Accelerated thymopoiesis and improved T-cell responses in HLA-A2/-DR2 transgenic BRGS-based human immune system mice.

Human immune system (HIS) mouse models provide a robust in vivo platform to study human immunity. Nevertheless, the signals that guide human lymphocyte differentiation in HIS mice remain poorly understood. Here, we have developed a novel Balb/c Rag2-/- Il2rg-/- SirpaNOD (BRGS) HIS mouse model expressing human HLA-A2 and -DR2 transgenes (BRGSA2DR2). When comparing BRGS and BRGSA2DR2 HIS mice engrafted with human CD34+ stem cells, a more rapid emergence of T cells in the circulation of hosts bearing human HLA was shown, which may reflect a more efficient human T-cell development in the mouse thymus. Development of CD4+ and CD8+ T cells was accelerated in BRGSA2DR2 HIS mice and generated more balanced B and T-cell compartments in peripheral lymphoid organs. Both B- and T-cell function appeared enhanced in the presence of human HLA transgenes with higher levels of class switched Ig, increased percentages of polyfunctional T cells and clear evidence for antigen-specific T-cell responses following immunization. Taken together, the presence of human HLA class I and II molecules can improve multiple aspects of human B- and T-cell homeostasis and function in the BRGS-based HIS mouse model.

Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear. METHODS: We performed studies with BALB/c Rag2-/-Il2rg-/-SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells. RESULTS: Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. CONCLUSION: In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

A functional DC cross talk promotes human ILC homeostasis in humanized mice.

Humanized mice harboring human hematopoietic systems offer a valuable small-animal model to assess human immune responses to infection, inflammation, and cancer. Human immune system (HIS) mice develop a broad repertoire of antigen receptor bearing B and T cells that can participate in adaptive immune responses after immunization. In contrast, analysis of innate immune components, including innate lymphoid cells (ILCs) and natural killer (NK) cells, is limited in current HIS mouse models, partly because of the poor development of these rare lymphoid subsets. Here we show that novel dendritic cell (DC)-boosted BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (BRGSF) HIS mice harbor abundant NK cells and tissue-resident ILC subsets in lymphoid and nonlymphoid mucosal sites. We find that human NK cells and ILCs are phenotypically and functionally mature and provide evidence that human DC activation in BRGSF-based HIS mice can "cross talk" to human NK cells and ILCs. This novel HIS mouse model should provide the opportunity to study the immunobiology of human NK cell and ILC subsets in vivo in response to various environmental challenges.

A novel mouse model for stable engraftment of a human immune system and human hepatocytes.

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.

Animal models to test hiPS-derived hepatocytes in the context of inherited metabolic liver diseases.

Human induced pluripotent stem (hiPS) cells are established following reprogramming of somatic cells from a wide variety of tissues. Given the scarcity of adult human hepatocytes, hiPS-derived hepatocytes would be a valuable source of cells to study differentiation programs, model patient-specific diseases, test drug toxicities, and cell transplantation therapies. Although hiPS-derived hepatocytes are extensively characterized in cell culture assays, testing these cells in animal models is necessary to fully evaluate their differentiation profile and their lack of tumorigenicity. Immunodeficient mouse models harboring liver damage are effective hosts in which xenogeneic hepatocytes can engraft, proliferate, and participate in liver regeneration, thus constituting a stringent test of hepatocyte functionality. The in vivo evaluation of disease-specific hiPS-derived hepatocytes should broaden our understanding of the cellular and molecular processes involved in inherited metabolic liver disease phenotypes. Herein, we detail our methods to test the functions of hiPS-derived hepatocytes in the context of the immunodeficient Rag2(-/-)IL2Rγc(-/-)Alb-uPAtg mouse model.

Interleukin-15-dependent NKp46+ innate lymphoid cells control intestinal inflammation by recruiting inflammatory monocytes.

