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Effective antibody responses are essential to generate protective humoral immunity. Different inflammatory signals polarize T cells towards appropriate effector phenotypes during an infection or immunization. Th1 and Th2 cells have been associated with the polarization of humoral responses. However, T follicular helper cells (Tfh) have a unique ability to access the B cell follicle and support the germinal center (GC) responses by providing B cell help. We investigated the specialization of Tfh cells induced under type-1 and type-2 conditions. We first studied homogenous Tfh cell populations generated by adoptively transferred TCR-transgenic T cells in mice immunized with type-1 and type-2 adjuvants. Using a machine learning approach, we established a gene expression signature that discriminates Tfh cells polarized towards type-1 and type-2 response, defined as Tfh1 and Tfh2 cells. The distinct signatures of Tfh1 and Tfh2 cells were validated against datasets of Tfh cells induced following lymphocytic choriomeningitis virus (LCMV) or helminth infection. We generated single-cell and spatial transcriptomics datasets to dissect the heterogeneity of Tfh cells and their localization under the two immunizing conditions. Besides a distinct specialization of GC Tfh cells under the two immunizations and in different regions of the lymph nodes, we found a population of Gzmk+ Tfh cells specific for type-1 conditions. In human individuals, we could equally identify CMV-specific Tfh cells that expressed Gzmk. Our results show that Tfh cells acquire a specialized function under distinct types of immune responses and with particular properties within the B cell follicle and the GC.

Objective: We study activation of T helper 17 (Th17) and regulatory T (Treg) cells and induction of apoptosis in cells from patients with systemic lupus erythematosus (SLE) compared with controls and effects of atorvastatin and its simulated interactions with other compounds. Methods: Mononuclear cells from 10 patients with SLE and 10 controls were cultured in conditions that induce Th17 and/or Treg cell polarization and/or apoptosis and were studied by FACScan. Gene expression was determined by quantitative real-time reverse transcription–polymerase chain reaction. Cytokines in plasma were determined by enzyme-linked immunosorbent assay. The Search Tool for Interactions of Chemicals (STITCH) was used to retrieve information regarding the binding properties of atorvastatin. Results: Among patients with SLE, the proportion of Th17 (CD4+IL17+) cells was higher compared with controls after activation, with Th17 or Treg polarizing cytokines, phorbol myristate acetate, and ionomycin. In contrast, Treg cells (CD4+CD25+CD127dim/−) frequencies were lower. CD95 stimulation induced relatively more apoptosis in Treg cells and less in Th17 cells, as compared with controls. Addition of atorvastatin normalized Th17/Treg cell balance and apoptosis induction. Accordingly, the ratio of RORC/FoxP3 decreased in patients with SLE. Interleukin 17 and interleukin 6 (IL-6) levels were increased in patients with SLE. Atorvastatin interacted strongly with C-reactive protein (CRP) and also significantly with IL-6. Conclusion: There is a higher proportion of Th17 cells and a lower proportion of Treg cells in patients with SLE after activation. Th17 cells were more resistant than Treg cells to CD95-induced apoptosis in SLE. Atorvastatin normalized these effects. Our findings reveal a novel mechanism behind the imbalance of Th17/Treg cells with implications for treatment in SLE. We determine for the first time simulated interaction between atorvastatin, CRP, and IL-6, implying a novel role of a, QC 20230308

Regulatory T cells (Tregs) and M2 macrophages have been hypothesized to contribute to tumor progression. We found that M2 macrophages and Tregs are associated with more aggressive renal cell carcinoma, and that they have a synergistic effect on clinical outcome. Background: It has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. Objective: To investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participants: A cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysis: Associations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitations: We found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. Conclusions: Infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summary: The aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in ren

Regulatory T cells (Tregs) and M2 macrophages have been hypothesized to contribute to tumor progression. We found that M2 macrophages and Tregs are associated with more aggressive renal cell carcinoma, and that they have a synergistic effect on clinical outcome. Background: It has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. Objective: To investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participants: A cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysis: Associations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitations: We found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. Conclusions: Infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summary: The aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in ren

