Transplantation of Donor-Origin Mouse Embryonic Stem Cell-Derived Thymic Epithelial Progenitors Prevents the Development of Chronic Graft-versus-Host Disease in Mice.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant and nonmalignant diseases. However, chronic graft-versus-host disease (cGVHD) remains a significant cause of late morbidity and mortality after allogeneic HSCT. cGVHD often manifests as autoimmune syndrome. Thymic epithelial cells (TECs) play a critical role in supporting negative selection and regulatory T-cell (Treg) generation. Studies have shown that damage in TECs is sufficient to induce cGVHD. We have previously reported that mouse embryonic stem cells (mESCs) can be selectively induced to generate thymic epithelial progenitors (TEPs) in vitro. When transplanted in vivo, mESC-TEPs further develop into TECs that support T-cell development. We show here that transplantation of donor-origin mESC-TEPs into cGVHD recipients induces immune tolerance to both donor and host antigens and prevents the development of cGVHD. This is associated with more TECs and Tregs. Our results suggest that embryonic stem cell-derived TEPs may offer a new tool to control cGVHD. Stem Cells Translational Medicine 2017;6:121-130.

In Vivo Antitumor Activity of a Recombinant IL7/IL15 Hybrid Cytokine in Mice.

Both IL7 and IL15 have become important candidate immunomodulators for cancer treatment. However, IL7 or IL15 used alone suffers from shortcomings, such as short serum half-life and limited antitumor effect. We have cloned and expressed a recombinant (r) IL7/IL15 fusion protein in which IL7 and IL15 are linked by a flexible linker. We then compared the antitumor effect of rIL7/IL15 with the individual factors rIL7 and/or rIL15. We show here that rIL7/IL15 has a higher antitumor activity than the combination of the individual factors in both murine B16F10 melanoma and CT-26 colon cancer models. This was associated with a significant increase in tumor infiltration of T cells, DCs, and NK cells and a decrease in regulatory T cells (Tregs). In addition, rIL7/IL15-treated DCs had higher expression of costimulatory molecules CD80 and CD86. The higher antitumor activity of rIL7/IL15 is likely due to its longer in vivo half-life and different effects on immune cells. Our results suggest that rIL7/IL15 may offer a new tool to enhance antitumor immunity and treat cancer. Mol Cancer Ther; 15(10); 2413-21. ©2016 AACR.

Recombinant IL-7/HGFß hybrid cytokine separates acute graft-versus-host-disease from graft-versus-tumour activity by altering donor T cell trafficking.

Given that donor T cells from a transplant contribute both the desired graft-versus-tumour (GVT) effect and detrimental graft-versus-host disease (GVHD), strategies to separate GVHD and GVT activity are a major clinical goal. We have previously demonstrated that in vivo administration of a recombinant (r)IL-7/HGFß hybrid cytokine, consisting of interleukin-7 (IL-7, IL7) and the ß-chain of hepatocyte growth factor (HGFß), significantly inhibits the growth of cancer cells in murine tumour models. The antit-umour effect of rIL-7/HGFß is related to a marked infiltration T cells in the tumour tissues. We have also shown that GVHD was not induced in rIL-7/HGFß-treated T cell-depleted allogeneic haematopoietic stem cell transplantation (HSCT) recipients. We show here that, in T cell-replete allogeneic HSCT murine models, rIL-7/HGFß attenuated acute GVHD (aGVHD), while promoting GVT activity. This was related to an alteration of donor T cell trafficking, with an increased infiltration of donor T cells into tumour tissues and the lympho-haematopoietic system but decreased number of the T cells in the GVHD target organs. Therefore, rIL-7/HGFß may offer a new tool to alleviate aGVHD while prompting GVT, and to study the molecular regulation of T cell trafficking.

FOXN1 recombinant protein enhances T-cell regeneration after hematopoietic stem cell transplantation in mice.

A prolonged period of T-cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T-cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cell-penetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T-cell regeneration in patients following HSCT.

Administration of embryonic stem cell-derived thymic epithelial progenitors expressing MOG induces antigen-specific tolerance and ameliorates experimental autoimmune encephalomyelitis.

Tolerance induction, and thus prevention or treatment of autoimmune disease, is not only associated with the persistent presence of self-antigen in the thymus, but also relies on a functional thymus; however, the thymus undergoes profound age-dependent involution. Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T cell development. We have reported that mouse embryonic stem cells (mESCs) can be induced in vitro to generate thymic epithelial progenitors (TEPs) that further develop into functional TECs in vivo. We show here that transplantation of mESC-TEPs expressing self-antigen myelin oligodendrocyte glycoprotein (MOG) in mice results in enhanced T cell regeneration, long-term expression of MOG in the thymus, prevention of experimental autoimmune encephalomyelitis (EAE) development, and remission of established EAE. Our findings indicate that transplantation of ESC-TEPs expressing disease-causative self-antigen(s) may provide an effective approach for the prevention and treatment of autoimmune disease.

c-Met signalling is required for efficient postnatal thymic regeneration and repair.

