Strongyloides stercoralis and HTLV-1 coinfection in CD34+ cord blood stem cell humanized mice: Alteration of cytokine responses and enhancement of larval growth.

Viral and parasitic coinfections are known to lead to both enhanced disease progression and altered disease states. HTLV-1 and Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. Independently, HTLV-1 induces a Th1 response and S. stercoralis infection induces a Th2 response. However, coinfection with the two pathogens has been associated with the development of S. stercoralis hyperinfection and an alteration of the Th1/Th2 balance. In this study, a model of HTLV-1 and S. stercoralis coinfection in CD34+ umbilical cord blood hematopoietic stem cell engrafted humanized mice was established. An increased level of mortality was observed in the HTLV-1 and coinfected animals when compared to the S. stercoralis infected group. The mortality was not correlated with proviral loads or total viral RNA. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. HTLV-1 infected and coinfection groups showed a significant, yet non-clonal expansion of the CD4+CD25+ T-cell population. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group and no autoinfective larvae were found. However, infective larvae recovered from the coinfection group showed an enhancement in growth, as was seen in mice with S. stercoralis hyperinfection caused by treatment with steroids. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections in vivo in the context of human immune cells.

Humoral Immunity in Mice Transplanted with Hematopoietic Stem Cells Derived from Human Umbilical Cord Blood Recapitulates That of Human Infants.

Immunodeficient mice transplanted with human hematopoietic stem cells (HSCs) have been referred to as "Human Immune System" (HIS) mice and are a translational platform for studying human immune responses in vivo. Human HSC sources used in generating HIS mice include fetal liver (FL), umbilical cord blood (CB), and adult bone marrow (BM). Since HSCs from FL, CB, and BM are produced at various stages of human development, we tested whether mice transplanted with these three HSCs differ in their immune responses. We found that compared with CB HSCs or FL HSCs, adult BM HSCs reconstitute the immune system poorly. The resulting HIS mice do not mount an antibody response to Borrelia hermsii infection and as a consequence suffer persistently high levels of bacteremia. While both CB and FL HSCs yield comparable levels of immune reconstitution of HIS mice resulting in robust anti-B. hermsii immune responses, FL HSC-transplanted mice exhibited a discernable difference in their human B cell maturity as identified by an increased frequency of CD10+ immature B cells and relatively smaller lymphoid follicles compared with CB HSC-transplanted mice. Although CB HSC-transplanted mice generated robust antibody responses to B. hermsii and specific protein antigens of B. hermsii, they failed to respond to Salmonella typhi Vi polysaccharide, a classical T cell-independent antigen. This situation resembles that seen in human infants and young children. Therefore, CB HSC-transplanted mice may serve as a translation platform to explore approaches to overcome the impaired antipolysaccharide responses characteristic of human infants.

Human B-1 and B-2 B Cells Develop from Lin-CD34+CD38lo Stem Cells.

The B-1 B cell population is an important bridge between innate and adaptive immunity primarily because B-1 cells produce natural Ab. Murine B-1 and B-2 cells arise from distinct progenitors; however, in humans, in part because it has been difficult to discriminate between them phenotypically, efforts to pinpoint the developmental origins of human B-1 and B-2 cells have lagged. To characterize progenitors of human B-1 and B-2 cells, we separated cord blood and bone marrow Lin-CD34+ hematopoietic stem cells into Lin-CD34+CD38lo and Lin-CD34+CD38hi populations. We found that transplanted Lin-CD34+CD38lo cells, but not Lin-CD34+CD38hi cells, generated a CD19+ B cell population after transfer into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1wjl/SxJ neonates. The emergent CD19+ B cell population was found in spleen, bone marrow, and peritoneal cavity of humanized mice and included distinct populations displaying the B-1 or the B-2 cell phenotype. Engrafted splenic B-1 cells exhibited a mature phenotype, as evidenced by low-to-intermediate expression levels of CD24 and CD38. The engrafted B-1 cell population expressed a VH-DH-JH composition similar to cord blood B-1 cells, including frequent use of VH4-34 (8 versus 10%, respectively). Among patients with hematologic malignancies who underwent hematopoietic stem cell transplantation, B-1 cells were found in the circulation as early as 8 wk posttransplantation. Altogether, our data demonstrate that human B-1 and B-2 cells develop from a Lin-CD34+CD38lo stem cell population, and engrafted B-1 cells in humanized mice exhibit an Ig-usage pattern comparable to B-1 cells in cord blood.

