IU Indianapolis ScholarWorks :: Browsing by Author "Bertolini, Thais B."

Browsing by Author "Bertolini, Thais B."

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Distinct functions and transcriptional signatures in orally induced regulatory T cell populations
(Frontiers Media, 2023-10-26) Biswas, Moanaro; So, Kaman; Bertolini, Thais B.; Krishnan, Preethi; Rana, Jyoti; Muñoz-Melero, Maite; Syed, Farooq; Kumar, Sandeep R. P.; Gao, Hongyu; Xuei, Xiaoling; Terhorst, Cox; Daniell, Henry; Cao, Sha; Herzog, Roland W.; Pediatrics, School of Medicine
Oral administration of antigen induces regulatory T cells (Treg) that can not only control local immune responses in the small intestine, but also traffic to the central immune system to deliver systemic suppression. Employing murine models of the inherited bleeding disorder hemophilia, we find that oral antigen administration induces three CD4+ Treg subsets, namely FoxP3+LAP-, FoxP3+LAP+, and FoxP3-LAP+. These T cells act in concert to suppress systemic antibody production induced by therapeutic protein administration. Whilst both FoxP3+LAP+ and FoxP3-LAP+ CD4+ T cells express membrane-bound TGF-β (latency associated peptide, LAP), phenotypic, functional, and single cell transcriptomic analyses reveal distinct characteristics in the two subsets. As judged by an increase in IL-2Rα and TCR signaling, elevated expression of co-inhibitory receptor molecules and upregulation of the TGFβ and IL-10 signaling pathways, FoxP3+LAP+ cells are an activated form of FoxP3+LAP- Treg. Whereas FoxP3-LAP+ cells express low levels of genes involved in TCR signaling or co-stimulation, engagement of the AP-1 complex members Jun/Fos and Atf3 is most prominent, consistent with potent IL-10 production. Single cell transcriptomic analysis further reveals that engagement of the Jun/Fos transcription factors is requisite for mediating TGFβ expression. This can occur via an Il2ra dependent or independent process in FoxP3+LAP+ or FoxP3-LAP+ cells respectively. Surprisingly, both FoxP3+LAP+ and FoxP3-LAP+ cells potently suppress and induce FoxP3 expression in CD4+ conventional T cells. In this process, FoxP3-LAP+ cells may themselves convert to FoxP3+ Treg. We conclude that orally induced suppression is dependent on multiple regulatory cell types with complementary and interconnected roles.
Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy
(Frontiers Media, 2021-05-31) Bertolini, Thais B.; Shirley, Jamie L.; Zolotukhin, Irene; Li, Xin; Kaisho, Tsuneyasu; Xiao, Weidong; Kumar, Sandeep R.P.; Herzog, Roland W.; Pediatrics, School of Medicine
Adeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector's DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.
Factor IX administration in the skin primes inhibitor formation and sensitizes hemophilia B mice to systemic factor IX administration
(Elsevier, 2023-11-04) Sherman, Alexandra; Bertolini, Thais B.; Arisa, Sreevani; Herzog, Roland W.; Kaczmarek, Radoslaw; Pediatrics, School of Medicine
Background: Factor IX inhibitor formation is the most serious complication of replacement therapy for the bleeding disorder hemophilia B, exacerbated by severe allergic reactions occurring in up to 60% of patients with inhibitors. Low success rates of immune tolerance induction therapy in hemophilia B necessitate the search for novel immune tolerance therapies. Skin-associated lymphoid tissues have been successfully targeted in allergen-specific immunotherapy. Objectives: We aimed to develop a prophylactic immune tolerance protocol based on intradermal administration of FIX that would prevent inhibitor formation and/or anaphylaxis in response to replacement therapy. Methods: We measured FIX inhibitor, anti-FIX immunoglobulin G1, and immunoglobulin E titers using the Bethesda assay and enzyme-linked immunosorbent assay after 4 weeks of twice-weekly intradermal FIX or FIX-Fc administration followed by 5 to 6 weeks of weekly systemic FIX injections in C3H/HeJ hemophilia B mice. We also measured skin antigen-presenting, follicular helper T, and germinal center B cell frequencies in skin-draining lymph nodes after a single or repeat intradermal FIX administration. Results: Intradermal administration enhanced FIX inhibitor formation in response to systemic administration. We further found that intradermal administration alone triggers inhibitor formation, even at a low dose of 0.4 IU/kg, which is 100-fold lower than the intravenous dose of 40 IU/kg typically required to induce inhibitor development in hemophilia B mice. Also, intradermal administration triggered germinal center formation in skin-draining lymph nodes and sensitized mice to systemic administration. Factor IX-Fc fusion protein did not modulate inhibitor formation. Conclusion: Intradermal FIX administration is highly immunogenic, suggesting that the skin compartment is not amenable to immune tolerance induction or therapeutic delivery of clotting factors.
Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells
(American Society of Hematology, 2023) Kaczmarek, Radoslaw; Piñeros, Annie R.; Patterson, Paige E.; Bertolini, Thais B.; Perrin, George Q.; Sherman, Alexandra; Born, Jameson; Arisa, Sreevani; Arvin, Matthew C.; Kamocka, Malgorzata M.; Martinez, Michelle M.; Dunn, Kenneth W.; Quinn, Sean M.; Morris, Johnathan J.; Wilhelm, Amelia R.; Kaisho, Tsuneyasu; Munoz-Melero, Maite; Biswas, Moanaro; Kaplan, Mark H.; Linnemann, Amelia K.; George, Lindsey A.; Camire, Rodney M.; Herzog, Roland W.; Pediatrics, School of Medicine
Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.
IL-15 blockade and rapamycin rescue multifactorial loss of factor VIII from AAV-transduced hepatocytes in hemophilia A mice
(Elsevier, 2022-12-07) Butterfield, John S. S.; Yamada, Kentaro; Bertolini, Thais B.; Syed, Farooq; Kumar, Sandeep R. P.; Li, Xin; Arisa, Sreevani; Piñeros, Annie R.; Tapia, Alejandro; Rogers, Christopher A.; Li, Ning; Rana, Jyoti; Biswas, Moanaro; Terhorst, Cox; Kaufman, Randal J.; de Jong, Ype P.; Herzog, Roland W.; Pediatrics, School of Medicine
Hepatic adeno-associated viral (AAV) gene transfer has the potential to cure the X-linked bleeding disorder hemophilia A. However, declining therapeutic coagulation factor VIII (FVIII) expression has plagued clinical trials. To assess the mechanistic underpinnings of this loss of FVIII expression, we developed a hemophilia A mouse model that shares key features observed in clinical trials. Following liver-directed AAV8 gene transfer in the presence of rapamycin, initial FVIII protein expression declines over time in the absence of antibody formation. Surprisingly, loss of FVIII protein production occurs despite persistence of transgene and mRNA, suggesting a translational shutdown rather than a loss of transduced hepatocytes. Some of the animals develop ER stress, which may be linked to hepatic inflammatory cytokine expression. FVIII protein expression is preserved by interleukin-15/interleukin-15 receptor blockade, which suppresses CD8+ T and natural killer cell responses. Interestingly, mice with initial FVIII levels >100% of normal had diminishing expression while still under immune suppression. Taken together, our findings of interanimal variability of the response, and the ability of the immune system to shut down transgene expression without utilizing cytolytic or antibody-mediated mechanisms, illustrate the challenges associated with FVIII gene transfer. Our protocols based upon cytokine blockade should help to maintain efficient FVIII expression.
