Non-coding autoimmune risk variant defines role for ICOS in T peripheral helper cell development.

Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid arthritis and type 1 diabetes. Here, using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated risk allele is functional, reducing affinity for the inhibitory chromosomal regulator SMCHD1 to enhance expression of inducible T-cell costimulator (ICOS) in memory CD4+ T cells from healthy donors. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells and, in rheumatoid arthritis patients, of blood and joint fluid Tph cells as well as circulating plasmablasts. Correspondingly, ICOS ligation and carriage of the rs117701653 risk allele accelerate T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13. Thus, mechanistic dissection of a functional non-coding variant in human autoimmunity discloses a previously undefined pathway through which ICOS regulates Tph development and abundance.

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus.

Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKϵ. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a new regulatory pathway involving rs2297550, Ikaros, and IKKϵ implicated by human genetics in risk for SLE.

Identification of a regulatory pathway governing TRAF1 via an arthritis-associated non-coding variant.

TRAF1/C5 was among the first loci shown to confer risk for inflammatory arthritis in the absence of an associated coding variant, but its genetic mechanism remains undefined. Using Immunochip data from 3,939 patients with juvenile idiopathic arthritis (JIA) and 14,412 control individuals, we identified 132 plausible common non-coding variants, reduced serially by single-nucleotide polymorphism sequencing (SNP-seq), electrophoretic mobility shift, and luciferase studies to the single variant rs7034653 in the third intron of TRAF1. Genetically manipulated experimental cells and primary monocytes from genotyped donors establish that the risk G allele reduces binding of Fos-related antigen 2 (FRA2), encoded by FOSL2, resulting in reduced TRAF1 expression and enhanced tumor necrosis factor (TNF) production. Conditioning on this JIA variant eliminated attributable risk for rheumatoid arthritis, implicating a mechanism shared across the arthritis spectrum. These findings reveal that rs7034653, FRA2, and TRAF1 mediate a pathway through which a non-coding functional variant drives risk of inflammatory arthritis in children and adults.

First-in-human therapy with Treg produced from thymic tissue (thyTreg) in a heart transplant infant.

Due to their suppressive capacity, regulatory T cells (Tregs) have attracted growing interest as an adoptive cellular therapy for the prevention of allograft rejection, but limited Treg recovery and lower quality of adult-derived Tregs could represent an obstacle to success. To address this challenge, we developed a new approach that provides large quantities of Tregs with high purity and excellent features, sourced from thymic tissue routinely removed during pediatric cardiac surgeries (thyTregs). We report on a 2-year follow-up of the first patient treated worldwide with thyTregs, included in a phase I/II clinical trial evaluating the administration of autologous thyTreg in infants undergoing heart transplantation. In addition to observing no adverse effects that could be attributed to thyTreg administration, we report that the Treg frequency in the periphery was preserved during the 2-year follow-up period. These initial results are consistent with the trial objective, which is to confirm safety of the autologous thyTreg administration and its capacity to restore the Treg pool.

Multidimensional analysis of immune cells from COVID-19 patients identified cell subsets associated with the severity at hospital admission.

BACKGROUND: SARS-CoV-2 emerged as a new coronavirus causing COVID-19, and it has been responsible for more than 760 million cases and 6.8 million deaths worldwide until March 2023. Although infected individuals could be asymptomatic, other patients presented heterogeneity and a wide range of symptoms. Therefore, identifying those infected individuals and being able to classify them according to their expected severity could help target health efforts more effectively. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we wanted to develop a machine learning model to predict those who will develop severe disease at the moment of hospital admission. We recruited 75 individuals and analysed innate and adaptive immune system subsets by flow cytometry. Also, we collected clinical and biochemical information. The objective of the study was to leverage machine learning techniques to identify clinical features associated with disease severity progression. Additionally, the study sought to elucidate the specific cellular subsets involved in the disease following the onset of symptoms. Among the several machine learning models tested, we found that the Elastic Net model was the better to predict the severity score according to a modified WHO classification. This model was able to predict the severity score of 72 out of 75 individuals. Besides, all the machine learning models revealed that CD38+ Treg and CD16+ CD56neg HLA-DR+ NK cells were highly correlated with the severity. CONCLUSIONS/SIGNIFICANCE: The Elastic Net model could stratify the uninfected individuals and the COVID-19 patients from asymptomatic to severe COVID-19 patients. On the other hand, these cellular subsets presented here could help to understand better the induction and progression of the symptoms in COVID-19 individuals.