With the goal in mind to define how interleukin-15 (IL-15) contributes to acute intestinal inflammation, we have used a mouse model of ileitis induced by oral infection with Toxoplasma gondii. We observed that a crosstalk between IL-15 and interleukin-18 (IL-18) promoted intestinal recruitment of inflammatory monocytes, where these cells participated in parasite control but also in tissue damage. A stromal source of IL-15 controlled the development of lamina propria NKp46(+)NK1.1(+) cells, whereas IL-18 produced during T. gondii infection stimulated their production of the chemokine CCL3. In turn, CCL3 attracted inflammatory monocytes via their chemokine receptor CCR1, which was indispensable for their recruitment into the inflamed gut. Collectively, these results identify the IL-15-dependent subset of intestinal NKp46(+) cells as an important source of CCL3, which can amplify intestinal inflammation via the recruitment of CCR1(+) inflammatory monocytes. Preliminary evidence suggests that this pathway might operate in Crohn's disease.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-gamma by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

B cells amplify IFN-gamma production by T cells via a TNF-alpha-mediated mechanism.

Aside from being the precursors of the Ab-secreting cells, B cells are engaged in other immune functions such as Ag presentation to T cells or cytokine production. These functions may contribute to the pathogenic role of B cells in a wide range of autoimmune diseases. We demonstrate that B cells acquire the capacity to amplify IFN-gamma production by CD4 and CD8 T cells during the course of the Th1 inflammatory response to Toxoplasma gondii infection. Using the two following different strategies, we observed that B cells from T. gondii-infected mice, but not from naive mice, induce higher IFN-gamma expression by splenic host T cells: 1) reconstitution of B cell-deficient mice with B cells expressing an alloantigen different from the recipients, and 2) adoptive transfer of B and T cells into RAG-/- mice. In vitro assays allowing the physical separation of T and B cells demonstrate that Ag-primed B cells enhance IFN-gamma production by T cells in a contact-dependent fashion. Using an OVA-transgenic strain of T. gondii and OVA-specific CD4 T cells, we observed that the proinflammatory effect of B cells is neither Ag specific nor requires MHCII expression. However, TNF-alpha expressed on the surface of B cells appears to mediate in part the up-regulation of IFN-gamma by the effector T cells.

Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways.

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lps(d)) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators as compared with infected wild-type C3H/HeOuJ (Lps(n)) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF-alpha still remained greater in UPEC-infected than in naive C3H/HeJ (Lps(d)) mice. Using primary cultures of microdissected Lps(n) MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-beta-independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lps(n) MCDs leads to the activation of NF-kappaB, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These findings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys.

TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii.

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.

CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain.

The protozoan parasite Toxoplasma gondii enters hosts through the intestinal mucosa and colonizes distant tissues such as the brain, where its progeny persists for a lifetime. We investigated the role of CD11c- and CD11b-expressing leukocytes in T. gondii transport during the early step of parasitism from the mouse small intestine and during subsequent parasite localization in the brain. Following intragastric inoculation of cyst-containing parasites in mice, CD11c+ dendritic cells from the intestinal lamina propria, the Peyer patches, and the mesenteric lymph nodes were parasitized while in the blood, parasites were associated with the CD11c- CD11b+ monocytes. Using adoptive transfer experiments, we demonstrated that these parasitized cells triggered a parasitic process in the brain of naive recipient mice. Ex vivo analysis of parasitized leukocytes showed that single tachyzoites remained at the cell periphery, often surrounded by the host cell plasma membrane, but did not divide. Using either a dye that labels circulating leukocytes or an antibody known to prevent CD11b+ circulating leukocytes from leaving the microvascular bed lumen, and chimeric mice in which the hematopoietic cells expressed the green fluorescent protein, we established that T. gondii zoites hijacked CD11b+ leukocytes to reach the brain extravascular space.

NKT cells are critical for the initiation of an inflammatory bowel response against Toxoplasma gondii.

We demonstrated in this study the critical role of NKT cells in the lethal ileitis induced in C57BL/6 mice after infection with Toxoplasma gondii. This intestinal inflammation is caused by overproduction of IFN-gamma in the lamina propria. The implication of NKT cells was confirmed by the observation that NKT cell-deficient mice (Jalpha281(-/-)) are more resistant than C57BL/6 mice to the development of lethal ileitis. Jalpha281(-/-) mice failed to overexpress IFN-gamma in the intestine early after infection. This detrimental effect of NKT cells is blocked by treatment with alpha-galactosylceramide, which prevents death in C57BL/6, but not in Jalpha281(-/-), mice. This protective effect is characterized by a shift in cytokine production by NKT cells toward a Th2 profile and correlates with an increased number of mesenteric Foxp3 lymphocytes. Using chimeric mice in which only NKT cells are deficient in the IL-10 gene and mice treated with anti-CD25 mAb, we identified regulatory T cells as the source of the IL-10 required for manifestation of the protective effect of alpha-galactosylceramide treatment. Our results highlight the participation of NKT cells in the parasite clearance by shifting the cytokine profile toward a Th1 pattern and simultaneously to immunopathological manifestation when this Th1 immune response remains uncontrolled.

