COMPARATIVE ANALYSIS OF ANTIGEN-BINDING T CELLS IN GENETIC HIGH AND LOW RESPONDER MICE

[125I](T,G)-A--L-binding T cells have been studied in mice whose ability to mount an immune response to (T,G)-A--L is under control of the H-2-linked Ir-1A gene. Nonimmunized high and low responder mice have approximately the same frequency of T-ABC. Following immunization, T-ABC proliferated only in high responders, but not in low responders, indicating expression of Ir-1A in T cells. When, for comparison, [125I]arsanyl-mouse serum albumin binding B and T cells were investigated in mice whose antibody response to the hapten arsanyl is controlled by an allotype-linked Ir gene, it was found that following immunization the number of B-ABC increased only in high responders. In contrast, T-ABC proliferated to the same extent in both high and low responders, suggesting exclusive expression of the allotype-linked Ir gene in the B-cell line. Preliminary studies indicate that anti-Ia sera inhibit neither B-ABC nor T-ABC

GENETIC CONTROL OF THE IMMUNE RESPONSE : FREQUENCY AND CHARACTERISTICS OF ANTIGEN-BINDING CELLS IN HIGH AND LOW RESPONDER MICE

The influence of immunization with (T,G)-A--L on the frequency and characteristics of [125I] (T,G)-A--L-binding cells (ABC) was investigated in high and low responder mice, whose ability to respond to (T,G)-A--L is under control of an H-2-linked immune response gene, Ir-1. Unimmunized high and low responder mice have about the same number of ABC in spleen and lymph nodes (6–12 ABC/104). However, after immunization with (T,G)-A--L in aqueous solution, ABC in high responders increase to a much greater extent than they do in low responders. By inhibition of ABC with class-specific anti-Ig sera, it was demonstrated that in nonimmune and primed mice antigen is bound to IgM receptors, which is in agreement with the exclusive production of 19S anti-(T,G)-A--L antibody in primed animals. In contrast, after secondary challenge with antigen, ABC in high and low responder mice have mainly IgG receptors, although under the conditions used for immunization, low responders are not able to produce detectable amounts of 7S anti-(T,G)-A--L antibody. From these results and from the evidence that low responders very probably have a T cell defect, it is suggested that the switchover from IgM to IgG precursor cells can be induced by antigen itself, without the action of specific T cells. Furthermore, the failure of marked proliferation of ABC in low responders after antigenic stimulation is explained by the lack of stimulation by specific T cells. By independent methods it has been shown that all ABC detected in this study are B cells. Preliminary experiments indicate that purified peripheral T cells bind antigen, but much less per cell than do B cells

Interference of Distinct Invariant Chain Regions with Superantigen Contact Area and Antigenic Peptide Binding Groove of HLA-DR

In the endoplasmic reticulum, MHC class II ab dimers associate with the trimeric invariant chain (li), generating a nine-subunit(abli)3 complex. In the presence of li, the peptide binding groove is blocked, so that loading with self or antigenic peptides can only occur after proteolytic removal of li in specialized post-Golgi compartments. The class 11-associated invariant chain peptide region of li (about residues 81-1 04) is known to mediate binding to class II molecules and blockade of the groove, but this does not exclude additional contact sites for li. Using a set of overlapping li peptides and recombinant soluble li, we demonstrate here that a large segment of Ii encompassing approximately residues 71 to 128 interacts with HLA-DR molecules. The N- and C-terminal regions of this Ii segment appear to bind outside the peptide groove to the contact area for the staphylococcal superantigen Staphylococcus aureus enterotoxin B on the a1 domain. The core region of this segment (residues 95-108)prevents binding of antigenic peptides, probably by interaction with the peptide groove. Occupation of the groove with antigenic peptides abolishes binding not only of the core region, but also that of those Ii peptides that bind outside the groove. These findings suggest the existence of distinct conformational states of class II molecules, with Ii binding preferentially to one form