Background: AK112 is a humanized IgG1 bispecific anti-PD-1/VEGF antibody. VEGF blockade potentiates PD-1 inhibition by, inter alia, opposing the immunosuppressive effects of VEGF-A, which include suppression of dendritic cell activity and enhancement of checkpoint molecule expression on CD8+ T cells and proliferation of regulatory T cells. Combination therapies involving PD-(L)1 and VEGF inhibitors have been approved for the treatment of selected patients with metastatic non-small cell lung carcinoma, advanced renal cell carcinoma and advanced endometrial carcinoma. A supplemental Biologics License Application has been submitted for an anti-PD-L1 + anti-VEGF combination for the firstline treatment of unresectable hepatocellular carcinoma. Given the strong correlation between VEGF and PD-1 expression in the tumor microenvironment, the simultaneous blockade of these 2 targets by AK112 as a single agent might achieve higher target binding specificities and synergistically produce enhanced antitumor activity compared to co-administration of anti-PD-(L)1 and anti-VEGF therapies. Method(s): This is a Phase 1a/1b, first-in-human, multicenter, open-label study in patients with advanced or metastatic solid tumor that is refractory/relapsed to standard therapies. The primary objective is to assess safety, tolerability and DLTs; and to determine the Maximum Tolerated Dose (MTD) or Maximum Administered Dose of AK112. Antitumor activity, PK and immunogenicity of AK112 will be studied as secondary objectives. As exploratory endpoints, tumor tissue samples may be evaluated for PD-L1 expression, mRNA expression profile and biomarkers (e.g. CD8+, FoxP3, Granzyme B, apelin and EPHB4). PD-1 receptor occupancy on circulating T-cells and serum VEGF level may also be measured as indications of target engagement. The dose-escalation phase will evaluate 5 dose levels of AK112 (up to 20 mg/kg Q2W IV) using a 3+3+3 study design. Additional subjects (up to 18 subjects) may be enrolled at any

Background: AK112 is a humanized IgG1 bispecific anti-PD-1/VEGF antibody. VEGF blockade potentiates PD-1 inhibition by, inter alia, opposing the immunosuppressive effects of VEGF-A, which include suppression of dendritic cell activity and enhancement of checkpoint molecule expression on CD8+ T cells and proliferation of regulatory T cells. Combination therapies involving PD-(L)1 and VEGF inhibitors have been approved for the treatment of selected patients with metastatic non-small cell lung carcinoma, advanced renal cell carcinoma and advanced endometrial carcinoma. A supplemental Biologics License Application has been submitted for an anti-PD-L1 + anti-VEGF combination for the firstline treatment of unresectable hepatocellular carcinoma. Given the strong correlation between VEGF and PD-1 expression in the tumor microenvironment, the simultaneous blockade of these 2 targets by AK112 as a single agent might achieve higher target binding specificities and synergistically produce enhanced antitumor activity compared to co-administration of anti-PD-(L)1 and anti-VEGF therapies. Method(s): This is a Phase 1a/1b, first-in-human, multicenter, open-label study in patients with advanced or metastatic solid tumor that is refractory/relapsed to standard therapies. The primary objective is to assess safety, tolerability and DLTs; and to determine the Maximum Tolerated Dose (MTD) or Maximum Administered Dose of AK112. Antitumor activity, PK and immunogenicity of AK112 will be studied as secondary objectives. As exploratory endpoints, tumor tissue samples may be evaluated for PD-L1 expression, mRNA expression profile and biomarkers (e.g. CD8+, FoxP3, Granzyme B, apelin and EPHB4). PD-1 receptor occupancy on circulating T-cells and serum VEGF level may also be measured as indications of target engagement. The dose-escalation phase will evaluate 5 dose levels of AK112 (up to 20 mg/kg Q2W IV) using a 3+3+3 study design. Additional subjects (up to 18 subjects) may be enrolled at any

Background: It has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. Objective: To investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participants: A cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysis: Associations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitations: We found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. Conclusions: Infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summary: The aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in renal cell carcinoma. We demonstrated that renal cell carcinoma patients with high infiltration of both these cell types are at an increased risk of poor clinical outcomes., Funding Agencies:Örebro County Council Research Committee, Sweden Lions Cancer Foundation, Sweden

