Mouse Models Of Autoimmune Diseases Research Articles

STING trafficking activates MAPK–CREB signaling to trigger regulatory T cell differentiation

The Type-I interferon (IFN-I) response is the major outcome of stimulator of interferon genes (STING) activation in innate cells. STING is more abundantly expressed in adaptive T cells; nevertheless, its intrinsic function in T cells remains unclear. Intriguingly, we previously demonstrated that STING activation in T cells activates widespread IFN-independent activities, which stands in contrast to the well-known STING-mediated IFN response. Here, we have identified that STING activation induces regulatory T cells (Tregs) differentiation independently of IRF3 and IFN. Specifically, the translocation of STING from the endoplasmic reticulum to the Golgi activates mitogen-activated protein kinase (MAPK) activity, which subsequently triggers transcription factor cAMP response element-binding protein (CREB) activation. The activation of the STING-MAPK-CREB signaling pathway induces the expression of many cytokine genes, including interleukin-2 (IL-2) and transforming growth factor-beta 2 (TGF-β2), to promote the Treg differentiation. Genetic knockdown of MAPK p38 or pharmacological inhibition of MAPK p38 or CREB markedly inhibits STING-mediated Treg differentiation. Administration of the STING agonist also promotes Treg differentiation in mice. In the Trex1-/- autoimmune disease mouse model, we demonstrate that intrinsic STING activation in CD4+ T cells can drive Treg differentiation, potentially counterbalancing the autoimmunity associated with Trex1 deficiency. Thus, STING-MAPK-CREB represents an IFN-independent signaling axis of STING that may have profound effects on T cell effector function and adaptive immunity.

High salt diet alleviates disease severity in native experimental autoimmune uveitis.

Recent studies reported a link between high salt diet (HSD) and clinical exacerbation in mouse models of autoimmune diseases, mainly through the induction of pathogenic Th17 cells and/or HSD-induced dysbiosis. However, the topic remains controversial and not fully understood. In this study, we investigated the effects of HSD on the development of experimental autoimmune uveitis (EAU) in C57BL/6J mice. Unexpectedly, our data showed a significant attenuating effect of HSD on disease severity of native EAU, induced by direct immunization with IRBP peptide. That said, HSD had no effect on EAU disease severity induced by adoptive transfer of semi-purified auto-reactive IRBP-specific T lymphocytes. Accordingly, HSD did not affect IRBP-specific systemic afferent immune response as attested by no HSD-linked changes in T lymphocytes proliferation, cytokine production and Treg proportion. Gut microbiota analysis from cecal samples in naïve and EAU mice demonstrated that HSD affected differentially α-diversity between groups, whereas β-diversity was significantly modified in all groups. Unknown Tannerellaceae was the only taxon associated to HSD exposure in all treatment groups. Interestingly, a significantly higher abundance of unknown Gastranaerophilales, with potential anti-inflammatory properties, appeared in HSD-fed native EAU mice, only. In conclusion, our study suggests a possible impact of HSD on gut microbiota composition and consequently on development and clinical severity of EAU. Further studies are required to investigate the potential beneficial role of Gastranaerophilales in EAU.

