Mouse Lymphoid Organs Research Articles

ScRNA-seq revealed the special TCR β & α V(D)J allelic inclusion rearrangement and the high proportion dual (or more) TCR-expressing cells

Allelic exclusion, one lymphocyte expresses one antigen receptor, is a fundamental mechanism of immunological self-tolerance and highly specific immune responses to pathogens. However, the phenomenon of V(D)J allelic inclusion (incomplete allelic exclusion or allelic escape) rearrangement and dual TCR T cells have been discovered by multiple laboratories. Despite continuous new discoveries, the proportion and underlying mechanism of dual TCR has been puzzling immunologists. In this study, we observed the presence of single T cells expressing multiple TCR chains in all samples, with the proportion of 15%, 10%, and 20% in the human thymus, human peripheral blood, and mouse lymphoid organs, respectively. The proportion of T cells possessing multiple T-cell receptors (TCR) varied significantly in different physiological states and developmental stages. By analyzing RSS category, RSS direction, and V(D)J gene position at TR locus of T cells which contain multiple TCR chains, we creatively found that one of TCR β (or TCR α) should originate from the transcription of V(D)J combination in T-cell receptor excision circle (TREC) formed after the twice successful rearrangement in the same chromosome. Moreover, human V30 (or mouse V31) gene may participate in reverse recombination and transcription to prevent allelic exclusion. In general, high proportion of T cells with multiple TCR at the transcriptome level was first made public, and we proposed a novel mechanism of secondary (or more) TCR rearrangement on a single chromosome. Our findings also indicated that the single-cell sequencing data should be classified according to the single, multiple, and abnormal TCR when analyzing the T-cell repertoire.

Intravital Longitudinal Three-Photon Imaging of Lymphocyte Migration in the Mouse Spleen.

Abstract For the last 20 years, intravital two-photon (2P) imaging has been a powerful tool to visualize lymphocyte dynamics in mouse lymphoid organs such as bone marrows and lymph nodes. Although spleen is a key lymphoid organ for adaptive immunity against blood-derived antigens, the lymphocyte dynamics in the spleen are poorly understood because intravital 2P imaging of spleen is challenging. Visualizing the white pulp in the spleen where most lymphocytes are located requires the excitation light to propagate through the red pulp which contains a lot of blood, causing high light-scattering. In addition, intravital spleen imaging suffers from motion artifact due to the mouse breathing motion. To overcome these challenges, we applied three-photon (3P) microscopy for spleen imaging, which can reduce light scattering and improve image contrast in deeper regions compared to 2P microscopy. We also designed a new implantable, chronic imaging window for reducing spleen motion in our up-light microscope. We could identify white pulp regions by in vivo labeling of the marginal zone of white pulp. Using 3P microscopy at 1300 nm wavelength, we could visualize EGFP+ lymphocytes in white pulps at approximately 400–500 μm depth below the surface while 2P microscopy at 920 nm could only reach imaging depths of 160–250 μm in the same region. We then determined the temperature of the imaging window that is required for maintaining normal lymphocyte motility during imaging by monitoring the lymphocyte motility at different temperatures. This intravital longitudinal 3P imaging technique has the potential to examine lymphocyte dynamics in blood-derived antigen-induced inflammatory process in the spleen.

Dynamic three-photon imaging through intact mouse lymphoid organs

Dynamic three-photon imaging through intact mouse lymphoid organs

Assessment of the TCR Repertoire of Human Circulating T Follicular Helper Cells.

