 21 individuals at risk for RA based on the presence of autoantibodies, the BCR repertoire of paired peripheral blood and synovial tissue samples was analysed using next-generation BCR sequencing. BCR clones that were expanded beyond 0.5% of the total repertoire were labelled dominant. The relative risk (RR) for onset of arthritis was assessed using the presence of ≥5 dominant BCR clones as cut-off. Findings in peripheral blood were validated in an independent prospective cohort of 50 at-risk individuals. Based on the test cohort, individuals in the validation cohort were considered positive if peripheral blood at study entry showed ≥5 dominant BCR clones. FINDINGS: Both in the test and validation cohort, the presence of ≥5 dominant BCR clones in peripheral blood was significantly associated with arthritis development after follow-up (validation cohort RR 6.3, 95% CI 2.7 to 15, p<1×10-4). Even when adjusted for a recently described clinical prediction rule the association remained intact (RR 5.0, 95% CI 1.2 to 20, p=0.024). When individuals developed arthritis, dominant BCR clones disappeared from peripheral blood and appeared in synovial tissue, suggesting a direct role of these clones in disease pathogenesis. INTERPRETATION: Dominant BCR clones in peripheral blood predict onset of clinical signs and symptoms of RA in at-risk individuals with high accuracy. Our data suggest that during onset of RA these clones shift from peripheral blood to the target tissue.

Computational Model Reveals Limited Correlation between Germinal Center B-Cell Subclone Abundancy and Affinity: Implications for Repertoire Sequencing.

Immunoglobulin repertoire sequencing has successfully been applied to identify expanded antigen-activated B-cell clones that play a role in the pathogenesis of immune disorders. One challenge is the selection of the Ag-specific B cells from the measured repertoire for downstream analyses. A general feature of an immune response is the expansion of specific clones resulting in a set of subclones with common ancestry varying in abundance and in the number of acquired somatic mutations. The expanded subclones are expected to have BCR affinities for the Ag higher than the affinities of the naive B cells in the background population. For these reasons, several groups successfully proceeded or suggested selecting highly abundant subclones from the repertoire to obtain the Ag-specific B cells. Given the nature of affinity maturation one would expect that abundant subclones are of high affinity but since repertoire sequencing only provides information about abundancies, this can only be verified with additional experiments, which are very labor intensive. Moreover, this would also require knowledge of the Ag, which is often not available for clinical samples. Consequently, in general we do not know if the selected highly abundant subclone(s) are also the high(est) affinity subclones. Such knowledge would likely improve the selection of relevant subclones for further characterization and Ag screening. Therefore, to gain insight in the relation between subclone abundancy and affinity, we developed a computational model that simulates affinity maturation in a single GC while tracking individual subclones in terms of abundancy and affinity. We show that the model correctly captures the overall GC dynamics, and that the amount of expansion is qualitatively comparable to expansion observed from B cells isolated from human lymph nodes. Analysis of the fraction of high- and low-affinity subclones among the unexpanded and expanded subclones reveals a limited correlation between abundancy and affinity and shows that the low abundant subclones are of highest affinity. Thus, our model suggests that selecting highly abundant subclones from repertoire sequencing experiments would not always lead to the high(est) affinity B cells. Consequently, additional or alternative selection approaches need to be applied.

Immunoglobulin G4(+) B-cell receptor clones distinguish immunoglobulin G 4-related disease from primary sclerosing cholangitis and biliary/pancreatic malignancies.

UNLABELLED: Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) of the biliary tree and pancreas is difficult to distinguish from sclerosing cholangitis and biliary/pancreatic malignancies (CA). An accurate noninvasive test for diagnosis and monitoring of disease activity is lacking. We demonstrate that dominant IgG4(+) B-cell receptor (BCR) clones determined by next-generation sequencing accurately distinguish patients with IgG4-associated cholangitis/autoimmune pancreatitis (n = 34) from those with primary sclerosing cholangitis (n = 17) and CA (n = 17). A novel, more affordable, and widely applicable quantitative polymerase chain reaction (qPCR) protocol analyzing the IgG4/IgG RNA ratio in blood also achieves excellent diagnostic accuracy (n = 125). Moreover, this qPCR test performed better than serum IgG4 levels in sensitivity (94% vs. 86%) and specificity (99% vs. 73%) and correlates with treatment response (n = 20). CONCLUSIONS: IgG4(+) BCR clones and IgG4/IgG RNA ratio markedly improve delineation, early diagnosis, and monitoring of IgG4-RD of the biliary tree and pancreas. (Hepatology 2016;64:501-507).

