Isolation of human lymphocytes with high yield and viability from the gastrointestinal and female reproductive tract of a humanized DRAG mouse.

The mucosal tissues of the gut and female reproductive tract (FRT) are susceptible to pathogen infections including bacteria, viruses, and parasites, and are also the targets for immune disorders such as Crohn's disease, inflammatory bowel disease (IBD), and many types of cancers. However, the role of the mucosal immune cells to control these diseases is largely unknown. The limited availability of human mucosal biopsy tissue and the low number of cells that can be isolated from these tissues hampers the characterization of the phenotype and function of human mucosal immune cell subsets. Therefore, human-immune-system humanized mice are surrogate models to investigate the human mucosal immune cell responses during the course of the disease. The current protocols used to harvest the immune cells from the mucosal tissues, however, result in low recovery of cells with poor viability. We have established a novel protocol, which results in a high yield of human lymphocytes with high viability to overcome this issue. The immune cells obtained from a single DRAG mouse by our protocol were sufficient for conducting functional assays and for flow cytometry analyses including phenotypic, exhaustion, and functional panels.

Tracking Human Immunodeficiency Virus-1 Infection in the Humanized DRAG Mouse Model.

Humanized mice are emerging as an alternative model system to well-established non-human primate (NHP) models for studying human immunodeficiency virus (HIV)-1 biology and pathogenesis. Although both NHP and humanized mice have their own strengths and could never truly reflect the complex human immune system and biology, there are several advantages of using the humanized mice in terms of using primary HIV-1 for infection instead of simian immunodeficiency virus or chimera simian/HIV. Several different types of humanized mice have been developed with varying levels of reconstitution of human CD45+ cells. In this study, we utilized humanized Rag1KO.IL2RγcKO.NOD mice expressing HLA class II (DR4) molecule (DRAG mice) infused with HLA-matched hematopoietic stem cells from umbilical cord blood to study early events after HIV-1 infection, since the mucosal tissues of these mice are highly enriched for human lymphocytes and express the receptors and coreceptors needed for HIV-1 entry. We examined the various tissues on days 4, 7, 14, and 21 after an intravaginal administration of a single dose of purified primary HIV-1. Plasma HIV-1 RNA was detected as early as day 7, with 100% of the animals becoming plasma RNA positive by day 21 post-infection. Single cells were isolated from lymph nodes, bone marrow, spleen, gut, female reproductive tissue, and brain and analyzed for gag RNA and strong stop DNA by quantitative (RT)-PCR. Our data demonstrated the presence of HIV-1 viral RNA and DNA in all of the tissues examined and that the virus was replication competent and spread rapidly. Bone marrow, gut, and lymph nodes were viral RNA positive by day 4 post-infection, while other tissues and plasma became positive typically between 7 and 14 days post-infection. Interestingly, the brain was the last tissue to become HIV-1 viral RNA and DNA positive by day 21 post-infection. These data support the notion that humanized DRAG mice could serve as an excellent model for studying the trafficking of HIV-1 to the various tissues, identification of cells harboring the virus, and thus could serve as a model system for HIV-1 pathogenesis and reservoir studies.

Improvements and Limitations of Humanized Mouse Models for HIV Research: NIH/NIAID "Meet the Experts" 2015 Workshop Summary.

The number of humanized mouse models for the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and other infectious diseases has expanded rapidly over the past 8 years. Highly immunodeficient mouse strains, such as NOD/SCID/gamma chain(null) (NSG, NOG), support better human hematopoietic cell engraftment. Another improvement is the derivation of highly immunodeficient mice, transgenic with human leukocyte antigens (HLAs) and cytokines that supported development of HLA-restricted human T cells and heightened human myeloid cell engraftment. Humanized mice are also used to study the HIV reservoir using new imaging techniques. Despite these advances, there are still limitations in HIV immune responses and deficits in lymphoid structures in these models in addition to xenogeneic graft-versus-host responses. To understand and disseminate the improvements and limitations of humanized mouse models to the scientific community, the NIH sponsored and convened a meeting on April 15, 2015 to discuss the state of knowledge concerning these questions and best practices for selecting a humanized mouse model for a particular scientific investigation. This report summarizes the findings of the NIH meeting.

Congenital Symmastia: A 3-Step Approach.

Congenital symmastia is a medial confluence of the breasts. It is a rare anomaly with few reports in the literature and no standard treatment. In this article, we present a case of congenital symmastia treated by 3 steps: liposuction, fixation of the skin to the chest wall in the area of the intermammary sulcus, and postoperative intermammary compression. A successful result was achieved with normal cleavage between the breasts. So, this is considered the ideal treatment for this condition.

TFH cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1.

CD4(+) T follicular helper cells (TFH) in germinal centers are required for maturation of B-cells. While the role of TFH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of TFH (CXCR5(+)PD-1(++)) and precursor-TFH (CXCR5(+)PD-1(+)) cells. The majority of TFH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of TFH-cells mainly in the Peyer's patches and FRT. The novel finding of TFH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study TFH-cells and to evaluate HIV-1 vaccines.

Dual function of CD70 in viral infection: modulator of early cytokine responses and activator of adaptive responses.

