The first-in-class treatment PF-06480605 targets the tumor necrosis factor-like ligand 1A (TL1A) molecule in humans. Results from the phase 2a TUSCANY trial highlighted the safety and efficacy of PF-06480605 in ulcerative colitis. Preclinical and in vitro models have identified a role for TL1A in both innate and adaptive immune responses, but the mechanisms underlying the efficacy of anti-TL1A treatment in inflammatory bowel disease (IBD) are not known.Here, we provide analysis of tissue transcriptomic, peripheral blood proteomic, and fecal metagenomic data from the recently completed phase 2a TUSCANY trial and demonstrate endoscopic improvement post-treatment with PF-06480605 in participants with ulcerative colitis.Our results revealed robust TL1A target engagement in colonic tissue and a distinct colonic transcriptional response reflecting a reduction in inflammatory T helper 17 cell, macrophage, and fibrosis pathways in patients with endoscopic improvement. Proteomic analysis of peripheral blood revealed a corresponding decrease in inflammatory T-cell cytokines. Finally, microbiome analysis showed significant changes in IBD-associated pathobionts, Streptococcus salivarius, S. parasanguinis, and Haemophilus parainfluenzae post-therapy.The ability of PF-06480605 to engage and inhibit colonic TL1A, targeting inflammatory T cell and fibrosis pathways, provides the first-in-human mechanistic data to guide anti-TL1A therapy for the treatment of IBD.
Donor T cell activation in response to alloantigens presented by host antigen presenting cells (APC) is a critical step in the pathophysiology of Graft Versus Host Disease. The macrolide Azithromycin (Azi) is studied in lung transplant and allogeneic hematopoietic cell transplant (HCT) recipients to decrease graft rejection and progression of lung injury, respectively. We now tested whether Azi plays a role in the modulation of adaptive immune responses by determining its effects on alloantigen-dependent T cell activation. In vitro: Mixed lymphocyte reactions (MLR) using C57BL/6 (H2b) T cells as responders and B6D2F1 (H2bd) or BALB/c (H2d) splenocytes as stimulators, as well as TCR dependent, alloantigen-independent CD3/CD28 or lectin-type stimulation (Concavalin) assays of C57BL/6 Tcells were performed in the absence or presence of Azi at varying concentrations 5μg/ml, 10μg/ml, 20μg/ml, 50μg/ml. T cell proliferation was assessed by 3HT-uptake. T cells were phenotyped and supernatant was tested for cytokine expression. In vivo: B6D2F1 were pretreated for 2 weeks with Azi at a dose of 3mg/ml drinking water or received untreated water as control. Then, animals were conditioned with 12Gy TBI and transplanted with 4X10E6 bone marrow cells and 6X10E6 splenocytes from allogeneic C57BL/6 donors. Recipients continued to receive Azi-supplemented or untreated water, and on day+7, splenic T cell expansion and T cell phenotyping were done. Alloreactive T cell proliferation in vitro showed an Azi dose-dependent decline at 96 hours along with reductions in TNF, IFNg, IL-2, IL-6, L-10, IL-17. Interestingly, a significant relative expansion of CD4+FOXP3+ T cells (Tregs) within the CD4+ T cell fraction was observed with increasing Azi-levels from 2.5±0.1% (0μg/ml) to 5.0±0.2% (50μg/ml). No differences in T cell proliferation were seen when T cell stimulation occurred APC-independent either via Concavalin A or via CD3/CD28 except for slight suppression at Azi 50 μg/ml.Tabled 1Azithromycin (μg/ml)05102050MLRcpm20053 ± 104117497 ± 1237ns15340 ± 1157∗p < 0.0512902 ± 1317P < .015637 ± 215P < .01IFNg (pg/ml)590.5 ± 59.9476.0 ± 42.6363.6 ± 18.6P < .01218.7 ± 19.2P < .0137.05 ± 4.5P < .01TNF (pg/ml)139.1 ± 8.0114.2 ± 5.4∗p < 0.05115.4 ± 5.6∗p < 0.05104.6 ± 2.5P < .0171.3 ± 3.7P < .01IL-2 (pg/ml)27.0 ± 0.623.4 ± 1.223.4 ± 0.225.2 ± 1.018.3 ± 1.2P < .01IL-10 (pg/ml)115.5 ± 8.899.1 ± 14.498.9 ± 4.365.0 ± 6.8P < .0119.7 ± 3.5P < .01IL-17 (pg/ml)24.7 ± 0.919.3 ± 3.317.6 ± 1.19.4 ± 1.5P < .014.2 ± 0.3P < .01IL-4 (pg/ml)—————IL-6 (pg/ml)169.3 ± 8.3139.6 ± 16.3120.7 ± 11.7∗p < 0.0596.7 ± 9.4P < .0149.9 ± 5.3P < .01%CD4+FOXP3+2.5 ± 0.12.9 ± 0.12.7 ± 0.34.2 ± 0.2P < .015.0 ± 0.1P < .01Con Acpm61791 ± 452964350 ± 369266065 ± 317440012 ± 2016∗p < 0.05CD3/CD28cpm54868 ± 187351182 ± 207151351 ± 144751659 ± 86242002 ± 633P < .01cpm = counts per minute— = below assay sensitivity∗ p < 0.05∗∗ P < .01 Open table in a new tab cpm = counts per minute — = below assay sensitivity In vivo, when compared to controls, pretreatment with Azi resulted in decreased splenic expansion of both CD4+ T cells (2.1X10E6±0.2 vs. 4.3X10E6±0.6, P < .01) and CD8+ T cells (4.9X10E6±0.5 vs. 6.4X10E6±0.4, P < .05) along with a slight and non-significant increase of Tregs (1.5± 0.3% vs 1.1± 0.1%). Azithromycin suppresses alloreactive T cell response in vitro and in vivo, paralleled by the induction of Tregs, supporting its use in lung transplantation and allogeneic HCT.