Abstract 6594: 4-1BB: A double-edged sword for T cells

Abstract Background: The costimulatory receptor 4-1BB (CD137) is expressed on the surface of activated T cells, playing a pivotal role in enhancing T cell responses and emerging as a promising target for cancer immunotherapy. Its primary ligand, 4-1BBL (CD137L), is found on antigen-presenting cells and various immune cells. The interaction between 4-1BBL and 4-1BB provides crucial costimulatory signals to both CD4+ and CD8+ T cells, amplifying their activation and effector functions. However, recent studies have shed light on a distinct role of 4-1BB in T cells. Prior work has revealed that 4-1BB can distinguish intratumoral regulatory T cells (Tregs) from peripheral Tregs and other CD4 lineage T cells in cancer patients, and the overexpression of 4-1BB on Tregs has been associated with unfavorable survival outcomes. Additionally, targeting 4-1BB with IgG2a isotype antibodies has shown significant anti-tumor efficacy in preclinical models by depleting Tregs. Furthermore, agonist monoclonal antibodies targeting 4-1BB have not only shown promise in inducing tumor regressions but also demonstrated improved outcomes in mouse models of autoimmune diseases induced by autoreactive CD4+ T cells. These findings indicate the existence of specific ligands or factors that modulate 4-1BB function on T cells that can vary depending on the disease state. Methods: To identify potential ligand(s) involved in the modulation of 4-1BB function, we developed an innovative CRISPR/Cas9-based transcriptional activation (CRISPRa) screening system that integrates CRISPR/Cas9-guide mediated gene activation, high avidity bead-based selection, serial enrichment, and flow cytometry-based monitoring. This screening platform provides enhanced sensitivity for identifying extracellular interactions between receptor-ligand partners that have not been fully characterized before, even with low-affinity. Results: Through our unbiased CRISPRa screen, we discovered that Myelin-Associated Glycoprotein (MAG), also known as siglec-4, a member of the siglec (sialic acid-binding immunoglobulin-like lectins) family, interacts with 4-1BB in trans. Our study demonstrated that MAG binds to activated T cells via 4-1BB and inhibits their activation and proliferation, suggesting a potential role of the MAG-4-1BB interaction in modulating T cell function. Notably, our data revealed that trans-binding to MAG-positive cells inhibits c-Jun signaling in activated T cells. Furthermore, the presence of MAG on target cells can hinder Chimeric Antigen Receptor (CAR) T-cell activity. Conclusion: Utilizing a highly efficient cell-based CRISPRa system, we discovered a novel interactor of 4-1BB capable of modulating T cell activation through its interaction with 4-1BB. This interaction suggests an immunosuppressive role of 4-1BB, explaining the paradoxical roles of 4-1BB in T cells. These findings provide crucial insights for the development of precise therapeutics targeting 4-1BB. Citation Format: Liping Yang, Timothy P. Sheets, Yang Feng, Kuo-Sheng Hsu, Guojun Yu, Steven Seaman, Pradip Bajgain, Daeho So, Jaewon Lee, Raj Chari, Brad St. Croix. 4-1BB: A double-edged sword for T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6594.

Epicutaneous immunization with TNP-Ig antigen induces CD11c+IL-10+ dendritic cells that promote suppression of Th1-mediated contact hypersensitivity in humanized HLA-DR4 transgenic mice

The contact hypersensitivity response (CHS) is a mouse model of allergic contact dermatitis in humans. The reaction is classified as type IV hypersensitivity and underlies many autoimmune disorders. Experiments employing the CHS model in wild-type mice showed that the protein antigen applied to the skin in the form of a gauze patch one week before the induction of Th1-dependent CHS was an effective strategy to reduce the inflammatory response in the skin. The approach of epicutaneous (EC) immunization also effectively suppressed the inflammatory response in various mouse models of autoimmune diseases. To evaluate the potential of EC immunization to suppress T cell-dependent immune response in humans, we used HLA-DR4 tg mice, which express the human DRB1*0401 allele and lack all endogenous mouse MHC class II genes. Our data show that EC immunization with TNP-conjugated protein antigen followed by induction of CHS to trinitrochlorobenzene (TNCB), effectively suppressed the CHS response as described by ear swelling, MPO activity in ear extracts, and the number of TCRβ+CD4+IFN-γ+ CHS T-effector cells in auxiliary and inguinal lymph nodes (ALN) and spleen (SPL) of HLA-DR4 tg mice. EC-induced suppression increases the frequency of CD11c+IL-10+ DCs in SPL. Their immunoregulatory role was confirmed by s.c. immunization with TNP-CD11c+DCs prior to CHS elicitation and induction. Our data in HLA-DR4 tg mice show that EC protein immunization induces IL-10-producing DCs, which suppress the development of CD4+IFN-γ+ T cell-dependent CHS, implying that EC protein immunization could be of therapeutic importance for T cell-mediated diseases in humans.

Small Molecule Drugs That Inhibit Phagocytosis.