A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy.

Due to their suppressive capacity, the adoptive transfer of regulatory T cells (Treg) has acquired a growing interest in controlling exacerbated inflammatory responses. Limited Treg recovery and reduced quality remain the main obstacles in most current protocols where differentiated Treg are obtained from adult peripheral blood. An alternate Treg source is umbilical cord blood, a promising source of Treg cells due to the higher frequency of naïve Treg and lower frequency of memory T cells present in the fetus' blood. However, the Treg number isolated from cord blood remains limiting. Human thymuses routinely discarded during pediatric cardiac surgeries to access the retrosternal operative field has been recently proposed as a novel source of Treg for cellular therapy. This strategy overcomes the main limitations of current Treg sources, allowing the obtention of very high numbers of undifferentiated Treg. We have developed a novel good manufacturing practice (GMP) protocol to obtain large Treg amounts, with very high purity and suppressive capacity, from the pediatric thymus (named hereafter thyTreg). The total amount of thyTreg obtained at the end of the procedure, after a short-term culture of 7 days, reach an average of 1,757 x106 (range 50 x 106 - 13,649 x 106) cells from a single thymus. The thyTreg product obtained with our protocol shows very high viability (mean 93.25%; range 83.35% - 97.97%), very high purity (mean 92.89%; range 70.10% - 98.41% of CD25+FOXP3+ cells), stability under proinflammatory conditions and a very high suppressive capacity (inhibiting in more than 75% the proliferation of activated CD4+ and CD8+ T cells in vitro at a thyTreg:responder cells ratio of 1:1). Our thyTreg product has been approved by the Spanish Drug Agency (AEMPS) to be administered as cell therapy. We are recruiting patients in the first-in-human phase I/II clinical trial worldwide that evaluates the safety, feasibility, and efficacy of autologous thyTreg administration in children undergoing heart transplantation (NCT04924491). The high quality and amount of thyTreg and the differential features of the final product obtained with our protocol allow preparing hundreds of doses from a single thymus with improved therapeutic properties, which can be cryopreserved and could open the possibility of an "off-the-shelf" allogeneic use in another individual.

A New Generation of Cell Therapies Employing Regulatory T Cells (Treg) to Induce Immune Tolerance in Pediatric Transplantation.

Kidney transplantation is the most common solid organ transplant and the preferred treatment for pediatric patients with end-stage renal disease, but it is still not a definitive solution due to immune graft rejection. Regulatory T cells (Treg) and their control over effector T cells is a crucial and intrinsic tolerance mechanism in limiting excessive immune responses. In the case of transplants, Treg are important for the survival of the transplanted organ, and their dysregulation could increase the risk of rejection in transplanted children. Chronic immunosuppression to prevent rejection, for which Treg are especially sensitive, have a detrimental effect on Treg counts, decreasing the Treg/T-effector balance. Cell therapy with Treg cells is a promising approach to restore this imbalance, promoting tolerance and thus increasing graft survival. However, the strategies used to date that employ peripheral blood as a Treg source have shown limited efficacy. Moreover, it is not possible to use this approach in pediatric patients due to the limited volume of blood that can be extracted from children. Here, we outline our innovative strategy that employs the thymus removed during pediatric cardiac surgeries as a source of therapeutic Treg that could make this therapy accessible to transplanted children. The advantageous properties and the massive amount of Treg cells obtained from pediatric thymic tissue (thyTreg) opens a new possibility for Treg therapies to prevent rejection in pediatric kidney transplants. We are recruiting patients in a clinical trial to prevent rejection in heart-transplanted children through the infusion of autologous thyTreg cells (NCT04924491). If its efficacy is confirmed, thyTreg therapy may establish a new paradigm in preventing organ rejection in pediatric transplants, and their allogeneic use would extend its application to other solid organ transplantation.

IL-1ß-driven osteoclastogenic Tregs accelerate bone erosion in arthritis.