YY1 Is Required for Germinal Center B Cell Development.

YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction.

Establishment and Characterization of Orthotopic Mouse Models for Human Uveal Melanoma Hepatic Colonization.

Uveal melanoma (UM) is a rare type of melanoma, although it is the most common primary ocular malignant tumor in adults. Nearly one-half the patients with primary UM subsequently develop systemic metastasis, preferentially to the liver. Currently, no treatment is effective for UM hepatic metastasis, and the prognosis is universally poor. The main challenge in designing a treatment strategy for UM hepatic metastasis is the lack of suitable animal models. We developed two orthotopic mouse models for human UM hepatic metastases: direct hepatic implantation model (intrahepatic dissemination model) and splenic-implantation model (hematogenous dissemination model) and investigated the tumorgenesis in the liver. A human UM cell line, established from a hepatic metastasis and nonobese diabetic severe combined immunodeficient γ mice, were used for development of in vivo tumor models. In the direct hepatic implantation model, a localized tumor developed in the liver in all cases and intrahepatic dissemination was subsequently seen in about one-half of cases. However, in the splenic implantation model, multiple hepatic metastases were observed after splenic implantation. Hepatic tumors subsequently seeded intra-abdominal metastasis; however, lung metastases were not seen. These findings are consistent with those observed in human UM hepatic metastases. These orthotopic mouse models offer useful tools to investigate the biological behavior of human UM cells in the liver.

Testing the role of the FcγRIIB immunoreceptor tyrosine-based inhibitory motif in regulation of the B cell immune response.

In vitro studies have demonstrated that the immunoreceptor tyrosine-based inhibitory motif (ITIM) of the inhibitory Fc receptor FcγRIIB is critical for mediating attenuation of signaling via immunoreceptor tyrosine-based activation motif (ITAM) containing receptors, such as the B cell antigen receptor (BCR), when FcγRIIB is co-cross-linked to these activation receptors. To test the role of the FcγRIIB ITIM motif in regulation of the B cell immune response in vivo, we constructed lines of transgenic mice expressing a form of FcγRIIB with an inactivating tyrosine (Y) to phenylalanine (F) mutation in the ITIM motif. Detailed studies of one of these lines, in which the mutant FcγRIIB was expressed on B cells and other cell types that normally express this receptor, were performed. No quantitative differences in germinal center (GC) B cell responses were observed between the mutant FcγRIIB transgenic line and control mice. However, serum antibody and antibody forming cell responses were often observed to be elevated in the ITIM mutant FcγRIIB transgenic mice as compared to controls, though not to the same extent as mice deficient in expression of FcγRIIB. Moreover, primary B cells from the ITIM mutant FcγRIIB line did not display the same level of augmented BCR signaling as primary FcγRIIB deficient B cells under conditions inducing co-cross-linking of FcγRIIB and the BCR. In total, these data suggest that a functional ITIM motif is not required for all in vivo inhibitory activity of this receptor. However, we also found that the transgenic ITIM mutant FcγRIIB receptor was expressed at abnormal levels in several hematopoietic lineages. Thus, confirmation of our findings will require the generation and analysis of mice in which an ITIM mutant form of FcγRIIB is expressed in vivo as is the endogenous receptor.

Evaluation of the efficiency of human immune system reconstitution in NSG mice and NSG mice containing a human HLA.A2 transgene using hematopoietic stem cells purified from different sources.

Severely immunodeficient mice such as the NOD/SCID/IL2rγ(null) (NSG) strain can be engrafted with human hematopoietic stem cells (HSCs), resulting in chimeric mice containing many components of the human immune system (Human Immune System mice or HIS mice). HIS mice can both support the replication of and recapitulate much of the immunological response to a variety of pathogens, including ones with strict human tropism, such as HIV-1. In an effort to develop a better mouse model for human infectious pathogen infection and possible immune resolution, we compared the human immune system reconstitution of NSG mice following injection with human CD34(+) HSCs purified from either fetal liver (FL) or umbilical cord blood (UCB). We analyzed reconstitution in standard NSG mice as well as a derivative of these mice containing an HLA.A2 encoding transgene (NSG.A2). HSCs from both sources effectively reconstituted hematopoietic lineages when injected into NSG mice. In marked contrast, total CD45(+) human hematopoietic cells in NSG.A2 mice were well reconstituted by HSCs from UCB but very poorly by HSCs purified from FL. Moreover, the reconstitution of T cell lineages in NSG.A2 mice by HSCs from UCB was inferior to that obtained using NSG mice. We also found that FL CD34(+) HSCs contain a much higher percentage of cells with a phenotype consistent with primitive progenitors than UCB HSCs. We discuss possible explanations for the influence of the HLA.A2 transgene on hematopoietic reconstitution using the two sources of HSCs.