Every T cell clone has its unique T cell receptor that results from somatic recombination of V(D)J genes in developing T cells. This process leads to a highly diverse TCR repertoire of naïve T cells, which is selected, upon antigenic recognition,to form the repertoires of effector and memory T cells. The advent of next-generation sequencing (NGS) technology allows for the high-throughput analysis of the TCR repertoires in the different T cell populations. TFH cells, since their initial discovery in human tonsils and in mouse lymphoid organs,have become the subject of intense investigations due to their essential role in regulating B cell responses and the process of antibody affinity maturation.Circulating follicular helper T cells (cTFH) are considered a helper T cell linage in the blood that to some extent relates with bona fide TFH cells in the germinal centers of secondary lymphoid organs. Due to the limited access to the secondary lymphoid organs, cTFHhave become a more accessible immunological readout.The assessment of the TCR repertoires of TFH and ofcTFH cells is of both fundamental and clinical importance being instrumental to define the linage relationship of cTFH with other T cell subsets and to monitor response to infections or vaccination or disease states. In this chapter, we will provide detailed methods for isolation of antigen-specific cTFH cells in vitro and subsequent protocols for the high-throughput TCR sequencing, followed by repertoire data analysis.

Isoflurane stress induces glucocorticoid production in mouse lymphoid organs.

Glucocorticoids (GCs) are secreted by the adrenal glands and locally produced by lymphoid organs. Adrenal GC secretion at baseline and in response to stressors is greatly reduced during the stress hyporesponsive period (SHRP) in neonatal mice (postnatal day (PND) 2-12). It is unknown whether lymphoid GC production increases in response to stressors during the SHRP. Here, we administered an acute stressor (isoflurane anesthesia) to mice before, during, and after the SHRP and measured systemic and local GCs via mass spectrometry. We administered isoflurane, vehicle control (oxygen), or neither (baseline) at PND 1, 5, 9, or 13 and measured progesterone and six GCs in blood, bone marrow, thymus, and spleen. At PND1, blood and lymphoid GC levels were high and did not respond to stress. At PND5, blood GC levels were very low and increased slightly after stress, while lymphoid GC levels were also low but increased greatly after stress. At PND9, blood and lymphoid GC levels were similar at baseline and increased similarly after stress. At PND13, blood GC levels were higher than lymphoid GC levels at baseline, and blood GC levels showed a greater response to stress. These data demonstrate the remarkable plasticity of GC physiology during the postnatal period, show that local steroid levels do not reflect systemic steroid levels, provide insight into the SHRP, and identify a potential mechanism by which early-life stressors can alter immunity in adulthood.

Predictive and Experimental Immunogenicity of Burkholderia Collagen-like Protein 8-Derived Antigens.

Burkholderia pseudomallei is an infectious bacterium of clinical and biodefense concern, and is the causative agent of melioidosis. The mortality rate can reach up to 50% and affects 165,000 people per year; however, there is currently no vaccine available. In this study, we examine the antigen-specific immune response to a vaccine formulated with antigens derived from an outer membrane protein in B. pseudomallei, Bucl8. Here, we employed a number of bioinformatic tools to predict Bucl8-derived epitopes that are non-allergenic and non-toxic, but would elicit an immune response. From these data, we formulated a vaccine based on two extracellular components of Bucl8, the β-barrel loops and extended collagen and non-collagen domains. Outbred CD-1 mice were immunized with vaccine formulations—composed of recombinant proteins or conjugated synthetic peptides with adjuvant—to assess the antigen-specific immune responses in mouse sera and lymphoid organs. We found that mice vaccinated with either Bucl8-derived components generated a robust TH2-skewed antibody response when antigen was combined with the adjuvant AddaVax, while the TH1 response was limited. Mice immunized with synthetic loop peptides had a stronger, more consistent antibody response than recombinant protein antigens, based on higher IgG titers and recognition of bacteria. We then compared peptide-based vaccines in an established C57BL/6 inbred mouse model and observed a similar TH2-skewed response. The resulting formulations will be applied in future studies examining the protection of Bucl8-derived vaccines.

Distinct Distribution of RTN1A in Immune Cells in Mouse Skin and Lymphoid Organs.