IL-1ß is a proinflammatory mediator with roles in innate and adaptive immunity. Here we show that IL-1ß contributes to autoimmune arthritis by inducing osteoclastogenic capacity in Tregs. Using mice with joint inflammation arising through deficiency of the IL-1 receptor antagonist (Il1rn-/-), we observed that IL-1ß blockade attenuated disease more effectively in early arthritis than in established arthritis, especially with respect to bone erosion. Protection was accompanied by a reduction in synovial CD4+Foxp3+ Tregs that displayed preserved suppressive capacity and aerobic metabolism but aberrant expression of RANKL and a striking capacity to drive RANKL-dependent osteoclast differentiation. Both Il1rn-/- Tregs and wild-type Tregs differentiated with IL-1ß accelerated bone erosion upon adoptive transfer. Human Tregs exhibited analogous differentiation, and corresponding RANKLhiFoxp3+ T cells could be identified in rheumatoid arthritis synovial tissue. Together, these findings identify IL-1ß-induced osteoclastogenic Tregs as a contributor to bone erosion in arthritis.

Ectopic FOXP3 Expression in Combination with TGF-ß1 and IL-2 Stimulation Generates Limited Suppressive Function in Human Primary Activated Thymocytes Ex Vivo.

Regulatory T cells (Tregs), which are characterized by the expression of the transcription factor forkhead box P3 (FOXP3), are the main immune cells that induce tolerance and are regulators of immune homeostasis. Natural Treg cells (nTregs), described as CD4+CD25+FOXP3+, are generated in the thymus via activation and cytokine signaling. Transforming growth factor beta type 1 (TGF-ß1) is pivotal to the generation of the nTreg lineage, its maintenance in the thymus, and to generating induced Treg cells (iTregs) in the periphery or in vitro arising from conventional T cells (Tconvs). Here, we tested whether TGF-ß1 treatment, associated with interleukin-2 (IL-2) and CD3/CD28 stimulation, could generate functional Treg-like cells from human thymocytes in vitro, as it does from Tconvs. Additionally, we genetically manipulated the cells for ectopic FOXP3 expression, along with the TGF-ß1 treatment. We demonstrated that TGF-ß1 and ectopic FOXP3, combined with IL-2 and through CD3/CD28 activation, transformed human thymocytes into cells that expressed high levels of Treg-associated markers. However, these cells also presented a lack of homogeneous suppressive function and an unstable proinflammatory cytokine profile. Therefore, thymocyte-derived cells, activated with the same stimuli as Tconvs, were not an appropriate alternative for inducing cells with a Treg-like phenotype and function.

The Presence of a Marked Imbalance Between Regulatory T Cells and Effector T Cells Reveals That Tolerance Mechanisms Could Be Compromised in Heart Transplant Children.

Regulatory T cells (Treg) are crucial for the induction and maintenance of graft tolerance. In pediatric heart transplant procedures, the thymus is routinely excised, removing the primary source of T-cell replenishment. Consequently, thymectomy joined to the effects of immunosuppression on the T-cell compartment may have a detrimental impact on Treg values, compromising the intrinsic tolerance mechanisms and the protective role of Treg preventing graft rejection in heart transplant children. METHODS: A prospective study including 7 heart transplant children was performed, and immune cell populations were evaluated periodically in fresh peripheral blood at different time points before and up to 3 y posttransplant. RESULTS: Treg counts decreased significantly from the seventh-month posttransplant. Furthermore, there was a significant increase in effector memory and terminally differentiated effector memory T cells coinciding with the fall of Treg counts. The Treg/Teffector ratio, a valuable marker of the tolerance/rejection balance, reached values around 90% lower than pretransplant values. Additionally, a negative correlation between Treg count and T effector frequency was observed. Particularly, when Treg count decreases below 50 or 75 cells/µL in the patients, the increase in the frequency of T effector CD4+ and CD8+, respectively, experiences a tipping point, and the proportion of T-effector cells increases dramatically. CONCLUSIONS: These results reveal that interventions employed in pediatric heart transplantation (immunosuppression and thymectomy) could induce, as an inevitable consequence, a dysregulation in the immunologic status characterized by a marked imbalance between Treg and T effector, which could jeopardize the preservation of tolerance during the period with the higher incidence of acute rejection.

Basiliximab impairs regulatory T cell (TREG) function and could affect the short-term graft acceptance in children with heart transplantation.