Abstract The objective of this study was to develop an ex vivo method to generate large numbers of Foxp3-expressing Tregs that can be used to evaluate their therapeutic efficacy in a T-cell-dependent model of chronic colitis. We found that polyclonal activation of CD4+CD25- Foxp3- T-cells in the presence of small amounts of IL-2, TGFβ and all trans retinoic acid (RA) converts >90% of these T-cells to Foxp3-expressing Tregs (termed iTregs) as well as induces a 4-5-fold increase in proliferation following a 4 day incubation period in vitro. Furthermore, we found that the presence of RA enhances the surface expression of the gut-homing adhesion molecules α4β7 and CCR9. Finally, we observed that these iTregs are significantly more potent at suppressing T-cell activation in vitro and reversing established colitis in vivo. This protocol routinely generates 30-40 million iTregs from one mouse spleen assuming that the spleen yields approximately 10 million CD4+Foxp3- T-cells and that >90% of these T-cells convert to Foxp3-expressing iTregs as well as undergo a 4-fold proliferation during the 4 day incubation period. We conclude that this protocol generates sufficient numbers of iTregs that can be used in vitro and in vivo to assess the therapeutic efficacy and immunological mechanisms responsible for suppression of inflammation in different models of autoimmune disease (Supported by PO143785).
The objectives of this study were to (a) evaluate and compare the ability of ex vivo-generated induced regulatory T cells (iTregs) and freshly isolated natural Tregs (nTregs) to reverse/attenuate preexisting intestinal inflammation in a mouse model of chronic colitis and (b) quantify the Treg-targeted gene expression profiles of these two Treg populations. We found that ex vivo-generated iTregs were significantly more potent than nTregs at attenuating preexisting colitis. This superior therapeutic activity was associated with increased accumulation of iTregs within the mesenteric lymph nodes and large and significant reductions in interleukin (IL)-6 and IL-17A expression in the colons of iTreg- versus nTreg-treated mice. The enhanced immunosuppressive activity of iTregs was not because of increased expression or stability of Foxp3 as iTregs and nTregs obtained from the mesenteric lymph nodes, and colons of reconstituted mice expressed similar levels of this important transcription factor. In addition, we observed a total of 27 genes that were either upregulated or downregulated in iTregs when compared with nTregs. Although iTregs were found to be superior at reversing established disease, their message levels of IL-10 and IL-35 and surface expression of the gut-homing molecules CCR9 and α4β7 were significantly reduced when compared with nTregs. Taken together, our data demonstrate that ex vivo-generated iTregs are significantly more potent than nTregs at attenuating preexisting gut inflammation despite reduced expression of classical regulatory cytokines and gut-homing molecules. Our data suggest that the immunosuppressive activity of iTregs may be because of their ability to directly or indirectly decrease expression of IL-6 and IL-17A within the inflamed bowel.
Dysregulated signaling from TNF and TNFR proteins is implicated in several immune-mediated inflammatory diseases (IMIDs). This review centers around seven IMIDs (rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, and asthma) with substantial unmet medical needs and sheds light on the signaling mechanisms, disease relevance, and evolving drug development activities for five TNF/TNFR signaling axes that garner substantial drug development interest in these focus conditions. The review also explores the current landscape of therapeutics, emphasizing the limitations of the approved biologics, and the opportunities presented by small-molecule inhibitors and combination antagonists of TNF/TNFR signaling.
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