IgG4-Associated Cholangitis--A Mimic of PSC.

IgG4-associated cholangitis (IAC) is an inflammatory disorder of the biliary tract representing a major manifestation of IgG4-related disease (IgG4-RD) often with elevation of serum IgG4 levels, infiltration of IgG4+ plasma cells in the affected tissue and good response to immunosuppressive treatment. Its first description may go back to 150 years ago. The clinical presentation of IAC is often misleading, mimicking other biliary diseases such as primary sclerosing cholangitis (PSC) or cholangiocarcinoma. The HISORt criteria--histopathological, imaging, and serological features (sIgG4), other organ manifestations of IgG4-RD and response to treatment--are the standard for the diagnosis of IAC. In this overview of a recent lecture, we summarize our original findings on IgG4-RD that (i) dominant IgG4+ B-cell clones identified by advanced next generation sequencing (NGS) are highly specific for IgG4-RD (meanwhile confirmed by others), are a highly accurate diagnostic marker to distinguish IgG4-RD from PSC and biliary/pancreatic malignancies and may be crucial in unravelling the pathophysiology of IgG4-RD; (ii) sIgG4/sIgG1 >0.24 have additional diagnostic value in comparison to sIgG4 in differentiating IAC from PSC; (iii) blood IgG4 mRNA is a highly accurate diagnostic marker comparable to NGS and may become an easily available and affordable diagnostic standard for distinguishing IgG4-RD from PSC and biliary/pancreatic malignancies; and (iv) 'blue collar work' with long-term exposure to solvents, paints, oil products or industrial gases may be a risk factor for development of IgG4-RD. These findings may contribute to the understanding of the pathophysiology and to the early diagnosis and adequate treatment of IgG4-RD.

Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction.

Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both α and ß chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and ß chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.

Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization, and function.

UNLABELLED: Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8(+) T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα(+) hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα(+) and IL-7Rα(-) subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8(+) T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8(+) T cell pool during latency or upon antigen recall. IL-7Rα(+) PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8(+) effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8(+) T cell pool. IMPORTANCE: Insight into the self-renewal properties of long-lived memory CD8(+) T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8(+) T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8(+) T cell pool.

IgG4-associated cholangitis: a comprehensive review.

IgG4-associated cholangitis (IAC) is a major manifestation of immunoglobulin G4-related disease (IgG4-RD), an inflammatory multiorgan disorder of unknown cause. IAC and autoimmune pancreatitis (AIP) may mimic sclerosing cholangitis, cholangiocarcinoma, or pancreatic carcinoma. Typically, elderly male patients present with abdominal discomfort, weight loss, jaundice, and itch. At present, no accurate diagnostic test for IAC and IgG4-RD is at hand, often causing significant diagnostic delay. Serum IgG4 is only diagnostic when markedly raised (>4× ULN). Imaging in IAC discloses mass-forming lesions and/or strictures in the biliary tract. Histology may show tissue infiltration of IgG4-expressing plasma cells. Diagnostic criteria for histologic and imaging findings, serum tests, organ manifestation pattern, and response to immunosuppressive therapy (HISORt) criteria are used for the diagnosis of IgG4-RD. Still, considering the difficulty in diagnosing IAC and AIP, unnecessary hepatic or pancreatic resections for presumed malignancies occur. The good response to corticosteroid therapy in IAC and other manifestations of IgG4-RD suggests an immune-mediated inflammatory disease. Maintenance immunosuppression after induction of remission is needed in the majority of patients to avoid relapse. The pathogenesis of IAC and IgG4-RD remains poorly understood. Unresolved questions include: (i) Does IgG4 have a pro- or anti-inflammatory role in IAC? (ii) Is IAC a B cell- and/or T cell-mediated disease? (iii) Which are the molecular targets attacked by the immune system in IgG4-RD? Here, we review the diagnostic and therapeutic management of the disease and discuss recent pathophysiological findings, which might help to better understand the molecular mechanisms contributing to IAC and other manifestations of IgG4-RD.

Regulatory T cells expanded from HIV-1-infected individuals maintain phenotype, TCR repertoire and suppressive capacity.

While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4(+) Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-ß) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.

PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis.