CD25, the alpha chain of the IL-2 receptor, is expressed on activated effector T cells that mediate immune graft damage. Induction immunosuppression is commonly used in solid organ transplantation and can include antibodies blocking CD25. However, regulatory T cells (Tregs) also rely on CD25 for their proliferation, survival, and regulatory function. Therefore, CD25-blockade may compromise Treg protective role against rejection. We analysed in vitro the effect of basiliximab (BXM) on the viability, phenotype, proliferation and cytokine production of Treg cells. We also evaluated in vivo the effect of BXM on Treg in thymectomized heart transplant children receiving BXM in comparison to patients not receiving induction therapy. Our results show that BXM reduces Treg counts and function in vitro by affecting their proliferation, Foxp3 expression, and IL-10 secretion capacity. In pediatric heart-transplant patients, we observed decreased Treg counts and a diminished Treg/Teff ratio in BXM-treated patients up to 6-month after treatment, recovering baseline values at the end of the 12-month follow up period. These results reveal that the use of BXM could produce detrimental effects on Tregs, and support the evidence suggesting that BXM induction could impair the protective role of Tregs in the period of highest incidence of acute graft rejection.

Early progression to active tuberculosis is a highly heritable trait driven by 3q23 in Peruvians.

Of the 1.8 billion people worldwide infected with Mycobacterium tuberculosis, 5-15% will develop active tuberculosis (TB). Approximately half will progress to active TB within the first 18 months after infection, presumably because they fail to mount an effective initial immune response. Here, in a genome-wide genetic study of early TB progression, we genotype 4002 active TB cases and their household contacts in Peru. We quantify genetic heritability ([Formula: see text]) of early TB progression to be 21.2% (standard error 0.08). This suggests TB progression has a strong genetic basis, and is comparable to traits with well-established genetic bases. We identify a novel association between early TB progression and variants located in a putative enhancer region on chromosome 3q23 (rs73226617, OR = 1.18; P = 3.93 × 10-8). With in silico and in vitro analyses we identify rs73226617 or rs148722713 as the likely functional variant and ATP1B3 as a potential causal target gene with monocyte specific function.

Implications of juvenile idiopathic arthritis genetic risk variants for disease pathogenesis and classification.

PURPOSE OF REVIEW: We assess the implications of recent advances in the genetics of juvenile idiopathic arthritis (JIA) for the evolving understanding of inflammatory arthritis in children. RECENT FINDINGS: JIA exhibits prominent genetic associations with the human leukocyte antigen (HLA) region, extending perhaps surprisingly even to the hyperinflammatory systemic JIA category. Some HLA associations resemble those for adult-onset inflammatory arthritides, providing evidence for pathogenic continuity across the age spectrum. Genome-wide association studies have defined an increasing number of JIA-linked non-HLA loci, many again shared with adult-onset arthritis. As most risk loci contain only noncoding variants, new experimental methods such as SNP-seq and innovative big-data strategies help identify responsible causative mutations, termed functional SNPs (fSNPs). Alternately, gene hunting in multiplex families implicates new genes in monogenic childhood arthritis, including MYD88 and the intriguing innate immune gene LACC1. SUMMARY: Genetic data indicate a continuity between JIA and adult arthritis poorly reflected in current nomenclature. Advancing methodologies will help to identify new pathogenic mechanisms that inform the understanding of biologic subdivisions within JIA. Resulting insights will facilitate the application of lessons learned across the age spectrum to the treatment of arthritis in children and adults.

Megakaryocyte emperipolesis mediates membrane transfer from intracytoplasmic neutrophils to platelets.

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the ß2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated murine marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of murine inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

HIV-1 increases extracellular amyloid-beta levels through neprilysin regulation in primary cultures of human astrocytes.

Since the success of combined antiretroviral therapy, HIV-1-infected individuals are now living much longer. This increased life expectancy is accompanied by a higher prevalence of HIV-1 associated neurocognitive disorders. Rising too is the incidence in these patients of pathological hallmarks of Alzheimer's disease such as increased deposition of amyloid beta protein (Aß). Although neurons are major sources of Aß in the brain, astrocytes are the most numerous glial cells, therefore, even a small level of astrocytic Aß metabolism could make a significant contribution to brain pathology. Neprilysin (NEP) is a decisive/crucial regulator of Aß levels. We evaluated the effects of HIV-1 on Aß deposition and the expression and activity of NEP in primary human astrocytes. Specifically, no differences in intracellular amyloid deposits were found between infected and control cells. However, primary cultures of infected astrocytes showed more extracellular Aß levels compared to controls. This was accompanied by reduced expression of NEP and to a significant decrease in its activity. These results indicate that the presence of HIV-1 in the brain could contribute to the increase in the total burden of cerebral Aß.