In our initial publication on the in vitro testing of more than 200 compounds, we demonstrated that small molecules can inhibit phagocytosis. We therefore theorized that a small molecule drug discovery-based approach to the treatment of immune cytopenias (ITP, AIHA, HTR, DHTR) is feasible. Those earlier studies showed that small molecules with anti-phagocytic groups, such as the pyrazole core, are good models for producing efficacious phagocytosis inhibitors with low toxicity. We recently screened a chemical library of 80 compounds containing pyrazole/isoxazole/pyrrole core structures and found four hit molecules for further follow-up, all having the pyrazole core structure. Subsequent evaluation via MTT viability, LDH release, and apoptosis, led to the selection of two lead compounds with negligible toxicity and high efficacy. In an in vitro assay for inhibition of phagocytosis, their IC50 values were 2-4 µM. The rational development of these discoveries from hit to lead molecule stage, viz. independent synthesis/scale up of hit molecules, and in vivo activities in mouse models of autoimmune disease, will result in the selection of a lead compound(s) for further pre-clinical evaluation.

Trichinella pseudospiralis-secreted 53 kDa protein ameliorates imiquimod-induced psoriasis by inhibiting the IL-23/IL-17 axis in mice

Trichinella infection can experimentally ameliorate many autoimmune diseases. However, the immune mechanism of the amelioration and the identification of corresponding Trichinella-derived molecule(s) are still not fully elucidated. Fifty-three kDa excretory-secretory (ES) protein from Trichinella pseudospiralis (Tpp53) is a molecule like TsP53 reported as a protein exerting immune-inhibitory effect in T. spiralis. In this study, we investigated the immunomodulatory effect of Tpp53 using imiquimod (IMQ)-induced psoriasis-like dermatitis model, which is a mouse model of autoimmune disease with the pathogenic interleukin 17 (IL-17) producing CD4+ T cells (Th17) via IL-23/IL17 axis. Administrating the recombinant Tpp53 (rTpp53) mixed with IMQ cream on the skin of mice ameliorated psoriatic lesions, as revealed by the improvement of erythema, scaling, skin thickening, epidermis hyperplasia and parakeratosis, thickening of acanthosis cell layer, epidermal extension of dermis, less infiltration of inflammatory cells, and decreased expression of inflammatory marker. The increased expression of the factors related to the IL-23/IL-17 axis, including IL-17A, IL-6, Il17F and Il23a, in the skins of IMQ-treated mice was inhibited by rTpp53 treatment. Moreover, the expression of activated keratinocyte-produced cytokines, chemokines, and antimicrobial peptides in the skin was also down-regulated in rTpp53-treated IMQ-treated mice. Co-culture of splenocytes with rTpp53 inhibited IL-17A and treatment of macrophages with rTpp53 reduced IL-6 production. Overall, our study revealed that the Trichinella-secreted 53 kDa ES protein could ameliorate IMQ-induced psoriasis by inhibiting the IL-23/IL-17 axis, suggesting that Tpp53 might involve in regulating host Th17 for immune evasion and have an alternative potential for psoriasis therapy.

The Ameliorative Effect of Dexamethasone on the Development of Autoimmune Lung Injury and Mediastinal Fat-Associated Lymphoid Clusters in an Autoimmune Disease Mouse Model.

In our previous study, we revealed the ameliorative therapeutic effect of dexamethasone (Dex) for Lupus nephritis lesions in the MRL/MpJ-Fas lpr/lpr (Lpr) mouse model. The female Lpr mice developed a greater number of mediastinal fat-associated lymphoid clusters (MFALCs) and inflammatory lung lesions compared to the male mice. However, the effect of Dex, an immunosuppressive drug, on both lung lesions and the development of MFALCs in Lpr mice has not been identified yet. Therefore, in this study, we compared the development of lung lesions and MFALCs in female Lpr mice that received either saline (saline group “SG”) or dexamethasone (dexamethasone group “DG”) in drinking water as a daily dose along with weekly intraperitoneal injections for 10 weeks. Compared to the SG group, the DG group showed a significant reduction in the levels of serum anti-dsDNA antibodies, the size of MFALCs, the degree of lung injury, the area of high endothelial venules (HEVs), and the number of proliferating and immune cells in both MFALCs and the lungs. A significant positive correlation was observed between the size of MFALCs and the cellular aggregation in the lungs of Lpr mice. Therefore, this study confirmed the ameliorative effect of Dex on the development of lung injury and MFALCs via their regressive effect on both immune cells’ proliferative activity and the development of HEVs. Furthermore, the reprogramming of MFALCs by targeting immune cells and HEVs may provide a therapeutic strategy for autoimmune-disease-associated lung injury.