The endoplasmic reticulum-associated protein reticulon 1A (RTN1A) is primarily expressed in neuronal tissues but was recently identified also specifically in cells of the dendritic cell (DC) lineage, including epidermal Langerhans cells (LCs) and dermal DCs in human skin. In this study, we found that in mice major histocompatibility complex class II (MHCII)+CD207+ LCs but not dermal DCs express RTN1A. Further, RTN1A expression was identified in CD45+MHCII+CD207+ cells of the lymph node and spleen but not in the thymus. Of note, RTN1A was expressed in CD207low LCs in adult skin as well as emigrated LCs and DCs in lymph nodes and marginally in CD207hi cells. Ontogeny studies revealed that RTN1A expression occurred before the appearance of the LC markers MHCII and CD207 in LC precursors, while dermal DC and T cell precursors remained negative during skin development. Analogous to the expression of RTN1A in neural tissue, we identified expression of RTN1A in skin nerves. Immunostaining revealed co-localization of RTN1A with nerve neurofilaments only in fetal but not in newborn or adult dermis. Our findings suggest that RTN1A might be involved in the LC differentiation process given its early expression in LC precursors and stable expression onward. Further analysis of the RTN1A expression pattern will enable the elucidation of the functional roles of RTN1A in both the immune and the nervous system of the skin.

SAT-360 Acute Stress Increases Local Corticosterone Levels In Lymphoid Organs Of Neonatal Mice: Analysis Using LC-MS/MS

Glucocorticoids (GCs) are steroids produced by the adrenal glands and also by lymphoid organs such as bone marrow, thymus, and spleen (Taves et al., 2015). GCs are critical regulators of immune system development. During early development (postnatal day (PND) 2 to 12), mice show decreased adrenal GC secretion at baseline and in response to stressors, which is termed the stress hyporesponsive period (SHRP) (D’Amato et al., 1992). Lymphoid organs locally produce GCs, particularly during the SHRP, suggesting that these organs might increase local GC production in response to stress. Here, using PND1, PND5, PND9, and PND13 mice, we administered 5% isoflurane (an anesthetic) in 21% oxygen as an acute stressor, 21% oxygen as a vehicle control, or neither (baseline). For both isoflurane and oxygen treated groups, pups were removed from their home cage and placed in an induction chamber with a heating pad and nesting material from their home cage. Animals were given either given isoflurane for 3min followed by 27min oxygen or 30min continuous oxygen and rapidly euthanized. Baseline animals were given neither isoflurane nor oxygen and were rapidly euthanized in less than 3min. We then measured a panel of 7 steroids, including corticosterone, in the blood and lymphoid organs using liquid chromatography tandem mass spectrometry (LC-MS/MS). Note that PND1 is pre-SHRP, PND5 and PND9 are within the SHRP, and PND13 is post-SHRP. At PND1, corticosterone levels were generally high in both blood and lymphoid organs and did not differ with treatment. At PND5, corticosterone levels were generally very low, but increased with stress in a tissue-dependent fashion, showing the greatest increase in bone marrow and the least increase in blood. At PND9, baseline corticosterone levels were very low, but increased in both blood and lymphoid organs in response to a stressor. At PND13, corticosterone levels were higher in blood than in lymphoid organs for all treatments and increased with stress. Taken together, these novel data indicate that during the SHRP, an acute stressor produces little to no increase in corticosterone levels in blood but produces a 100-fold increase in bone marrow and a 20-fold increase in thymus and spleen. These data support the exciting possibility that mouse lymphoid organs can locally produce corticosterone, even when the adrenal glands produce little corticosterone. More generally, it is important to recognize that local steroid levels often do not match systemic steroid levels and that local steroid synthesis is particularly important in certain developmental and physiological contexts.

Altered frequency of CD8+ CD11c+ T cells and expression of immunosuppressive molecules in lymphoid organs of mouse model of colorectal cancer.

