Lentiviral-mediated gene transfer to the sheep brain: Implications for gene therapy in batten disease

The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are inherited neurodegenerative lysosomal storage diseases with common clinical features of blindness and seizures culminating in premature death. Gene-therapy strategies for these diseases depend on whether the missing activity is a secreted lysosomal protein taken up by neighboring cells, or an intramembrane protein that requires careful targeting. Therapies are best developed in animal models with large complex human-like brains. Lentiviral-mediated gene delivery to neural cell cultures from normal sheep and sheep affected with an NCL resulted in green fluorescent protein (GFP) expression in neurons and neuroblasts, more efficiently than in astrocytes. Similar transgene expression was obtained from two constitutive promoters, the viral MND promoter and the human EF1α promoter. In vivo studies showed stable and persistent GFP expression throughout the cell bodies, axons, and dendrites from intracortical injections and indicated ependymal and subependymal transduction. The sheep showed no ill effects from the injections. These data support continuing gene-therapy trials in the sheep models of Batten disease

Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd-/-) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2-/- recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd-/- recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2-/- recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a-/- TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd-/- recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019

Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging.

The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines

Data regarding transplant induced germinal center humoral autoimmunity.

This data is related to the research article entitled "Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy" (Harper et al., 2016) [2]. The data presented here focuses on the humoral autoimmune response triggered by transferred allogeneic CD4 T cells and includes details on: (a) the recipient splenic germinal center (GC) response; (b) augmentation of humoral autoimmunity and accelerated heart allograft rejection following transplantation from donors primed against recipient; (c) flow cytometric analysis of donor and recipient CD4 T cells for signature markers of T follicular helper cell differentiation; (d) in vitro donor endothelial cell migration in response to column purified autoantibody from recipient sera; (e) analysis of development of humoral responses in recipients following adoptive transfer of donor CD4 T cells and; (f) the development of humoral autoimmunity in mixed haematopoietic chimeric mice.British Heart Foundatio

Human SNP links differential outcomes in inflammatory and infectious disease to a FOXO3-Regulated Pathway

The clinical course and eventual outcome, or prognosis, of complex diseases varies enormously between affected individuals. This variability critically determines the impact a disease has on a patient’s life but is very poorly understood. Here, we exploit existing genome-wide association study data to gain insight into the role of genetics in prognosis. We identify a noncoding polymorphism in FOXO3A (rs12212067: T > G) at which the minor (G) allele, despite not being associated with disease susceptibility, is associated with a milder course of Crohn’s disease and rheumatoid arthritis and with increased risk of severe malaria. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGFb1 reduces production of proinflammatory cytokines, including TNFa, and increases production of anti-inflammatory cytokines, including IL-10. Thus, we uncover a shared genetic contribution to prognosis in distinct diseases that operates via a FOXO3-driven pathway modulating inflammatory responses

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response

Type I interferon induces CXCL13 to support ectopic germinal center formation.

Ectopic lymphoid structures form in a wide range of inflammatory conditions, including infection, autoimmune disease, and cancer. In the context of infection, this response can be beneficial for the host: influenza A virus infection-induced pulmonary ectopic germinal centers give rise to more broadly cross-reactive antibody responses, thereby generating cross-strain protection. However, despite the ubiquity of ectopic lymphoid structures and their role in both health and disease, little is known about the mechanisms by which inflammation is able to convert a peripheral tissue into one that resembles a secondary lymphoid organ. Here, we show that type I IFN produced after viral infection can induce CXCL13 expression in a phenotypically distinct population of lung fibroblasts, driving CXCR5-dependent recruitment of B cells and initiating ectopic germinal center formation. This identifies type I IFN as a novel inducer of CXCL13, which, in combination with other stimuli, can promote lung remodeling, converting a nonlymphoid tissue into one permissive to functional tertiary lymphoid structure formation

Human SNP links differential outcomes in inflammatory and infectious disease to a FOXO3-regulated pathway

The clinical course and eventual outcome, or prognosis, of complex diseases varies enormously between affected individuals. This variability critically determines the impact a disease has on a patient’s life but is very poorly understood. Here, we exploit existing genome-wide association study data to gain insight into the role of genetics in prognosis. We identify a noncoding polymorphism in FOXO3A (rs12212067: T > G) at which the minor (G) allele, despite not being associated with disease susceptibility, is associated with a milder course of Crohn’s disease and rheumatoid arthritis and with increased risk of severe malaria. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGFβ1 reduces production of proinflammatory cytokines, including TNFα, and increases production of anti-inflammatory cytokines, including IL-10. Thus, we uncover a shared genetic contribution to prognosis in distinct diseases that operates via a FOXO3-driven pathway modulating inflammatory responses. PAPERCLIP

Germinal Center Alloantibody Responses Mediate Progression of Chronic Allograft Injury

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient (Tcrbd−/−) C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 103 “TCR75” CD4 T cells that recognize self-restricted allopeptide derived from the H-2Kd MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-Kd alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2Kd-allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (TFH) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with Sh2d1a−/− TCR75 CD4 T cells that were genetically incapable of providing TFH cell function. The GC response was associated with affinity maturation of the anti-Kd alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses in vitro than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by TFH cells. Clinical strategies that target the TFH cell subset may hold therapeutic potential.This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019

High Frequency of CD4+CXCR5+ TFH Cells in Patients with Immune-Active Chronic Hepatitis B

BACKGROUND: T follicular helper (TFH) cells are a special subpopulation of T helper cells and can regulate humoral immune responses. This study examined whether the frequency of CD4(+)CXCR5(+) TFH cells could be associated with active immunity in chronic hepatitis B (CHB) patients. METHODOLOGY AND FINDINGS: The frequencies of peripheral blood CD4(+)CXCR5(+) TFH cells, inducible T cell costimulator (ICOS), and/or programmed death 1 (PD-1) positive CD4(+)CXCR5(+) TFH cells in immune-active (IA), immune-tolerant (IT) CHB, and healthy controls (HC) were characterized by flow cytometry analysis. The effect of adevofir dipivoxil treatment on the frequency of CD4(+)CXCR5(+) TFH cells, the concentrations of serum IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-21, ALT, AST, HBsAg, HBsAb, HBeAg, HBeAb and HBV loads in IA patients were determined. The potential association of the frequency of CD4(+)CXCR5(+) TFH cells with clinical measures was analyzed. In addition, the frequency of splenic and liver CD4(+)CXCR5(+) TFH cells in HBV-transgenic mice was examined. We found that the frequency of CD4(+)CXCR5(+) TFH cells in IA patients was significantly higher than that of IT patients and HC, and the percentages of CD4(+)CXCR5(+) TFH in IA patients were positively correlated with AST. Furthermore, the percentages of ICOS(+), PD-1(+), and ICOS(+)PD-1(+) in CD4(+)CXCR5(+) TFH cells in CHB patients were significantly higher than that of HC. Treatment with adefovir dipivoxil reduced the frequency of CD4(+)CXCR5(+) TFH, PD-1(+)CD4(+)CXCR5(+) TFH cells and the concentrations of HBsAg and HBeAg, but increased the concentrations of HBsAb, HBeAb, IL-2 and IFN-γ in IA patients. Moreover, the frequency of splenic and liver CD4(+)CXCR5(+) TFH cells in HBV-transgenic mice was higher than that of wild-type controls. CONCLUSIONS: These data indicate that CD4(+)CXCR5(+) TFH cells may participate in the HBV-related immune responses and that high frequency of CD4(+)CXCR5(+) TFH cells may be a biomarker for the evaluation of active immune stage of CHB patients