CD6-Targeted Antibody-Drug Conjugate As a Potential Therapeutic Agent for T Cell Lymphomas

Introduction: Peripheral T-cell lymphomas (PTCLs) comprise a subset of blood cancers that are molecularly heterogeneous and often associated with poor survival. While brentuximab vedotin has shown efficacy in CD30-expressing lymphomas such as anaplastic large cell lymphoma, effective targeted therapies for PTCLs are lacking and this represents an unmet medical need.CD6, an established surface marker for T cells, is a key regulator of T cell responses. CD6 has been suggested as a target for treating T cell-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. We previously reported that CD6 knockout mice show reduced pathogenic T-cell responses and thus are protected against several models of autoimmune diseases. We also found that CD6 could be targeted therapeutically by anti-CD6 mAbs in these mouse models of autoimmune diseases. Although the distribution of CD6 and its role in regulating normal, non-malignant T-cells has been established, the expression of this marker on PTCL cells and its potential use as a selective targeted therapeutic agent for PTCLs has not been explored.METHODS: To determine the expression profile of CD6 on the malignant T-cells, different specimens including established T-cell lymphoma/leukemia cell lines HH, MOLT-4, HUT-78 and JurkaT-cells, as well as PTCL patient-derived xenografts (PDX) were analyzed for CD6 expression by flow cytometry using UMCD6, a high-affinity anti-human CD6 mAb. Besides, a custom-made tissue microarray containing biopsy samples from different PTCL patients was also evaluated by immunohistochemical staining using the same mAb.A CD6-targeted antibody-drug conjugate (CD6-ADC) was prepared by conjugating monomethyl auristatin E (MMAE), a potent antimitotic toxin successfully used in other FDA-approved ADCs, to UMCD6. Control non-binding ADC was prepared the same way using purified mouse IgGs. The potency of the CD6-ADC of killing PTCL was first evaluated in vitro using HH and HUT78 cells with trypan blue staining. For in vivo treatment studies, NSG mice were subcutaneously inoculated with HH cells, then treated with CD6-ADC or controls at different time points either systemically by intraperitoneal injections or locally by intratumor injections. Treatment efficacies were evaluated by measuring subcutaneous tumor volumes and detecting metastasized tumors in bone marrows, spleens and livers.RESULTS: Our flow cytometric and immunohistochemical staining studies showed that most, if not all the specimens examined expressed high levels of CD6 on the surface, suggesting that CD6 could be a novel therapeutic target for this molecularly heterogeneous blood cancer. Besides, our anti-CD6 mAb, was quickly internalized by PTCL cells after binding to CD6 on their cell surface in an in vitro assay. In contrast to either the parental anti-CD6 mAb, or the non-binding control ADC, the CD6-ADC potently and selectively killed T lymphoma cells in vitro in a time- and concentration-dependent manner. It further prevented the development of tumors in vivo when given 1 day after PTCL cell inoculations. More importantly, systemic or local administration of CD6-ADC, but not the control ADC, significantly shrank existing tumors, reduced tumor metastases from the already developed tumors, and prolonged overall survival in the preclinical mouse model of PTCL without apparent adverse effects.CONCLUSIONS: These results suggest that CD6 is a novel therapeutic target for PTCLs and provide a strong rationale for the further development of CD6-ADC as a promising therapy for patients with this aggressive blood cancer. [Display omitted] DisclosuresHsi: AbbVie Inc, Eli Lilly: Research Funding. Lin: takeda Pharma: Consultancy, Research Funding.

018 Cutaneous kinase activity correlates with treatment outcomes following PI3K delta inhibition in mice with experimental pemphigoid diseases

Pemphigoid diseases (PD) comprise a group of autoimmune disorders clinically characterized by (muco)-cutaneous inflammation and subepidermal blistering. PD are caused by autoantibodies targeting structural proteins of the dermal-epidermal junction. Skin-bound immune complexes trigger recruitment of myeloid cells, engagement of Fcγ receptors, and activation of specific kinases. Selective blockade of PI3K can alleviate disease manifestation in mouse models of autoimmune diseases. Here, we evaluated the treatment efficacy of parsaclisib, a selective PI3KF064 inhibitor, in in-vitro and in-vivo models of epidermolysis bullosa acquisita (EBA) and mucous membrane pemphigoid (MMP).