Background: It has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. Objective: To investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participants: A cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysis: Associations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitations: We found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. Conclusions: Infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summary: The aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in renal cell carcinoma. We demonstrated that renal cell carcinoma patients with high infiltration of both these cell types are at an increased risk of poor clinical outcomes., Funding Agencies:Örebro County Council Research Committee, Sweden Lions Cancer Foundation, Sweden

Regulatory T cells (Tregs) and M2 macrophages have been hypothesized to contribute to tumor progression. We found that M2 macrophages and Tregs are associated with more aggressive renal cell carcinoma, and that they have a synergistic effect on clinical outcome. Background: It has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. Objective: To investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participants: A cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysis: Associations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitations: We found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. Conclusions: Infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summary: The aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in ren

Regulatory T cells (Tregs) and M2 macrophages have been hypothesized to contribute to tumor progression. We found that M2 macrophages and Tregs are associated with more aggressive renal cell carcinoma, and that they have a synergistic effect on clinical outcome. Background: It has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. Objective: To investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participants: A cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysis: Associations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitations: We found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. Conclusions: Infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summary: The aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in ren

Regulatory T cells (Tregs) and M2 macrophages have been hypothesized to contribute to tumor progression. We found that M2 macrophages and Tregs are associated with more aggressive renal cell carcinoma, and that they have a synergistic effect on clinical outcome. Background: It has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. Objective: To investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participants: A cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysis: Associations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitations: We found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. Conclusions: Infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summary: The aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in ren

Regulatory T cells (Tregs) play important roles in limiting inflammatory responses in the skin. During these responses, Treg abundance increases and interfering with their recruitment results in exacerbation of inflammation and increased endothelial activation. However, the mechanisms whereby Tregs enter the skin remain poorly understood. The aim of this study was to use intravital microscopy to investigate adhesion and transmigration of Tregs in the dermal microvasculature in a two-challenge model of contact sensitivity. Using intravital confocal microscopy of Foxp3-GFP mice, we visualised endogenous Tregs and assessed their interactions in the dermal microvasculature at different stages of the response. Four hours after hapten challenge, Tregs underwent adhesion with ~25% of these cells proceeding to transmigrate, a process dependent on CCR4. At 24 h, Tregs adhered but no longer underwent transmigration, instead remaining in prolonged contact with the endothelium, migrating over the endothelial surface. Fours after a second challenge, Treg transmigration was restored, although in this case transmigration was CCR4-independent. Notably, at 24 hours but not 4 hours after challenge, endothelial cells expressed MHCII. Moreover, inhibition of MHCII reduced Treg adhesion, demonstrating an unexpected role for MHCII in Treg attachment to the endothelium. Together these data show that Treg adhesion and transmigration can be driven by different molecular mechanisms at different stages of an antigen-driven inflammatory response. In addition, Tregs can undergo prolonged migration on the inflamed endothelium, potentially affording them the opportunity to modulate endothelial adhesive function. Animal work was approved by the Monash Medical Centre Animal Ethics Committee.

Regulatory T cells (Tregs) play important roles in limiting inflammatory responses in the skin. During these responses, Treg abundance increases and interfering with their recruitment results in exacerbation of inflammation and increased endothelial activation. However, the mechanisms whereby Tregs enter the skin remain poorly understood. The aim of this study was to use intravital microscopy to investigate adhesion and transmigration of Tregs in the dermal microvasculature in a two-challenge model of contact sensitivity. Using intravital confocal microscopy of Foxp3-GFP mice, we visualised endogenous Tregs and assessed their interactions in the dermal microvasculature at different stages of the response. Four hours after hapten challenge, Tregs underwent adhesion with ~25% of these cells proceeding to transmigrate, a process dependent on CCR4. At 24 h, Tregs adhered but no longer underwent transmigration, instead remaining in prolonged contact with the endothelium, migrating over the endothelial surface. Fours after a second challenge, Treg transmigration was restored, although in this case transmigration was CCR4-independent. Notably, at 24 hours but not 4 hours after challenge, endothelial cells expressed MHCII. Moreover, inhibition of MHCII reduced Treg adhesion, demonstrating an unexpected role for MHCII in Treg attachment to the endothelium. Together these data show that Treg adhesion and transmigration can be driven by different molecular mechanisms at different stages of an antigen-driven inflammatory response. In addition, Tregs can undergo prolonged migration on the inflamed endothelium, potentially affording them the opportunity to modulate endothelial adhesive function. Animal work was approved by the Monash Medical Centre Animal Ethics Committee.