The role of the TNF family member CD70 in adaptive T cell responses has been intensively studied, but its function in innate responses is still under investigation. In this study, we show that CD70 inhibits the early innate response to murine CMV (MCMV) but is essential for the optimal generation of virus-specific CD8 T cells. CD70(-/-) mice reacted to MCMV infection with a robust type I IFN and proinflammatory cytokine response. This response was sufficient for initial control of MCMV, although at later time points, CD70(-/-) mice became more susceptible to MCMV infection. The heightened cytokine response during the early phase of MCMV infection in CD70(-/-) mice was paralleled by a reduction in regulatory T cells (Treg). Treg from naive CD70(-/-) mice were not as efficient at suppressing T cell proliferation compared with Treg from naive wild-type mice, and depletion of Treg during MCMV infection in Foxp3-diphtheria toxin receptor mice or in wild-type mice recapitulated the phenotype observed in CD70(-/-) mice. Our study demonstrates that although CD70 is required for the activation of the antiviral adaptive response, it has a regulatory role in early cytokine responses to viruses such as MCMV, possibly through maintenance of Treg survival and function.

CD70 deficiency impairs effector CD8 T cell generation and viral clearance but is dispensable for the recall response to lymphocytic choriomeningitis virus.

CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far, all studies addressing the function of the CD27-CD70 axis have been performed in mice lacking CD27, in those overexpressing CD70, or in those in which these molecules were blocked or mimicked by Abs or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to lymphocytic choriomeningitis virus lowered the magnitude of CD8 Ag-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70-CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to lymphocytic choriomeningitis virus. Adoptive transfers of wild-type memory T cells into CD70(-/-) or wild-type hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70 expression by CD8 T cells could not rescue endogenous CD70(-/-) cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses.

The CD8+ memory T-cell state of readiness is actively maintained and reversible.

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.

TCR trans-rearrangements: biological significance in antigen recognition vs the role as lymphoma biomarker.

V(D)J rearrangements occur within loci of TCR and BCR genes, thus generating the diversity of the AgR repertoire. In addition, interlocus V(D)J rearrangements occur, giving rise to so-called "trans-rearrangements." Such trans-rearrangements increase the diversity of the immune receptor repertoire and can be expressed as functional chimeric TCR proteins on the surface of T cells. Although chimeric receptors are not pathogenic per se, the frequency of AgR trans-rearrangements correlates with the level of genetic instability and thus could be used as a predictive biomarker for lymphoma risk.

Role of the adaptor proteins Bam32, TAPP1 and TAPP2 in lymphocyte activation.

Adaptor proteins play critical roles in lymphocyte activation by mediating intermolecular interactions and assembling signaling complexes at the activated plasma membrane. Bam32/DAPP1 and the related adaptor proteins TAPP1 and TAPP2 were identified by multiple groups about 5 years ago and considerable progress has been made in elucidating the structure, interaction partners and function of these molecules. These cytoplasmic adaptor proteins are recruited to the plasma membrane through interaction of their PH domains with the lipid products of phosphatidylinositol 3-kinases. They share a unique mode of regulation in that they bind with high affinity to phosphatidylinositol-3,4-bisphosphate and their recruitment is enhanced rather than inhibited by the lipid phosphatase SHIP. Two knockout mouse studies and several gain-and-loss of function studies in cell lines have recently been published, demonstrating multiple functions of Bam32 in B cell activation. Bam32 is required for biological responses including B cell antigen receptor (BCR)-induced proliferation and antibody responses to type II T-independent antigens. Bam32 regulates multiple BCR signaling events including activation of the mitogen activated protein kinases ERK and JNK, remodeling of the actin cytoskeleton through the GTPase Rac1 and BCR internalization. Several studies have emerged suggesting that TAPP1 and TAPP2 may play roles in B and T cell activation; however, the biological functions regulated by these molecules remain to be defined. Here we will comprehensively review the available data on the structure and function of Bam32, TAPP1 and TAPP2 and present an integrated working model for Bam32 function in B cell activation and a general model for distinct effector pathways of PI 3-kinases.

The B lymphocyte adaptor molecule of 32 kilodaltons (Bam32) regulates B cell antigen receptor internalization.

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32-/- and Lyn-/- cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32-/- cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.

The adaptor protein Bam32 regulates Rac1 activation and actin remodeling through a phosphorylation-dependent mechanism.

The B cell adaptor molecule of 32 kDa (Bam32) is an adaptor that links the B cell antigen receptor (BCR) to ERK and JNK activation and ultimately to mitogenesis. After BCR cross-linking, Bam32 is recruited to the plasma membrane and accumulates within F-actin-rich membrane ruffles. Bam32 contains one Src homology 2 and one pleckstrin homology domain and is phosphorylated at a single site, tyrosine 139. To define the function of Bam32 in membrane-proximal signaling events, we established human B cell lines overexpressing wild-type or mutant Bam32 proteins. The basal level of F-actin increased in cells expressing wild-type or myristoylated Bam32 but decreased in cells expressing either an Src homology-2 or Tyr-139 Bam32 mutant. Overexpression of wild-type Bam32 also affected BCR-induced actin remodeling, which was visualized as increases in F-actin-rich membrane ruffles. In contrast, Bam32 mutants largely blocked the BCR-induced increase in cellular F-actin. The positive and negative effects of Bam32 variants on F-actin levels were closely mirrored by their effects on the activation of the GTPase Rac1, which is known to regulate actin remodeling in lymphocytes. Bam32-deficient DT40 B cells showed decreased Rac1 activation and a failure of Rac1 to co-localize with the BCR, whereas cells overexpressing Bam32 had increased constitutive Rac1 activation. These results suggest that Bam32 regulates the cytoskeleton through Rac1. Bam32 variants also affected downstream signaling to JNK in a manner similar to that of Rac1, suggesting that the effect of Bam32 on JNK activation may be at least partially mediated through Rac1. Our results demonstrate a novel phosphorylation-dependent function of Bam32 in regulating Rac1 activation and actin remodeling.