Dual Effect of Bleomycin on Histopathological Features of Lungs and Mediastinal Fat-Associated Lymphoid Clusters in an Autoimmune Disease Mouse Model.

Mediastinal fat-associated lymphoid clusters (MFALCs) are novel immune clusters that function in the pathogenesis of bleomycin (BLM)-induced pneumonitis in a C57BL/6 mouse model. However, we lack literature on the effects of BLM in an autoimmune disease mouse model (AIDM). In the present study, BLM sulfate (BLM group) or phosphate-buffered saline (PBS group) were intranasally administered in BXSB/MpJ-Yaa (Yaa) AIDM and its wild-type strains (BXSB/MpJ “BXSB”) and the histopathology of MFALCs and lungs were examined on days 7 and 21 days. Immunohistochemical analysis was performed to detect lymphatic vessels (LVs), high endothelial venules (HEVs), proliferating, and immune cells. Furthermore, the mRNA expression of Yaa locus genes (TLR7, TLR8, Arhgap6, Msl3, and Tceanc) was detected in the lung tissues. Here, we show a dual effect of BLM on intra-thoracic immune hemostasis among Yaa AIDM and its corresponding wild-type strain (BXSB mice). The BLM group of BXSB mice displayed significantly higher values of lung injury scores (LIS) and size of MFALCs as compared with the corresponding PBS group. However, an opposite effect was detected in Yaa mice. Furthermore, Yaa mice displayed decreased serum autoantibody titers and downregulated expression of TLR7, TLR8, Msl3, and Tceanc in the lungs following BLM administration, especially on day 21. Interestingly, significant positive correlations were detected in both strains between the LIS and the size of MFALCs, LVs, HEVs, and proliferating cells. Conclusively, our findings revealed a crucial function of HEVs on the extent of lung injury and the development of MFALCs in BLM-administered Yaa AIDM and control BXSB mice with dual effects. Moreover, our data suggest that down regulation of Yaa locus genes could contribute as an important attributing factor leading to decrease in the degree of autoimmunity and lung injury in AIDM. Therefore, we suggest that genetic background contributes to BLM diversity among AIDM and the wild-type strain. Targeting some genes or venules could provide novel therapeutic approaches for some autoimmune-associated respiratory diseases via controlling the MFALCs development.

Human Gut Bacterial Metabolism Drives Th17 Activation and Colitis

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmune disease, yet the relevant human-associated bacteria for Th17-driven disease remain elusive. Here, we show the prevalent human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by upregulating interleukin (IL)-17 expression through a cell- and antigen-independent mechanism. E. lenta is enriched in inflammatory bowel disease patients and worsens colitis severity in a Rorc-dependent manner. We exploited natural E. lenta strain-level variation to identify a bacterial enzyme (Cardiac glycoside reductase 2, Cgr2) necessary and sufficient to induce IL-17a. The cgr2 gene is enriched in the gut microbiomes of autoimmune subjects and correlates with disease severity. Finally, we leveraged the sensitivity of Cgr2 to dietary arginine to prevent E. lenta-induced intestinal inflammation. Together, these results expand the mechanisms through which bacteria shape mucosal immunity and support a role of human gut bacterial metabolism in driving Th17-dependent autoimmune diseases.

Noncytotoxic Inhibition of the Immunoproteasome Regulates Human Immune Cells In Vitro and Suppresses Cutaneous Inflammation in the Mouse.