Aim: To use Goodpasture's disease (GPD), a classical form of HLA-linked autoimmunity, to define the mechanism of HLA-mediated risk, and dominant protection from risk of disease. Background(s): The mechanisms underpinning HLA-mediated susceptibility to, and protection from autoimmune diseases are unknown. GPD is HLAlinked and characterized by an immunodominant CD4+ self-epitope derived from the alpha3 chain of Type IV collagen (alpha3135-145). Method(s): HLA-DR transgenic mice, cells from HLA-typed healthy humans and from patients with GPD were used, with HLA-DR-alpha3135-145 tetramers, Xray crystallography, and in vivo/in vitro models of autoimmunity. Result(s): Autoreactive alpha3135-145-specific T cells clonally expanded in GPD patients. In alpha3135-145-immunized HLA-DR15 transgenic mice with GPD, alpha3135-145-specific T cells infiltrated the kidney. Structurally, HLA-DR15 and HLA-DR1 presented alpha3135-145 in different binding registers, resulting in differential T cell receptor (TCR) usage. HLA-DR15-alpha3135-145 tetramer+ CD4+ T cells in disease susceptible HLA-DR15 transgenic mice exhibited a conventional phenotype (Tconv), secreting pro-inflammatory cytokines. In contrast, HLA-DR1-alpha3135-145 tetramer+ cells in disease resistant HLA-DR1 and HLADR15/ DR1 transgenic mice were largely CD4+Foxp3+ regulatory T cells (Tregs) expressing tolerogenic cytokines. In HLA-DR15/DR1 transgenic mice, HLA-DR1-alpha3135-145 specific Tregs conferred protection to alpha3135-145-specific autoimmunity and protected from GPD (alpha3135-145-immunized, Treg intact vs Treg depleted: segmental necrosis 0+/-0 vs 50+/-13% P<0.01, serum urea 9+/-1 vs 31+/-10 P<0.001). HLA-DR15+ and HLA-DR1+ healthy human donors displayed altered alpha3135-145-specific TCRs, with HLA-DR15-alpha3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-alpha3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes (alpha3135-145 tetramer+ Treg:Tconv ratios: DR15 0.04+/-0.00, DR1 9.61+/-1.79, P<0.001). Conclusio

Objectives: Anti-glomerular basement membrane (GBM) disease is a small vessel vasculitis caused by anti-a3(IV)NC1 T and B cell autoreactivity. An immunodominant T cell epitope, a3135-145, has been defined. HLA-DR15 confers an increased disease risk (OR 8.5) while HLA-DR1 is dominantly protective from this risk. These studies aimed to define the mechanisms by which different HLA-DR alleles lead to susceptibility or protection from disease. Method(s): a3135-145-specific CD4+ T cells were enumerated in anti-GBM patients, HLA-typed healthy humans and HLA-DR transgenic mice using HLA-DR-a3135-145 tetramers. The crystal structures of HLADR15-a3135-145 and HLA-DR1-a3135-145 were solved. a3135-145-specific T cell receptor (TCR) usage was determined by single cell sequencing. T cell phenotype was determined by flow cytometry. T cell responses were determined in vivo, ex vivo and by culturing naive T cells with a3135-145 in vitro. Experimental autoimmune anti-GBM disease was induced by a3135-145 immunization of HLA-DR transgenic mice (Treg intact or Treg depleted). Result(s): a3135-145-specific CD4+ T cells were expanded in anti-GBM patients and infiltrated kidneys of a3135-145-immunized HLA-DR15 transgenic mice. Mice transgenic for both HLA-DR15 and HLA-DR1 showed similar reactivity to most a3 peptides, but lacked proinflammatory responses to a3135-145. Structurally, HLA-DR15 and HLA-DR1 presented a3135-145 in different binding registers. Compared to the conformation of a3135-145 in HLA-DR15, in HLA-DR1 a3135-145 adopted a more pronounced conformation with a flipped backbone. These different HLA-peptide conformations altered a3135-145-specific TCR usage and T cell phenotypes. HLA-DR15-a3135-145-specific T cells exhibited a Foxp3-conventional T cell phenotype resulting in proinflammatory T cell autoreactivity, anti-GBM antibody production and disease. In contrast, HLA-DR1-a3135-145-specific T cells were predominantly anti-inflammatory Foxp3+ Tregs that proliferated and respon