Fine-mapping and functional studies highlight potential causal variants for rheumatoid arthritis and type 1 diabetes.

To define potentially causal variants for autoimmune disease, we fine-mapped1,2 76 rheumatoid arthritis (11,475 cases, 15,870 controls)3 and type 1 diabetes loci (9,334 cases, 11,111 controls)4. After sequencing 799 1-kilobase regulatory (H3K4me3) regions within these loci in 568 individuals, we observed accurate imputation for 89% of common variants. We defined credible sets of ≤5 causal variants at 5 rheumatoid arthritis and 10 type 1 diabetes loci. We identified potentially causal missense variants at DNASE1L3, PTPN22, SH2B3, and TYK2, and noncoding variants at MEG3, CD28-CTLA4, and IL2RA. We also identified potential candidate causal variants at SIRPG and TNFAIP3. Using functional assays, we confirmed allele-specific protein binding and differential enhancer activity for three variants: the CD28-CTLA4 rs117701653 SNP, MEG3 rs34552516 indel, and TNFAIP3 rs35926684 indel.

High-throughput identification of noncoding functional SNPs via type IIS enzyme restriction.

Genome-wide association studies (GWAS) have identified many disease-associated noncoding variants, but cannot distinguish functional single-nucleotide polymorphisms (fSNPs) from others that reside incidentally within risk loci. To address this challenge, we developed an unbiased high-throughput screen that employs type IIS enzymatic restriction to identify fSNPs that allelically modulate the binding of regulatory proteins. We coupled this approach, termed SNP-seq, with flanking restriction enhanced pulldown (FREP) to identify regulation of CD40 by three disease-associated fSNPs via four regulatory proteins, RBPJ, RSRC2 and FUBP-1/TRAP150. Applying this approach across 27 loci associated with juvenile idiopathic arthritis, we identified 148 candidate fSNPs, including two that regulate STAT4 via the regulatory proteins SATB2 and H1.2. Together, these findings establish the utility of tandem SNP-seq/FREP to bridge the gap between GWAS and disease mechanism.

Impact of intensification with raltegravir on HIV-1-infected individuals receiving monotherapy with boosted PIs.

Background: Monotherapy with ritonavir-boosted PIs (PI/r) has been used to simplify treatment of HIV-1-infected patients. In previous studies raltegravir intensification evidenced ongoing viral replication and reduced T cell activation, preferentially in subjects receiving PI-based triple ART. However, data about low-level viral replication and its consequences in patients receiving PI/r monotherapy are scarce. Methods: We evaluated the impact of 24 weeks of intensification with raltegravir on markers of viral persistence, cellular immune activation and inflammation biomarkers in 33 patients receiving maintenance PI/r monotherapy with darunavir or lopinavir boosted with ritonavir. ClinicalTrials.gov identifier: NCT01480713. Results: The addition of raltegravir to PI/r monotherapy resulted in a transient increase in 2-LTR (long-terminal repeat) circles in a significant proportion of participants, along with decreases in CD8+ T cell activation levels and a temporary increase in the expression of the exhaustion marker CTLA-4 in peripheral T lymphocytes. Intensification with raltegravir also reduced the number of samples with intermediate levels of residual viraemia (10-60 HIV-1 RNA copies/mL) compared with samples taken during PI/r monotherapy. However, there were no changes in cell-associated HIV-1 DNA in peripheral CD4+ T cells or soluble inflammatory biomarkers (CD14, IP-10, IL-6, C-reactive protein and D-dimer). Conclusions: Intensification of PI/r monotherapy with raltegravir revealed persistent low-level viral replication and reduced residual viraemia in some patients during long-term PI/r monotherapy. The concomitant change in T cell phenotype suggests an association between active viral production and T cell activation. These results contribute to understanding the lower efficacy rates of PI/r monotherapies compared with triple therapies in clinical trials.