Autoimmune disease results from a loss of tolerance to self-antigens in genetically susceptible individuals. Completely understanding this process requires that targeted antigens be identified, and so a number of techniques have been developed to determine immune receptor specificities. We previously reported the construction of a phage-displayed synthetic human peptidome and a proof-of-principle analysis of antibodies from three patients with neurological autoimmunity. Here we present data from a large-scale screen of 298 independent antibody repertoires, including those from 73 healthy sera, using phage immunoprecipitation sequencing. The resulting database of peptide-antibody interactions characterizes each individual's unique autoantibody fingerprint, and includes specificities found to occur frequently in the general population as well as those associated with disease. Screening type 1 diabetes (T1D) patients revealed a prematurely polyautoreactive phenotype compared with their matched controls. A collection of cerebrospinal fluids and sera from 63 multiple sclerosis patients uncovered novel, as well as previously reported antibody-peptide interactions. Finally, a screen of synovial fluids and sera from 64 rheumatoid arthritis patients revealed novel disease-associated antibody specificities that were independent of seropositivity status. This work demonstrates the utility of performing PhIP-Seq screens on large numbers of individuals and is another step toward defining the full complement of autoimmunoreactivities in health and disease.

Immunoglobulin G4+ clones identified by next-generation sequencing dominate the B cell receptor repertoire in immunoglobulin G4 associated cholangitis.

UNLABELLED: Immunoglobulin G4 (IgG4)-associated cholangitis (IAC) is a manifestation of the recently discovered idiopathic IgG4-related disease. The majority of patients have elevated serum IgG4 levels and/or IgG4-positive B-cell and plasma cell infiltrates in the affected tissue. We hypothesized that clonally expanded, class-switched IgG4-positive B cells and plasma cells could be causal to these poorly understood phenomena. In a prospective cohort of six consecutive IAC patients, six healthy controls, and six disease controls, we used a novel next-generation sequencing approach to screen the B-cell receptor (BCR) repertoires, in blood as well as in affected tissue, for IgG4+ clones. A full repertoire analysis of the BCR heavy chain was performed using GS-FLX/454 and customized bioinformatics algorithms (>10,000 sequences/sample; clones with a frequency ≥0.5% were considered dominant). We found that the most dominant clones within the IgG+ BCRheavy repertoire of the peripheral blood at baseline were IgG4+ only in IAC patients. In all IAC patients, but none of the controls, IgG4+ BCR clones were among the 10 most dominant BCR clones of any immunoglobulin isotype (IgA, IgD, IgM, and IgG) in blood. The BCR repertoires of the duodenal papilla comprised the same dominant IgG4+ clones as the paired peripheral blood samples. In all IAC patients, after 4 and 8 weeks of corticosteroid therapy the contribution of these IgG4+ clones to the IgG+ repertoire as well as to total BCR repertoire was marginalized, mirroring sharp declines in serum IgG4 titers and regression of clinical symptoms. CONCLUSION: The novel finding of highly abundant IgG4+ BCR clones in blood and tissue of patients with active IAC, which disappear upon corticosteroid treatment, suggests that specific B cell responses are pivotal to the pathogenesis of IAC. (HEPATOLOGY 2013 ).

Pro-apoptotic protein Noxa regulates memory T cell population size and protects against lethal immunopathology.

Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious agent. This is because each T cell response harbors multiple clones with slightly different affinities, thereby creating T cell memory with a certain degree of diversity. Currently, the mechanisms that control size, diversity, and cross-reactivity of the memory T cell pool are incompletely defined. Previously, we established a role for apoptosis, mediated by the BH3-only protein Noxa, in controlling diversity of the effector T cell population. This function might positively or negatively impact T cell memory in terms of function, pool size, and cross-reactivity during recall responses. Therefore, we investigated the role of Noxa in T cell memory during acute and chronic infections. Upon influenza infection, Noxa(-/-) mice generate a memory compartment of increased size and clonal diversity. Reinfection resulted in an increased recall response, whereas cross-reactive responses were impaired. Chronic infection of Noxa(-/-) mice with mouse CMV resulted in enhanced memory cell inflation, but no obvious pathology. In contrast, in a model of continuous, high-level T cell activation, reduced apoptosis of activated T cells rapidly led to severe organ pathology and premature death in Noxa-deficient mice. These results establish Noxa as an important regulator of the number of memory cells formed during infection. Chronic immune activation in the absence of Noxa leads to excessive accumulation of primed cells, which may result in severe pathology.