We have reported that in vivo administration of the hybrid cytokine rIL-7/HGFß or rIL-7/HGFα, which contains interleukin-7 (IL-7) and the ß- or α-chain of hepatocyte growth factor (HGF), significantly enhances thymopoiesis in mice after bone marrow transplantation. We have shown that the HGF receptor, c-Met, is involved in the effect of the hybrid cytokines. To address the role of c-Met signalling in thymocyte development and recovery, we generated conditional knockout (cKO) mice in which c-Met was specifically deleted in T cells by crossing c-Met(ft/ft) mice with CD4-Cre transgenic mice. We show here that although the number of total thymocytes and thymocyte subsets in young c-Met cKO mice is comparable to age-matched control (Ctrl) mice, the cKO mice were more susceptible to sub-lethal irradiation and dexamethasone treatment. This was demonstrated by low recovery in thymic cellularity in c-Met cKO mice after insult. Furthermore, the number of total thymocytes and thymocyte subsets was markedly reduced in 6- to 12-month-old cKO mice compared with age-matched Ctrl mice, and the thymic architecture of 12-month-old cKO mice was similar to that of 20-month-old wild-type mice. In addition, c-Met deficiency reduced cell survival and the expression of Bcl-xL in double-positive thymocytes, and decreased cell proliferation and the expression of cyclin E and cyclin-dependent kinase 5 in single-positive thymocytes. Our data indicate that c-Met signalling plays an important role in thymic regeneration after thymic insult. In addition, T-cell-specific inactivation of c-Met accelerates age-related thymic involution.

Tbx1 modulates endodermal and mesodermal differentiation from mouse induced pluripotent stem cells.

The T-box transcriptional factor (Tbx) family of transcriptional factors has distinct roles in a wide range of embryonic differentiation or response pathways. Tbx1, a T-box transcription factor, is an important gene for the human congenital disorder 22q11.2 deletion syndrome. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem-cell-based therapies. In this study, we generated iPSCs from Tbx1(-/-) and Tbx1(+/+) fibroblasts and investigated the spontaneous differentiation potential of iPSCs by detailed lineage analysis of the iPSC-derived embryoid bodies. Undifferentiated Tbx1(-/-) and Tbx1(+/+) iPSCs showed similar expression levels of pluripotent markers. The ability of the Tbx1(-/-) iPSCs to generate endodermal and mesodermal lineages was compromised upon spontaneous differentiation into embryonic bodies. Restoration of Tbx1 expression in the Tbx1(-/-) iPSCs to normal levels using an inducible lentiviral system rescued these cells from the potential of defective differentiation. Interestingly, overexpression of Tbx1 in the Tbx1(-/-) iPSCs to higher levels than in the Tbx1(+/+) iPSCs again led to a defective differentiation potential. Additionally, we observed that expression of fibroblast growth factor (FGF) 10 and FGF8 was downregulated in the Tbx1(-/-) iPSC-derived cells, which suggests that Tbx1 regulates the expression of FGFs. Taken together, our results implicated the Tbx1 level as an important determinant of endodermal and mesodermal lineage differentiation during embryonic development.

Recombinant IL-7/HGFß hybrid cytokine enhances T cell recovery in mice following allogeneic bone marrow transplantation.

T cell immunodeficiency is a major complication of bone marrow (BM) transplantation (BMT). Therefore, approaches to enhance T cell reconstitution after BMT are required. We have purified a hybrid cytokine, consisting of IL-7 and the ß-chain of hepatocyte growth factor (HGFß) (IL-7/HGFß), from a unique long-term BM culture system. We have cloned and expressed the IL-7/HGFß gene in which the IL-7 and HGFß genes are connected by a flexible linker to generate rIL-7/HGFß protein. Here, we show that rIL-7/HGFß treatment enhances thymopoiesis after allogeneic BMT. Although rIL-7 treatment also enhances the number of thymocytes, rIL-7/HGFß hybrid cytokine was more effective than was rIL-7 and the mechanisms by which rIL-7 and rIL-7/HGFß increase the numbers of thymocytes are different. rIL-7 enhances the survival of double negative (DN), CD4 and CD8 single positive (SP) thymocytes. In contrast, rIL-7/HGFß enhances the proliferation of the DN, SP thymocytes, as well as the survival of CD4 and CD8 double positive (DP) thymocytes. rIL-7/HGFß treatment also increases the numbers of early thymocyte progenitors (ETPs) and thymic epithelial cells (TECs). The enhanced thymic reconstitution in the rIL-7/HGFß-treated allogeneic BMT recipients results in increased number and functional activities of peripheral T cells. Graft-versus-host-disease (GVHD) is not induced in the rIL-7/HGFß-treated BMT mice. Therefore, rIL-7/HGFß may offer a new tool for the prevention and/or treatment of T cell immunodeficiency following BMT.