CD11c is a member of the β2-integrin family typically used to define myeloid dendritic cells (DCs). Recent reports identify CD11c-expressing CD8+ T cells as a new subset of CD8+ regulatory T cells (Treg). Evidence exists that CD11c+ CD8+ T cells may exert their effector or regulatory functions under different conditions. To date, no studies have addressed the frequency of CD11c+ T cells in cancer. Limited evidence exists in terms of expression of immune-checkpoint receptors, programmed cell death protein 1 (PD-1) and T-lymphocyte-associated antigen 4 (CTLA-4), as well as forkhead box P3 (Foxp3) in mouse lymphoid organs. Here, we have assessed CD11c+ CD8+ and CD11c+ CD4+ T cells, Foxp3, PD-1, and CTLA-4 expressing CD4+ T cells and CD8+ T cells in different tissues from three groups of male BALB/c mice-young, mature, and those with colorectal cancer (CRC). Analysis of CD3+ CD11c+ T cells in the bone marrow (BM), spleen, and lymph nodes (LN) in each group showed a higher percentage of CD3+ CD11c+ T cells in the BM from all groups and in the lymphoid organs of the cancer group compared with the young and mature groups. CD4low and CD4high cell fractions in mice BM have different expression patterns for Foxp3 and CTLA-4. We have observed a higher frequency of CD8+ PD-1+ T cells in the BM, spleen, and LN of CRC mice compared with normal mice. T-cell exhaustion is associated with inhibitory receptor PD-1. According to the regulatory roles of CD11c expression in CD8+ T cells, we have proposed that the elevated percentage of CD11c, Foxp3, CTLA-4, and PD-1 expressing T cells were associated with immune response dysregulation in CRC.

Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR.

CD4+ T regulatory (Treg) cells are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells they regulate. To understand this heterogeneity, we combined single-cell RNAseq, activation reporter and TCR analysis to profile thousands of Tregs or Tconvs from mouse lymphoid organs or human blood. Treg and Tconv pools showed areas of overlap, as resting “furtive” Tregs with overall similarity to Tconv, or as a convergence of activated states. All Tregs express a small core of FoxP3-dependent transcripts, onto which additional programs are added less uniformly. Among suppressive functions, Il2ra and Ctla4 were quasi-constant, inhibitory cytokines being more sparsely distributed. TCR signal intensity didn’t affect resting/activated Treg proportions, but molded activated Treg programs. The main lines of Treg heterogeneity in mice were strikingly conserved in human blood. These results reveal unexpected TCR-shaped states of activation, providing a framework to synthesize previous observations about Treg heterogeneity.

Chronic Consumption of Sweeteners Increases Carbonylated Protein Production in Lymphocytes from Mouse Lymphoid Organs

Articulo derivado de un proyecto de investigacion para evaluar el efecto del consumo de edulcorantes sobre el sistema inmunitario

For3D: Full organ reconstruction in 3D, an automatized tool for deciphering the complexity of lymphoid organs

To decipher the complex topology of lymphoid structures, we developed an automated process called Full Organ Reconstruction in 3D (For3D). A dedicated image-processing pipeline is applied to entire collections of immunolabeled serial sections, acquired with a slide-scanning microscope. This method is automated, flexible and readily applicable in two days to frozen or paraffin-embedded organs stained by fluorescence or brightfield immunohistochemistry. 3D-reconstructed organs can be visualized, rotated and analyzed to quantify substructures of interest. Usefulness of For3D is exemplified here through topological analysis of several mouse lymphoid organs exhibiting a complex organization: (i) the thymus, composed of two compartments, a medulla intricately imbricated into a surrounding cortex, (ii) lymph nodes, also highly compartmentalized into cortex, paracortex and medulla and (iii) the vascularization of an EG7 primary thymoma. This open-source algorithm, based on ImageJ and Matlab scripts, offers a user-friendly interface and is widely applicable to any organ or tissue, hence readily adaptable to a broad range of biomedical samples.

Effect of sulfated polysaccharides from brown seaweed Laminaria japonica on the morfology of lymfoid organs and functional characteristics of immunocompetent cells

The effect of sulfated polysaccharide fucoidan from the brown alga Laminaria japonica on morphological characteristics of mouse lymphoid organs, subpopulations of spleen mononuclear leukocytes, cytokine production and cytotoxic activity of splenocytes has been investigated. Fucoidan promoted activation and proliferation of lymphoid hematopoietic cells in primary and secondary immunogenesis bodies, increased expression of markers CD19, NK, NKT, CD25, MHC II, TCR, TLR2 and TLR4, the cytotoxic activity of splenocytes and production of immunoregulatory and proinflammatory cytokines (IL- 2, IL-12, IFN-g, TNF-a, IL-6). This suggests activation of effector mechanisms of innate immunity and adaptive immune responses via the Th-1 type.