Inhibitors of the immunoproteasome (i-20S) have shown promise in mouse models of autoimmune diseases and allograft rejection. In this study, we used a novel inhibitor of the immunoproteasome, PKS3053, that is reversible, noncovalent, tight-binding, and highly selective for the β5i subunit of the i-20S to evaluate the role that i-20S plays in regulating immune responses in vitro and in vivo. In contrast to irreversible, less-selective inhibitors, PKS3053 did not kill any of the primary human cell types tested, including plasmacytoid dendritic cells, conventional dendritic cells, macrophages, and T cells, all of which expressed genes encoding both the constitutive proteasome (c-20S) and i-20S. PKS3053 reduced TLR-dependent activation of plasmacytoid dendritic cells, decreasing their maturation and IFN-α response and reducing their ability to activate allogenic T cells. In addition, PKS3053 reduced T cell proliferation directly and inhibited TLR-mediated activation of conventional dendritic cells and macrophages. In a mouse model of skin injury that shares some features of cutaneous lupus erythematosus, blocking i-20S decreased inflammation, cellular infiltration, and tissue damage. We conclude that the immunoproteasome is involved in the activation of innate and adaptive immune cells, that their activation can be suppressed with an i-20S inhibitor without killing them, and that selective inhibition of β5i holds promise as a potential therapy for inflammatory skin diseases such as psoriasis, cutaneous lupus erythematosus, and systemic sclerosis.

HSP70, a Novel Regulatory Molecule in B Cell-Mediated Suppression of Autoimmune Diseases

B cells have recently emerged as playing regulatory role in autoimmune diseases. We have previously demonstrated that human peripheral blood CD19+CD24hiCD27+ B cells have regulatory function both in healthy donors and in patients with autoimmune disease. However, the mechanism of this regulation is still not fully understood. In this study, microarrays were utilized to compare gene expression of CD19+CD24hiCD27+ B cells (regulatory B cells, Bregs) with CD19+CD24loCD27− B cells (non-Bregs) in human peripheral blood. We found that heat shock protein 70 (HSP70) expression was significantly upregulated in Bregs. In vitro studies explored that HSP70 inhibition impaired the regulatory function of peripheral blood Bregs. In mouse models of autoimmune disease, using HSP70-deficient mice or HSP70 inhibitors, Bregs suppressed effector cells and rescued disease-associated phenotypes that were dependent on HSP70. Mechanistically, Bregs secreted HSP70, directly suppressing effector cells, such as T effect cells. These findings reveal that HSP70 is a novel factor that modulates Breg function and suggest that enhancing Breg-mediated production of HSP70 could be a viable therapy for autoimmune disease.

Celastrol ameliorates autoimmune disorders in Trex1-deficient mice

Celastrol is one of most potent bioactive molecule isolated from the medicinal plant Tripterygium wilfordii (Thunder God Vine) and is well known for its potential therapeutic value against various chronic diseases including the autoimmune diseases, such as systemic lupus erythematosus and Aicardi-Goutieres syndrome, or other interferonopathies. However, the underlying mechanism of celastrol function remains unclear. Here we showed that celastrol caused inhibition of interferon regulatory factor 3 (IRF3) activation leading to the down-regulation of the interferon response triggered by cytosolic nucleic acids in vitro and in vivo. Moreover, celastrol treatment markedly ameliorates the autoimmune phenotypes including myocarditis, aberrant interferon response and autoantibody production, as well as the excessive T-cell activation in Trex1−/− autoimmune disease mouse model. Collectively, our results indicate that celastrol inhibits interferon response by targeting IRF3 activation and may be used as an effective treatment for interferon response-dependent autoimmune diseases.

Histopathological changes in tear-secreting tissues and cornea in a mouse model of autoimmune disease.

Cornea, an outermost layer of mammalian eye, is protected by tear film and abnormalities of tear film causes dry eye. Dry eye injures the cornea which results lower vision in patients. Several factors cause dry eye, including altered systemic conditions, environment, and immunological abnormality of the patient in autoimmune disease like Sjögren's syndrome (SS). However, the detailed pathology of autoimmune abnormality-mediated dry eye is unclear. Here we demonstrated that systemic autoimmune abnormality in BXSB-Yaa mice was associated with histological changes in the exocrine glands and cornea of the eyes. We also showed that BXSB-Yaa mice developed mild or early stage dry eye-like disease and explain the existence of a compensatory mechanism associated with the dysfunction of these tissues. Thus, BXSB-Yaa could be a model for SS-like disease-associated dry eye and these data would contribute to the understanding of the pathogenesis of autoimmune-related dry eye disease.

Roles of Endogenous IL-10 and IL-10-Competent and CD5+ B Cells in Autoimmune Thyroiditis in NOD.H-2h4 Mice.