Background Bronchopulmonary dysplasia (BPD) is a common neonatal lung disease that has been linked to pulmonary inflammation. Previous studies have suggested involvement of various cell types and soluble factors but have thus far failed to reveal a dominant pathway that underpins disease progression. It remains unknown that which T helper (Th) cell polarization, if any, contributes to the pathogenesis of BPD. Methods Citrated whole cord and peripheral blood were collected from infants born between 24+0-28+6 gestational weeks at five different time points, namely at birth, on days 1, 7 and 14 and at 36 weeks corrected gestational age (CGA). Cells were stimulated with either PMA, ionomycin and brefeldin A or vehicle and brefeldin A overnight. Cells were then stained for multi-color flow cytometry to enumerate Th cell subsets including Th1, Th2 and Th17 and regulatory T cells. Results were analyzed against BPD disease status at 36 weeks CGA and corrected for confounders. Results 50 infants were enrolled and 228 unique samples were collected. 36 enrolled infants (72%) had BPD (mild to severe) at 36 weeks CGA. When compared to infants that did not have the disease, the frequency of interleukin(IL)-4 in CD4+ Th cells in peripheral blood was increased in infants with BPD on days 1 (p=0.0449), 7 (p=0.0423), 14 (p=0.0489) and 36 weeks CGA but not within cord blood. There was no difference in the frequency of cells positive for interferongamma (IFNgamma), IL-17A or FOXP3 among CD4+ cells in blood from BPD and non-BPD infants across all time points. Conclusions T helper cells are polarized towards a Th2 phenotype in infants with BPD based on elevated levels of IL-4 in CD4+ cells. We hypothesize that BPD infants may benefit from therapies that target the Th2 pathway.

Aim: To use Goodpasture's disease (GPD), a classical form of HLA-linked autoimmunity, to define the mechanism of HLA-mediated risk, and dominant protection from risk of disease. Background(s): The mechanisms underpinning HLA-mediated susceptibility to, and protection from autoimmune diseases are unknown. GPD is HLAlinked and characterized by an immunodominant CD4+ self-epitope derived from the alpha3 chain of Type IV collagen (alpha3135-145). Method(s): HLA-DR transgenic mice, cells from HLA-typed healthy humans and from patients with GPD were used, with HLA-DR-alpha3135-145 tetramers, Xray crystallography, and in vivo/in vitro models of autoimmunity. Result(s): Autoreactive alpha3135-145-specific T cells clonally expanded in GPD patients. In alpha3135-145-immunized HLA-DR15 transgenic mice with GPD, alpha3135-145-specific T cells infiltrated the kidney. Structurally, HLA-DR15 and HLA-DR1 presented alpha3135-145 in different binding registers, resulting in differential T cell receptor (TCR) usage. HLA-DR15-alpha3135-145 tetramer+ CD4+ T cells in disease susceptible HLA-DR15 transgenic mice exhibited a conventional phenotype (Tconv), secreting pro-inflammatory cytokines. In contrast, HLA-DR1-alpha3135-145 tetramer+ cells in disease resistant HLA-DR1 and HLADR15/ DR1 transgenic mice were largely CD4+Foxp3+ regulatory T cells (Tregs) expressing tolerogenic cytokines. In HLA-DR15/DR1 transgenic mice, HLA-DR1-alpha3135-145 specific Tregs conferred protection to alpha3135-145-specific autoimmunity and protected from GPD (alpha3135-145-immunized, Treg intact vs Treg depleted: segmental necrosis 0+/-0 vs 50+/-13% P<0.01, serum urea 9+/-1 vs 31+/-10 P<0.001). HLA-DR15+ and HLA-DR1+ healthy human donors displayed altered alpha3135-145-specific TCRs, with HLA-DR15-alpha3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-alpha3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes (alpha3135-145 tetramer+ Treg:Tconv ratios: DR15 0.04+/-0.00, DR1 9.61+/-1.79, P<0.001). Conclusio