Analysis of ligands for CD83, a marker of activated immune cells

CD83, a member of the immunoglobulin superfamily, is a type‐1 transmembrane glycoprotein and expressed on various activated immune cells. The soluble form of CD83 strongly suppresses mixed lymphocyte reaction (MLR) and experimental autoimmune encephalomyelitis (EAE), however, the mechanism of CD83‐mediated immune regulation including ligands for CD83 (CD83L) has not been elucidated. By flow cytometry using the CD83‐Fc fusion protein, we screened immune cells from mouse lymphoid organs to identify CD83L+ cells and found that CD83‐Fc bound to B220int IgMhi CD11b+ peritoneal B‐1 B cells, but not to cells from spleen, bone marrow, or thymus. When we tested the binding of CD83‐Fc to various B lymphocytic cell lines, the fusion protein bound to BCL1‐B20 cells which are known to be derived from B‐1 B cells. The pull‐down assay using CD83‐Fc revealed that a part of glycosylated IgM heavy chain was precipitated from the crude membrane fraction of BCL1‐B20 cells. Furthermore, anti‐mouse IgM antibodies blocked binding of CD83‐Fc to B‐1 B cells and BCL1‐B20 cells. These results suggest that CD83+ cells might recognize surface IgM with unique glycosylation expressed on B‐1 B cells during immune responses.

Three-Dimensional Visualization of the Mouse Thymus Organization in Health and Immunodeficiency

Lymphoid organs exhibit complex structures tightly related to their function. Surprisingly, although the thymic medulla constitutes a specialized microenvironment dedicated to the induction of T cell tolerance, its three-dimensional topology remains largely elusive because it has been studied mainly in two dimensions using thymic sections. To overcome this limitation, we have developed an automated method for full organ reconstruction in three dimensions, allowing visualization of intact mouse lymphoid organs from a collection of immunolabeled slices. We validated full organ reconstruction in three dimensions by reconstructing the well-characterized structure of skin-draining lymph nodes, before revisiting the complex and poorly described corticomedullary organization of the thymus. Wild-type thymi contain ~200 small medullae that are connected to or separated from a major medullary compartment. In contrast, thymi of immunodeficient Rag2(-/-) mice exhibit only ~20 small, unconnected medullary islets. Upon total body irradiation, medullary complexity was partially reduced and then recovered upon bone marrow transplantation. This intricate topology presents fractal properties, resulting in a considerable corticomedullary area. This feature ensures short distances between cortex and medulla, hence efficient thymocyte migration, as assessed by mathematical models. Remarkably, this junction is enriched, particularly in neonates, in medullary thymic epithelial cells expressing the autoimmune regulator. The emergence of a major medullary compartment is induced by CD4(+) thymocytes via CD80/86 and lymphotoxin-α signals. This comprehensive three-dimensional view of the medulla emphasizes a complex topology favoring efficient interactions between developing T cells and autoimmune regulator-positive medullary thymic epithelial cells, a key process for central tolerance induction.

Endogenous IL-33 Is Highly Expressed in Mouse Epithelial Barrier Tissues, Lymphoid Organs, Brain, Embryos, and Inflamed Tissues: In Situ Analysis Using a Novel Il-33–LacZ Gene Trap Reporter Strain

IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.

Alpha Interferon and HIV Infection Cause Activation of Human T Cells in NSG-BLT Mice