Interleukin (IL)-10 is a highly important anti-inflammatory cytokine in the immune system. CD1dhi and CD5+ B cells are both traditionally defined IL-10-secreting B cells. In recent years, a B cell group with combined markers of CD1dhi and CD5+ has been widely studied as it has been reported to suppress autoimmunity in mouse models of autoimmune diseases through IL-10 mechanisms. From the perspective of origination, CD1dhi and CD5+ B cells are developed from different B cell lineages. Whether the regulatory capacity of these 2 B cell groups is consistent with their ability to secrete IL-10 has not been determined. In this study, we generated IL-10 knockout NOD.H-2h4 mice to investigate the function of endogenous IL-10 in autoimmune thyroiditis and conducted adoptive transfer experiments to explore the respective roles of CD5+ and CD1dhi B cells. In our results, the IL-10-/- NOD.H-2h4 mice developed thyroiditis, similar to wild-type NOD.H-2h4 mice. The CD5+ B cells were more capable of secreting IL-10 than CD1dhi B cells in flow cytometric analysis, but the CD1dhi B cells showed more suppressive effects on thyroiditis development and autoantibody production, as well as Th17 cell response. In conclusion, endogenous IL-10 does not play an important role in autoimmune thyroiditis. CD1dhi B cells may play regulatory roles through mechanisms other than secreting IL-10.

A Novel Targeted Approach to Achieve Immune System Reset: CD45-Targeted Antibody Drug Conjugates Enable Autologous HSCT and Ameliorate Disease in Preclinical Autoimmune Disease Models

Resetting the immune system through autologous hematopoietic stem cell transplant (autoHSCT) is a highly effective treatment in patients with autoimmune diseases (AID). AutoHSCT achieved long-term remission in patients with relapsed refractory and secondary progressive multiple sclerosis (Muraro 2017), superior to their previous standard of care (Burt 2019). AutoHSCT in scleroderma patients achieved superior outcomes in two randomized studies (Tyndall 2014, Sullivan 2018). These impressive results are achieved by both eradication of autoreactive immune effector cells and re-establishment of a self-tolerant immune system, i.e., immune system reset. However, only a fraction of eligible patients undergo autoHSCT, in part due to toxicity associated with current conditioning regimens that remove the disease-causing cells. To enable more patients to benefit from immune reset without debilitating chemotherapy, we generated novel anti-human CD45 ADCs that cross react with NHP and evaluated these for the ability to deplete hematopoietic cells in vitro and in vivo (Fig.1). In vitro the CD45-ADC efficiently killed human CD34+ progenitors and peripheral CD3+ T cells from both healthy donors and MS patients. In vivo in humanized NSG mice, single doses of the CD45-ADCs were well-tolerated and led to depletion of human hematopoietic cells in BM (Fig. 1A) and periphery. In NHPs, single doses of CD45-ADCs were well tolerated and depleted BM HSCs and peripheral lymphocytes (Fig.1B). CD45-ADC treatment of hNSGs with sclerodermatous xenoGVHD resulted in resolution of symptoms (Fig. 1C). To model the complete immune reset approach in mouse models of AID, we generated a tool anti-mouse CD45 ADC. A single-dose enabled full myeloablation (>99% depletion of LT-HSCs) and complete donor chimerism with congenic HSCT (>90% chimerism at 16 weeks). In an adoptive transfer model of type I diabetes, treatment with a single dose of CD45-ADC and congenic HSCT led to disease prevention. In a murine model of MS EAE, a single dose of the CD45-ADC followed by congenic HSCT enabled full donor chimerism; treatment prior to disease onset significantly delayed disease onset and reduced disease severity; treatment after disease onset also halted progression of symptoms. These data demonstrate that CD45-ADC conditioning followed by congenic HSCT is sufficient for full myeloablation and immune reset. Experiments are ongoing, and evaluation of this ADC in murine models of diabetes and arthritis will be presented. These results indicate that targeted immune depletion with a single dose of CD45-ADC may be sufficient to enable auto-HSCT and immune reset in multiple AID indications. Targeted conditioning with CD45-ADC may enable more patients to benefit from immune reset through removal of pathogenic cells and autoHSCT without the morbidity and mortality associated with current chemotherapeutic conditioning.