Cloning and characterization of a hybridoma secreting a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-specific monoclonal antibody and recombinant F(ab).

Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine.

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry.

Xenoepitope substitution avoids deceptive imprinting and broadens the immune response to foot-and-mouth disease virus.

Many RNA viruses encode error-prone polymerases which introduce mutations into B and T cell epitopes, providing a mechanism for immunological escape. When regions of hypervariability are found within immunodominant epitopes with no known function, they are referred to as "decoy epitopes," which often deceptively imprint the host's immune response. In this work, a decoy epitope was identified in the foot-and-mouth disease virus (FMDV) serotype O VP1 G-H loop after multiple sequence alignment of 118 isolates. A series of chimeric cyclic peptides resembling the type O G-H loop were prepared, each bearing a defined "B cell xenoepitope" from another virus in place of the native decoy epitope. These sequences were derived from porcine respiratory and reproductive syndrome virus (PRRSV), from HIV, or from a presumptively tolerogenic sequence from murine albumin and were subsequently used as immunogens in BALB/c mice. Cross-reactive antibody responses against all peptides were compared to a wild-type peptide and ovalbumin (OVA). A broadened antibody response was generated in animals inoculated with the PRRSV chimeric peptide, in which virus binding of serum antibodies was also observed. A B cell epitope mapping experiment did not reveal recognition of any contiguous linear epitopes, raising the possibility that the refocused response was directed to a conformational epitope. Taken together, these results indicate that xenoepitope substitution is a novel method for immune refocusing against decoy epitopes of RNA viruses such as FMDV as part of the rational design of next-generation vaccines.

Use of inactivated Escherichia coli enterotoxins to enhance respiratory mucosal adjuvanticity during vaccination in swine.

In order to augment responses to respiratory vaccines in swine, various adjuvants were intranasally coadministered with a foot-and-mouth disease virus (FMDV) antigen to pigs. Detoxified Escherichia coli enterotoxins LTK63 and LTR72 enhanced antigen-specific mucosal and systemic immunity, demonstrating their efficacy as adjuvants for nonreplicating antigens upon intranasal immunization in swine.

Bacterial Toxin Fusion Proteins Elicit Mucosal Immunity against a Foot-and-Mouth Disease Virus Antigen When Administered Intranasally to Guinea Pigs.

Peptides corresponding to the foot-and-mouth disease virus VP1 G-H loop are capable of inducing neutralizing antibodies in some species but are considered relatively poor immunogens, especially at mucosal surfaces. However, intranasal administration of antigens along with the appropriate delivery vehicle/adjuvant has been shown to induce mucosal immune responses, and bacterial enterotoxins have long been known to be effective in this regard. In the current study, two different carrier/adjuvant approaches were used to augment mucosal immunity to the FMDV O(1) BFS G-H loop epitope, in which the G-H loop was genetically coupled to the E. coli LT-B subunit and coexpressed with the LTA2 fragment (LTA2B-GH), or the nontoxic pseudomonas exotoxin A (ntPE) was fused to LTA2B-GH at LT-A2 to enhance receptor targeting. Only guinea pigs that were inoculated intranasally with ntPE-LTA2B-GH and LTA2B-GH induced significant anti-G-H loop IgA antibodies in nasal washes at weeks 4 and 6 when compared to ovalbumin or G-H loop immunized animals. These were also the only groups that exhibited G-H loop-specific antigen-secreting cells in the nasal mucosa. These data demonstrate that fusion of nonreplicating antigens to LTA2B and ntPE-LTA2B has the potential to be used as carriers/adjuvants to induce mucosal immune responses against infectious diseases.

Chemokine and cytokine gene expression profiles in chickens inoculated with Mycoplasma gallisepticum strains Rlow or GT5.