Role of Orally Induced Regulatory T Cells in Immunotherapy and Tolerance
(Elsevier, 2021) Bertolini, Thais B.; Biswas, Moanaro; Terhorst, Cox; Daniell, Henry; Herzog, Roland W.; Piñeros, Annie R.; Pediatrics, School of Medicine
Oral antigen administration to induce regulatory T cells (Treg) takes advantage of regulatory mechanisms that the gastrointestinal tract utilizes to promote unresponsiveness against food antigens or commensal microorganisms. Recently, antigen-based oral immunotherapies (OITs) have shown efficacy as treatment for food allergy and autoimmune diseases. Similarly, OITs appear to prevent anti-drug antibody responses in replacement therapy for genetic diseases. Intestinal epithelial cells and microbiota possibly condition dendritic cells (DC) toward a tolerogenic phenotype that induces Treg via expression of several mediators, e.g. IL-10, transforming growth factor-β, retinoic acid. Several factors, such as metabolites derived from microbiota or diet, impact the stability and expansion of these induced Treg, which include, but are not limited to, FoxP3+ Treg, LAP+ Treg, and/or Tr1 cells. Here, we review various orally induced Treg, their plasticity and cooperation between the Treg subsets, as well as underlying mechanisms controlling their induction and role in oral tolerance.
Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia
(Frontiers, 2020-05) Kumar, Sandeep R. P.; Wang, Xiaomei; Avuthu, Nagavardhini; Bertolini, Thais B.; Terhorst, Cox; Guda, Chittibabu; Daniell, Henry; Herzog, Roland W.; Pediatrics, School of Medicine
Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells in the lamina propria of the small but not large intestine. By contrast, no changes in frequencies of CD4+CD25+FoxP3+ T cells were observed. Here we demonstrate that, surprisingly, the adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice significantly suppress antibodies directed against FIX. By contrast, equal numbers of splenic CD4+CD25+ T cells do not have an effect on antibody formation. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine and that suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg. The observation that CD4+CD25-LAP+ Treg migrate to the spleen are useful for the design of clinical protocols.
Suppression of anti-drug antibody formation against coagulation factor VIII by oral delivery of anti-CD3 monoclonal antibody in hemophilia A mice
(Elsevier, 2023) Bertolini, Thais B.; Herzog, Roland W.; Kumar, Sandeep; Sherman, Alexandra; Rana, Jyoti; Kaczmarek, Radoslaw; Yamada, Kentaro; Arisa, Sreevani; Lillicrap, David; Terhorst, Cox; Daniell, Henry; Biswas, Moanaro; Pediatrics, School of Medicine
Active tolerance to ingested dietary antigens forms the basis for oral immunotherapy to food allergens or autoimmune self-antigens. Alternatively, oral administration of anti-CD3 monoclonal antibody can be effective in modulating systemic immune responses without T cell depletion. Here we assessed the efficacy of full length and the F(ab')2 fragment of oral anti-CD3 to prevent anti-drug antibody (ADA) formation to clotting factor VIII (FVIII) protein replacement therapy in hemophilia A mice. A short course of low dose oral anti-CD3 F(ab')2 reduced the production of neutralizing ADAs, and suppression was significantly enhanced when oral anti-CD3 was timed concurrently with FVIII administration. Tolerance was accompanied by the early induction of FoxP3+LAP-, FoxP3+LAP+, and FoxP3-LAP+ populations of CD4+ T cells in the spleen and mesenteric lymph nodes. FoxP3+LAP+ Tregs expressing CD69, CTLA-4, and PD1 persisted in spleens of treated mice, but did not produce IL-10. Finally, we attempted to combine the anti-CD3 approach with oral intake of FVIII antigen (using our previously established method of using lettuce plant cells transgenic for FVIII antigen fused to cholera toxin B (CTB) subunit, which suppresses ADAs in part through induction of IL-10 producing FoxP3-LAP+ Treg). However, combining these two approaches failed to improve suppression of ADAs. We conclude that oral anti-CD3 treatment is a promising approach to prevention of ADA formation in systemic protein replacement therapy, albeit via mechanisms distinct from and not synergistic with oral intake of bioencapsulated antigen.

Browsing by Author "Bertolini, Thais B."

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