Cutting edge: Macrophages are required for localization of antigen-activated B cells to the follicular perimeter and the subsequent germinal center response.

We demonstrated recently that, after accumulation of Ag-engaged B cells at the T cell zone boundaries in the spleen, these B cells migrate to the perimeter of follicles adjacent to the marginal zone. They undergo rapid proliferation at this site prior to coalescence into germinal centers (GCs). In this article, we report that this phase of migration and expansion of activated Ag-specific B cells, as well as subsequent formation of GCs, does not take place in the absence of splenic macrophages. Our data suggest a previously unappreciated function for macrophages in orchestrating the early phases of T cell-dependent B cell responses and formation of GCs distinct from their potential role in Ag presentation to T cells.

A germline-competent embryonic stem cell line from NOD.Cg-Prkdc ( scid ) Il2rg ( tm1Wjl )/SzJ (NSG) mice.

The NOD.Cg-Prkdc ( scid ) Il2rg ( tm1Wjl )/SzJ mouse strain, commonly known as NSG (for NOD SCID Gamma) is severely immunodeficient and thus is an excellent recipient for xenografts, and in particular for engrafting human tumor cells and human hematopoietic stem cells. In the latter case, these cells give rise to many human hematopoetic lineages in their NSG hosts, resulting in recapitulation of many of the features of a human immune system. However, the immune system of these "humanized mice" (huMice) is not completely functional, in part because of a lack of expression of necessary human cytokines and HLA molecules by NSG host tissues. In order to facilitate the genetic modification of this strain in order to improve the huMouse model, we have created germline competent ES cells of this strain in which such modifications can be carried out.

Direct reduction of antigen receptor expression in polyclonal B cell populations developing in vivo results in light chain receptor editing.

Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.

Characteristics of Borrelia hermsii infection in human hematopoietic stem cell-engrafted mice mirror those of human relapsing fever.

Rodents are natural reservoirs for a variety of species of Borrelia that cause relapsing fever (RF) in humans. The murine model of this disease recapitulates many of the clinical manifestations of the human disease and has revealed that T cell-independent antibody responses are required to resolve the bacteremic episodes. However, it is not clear whether such protective humoral responses are mounted in humans. We examined Borrelia hermsii infection in human hematopoietic stem cell-engrafted nonobese diabetic/SCID/IL-2Rγ(null) mice: "human immune system mice" (HISmice). Infection of these mice, which are severely deficient in lymphoid and myeloid compartments, with B. hermsii resulted in persistent bacteremia. In contrast, this infection in HISmice resulted in recurrent episodes of bacteremia, the hallmark of RF. The resolution of the primary episode of bacteremia was concurrent with the generation of B. hermsii-specific human IgM. Remarkably, HISmice generated antibody responses to the B. hermsii outer-membrane protein Factor H binding protein A. Sera from humans infected by B. hermsii have a similar reactivity, and studies in mice have shown that this response is generated by the B1b cell subset. HISmice contain several B-cell subsets, including those with the phenotype CD20(+)CD27(+)CD43(+)CD70(-), a proposed human equivalent of mouse B1 cells. Reduction of B cells by administration of anti-human CD20 antibody resulted in diminished anti-B. hermsii responses and persistent bacteremia in HISmice. These data indicate that analysis of B. hermsii infection in HISmice will serve as a model in which to study the cellular and molecular mechanisms involved in controlling human RF.

Human immune system mice: current potential and limitations for translational research on human antibody responses.