Objectives: Anti-glomerular basement membrane (GBM) disease is a small vessel vasculitis caused by anti-a3(IV)NC1 T and B cell autoreactivity. An immunodominant T cell epitope, a3135-145, has been defined. HLA-DR15 confers an increased disease risk (OR 8.5) while HLA-DR1 is dominantly protective from this risk. These studies aimed to define the mechanisms by which different HLA-DR alleles lead to susceptibility or protection from disease. Method(s): a3135-145-specific CD4+ T cells were enumerated in anti-GBM patients, HLA-typed healthy humans and HLA-DR transgenic mice using HLA-DR-a3135-145 tetramers. The crystal structures of HLADR15-a3135-145 and HLA-DR1-a3135-145 were solved. a3135-145-specific T cell receptor (TCR) usage was determined by single cell sequencing. T cell phenotype was determined by flow cytometry. T cell responses were determined in vivo, ex vivo and by culturing naive T cells with a3135-145 in vitro. Experimental autoimmune anti-GBM disease was induced by a3135-145 immunization of HLA-DR transgenic mice (Treg intact or Treg depleted). Result(s): a3135-145-specific CD4+ T cells were expanded in anti-GBM patients and infiltrated kidneys of a3135-145-immunized HLA-DR15 transgenic mice. Mice transgenic for both HLA-DR15 and HLA-DR1 showed similar reactivity to most a3 peptides, but lacked proinflammatory responses to a3135-145. Structurally, HLA-DR15 and HLA-DR1 presented a3135-145 in different binding registers. Compared to the conformation of a3135-145 in HLA-DR15, in HLA-DR1 a3135-145 adopted a more pronounced conformation with a flipped backbone. These different HLA-peptide conformations altered a3135-145-specific TCR usage and T cell phenotypes. HLA-DR15-a3135-145-specific T cells exhibited a Foxp3-conventional T cell phenotype resulting in proinflammatory T cell autoreactivity, anti-GBM antibody production and disease. In contrast, HLA-DR1-a3135-145-specific T cells were predominantly anti-inflammatory Foxp3+ Tregs that proliferated and respon

Background Bronchopulmonary dysplasia (BPD) is a common neonatal lung disease that has been linked to pulmonary inflammation. Previous studies have suggested involvement of various cell types and soluble factors but have thus far failed to reveal a dominant pathway that underpins disease progression. It remains unknown that which T helper (Th) cell polarization, if any, contributes to the pathogenesis of BPD. Methods Citrated whole cord and peripheral blood were collected from infants born between 24+0-28+6 gestational weeks at five different time points, namely at birth, on days 1, 7 and 14 and at 36 weeks corrected gestational age (CGA). Cells were stimulated with either PMA, ionomycin and brefeldin A or vehicle and brefeldin A overnight. Cells were then stained for multi-color flow cytometry to enumerate Th cell subsets including Th1, Th2 and Th17 and regulatory T cells. Results were analyzed against BPD disease status at 36 weeks CGA and corrected for confounders. Results 50 infants were enrolled and 228 unique samples were collected. 36 enrolled infants (72%) had BPD (mild to severe) at 36 weeks CGA. When compared to infants that did not have the disease, the frequency of interleukin(IL)-4 in CD4+ Th cells in peripheral blood was increased in infants with BPD on days 1 (p=0.0449), 7 (p=0.0423), 14 (p=0.0489) and 36 weeks CGA but not within cord blood. There was no difference in the frequency of cells positive for interferongamma (IFNgamma), IL-17A or FOXP3 among CD4+ cells in blood from BPD and non-BPD infants across all time points. Conclusions T helper cells are polarized towards a Th2 phenotype in infants with BPD based on elevated levels of IL-4 in CD4+ cells. We hypothesize that BPD infants may benefit from therapies that target the Th2 pathway.