A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells

Dendritic cells (DC) are known to support the activation of natural killer (NK) cells. However, little is known about the role for DC in NK-cell homeostasis. In order to investigate this question, a novel bacterial artificial chromosome transgenic mouse model was generated in which the diphtheria toxin receptor is expressed under the CD11c promoter. In these mice efficient DC depletion can be achieved over prolonged periods of time by multiple injections of diphtheria toxin. We show here that NK cells require DC for full acquisition of effector function in vivo in response to the bacterial-derived TLR ligand CpG. Importantly, DC were found to play an instrumental role for maintaining normal homeostasis of NK cells. This is achieved by IL-15 production by DC, which supports the homeostatic proliferation of NK cells. . There is much known about the molecular mechanisms of NK-cell functions, but the factors influencing NK-cell numbers are only beginning to be elucidated. Mature NK cells were typically thought to be a terminally differentiated population, with a very limited selfrenewal capacity. However, it was recently shown that a small percentage of NK cells actively proliferate in the steady state, resulting in a half life of the NK-cell population of about 17 days SHORT COMMUNICATION Ã These authors contributed equally to this study. Correspondence: Dr. Natalio Garbi e-mail: n. 2776 Importantly, we observe a previously unrecognized role for DC in optimal homeostatic proliferation of NK cells in lymphopenic conditions. In this process, DC-derived IL-15 appears to play an important role. Our data indicate that not only T lymphocytes, but also NK cells require DC for homeostatic proliferation. Results CD11c.DOG mice allow long-term ablation of DC in vivo without toxicity effects In order to study the effect of in vivo interaction between DC and NK cells, a system that permits long-term depletion of DC is required. The diphtheria toxin/diphtheria toxin receptor (DT/DTR) system introduced by Saito et al. Injection of 8 ng/gram body weight (gbw) DT induced depletion of DC (CD11c high MHC class II 1 ) in spleen, lymph nodes, thymus and bone marrow, albeit efficiency of depletion varied in the different organs tested The data shown in Although mice tolerated well 8 ng/gbw for prolonged periods of time, DC depletion was limited to a period of 11-12 days In order to confirm faithful expression of the BAC, we thoroughly examined CD11c expression and depletion of B cells, T cells, NK cells and NKT cells in spleens after 8 ng/gbw DT administration. The minor CD11c hi subpopulations of these cell types were effectively depleted, whereas CD11c int/lo cells were only partially depleted (Supporting Information In conclusion, the new BAC transgenic CD11c.DOG mouse model introduced here allows effective long-term depletion of CD11c 1 cells. DC are required for optimal NK-cell activation and homeostatic proliferation in vivo of NK cells. NK cells were activated by administration of the TLR-9 ligand CpG and the in vivo lytic activity determined by injection of CFSE-labelled tapasin-deficient splenocytes that served as NK targets. CpG administration in mice with a normal DC compartment lead to increased NK cytotoxicity in vivo ( The homeostasis of T cells has been well studied and found to require several factors including the presence of DC, self MHC molecules and the cytokine IL-7 for proliferation and survival ) in DC-depleted CD11c.DOG mice compared with DC-sufficient B6 mice ( DC-derived IL-15 contributes to lymphopenia-induced proliferation of NK cells The cytokine IL-15 has been reported to be crucial for activation and survival of NK cells Concluding remarks In summary, this study introduces a new BAC transgenic CD11c.DTR mouse model for prolonged in vivo depletion of DC by application of DT, without any apparent signs of toxicity. Experiments using this mouse line demonstrated a previously unrecognized role for DC in the homeostasis of NK cells, and showed that local production of IL-15 by DC is required for the maintenance of the NK-cell compartment. Thus, the present study adds an additional function to the long list of DC functions, which so far encompass antigen presentation to and activation of CD4 and CD8 T cells, deletion of T cells and induction of regulatory 1 ) NK cells were quantified 7 days after transfer into irradiated chimeras. Three mice were used in each group. The experiment was repeated three times with similar results. Ã po0.05, Student's t-test

Innate type 2 immunity in helminth infection is induced redundantly and acts autonomously following CD11c+ cell depletion