It has recently become possible to generate chimeric mice durably engrafted with many components of the human immune system (HIS mice). We have characterized the maturation and function of the B cell compartment of HIS mice. The antibody response of HIS mice to T cell-dependent B cell antigens is limited, and contributing factors may be the general immaturity of the B cell compartment, infrequent helper T cells selected on human MHC class II antigens, and incomplete reconstitution of secondary lymphoid organs and their microenvironments. In contrast, HIS mice generate protective antibody responses to the bacterium Borrelia hermsii, which acts as a T cell-independent antigen in mice, but do not respond to purified polysaccharide antigens (PPS). We speculate that the anti-B. hermsii response of HIS mice is derived from an abundant B cell subset that may be analogous to B1 B cells in mice. We suggest that failure of HIS mice to respond to PPS is due to the lack of a B cell subset that may originate from adult bone marrow and is highly dependent on human interleukin-7 for development.

Cellular competition independent of BAFF/B lymphocyte stimulator results in low frequency of an autoreactive clonotype in mature polyclonal B cell compartments.

The peripheral B cell prosurvival cytokine BAFF/B lymphocyte stimulator (BLyS) has been proposed to participate in the regulation of immunological tolerance. Selective elimination or reconstitution of B cells expressing transgene-encoded, autoreactive BCRs upon systemic BLyS depletion or supplementation, respectively, was observed in two separate studies. Such findings led to a model positing a higher dependency of autoreactive B cells on BLyS. We tested this model by exploiting two targeted IgH transgenic mice (H chain knock-in [HKI]) that produce large numbers of follicular (FO) B cells that are either weakly or strongly autoreactive with nuclear autoantigens. Even though HKI B cells do not exhibit classical features of anergy, we found that mature, naive, autoreactive HKI B cells are outcompeted for representation in the periphery by a polyclonal B cell population. However, this is not due to a higher dependency of HKI B cells on BLyS for survival. Additionally, excess BLyS does not rescue HKI B cells from selective elimination. These findings suggest that some autoreactive FO B cells can fully develop while in competition with non-autoreactive cells for BLyS, but remain at a competitive disadvantage for other trophic factors that regulate peripheral stability. As such, our data indicate the existence of peripheral tolerance mechanisms that regulate the frequency of autoreactive FO B cells independent of the BLyS pathway.

Expression of cellular FLIP by B cells is required for their participation in an immune response.

High levels of the Fas-signaling antagonist cellular FLIP (cFLIP) in germinal center (GC) B cells suggests an important role for this factor during this stage of the T cell-dependent B cell immune response. To test this idea, we used mice with B cell-specific deletion of a floxed cFLIP allele. Although deletion of cFLIP did not alter their primary development, participation of cFLIP-deficient B cells in the immune response was severely perturbed. Using previously characterized IgH locus-targeted BCR transgenic mice, we showed that adoptively transferred cFLIP-deficient follicular B cells do not effectively participate in the GC response in wild-type hosts. However, this failure was accompanied by severe defects in the initial activation and proliferation of these B cells in vivo. In addition, immunization of mice with B cell-specific cFLIP deletion resulted in selective recruitment into GCs and Ab-forming cell responses of B cells that had not deleted the floxed cFLIP allele. Together, these findings demonstrate that expression of cFLIP is a prerequisite for participation of B cells in all stages of Ag-driven immune responses.

Germinal center reutilization by newly activated B cells.

Germinal centers (GCs) are specialized structures in which B lymphocytes undergo clonal expansion, class switch recombination, somatic hypermutation, and affinity maturation. Although these structures were previously thought to contain a limited number of isolated B cell clones, recent in vivo imaging studies revealed that they are in fact dynamic and appear to be open to their environment. We demonstrate that B cells can colonize heterologous GCs. Invasion of primary GCs after subsequent immunization is most efficient when T cell help is shared by the two immune responses; however, it also occurs when the immune responses are entirely unrelated. We conclude that GCs are dynamic anatomical structures that can be reutilized by newly activated B cells during immune responses.

The lupus susceptibility locus Sle1 breaches peripheral B cell tolerance at the antibody-forming cell and germinal center checkpoints.