Bivalent anti-CD3 Fab dimers drive T cells into a highly efficient fratricide with potential clinical application

Abstract Non-covalent anti-CD3 Fab dimers (Bi-Fabs) bind to the TCR/CD3 complex and activate T cells. In the presence of proper co-stimulation, anti-CD3 Bi-Fabs promote T cell cycling and acquisition of effector functions on CD4 and CD8 T cells more robustly than other bivalent antibody species like F(ab′)2 and mAb with the same specificity. Surprisingly, the net effect of anti-CD3 Bi-Fab treatment on T cells in vitro is contraction of CD4 and CD8 T cells, via AICD. This is in contrast with the net T cell proliferation observed with soluble anti-CD3 mAbs that are stimulatory in the presence of proper co-stimulation. Bi-Fab binding to the TCR/CD3 complex, and direct cellular contact with Bi-Fab engaged T cells, are required to observe the net contraction of treated T cells. Using perforin-deficient T cells, and Fas-FasL blockade, we show this net contraction results from T cell apoptosis via Fas-FasL and CTL degranulation. Structural studies on Bi-Fabs suggest a rigid rod like structure. We hypothesize that the rigid rod allows for a potent crosslinking structure with both paratopes from the anti-CD3 Fabs capable to engage effector T cells on fratricide. In vivo, mice injected with anti-CD3 Bi-Fabs present depletion of T cells from blood, and absence of any adverse effects derived from T cell activation like cytokine storm. We have begun exploring the therapeutic potential of T cell depletion in mouse models of autoimmune disease with promising results.

CCR2 mediates transendothelial migration of human pathogenic Th17 cells

Abstract Leukocyte extravasation is characterized by rolling and arrest on the endothelium followed by transendothelial migration (TEM). Leukocyte arrest and TEM typically require chemokines and their receptors. Previously, we showed that CD4+CCR5+CCR2+ T cells are highly differentiated, readily activated memory cells in human blood that exhibit diverse effector capabilities. We and others have also shown that CCR6 is the signature chemokine receptor for Th17 cells. Here we describe studies of the migratory capabilities and effector profiles of CD4+CCR6+CCR2+ as compared with CD4+CCR6+CCR2− and CD4+CCR6−CCR2− effector-memory T cells from human blood. Using flow chamber assays with TNFa-activated human umbilical vein endothelial cells, we found that among the CCR6+ cells, only the CCR2+ subset was able to cross the endothelial monolayer efficiently. We found that CCR6 and CCR5 were important for arrest whereas CCR2 was critical for subsequent TEM. Our RNA-Seq data and intracellular staining data of ex vivo activated T cells showed that the CCR6+CCR2+ cells were distinguished by their expression not only of the cytokines typical of Th17-cells, but also of cytokines, including IFNg and GM-CSF, and transcription factors characteristic of the pathogenic Th17 cells that mediate tissue injury in mouse models of autoimmune disease. Taken together, our studies suggest that chemokine receptors on memory Th cells play non-redundant roles in the egress from blood, that providing T cells with a combination of CCR6 CCR5 and CCR2 could enhance their trafficking into tissue, and that, on the other hand, blocking CCR2 alone could prevent the extravasation of pathogenic Th17 cells and potentially diminish tissue damage in Th17-cell driven disease.

The pathogenicity of Th17 cells in autoimmune diseases.

IL-17-producing T helper (Th17) cells have been implicated in the pathogenesis of many inflammatory and autoimmune diseases. Targeting the effector cytokines IL-17 and GM-CSF secreted by autoimmune Th17 cells has been shown to be effective for the treatment of the diseases. Understanding a molecular basis of Th17 differentiation and effector functions is therefore critical for the regulation of the pathogenicity of tissue Th17 cells in chronic inflammation. Here, we discuss the roles of proinflammatory cytokines and environmental stimuli in the control of Th17 differentiation and chronic tissue inflammation by pathogenic Th17 cells in humans and in mouse models of autoimmune diseases. We also highlight recent advances in the regulation of pathogenic Th17 cells by gut microbiota and immunometabolism in autoimmune arthritis.