The development of small animal models for the study of HIV transmission is important for evaluation of HIV prophylaxis and disease pathogenesis. In humanized bone marrow-liver-thymus (BLT) mice, hematopoiesis is reconstituted by implantation of human fetal liver and thymus tissue (Thy/Liv) plus intravenous injection of autologous liver-derived hematopoietic stem progenitor cells (HSPC). This results in reconstitution of human leukocytes in the mouse peripheral blood, lymphoid organs, and mucosal sites. NOD-scid interleukin-2 receptor-negative (IL-2Rγ(-/-)) (NSG)-BLT mice were inoculated intravaginally with HIV and were monitored for plasma viremia by a branched DNA assay 4 weeks later. T-cell activation was determined by expression of CD38 and HLA-DR on human CD4(+) and CD8(+) T cells in mouse peripheral blood at the time of inoculation and 4 weeks later. Additional BLT mice were treated with human alpha interferon 2b (IFN-α2b) (intron A) and assessed for T-cell activation. Productive HIV infection in BLT mice was associated with T-cell activation (increases in CD38 mean fluorescence intensity and both the frequency and absolute number of CD38(+) HLA-DR(+) T cells) that correlated strongly with plasma viral load and was most pronounced in the CD8(+) T-cell compartment. This T-cell activation phenotype was recapitulated in NSG-BLT mice treated with intron A. HIV susceptibility correlated with the number of HSPC injected, yet a number of mice receiving the Thy/Liv implant alone, with no HSPC injection, were also susceptible to intravaginal HIV. These results are consistent with studies linking T-cell activation to progressive disease in humans and lend support for the use of NSG-BLT mice in studies of HIV pathogenesis.

CD69 Modulates Sphingosine-1-Phosphate-Induced Migration of Skin Dendritic Cells

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

Expression of HLA Class II Molecules in Humanized NOD.Rag1KO.IL2RgcKO Mice Is Critical for Development and Function of Human T and B Cells

BackgroundHumanized mice able to reconstitute a surrogate human immune system (HIS) can be used for studies on human immunology and may provide a predictive preclinical model for human vaccines prior to clinical trials. However, current humanized mouse models show sub-optimal human T cell reconstitution and limited ability to support immunoglobulin class switching by human B cells. This limitation has been attributed to the lack of expression of Human Leukocyte Antigens (HLA) molecules in mouse lymphoid organs. Recently, humanized mice expressing HLA class I molecules have been generated but showed little improvement in human T cell reconstitution and function of T and B cells.MethodsWe have generated NOD.Rag1KO.IL2RγcKO mice expressing HLA class II (HLA-DR4) molecules under the I-Ed promoter that were infused as adults with HLA-DR-matched human hematopoietic stem cells (HSC). Littermates lacking expression of HLA-DR4 molecules were used as control.ResultsHSC-infused HLA-DR4.NOD.Rag1KO.IL-2RγcKO mice developed a very high reconstitution rate (>90%) with long-lived and functional human T and B cells. Unlike previous humanized mouse models reported in the literature and our control mice, the HLA-DR4 expressing mice reconstituted serum levels (natural antibodies) of human IgM, IgG (all four subclasses), IgA, and IgE comparable to humans, and elicited high titers of specific human IgG antibodies upon tetanus toxoid vaccination.ConclusionsOur study demonstrates the critical role of HLA class II molecules for development of functional human T cells able to support immunoglobulin class switching and efficiently respond to vaccination.

Mycobacterium tuberculosis attenuated by multiple deletions of rpf genes effectively protects mice against TB infection

In this study, we investigated the residual virulence of mutants of Mycobacterium tuberculosis that are defective in 4 of the 5 rpf-like genes, their capacity to persist in the murine host and the utility present in these mutants to serve as novel vaccine candidates. Our data indicate that the two quadruple rpf deletion mutants, ΔACBD and ΔACDE, both display significant attenuation in the mouse lungs after aerosol infection, with no observable increase in bacillary loads upon aminoguanidine-induced immune suppression. However, after subcutaneous injection these strains were able to persist at the low level, similar to that of BCG, in the mouse lungs and lymphoid organs. Furthermore, both rpf quadruple mutants were able to enhance the numbers of IFN-γ-producing T-cells in spleens to a level comparable to that of BCG, and conferred protection upon subsequent challenge with virulent M. tuberculosis in terms of mycobacterial multiplication in organs and survival time. The reduction in organ bacillary loads after vaccination with ΔACDE was comparable to that of BCG, while ΔACBD displayed a small but statistically significant enhancement in protection compared to BCG. Collectively, these data suggest that rpf deletion mutants show potential for further development as novel vaccine candidates for tuberculosis.