We have described a line of V(H) knock-in mice termed HKIR in which the transgenic Igh locus partially encodes "dual-reactive" antichromatin and anti-p-azophenylarsonate (Ars) BCRs. HKIR B cells termed canonical, expressing a particular Vkappa L chain, evade central tolerance by down-regulating BCR levels. Canonical HKIR B cells can be recruited into the primary germinal center (GC) and Ab-forming cell (AFC) compartments via Ars immunization. However, their participation in the GC response rapidly wanes and they do not efficiently contribute to the memory compartment, indicating that they are regulated by a GC tolerance checkpoint. We analyzed the influence of the Sle1 genetic interval, shown to break tolerance of chromatin-reactive B cells, on the behavior of HKIR B cells during the anti-Ars response. Canonical B cells from congenic HKIR.Sle1 mice gave rise to elevated short and long-lived AFC responses, and the attenuated GC and memory responses characteristic of these B cells were relieved in adoptive, wild-type recipients. HKIR GC B cells containing Sle1 expressed increased levels of Bcl-2 and c-FLIP and decreased levels of Fas RNA compared with HKIR controls, suggesting direct alteration of the regulation of the GC response by Sle1. High titers of canonical and anti-dsDNA Abs spontaneously developed in many aged HKIR.Sle1 mice. Together, these data indicate that Sle1 perturbs the action of peripheral tolerance checkpoints operative on antinuclear Ag B cells in both the AFC and GC pathways in a cell autonomous fashion.

Initial clonal expansion of germinal center B cells takes place at the perimeter of follicles.

Current models of the germinal center (GC) response propose that after stimulation at the edges of T cell zones, pre-GC B cells directly migrate to the center of follicles and proliferate to form GCs. We followed the interrelationship of proliferation, differentiation, and microenvironmental locale in populations of pre-GC B cells responding to antigen. In contrast to the predictions of current models, after accumulation at the T-B interface, these cells appeared at the perimeter of follicles adjacent to the marginal zone. There, they rapidly proliferated for several days but underwent no V gene hypermutation and little heavy-chain class switching. Their chemokine receptor expression pattern indicated that these cells were sessile, yet they had begun to acquire many phenotypic characteristics of GC B cells. The expanded clones were subsequently observed in the center of follicles, suggesting that GCs are created by coalescence of B cells from this follicular perimeter response.

Influence of B cell antigen receptor expression level on pathways of B cell tolerance induction.

We have described an Ig-transgenic, autoreactive B cell clonotype that undergoes a novel tolerance pathway. Early in development this clonotype expresses average BCR levels, but these levels are progressively down-regulated as development proceeds efficiently to the mature, follicular compartment. This clonotype does not display conventional features of anergy and can be induced to undergo apoptosis and receptor editing in in vitro bone marrow cultures, but these pathways are not taken in vivo. These data suggested that autoantigen-driven down-regulation of BCR levels and, hence, avidity for autoantigen allows this clonotype to bypass conventional tolerance mechanisms. To test this idea, we enforced elevated levels of expression of BCR in this clonotype by making the transgenic Igh locus homozygous. This resulted in retarded clonotype development and L chain receptor editing in vivo. These data support a pivotal role for adaptive, autoantigen-induced adjustment of BCR expression levels in the regulation of primary B cell development and tolerance.

Influence of Fas on the regulation of the response of an anti-nuclear antigen B cell clonotype to foreign antigen.

A peripheral B cell tolerance checkpoint appears to be operative during the germinal center (GC) reaction. We previously showed that a transgenic BCR clonotype that is 'dual reactive' for the hapten arsonate (Ars) and nuclear auto-antigens is stimulated to enter the GC response via Ars immunization. However, the participation of this clonotype in this response wanes with time and it gives rise to few memory B cells capable of mounting a secondary anti-Ars IgG response. Enforced expression of Bcl-2 partially rescues the GC and memory B cell responses of this clonotype, suggesting that apoptotic pathways are involved in the action of the GC tolerance checkpoint. Since GC B cells substantially up-regulate levels of expression of the Fas apoptotic death receptor, we determined whether an intrinsic Fas deficient could rescue the participation of this clonotype in the GC response. It could not, strongly indicating that Fas expression by autoreactive GC B cells is not necessary for their elimination. In addition, experiments in which Fas-sufficient dual reactive clonotype B cells were transferred to Fas-deficient hosts revealed an absence of participation of these B cells in the GC and IgG anti-Ars responses. We present data consistent with the idea that T cells in Fas-deficient hosts are primed to express elevated levels of FasL and eliminate antigen-activated B cells that up-regulate Fas.