Mycoplasma gallisepticum infection in chickens leads to tracheitis, airsacculitis, poor feed conversion and reduced egg production, resulting in considerable economic hardship on the poultry industry. The chemokines and cytokines responsible for recruitment, activation and proliferation of leukocytes in affected tissues have not been described. In the current study, chemokine and cytokine gene expression profiles were investigated in tracheas of chickens inoculated with M. gallisepticum strains R(low) (pathogenic) and GT5 (attenuated) at days 1, 4 and 8 post-inoculation. Expression of lymphotactin mRNA was higher in R(low)-inoculated chickens than GT5- or PBS-inoculated chickens, while CXCL13/BCA1 mRNA expression level was higher in both GT5- or R(low)-inoculated chickens than in PBS-inoculated controls on day 1 post-inoculation. However, both R(low) and GT5 strains induced a down-regulation in mRNA expression of CCL20, IL-1beta, IL-8 and IL-12p40 genes, with CCL20 and IL-12 mRNA levels remaining lower on days 4 and 8 post-inoculation. On day 4, R(low)-inoculated chickens exhibited significantly higher tracheal lesion scores and higher levels of lymphotactin, CXCL13, CXCL14, RANTES, MIP-1beta, IL-1beta and IFN-gamma mRNA compared to PBS-inoculated controls. The mRNA levels of these genes were also higher in R(low)-inoculated chickens that had moderate to severe tracheal lesion scores on day 8 post-inoculation. These results reflect the importance of lymphocyte and monocyte chemotactic factors in the development of tracheal lesions in chickens inoculated with M. gallisepticum strain R(low). Our data also suggest that M. gallisepticum may modulate the host response causing dramatic decreases in CCL20, IL-8 and IL-12 mRNA levels in GT5- or R(low)-inoculated chickens as early as one day post-inoculation.

Non-toxic Pseudomonas aeruginosa exotoxin A expressing the FMDV VP1 G-H loop for mucosal vaccination of swine against foot and mouth disease virus.

Synthetic peptides derived from the G-H loop of the foot and mouth disease virus (FMDV) capsid protein VP1 are relatively poor at recapitulating the native conformation present in the virus, and thus are often poor immunogens. We hypothesized that a candidate mucosal vaccine against FMDV could be developed using the non-toxic Pseudomonas aeruginosa exotoxin A (ntPE) to deliver the G-H loop in its native conformation. An added benefit of this approach is the potential for ntPE to serve as an effective carrier/adjuvant molecule for delivery of the fusion protein across the epithelial barrier by virtue of its capacity to bind to CD91. A chimeric protein (ntPE-GH) was generated by inserting the coding sequence of the G-H loop into an expression plasmid encoding ntPE, in place of the native Ib loop. Recombinant ntPE-GH and wild-type ntPE were each expressed in Escherichia coli, purified over a nickel resin, then administered intranasally to the pigs, with or without the mucosal adjuvant cholera toxin (CT). Both the ntPE and ntPE-GH induced mucosal and systemic immune responses against ntPE; moreover, ntPE-GH administered without CT induced anti-GH loop serum IgG antibodies. In conclusion, these data demonstrate that ntPE can be used as a mucosal carrier/adjuvant to induce an immune response against the VP1 G-H loop of FMDV.

Correlates of immune protection in chickens vaccinated with Mycoplasma gallisepticum strain GT5 following challenge with pathogenic M. gallisepticum strain R(low).

Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the trachea, air sacs, conjunctiva, and lungs. A live attenuated M. gallisepticum vaccine strain, GT5, was previously shown to be protective in chickens upon challenge; however, the mechanisms by which this vaccine and others confer protection remain largely unknown. The current study evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum strain R(low). GT5-vaccinated chickens developed mild tracheal lesions, consisting of few and scattered, discrete, lymphofollicular aggregates in the lamina propria. In addition, low numbers of aggregated B, CD4(+), and CD8(+) cells were observed to infiltrate the trachea, in stark contrast to the large numbers infiltrating the tracheas of sham-vaccinated chickens challenged with R(low). Lymphofollicular aggregates were rarely observed prior to day 12 postchallenge in sham-vaccinated chickens. Instead, they contained an increasingly more cellular inflammatory response characterized by expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This was due in part to expansion of interfollicular zones by large numbers of infiltrating CD4(+) and CD8(+) cells and a sizeable population of immunoglobulin A (IgA)- and IgG-secreting plasma cells. GT5-vaccinated chickens also had higher serum IgG concentrations, and significantly higher numbers of M. gallisepticum-specific IgG- and IgA-secreting plasma/B cells within the trachea, than did sham-vaccinated chickens. These responses were observed as early as day 4 postchallenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5-vaccinated chickens.