The TL MHC class Ib molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes.

Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8alphaalpha homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in beta2m-/- mice but unaffected in TCR-/- mice or CD8alpha chain-/- mice. We demonstrate that the interaction between TL and CD8alphaalpha is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of alpha-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-gamma in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-gamma production suggest that the functional consequences of TL interaction with CD8alphaalpha as well as the more general biological role of TL in mucosal immunity remains to be discovered.

Toxoplasma gondii and mucosal immunity.

Toxoplasma gondii, an intracellular parasite infects the host through the oral route. Infection induces a cascade of immunological events that involve both the components of the innate and adaptative immune responses. Alteration of the homeostatic balance of infected intestine results in an acute inflammatory ileitis in certain strains of inbred mice. Both the infected enterocytes as well as the CD4 T cells from the lamina propria produce chemokines and cytokines that are necessary to clear the parasite whereas CD8 intraepithelial lymphocytes secrete transforming growth factor beta that reduces the inflammation. In this review, we describe the salient features of this complex network of interactions among the different components of the gut-associated lymphoid tissue cell population that are induced after oral infection with T. gondii.

Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice.

In the absence of thymopoiesis, T lymphocytes are nevertheless present, mainly in the gut epithelium. Ontogeny of the extrathymic pathway and the extent of its involvement in euthymic mice are controversial. These questions have been addressed by assessing the expression of recombinase activating gene (RAG) through the use of green fluorescent protein RAG2 transgenic mouse models. In athymic mice, T lymphopoiesis occurs mainly in the mesenteric lymph node and less in the Peyer's patches. Ontogenic steps of this lymphopoiesis resemble those of thymopoiesis, but with an apparent bias toward gamma delta T cell production and with a paucity of oligoclonal alpha beta T cells possibly resulting from a deficit in positive selection. Whether in athymic or euthymic mice, neither T intraepithelial lymphocytes (IEL) nor cryptopatch cells (reported to contain precursors of IEL) displayed fluorescence indicating recent RAG protein synthesis. Newly made T cells migrate from the mesenteric node into the thoracic duct lymph to reach the gut mucosa. In euthymic mice, this extrathymic pathway is totally repressed, except in conditions of severe lymphocytic depletion. Thus, in normal animals, all gut T IEL, including CD8 alpha alpha(+) cells, are of thymic origin, CD8 alpha alpha(+) TCR alpha beta(+) IEL being the likely progeny of double negative NK1-1(-) thymocytes, which show polyclonal V alpha and V beta repertoires.

Comparative analysis of the CD8(+) T cell repertoires of H-2 class I wild-type/HLA-A2.1 and H-2 class I knockout/HLA-A2.1 transgenic mice.

HHD transgenic mice which express HLA-A2.1 monochain molecules in a H-2 class I(-) context have an improved capacity to develop HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) responses as compared with classical A2.1/K(b) transgenic mice, which express heterodimeric HLA-A2.1 molecules in a H-2 class I wild-type context. A detailed TCR analysis of HLA-A2.1-restricted CD8(+) T cells educated and mobilized in both strains of mice was undertaken. Focusing on TCR beta chains, comparative PCR analysis of naive and immune CD8(+) T cell repertoires were performed. In spite of lower cell surface expression of HLA class I molecules and lower overall number of CD8(+) T cells, HHD mice educate a qualitatively normally diversified CD8(+) T cell repertoire and mobilize a larger variety of CD8(+) T cells in response to HLA-A2.1-restricted antigens compared with A2.1/K(b) mice. These observations provide the molecular bases accounting for the fact that HHD mice represent the most versatile animal model currently available for preclinical studies of HLA-A2.1-restricted CTL responses.