Infection with gastrointestinal helminths generates a dominant type 2 response among both adaptive (Th2) and innate (macrophage, eosinophil, and innate lymphoid) immune cell types. Two additional innate cell types, CD11chigh dendritic cells (DCs) and basophils, have been implicated in the genesis of type 2 immunity. Investigating the type 2 response to intestinal nematode parasites, including Heligmosomoides polygyrus and Nippostrongylus brasiliensis, we first confirmed the requirement for DCs in stimulating Th2 adaptive immunity against these helminths through depletion of CD11chigh cells by administration of diphtheria toxin to CD11c.DOG mice. In contrast, responsiveness was intact in mice depleted of basophils by antibody treatment. Th2 responses can be induced by adoptive transfer of DCs, but not basophils, exposed to soluble excretory-secretory products from these helminths. However, innate type 2 responses arose equally strongly in the presence or absence of CD11chigh cells or basophils; thus, in CD11c.DOG mice, the alternative activation of macrophages, as measured by expression of arginase-1, RELM-α, and Ym-1 (Chi3L3) in the intestine following H. polygyrus infection or in the lung following N. brasiliensis infection, was unaltered by depletion of CD11c-expressing DCs and alveolar macrophages or by antibody-mediated basophil depletion. Similarly, goblet cell-associated RELM-β in lung and intestinal tissues, lung eosinophilia, and expansion of innate lymphoid (“nuocyte”) populations all proceeded irrespective of depletion of CD11chigh cells or basophils. Thus, while CD11chigh DCs initiate helminth-specific adaptive immunity, innate type 2 cells are able to mount an autonomous response to the challenge of parasite infection

CD80 and CD86 Differentially Regulate Mechanical Interactions of T-Cells with Antigen-Presenting Dendritic Cells and B-Cells

10.1371/journal.pone.0045185PLoS ONE79

Control of Uterine Microenvironment by Foxp3+ Cells Facilitates Embryo Implantation

Implantation of the fertilized egg into the maternal uterus depends on the fine balance between inflammatory and anti-inflammatory processes. Whilst regulatory T cells (Tregs) are reportedly involved in protection of allogeneic fetuses against rejection by the maternal immune system, their role for pregnancy to establish, e.g., blastocyst implantation, is not clear. By using 2-photon imaging we show that Foxp3(+) cells accumulated in the mouse uterus during the receptive phase of the estrus cycle. Seminal fluid further fostered Treg expansion. Depletion of Tregs in two Foxp3.DTR-based models prior to pairing drastically impaired implantation and resulted in infiltration of activated T effector cells as well as in uterine inflammation and fibrosis in both allogeneic and syngeneic mating combinations. Genetic deletion of the homing receptor CCR7 interfered with accumulation of Tregs in the uterus and implantation indicating that homing of Tregs to the uterus was mediated by CCR7. Our results demonstrate that Tregs play a critical role in embryo implantation by preventing the development of a hostile uterine microenvironment.DFG grants: (ZE526/4-2, SFB854TP7), Wilhelm Sander Stiftung Germany grant: (2009.022.1), Helmholtz Alliance for Immunotherapy, FCT, Medical Faculty Otto-von-Guericke University PhD grant

Metastasis of prostate cancer and melanoma cells in a preclinical in vivo mouse model is enhanced by L-plastin expression and phosphorylation

BACKGROUND: Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. METHODS: We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. RESULTS: Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. CONCLUSIONS: Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable

Interleukin-7 Links T Lymphocyte and Intestinal Epithelial Cell Homeostasis

Interleukin-7 (IL-7) is a major survival factor for mature T cells. Therefore, the degree of IL-7 availability determines the size of the peripheral T cell pool and regulates T cell homeostasis. Here we provide evidence that IL-7 also regulates the homeostasis of intestinal epithelial cells (IEC), colon function and the composition of the commensal microflora. In the colon of T cell-deficient, lymphopenic mice, IL-7-producing IEC accumulate. IEC hyperplasia can be blocked by IL-7-consuming T cells or the inactivation of the IL-7/IL-7R signaling pathway. However, the blockade of the IL-7/IL-7R signaling pathway renders T cell-deficient mice more sensitive to chemically-induced IEC damage and subsequent colitis. In summary, our data demonstrate that IL-7 promotes IEC hyperplasia under lymphopenic conditions. Under non-lymphopenic conditions, however, T cells consume IL-7 thereby limiting IEC expansion and survival. Hence, the degree of IL-7 availability regulates both, T cell and IEC homeostasis

Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response

This is the publisher's version, also available electronically from http://www.jci.org/articles/view/18704While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation