High levels of gut-homing immunoglobulin A+ B lymphocytes support the pathogenic role of intestinal mucosal hyperresponsiveness in immunoglobulin A nephropathy patients.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulonephritis. The role of the microbiota and mucosal immunity in the pathogenesis of IgAN remains a key element. To date, the hypothetical relationship between commensal bacteria, elevated tumour necrosis factor (TNF) superfamily member 13 [also known as B-cell activating factor (BAFF)] levels, perturbed homoeostasis of intestinal-activated B cells and intestinal IgA class switch has not been clearly shown in IgAN patients. METHODS: We studied the intestinal-renal axis connections, analysing levels of BAFF, TNF ligand superfamily member 13 (APRIL) and intestinal-activated B cells in IgAN patients, healthy subjects (HSs) and patients with non-IgA glomerulonephritides. RESULTS: IgAN patients had increased serum levels of BAFF cytokine, correlating with higher amounts of five specific microbiota metabolites, and high APRIL cytokine serum levels. We also found that subjects with IgAN have a higher level of circulating gut-homing (CCR9+ ß7 integrin+) regultory B cells, memory B cells and IgA+ memory B cells compared with HSs. Finally, we found that IgAN patients had high levels of both total plasmablasts (PBs) and intestinal-homing PBs. Interestingly, PBs significantly increased in IgAN but not in patients with other glomerulonephritides. CONCLUSIONS: Our results demonstrate a significant difference in the amount of intestinal-activated B lymphocytes between IgAN patients and HSs, confirming the hypothesis of the pathogenic role of intestinal mucosal hyperresponsiveness in IgAN. The intestinal-renal axis plays a crucial role in IgAN and several factors may contribute to its complex pathogenesis and provide an important area of research for novel targeted therapies to modulate progression of the disease.

Direct and Indirect Effect of TGFß on Treg Transendothelial Recruitment in HCC Tissue Microenvironment.

The balance between anti-tumor and tumor-promoting immune cells, such as CD4+ Th1 and regulatory T cells (Tregs), respectively, is assumed to dictate the progression of hepatocellular carcinoma (HCC). The transforming growth factor beta (TGFß) markedly shapes the HCC microenvironment, regulating the activation state of multiple leukocyte subsets and driving the differentiation of cancer associated fibroblasts (CAFs). The fibrotic (desmoplastic) reaction in HCC tissue strongly depends on CAFs activity. In this study, we attempted to assess the role of TGFß on transendothelial migration of Th1-oriented and Treg-oriented CD4+ T cells via a direct or indirect, CAF-mediated mechanisms, respectively. We found that the blockage of TGFß receptor I-dependent signaling in Tregs resulted in impaired transendothelial migration (TEM) of these cells. Interestingly, the secretome of TGFß-treated CAFs inhibited the TEM of Tregs but not Th1 cells, in comparison to the secretome of untreated CAFs. In addition, we found a significant inverse correlation between alpha-SMA and FoxP3 (marker of Tregs) mRNA expression in a microarray analysis involving 78 HCCs, thus suggesting that TGFß-activated stromal cells may counteract the trafficking of Tregs into the tumor. The apparent dual behavior of TGFß as both pro- and anti-tumorigenic cytokines may add a further level of complexity to the mechanisms that regulate the interactions among cancerous, stromal, and immune cells within HCC, as well as other solid tumors, and contribute to better manipulation of the TGFß signaling as a therapeutic target in HCC patients.

Safety of measles, mumps, and rubella vaccine in egg allergy: in vivo and in vitro management.

BACKGROUND: Egg allergy is the second most prevalent form of food allergy in childhood. In spite of the evidence accumulated, inoculating egg allergy children with attenuated vaccines grown on chick embryo cell cultures, such as the measles, mumps, and rubella (MMR) vaccine, is regarded (erroneously) as potentially dangerous or even anaphylactogenic, by many. An issue perceived as particularly conflicting also by Health Professionals. CASE PRESENTATION: A 15-year-old boy, with a history of severe egg allergy in early infancy, who was still sensitized to egg allergens, including baked egg, had never received MMR vaccination, in fear of possible anaphylaxis, in spite of the fact that this vaccination is mandatory in the first year of life, in Italy. Because of that, he was not allowed to attend school, longer, and was referred to us in order to assess the potential risk of MMR vaccination. Upon thorough allergologic workup, sensitization to MMR vaccine components was excluded by an in vivo approach, consisting in skin prick tests, intradermal tests, and subcutaneous injection test, corroborated by vaccine-specific B-lymphocyte proliferation assay, ex vivo. T-cell proliferation in response to MMR vaccine was also excluded. Eventually, the boy was inoculated with MMR vaccine and was readmitted to school. CONCLUSIONS: The diagnostic strategy adopted appears feasible and easy-to-perform and may be adopted in controversial cases (as the one reported), characterized by previous severe allergic reactions to egg. The B-lymphocyte proliferation assay we developed may represent a useful and reliable tool not only in research but also in clinical practice.

Adult Renal Stem/Progenitor Cells Can Modulate T Regulatory Cells and Double Negative T Cells.

Adult Renal Stem/Progenitor Cells (ARPCs) have been recently identified in the human kidney and several studies show their active role in kidney repair processes during acute or chronic injury. However, little is known about their immunomodulatory properties and their capacity to regulate specific T cell subpopulations. We co-cultured ARPCs activated by triggering Toll-Like Receptor 2 (TLR2) with human peripheral blood mononuclear cells for 5 days and 15 days and studied their immunomodulatory capacity on T cell subpopulations. We found that activated-ARPCs were able to decrease T cell proliferation but did not affect CD8+ and CD4+ T cells. Instead, Tregs and CD3+ CD4- CD8- double-negative (DN) T cells decreased after 5 days and increased after 15 days of co-culture. In addition, we found that PAI1, MCP1, GM-CSF, and CXCL1 were significantly expressed by TLR2-activated ARPCs alone and were up-regulated in T cells co-cultured with activated ARPCs. The exogenous cocktail of cytokines was able to reproduce the immunomodulatory effects of the co-culture with activated ARPCs. These data showed that ARPCs can regulate immune response by inducing Tregs and DN T cells cell modulation, which are involved in the balance between immune tolerance and autoimmunity.

Maintenance-Phase Subcutaneous Immunotherapy with House Dust Mites Induces Cyclic Immunologic Effects.

BACKGROUND: Subcutaneous immunotherapy (SCIT) is an effective treatment of respiratory allergies including house dust mite (HDM) and Hymenoptera venom allergy. During the build-up phase, the allergen is administered weekly at increasing doses, while during the maintenance phase, it is administered at a fixed high dose every 4 weeks. Upon SCIT injection, the allergen is driven to the draining lymph nodes where it most likely induces an immune response. Immunologic changes are thus supposedly induced at each injection. OBJECTIVES: It is now established that SCIT induces tolerance in the long term, but the precise underlying immunologic mechanisms remain unclear. Therefore, we wanted to analyze the immunologic changes induced in both innate and adaptive immune cells at each individual SCIT administration during the maintenance phase in HDM-allergic patients. More specifically, we wondered whether the changes in regulatory T cell (Treg) and IgE+ B cell percentages, which are observed at the end of a 3-year course of SCIT, already occurred during the maintenance phase and whether these possible changes were sustained. METHODS: We enrolled 6 patients suffering from HDM allergic rhinitis and undergoing maintenance HDM SCIT for 18-24 months. The same SCIT extract was used for all patients. We collected blood samples at 5 time points: T1 (immediately before a given SCIT injection), T2 (9 days after T1), T3 (29 days after T1 and right before the successive administration), T4 (39 days after T1), and T5 (61 days after T1 and just before the next injection). Six non-allergic age-matched healthy individuals were used as controls. Using flow cytometry, we assessed the following cell subsets in peripheral blood mononuclear cells: CD4 and CD8 T cells, Tregs, B cells, IgE+ B cells, NK and NKT cells, and total and activated basophils. RESULTS: HDM-allergic patients displayed increased percentages of CD4 and CD8 T cells and NK cells compared to healthy controls. In contrast, NKT cells, total B cells, and basophils were diminished. These differences were maintained throughout the time course and seemed to be independent of the periodical SCIT injections. On the contrary, Treg percentages were significantly reduced in all HDM-allergic patients at T1. However, they increased at T2 and T4 (9 days after each SCIT injection) but decreased again at T3 and T5, just before the next one, resulting in cyclic changes. IgE+ B cells were significantly increased at T1, even more increased after each administration (T2, T4), and went back to their initial levels at T3 and T5, also resulting in a cyclic pattern. CONCLUSIONS: Our data suggest that during the SCIT maintenance phase, cycles of expansion/contraction of Tregs and IgE+ B cells occur at each SCIT injection. Therefore, the sustained induction of immune tolerance by SCIT, through the increase of Tregs, seems to depend on the periodical exposure to the allergen, at least during the early steady state.

Inflammation induces osteoclast differentiation from peripheral mononuclear cells in chronic kidney disease patients: crosstalk between the immune and bone systems.

Background: Inflammation and immune system alterations contribute to bone damage in many pathologies by inducing the differentiation of osteoclasts (OCs), the bone resorbing cells. This link is largely unexplored in chronic kidney disease (CKD) and haemodialysis (HD) patients, in which reduced renal function is accompanied by an increased inflammatory state and skeletal abnormality. Methods: We used ex vivo culture experiments to investigate the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs) of CKD and HD patients, focusing on immune cell subsets and inflammatory cytokines such as LIGHT and receptor activator of nuclear factor κB ligand (RANKL). Results: We observed spontaneous osteoclastogenesis with a significant increase in OC formation and bone resorbing activity in late-stage CKD and HD patients when compared with early-stage CKD patients and healthy donors, likely due to an increased expression of RANKL and LIGHT (homologous to Lymphotoxins exhibiting Inducible expression and competing with herpes simplex virus Glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes) in PBMCs. Specific inhibition of these cytokines in PBMCs isolated from CKD stages 3b-5 and HD patients induced the reduction of OC formation in vitro. The phenotypic characterization of peripheral blood cells revealed a significant increase of OC precursors (CD14+CD11b+CD51/61+) and CD14+CD16+ monocytes in advanced CKD and HD patients compared with the control group. Conclusions: Our results suggest that circulating inflammatory monocytes from advanced CKD or HD patients trans differentiate into OCs in vitro and play a relevant role in mineral bone disorders and that LIGHT and RANKL represent new potential therapeutic targets in these settings.

Default in plasma and intestinal IgA responses during acute infection by simian immunodeficiency virus.

BACKGROUND: Conflicting results regarding changes in mucosal IgA production or in the proportions of IgA plasma cells in the small and large intestines during HIV-infection have been previously reported. Except in individuals repeatedly exposed to HIV-1 but yet remaining uninfected, HIV-specific IgAs are frequently absent in mucosal secretions from HIV-infected patients. However, little is known about the organization and functionality of mucosal B-cell follicles in acute HIV/SIV infection during which a T-dependent IgA response should have been initiated. In the present study, we evaluated changes in B-cell and T-cell subsets as well as the extent of apoptosis and class-specific plasma cells in Peyer's Patches, isolated lymphoid follicles, and lamina propria. Plasma levels of IgA, BAFF and APRIL were also determined. RESULTS: Plasma IgA level was reduced by 46% by 28 days post infection (dpi), and no IgA plasma cells were found within germinal centers of Peyer's Patches and isolated lymphoid follicles. This lack of a T-dependent IgA response occurs although germinal centers remained functional with no sign of follicular damage, while a prolonged survival of follicular CD4+ T-cells and normal generation of IgG plasma cells is observed. Whereas the average plasma BAFF level was increased by 4.5-fold and total plasma cells were 1.7 to 1.9-fold more numerous in the lamina propria, the relative proportion of IgA plasma cells in this effector site was reduced by 19% (duodemun) to 35% (ileum) at 28 dpi. CONCLUSION: Our data provide evidence that SIV is unable to initiate a T-dependent IgA response during the acute phase of infection and favors the production of IgG (ileum) or IgM (duodenum) plasma cells at the expense of IgA plasma cells. Therefore, an early and generalized default in IgA production takes place during the acute of phase of HIV/SIV infection, which might impair not only the virus-specific antibody response but also IgA responses to other pathogens and vaccines as well. Understanding the mechanisms that impair IgA production during acute HIV/SIV infection is crucial to improve virus-specific response in mucosa and control microbial translocation.

Angiogenesis in NENs, with a focus on gastroenteropancreatic NENs: from biology to current and future therapeutic implications.

Neuroendocrine neoplasms (NENs) are highly vascularized malignancies arising from cells of the diffuse neuroendocrine system. An intricated cross-talk exists between NEN cells and the tumor microenvironment, and three main molecular circuits (VEGF/VEGFR pathway, FGF-dependent signaling and PDGF/PDGFR axis) have been shown to regulate angiogenesis in these neoplasms. Multiple randomized trials have investigated antiangiogenic agents over the past two decades, and sunitinib is currently approved for the treatment of advanced, progressive, G1/G2 pancreatic NENs. In recent years, two phase III clinical trials have demonstrated the efficacy and safety of surufatinib, a multi-tyrosine kinase angioimmune inhibitor, in patients with well-differentiated pancreatic and extrapancreatic NENs, and two studies of this agent are currently underway in Europe and US. The HIF-2α inhibitor belzutifan has recently received regulatory approval for the treatment of tumors arising in the context of Von-Hippel Lindau syndrome including pancreatic NENs, and a study of this drug in patients with sporadic tumors is presently ongoing. Combinations of antiangiogenic agents with chemotherapeutics and targeted drugs have been tested, with accumulating toxicities being a matter of concern. The potential of antiangiogenic agents in fine-tuning the immune microenvironment of NENs to enhance the activity of immune checkpoint inhibitors has been only partially elucidated, and further research should be carried out at this regard. Here, we review the current understanding of the biology of angiogenesis in NENs and provide a summary of the latest clinical investigations on antiangiogenic drugs in this malignancy.

Tumor Infiltrating T Cell Cytotoxicity Assay.

Cytotoxicity is the primary function of CD8+ T-cells, also called cytotoxic CD8+ T lymphocytes (or CTLs). Quantification of this capacity is of major importance in diagnostic and research tools. While phenotypic characterization of CTLs is frequent and easily performed, their function is indeed more difficult to assess. CTLs are responsible for the lysis of cells expressing foreign or modified antigen peptides on their MHC class I molecules. Here we describe the detailed protocol used for the in vitro specific lysis of target cells.

Antigen-Specific In Vivo Killing Assay.

The in vivo killing assay allows the quantification of the antigen-specific killing capacity of Cytotoxic CD8+ T Lymphocytes (CTLs) in mice. CTLs are indeed known for the lysis of cells expressing foreign or modified antigen peptides on their MHC class I molecules. Here we describe the detailed protocol used for the in vivo specific lysis of cells expressing the H-2 Kb immunodominant CD8+ T-cell epitope of the OVA protein: an 8 amino acid peptide corresponding to the 257-264 region of OVA (SIINFEKL).

Impaired Anti-Tumor T cell Response in Hepatocellular Carcinoma.

Different subsets of lymphocytes have the capacity to promote or counteract the progression of solid cancers, including hepatocellular carcinoma (HCC). Therefore, to determine the infiltrative ability and functional status of major immune cell subtypes into tumor may lead to novel insights from the perspective of immunotherapy. After obtaining single cell suspensions from freshly collected specimens of HCC tumor, along with paired peritumor tissues and peripheral blood mononuclear cells (PBMCs) from 14 patients, we flow-cytometrically identified and quantified the relative frequencies of lymphocyte subsets within the tissues of origin. We found that the recruitment in the tumor of cytotoxic cells, namely the terminally differentiated CD4+ and CD8+ T cells (TEFF), is impaired, whereas the effector memory CD4+ T cells (TEM) are more attracted in this site. Concerning the other subsets, the frequency of NK CD56hi and NKT CD56hi cells infiltration in the tumor is increased, whereas that of NKT CD56low is reduced. Although CD4+ and CD8+ T cells settled in the tumor show a higher degree of activation than the circulating counterpart, they occur with a more exhausted phenotype. Overall, these data demonstrate the prevalently immunosuppressive nature of HCC microenvironment, and prompt us to search for strategies to enhance the activity of anti-tumor immune cell subsets.

Lack of MHC class II molecules favors CD8+ T-cell infiltration into tumors associated with an increased control of tumor growth.

Regulatory T-cells (Tregs) are crucial for the maintenance of immune tolerance and homeostasis as well as for preventing autoimmune diseases, but their impact on the survival of cancer patients remains controversial. In the TC-1 mouse model of human papillomavirus (HPV)-related carcinoma, we have previously demonstrated that the therapeutic efficacy of the CyaA-E7-vaccine, targeting the HPV-E7 antigen, progressively declines with tumor growth, in correlation with increased intratumoral recruitment of Tregs. In the present study, we demonstrated that these TC-1 tumor-infiltrating Tregs were highly activated, with increased expression of immunosuppressive molecules. Both intratumoral effector CD4+ T-cells (Teffs) and Tregs expressed high levels of PD-1, but anti-PD-1 antibody treatment did not impact the growth of the TC-1 tumor nor restore the therapeutic effect of the CyaA-E7 vaccine. To analyze the mechanisms by which Tregs are recruited to the tumor site, we used MHC-II KO mice with drastically reduced numbers of CD4+ effector T-cells. We demonstrated that these mice still had significant numbers of Tregs in their lymphoid organs which were recruited to the tumor. In MHC-II KO mice, the growth of the TC-1 tumor was delayed in correlation with a strong increase in the intratumoral recruitment of CD8+ T-cells. In addition, in mice that spontaneously rejected their tumors, the infiltration of E7-specific CD8+ T-cells was significantly higher than in MHC-II KO mice with a growing tumor. These results demonstrate that tumor-specific CD8+ T-cells can be efficiently activated and recruited in the absence of MHC class II molecules and of CD4+ T-cell help.

B-Cell-Activating Factor and the B-Cell Compartment in HIV/SIV Infection.

With the goal to design effective HIV vaccines, intensive studies focused on broadly neutralizing antibodies, which arise in a fraction of HIV-infected people. Apart from identifying new vulnerability sites in the viral envelope proteins, these studies have shown that a fraction of these antibodies are produced by self/poly-reactive B-cells. These findings prompted us to revisit the B-cell differentiation and selection process during HIV/SIV infection and to consider B-cells as active players possibly shaping the helper T-cell program within germinal centers (GCs). In this context, we paid a particular attention to B-cell-activating factor (BAFF), a key cytokine in B-cell development and immune response that is overproduced during HIV/SIV infection. As it does in autoimmune diseases, BAFF excess might contribute to the abnormal rescue of self-reactive B-cells at several checkpoints of the B-cell development and impair memory B-cell generation and functions. In this review, we first point out what is known about the functions of BAFF/a proliferation-inducing ligand and their receptors [B-cell maturation, transmembrane activator and CAML interactor (TACI), and BAFF-R], in physiological and pathophysiological settings, in mice and humans. In particular, we highlight recent results on the previously underappreciated regulatory functions of TACI and on the highly regulated production of soluble TACI and BAFF-R that act as decoy receptors. In light of recent data on BAFF, TACI, and BAFF-R, we then revisit the altered phenotypes and functions of B-cell subsets during the acute and chronic phase of HIV/SIV infection. Given the atypical phenotype and reduced functions of memory B-cells in HIV/SIV infection, we particularly discuss the GC reaction, a key checkpoint where self-reactive B-cells are eliminated and pathogen-specific memory B-cells and plasmablasts/cells are generated in physiological settings. Through its capacity to differentially bind and process BAFF-R and TACI on GC B-cells and possibly on follicular helper T-cells, BAFF appears as a key regulator of the physiological GC reaction. Its local excess during HIV/SIV infection could play a key role in B-cell dysregulations.

Plasmacytoid dendritic cells and myeloid cells differently contribute to B-cell-activating factor belonging to the tumor necrosis factor superfamily overexpression during primary HIV infection.

BACKGROUND: After describing heightened levels of circulating B-cell-activating factor belonging to the tumor necrosis factor superfamily (BAFF) as well as changes in B-cell phenotype and functions during acute infection by simian immunodeficiency virus, we wanted to determine whether and by which cells BAFF was over-expressed in primary HIV-infected (PHI) patients. DESIGN AND METHODS: We simultaneously examined circulating BAFF levels by ELISA and membrane-bound BAFF (mBAFF) expression by flow cytometry in peripheral blood mononuclear cells of healthy donors and PHI patients followed for 6 months. We also examined whether HIV-1 modifies BAFF expression or release in various myeloid cells and plasmacytoid dendritic cells (pDC) in vitro. RESULTS: Circulating BAFF levels were transiently increased at enrolment. They positively correlated with CXCL10 levels and inversely with B-cell counts. Whereas mBAFF was expressed by most pDC and on a fraction of intermediate monocytes in healthy donors, the frequency of mBAFF cells significantly increased among nonclassical monocytes and CD1c dendritic cells but decreased among pDC in PHI patients. In contrast to myeloid cells, pDC never released BAFF upon stimulation. Their mBAFF expression was enhanced by HIV-1, independently of type I IFN. CONCLUSION: Our findings reveal that the pattern of BAFF expression by myeloid cells and pDC is altered in PHI patients and constitutes a valuable marker of immune activation whose circulating levels correlate with CXCL10 levels. Due to their homing in different tissue areas, pDC and myeloid cells might target different B-cell subsets through their mBAFF expression or soluble BAFF release.

Rapamycin Impairs Antitumor CD8+ T-cell Responses and Vaccine-Induced Tumor Eradication.

The metabolic sensor mTOR broadly regulates cell growth and division in cancer cells, leading to a significant focus on studies of rapamycin and its analogues as candidate anticancer drugs. However, mTOR inhibitors have failed to produce useful clinical efficacy, potentially because mTOR is also critical in T cells implicated in immunosurveillance. Indeed, recent studies using rapamycin have demonstrated the important role of mTOR in differentiation and induction of the CD8+ memory in T-cell responses associated with antitumor properties. In this study, we demonstrate that rapamycin harms antitumor immune responses mediated by T cells in the setting of cancer vaccine therapy. Specifically, we analyzed how rapamycin affects the antitumor efficacy of a human papilloma virus E7 peptide vaccine (CyaA-E7) capable of eradicating tumors in the TC-1 mouse model of cervical cancer. In animals vaccinated with CyaA-E7, rapamycin administration completely abolished recruitment of CD8+ T cells into TC-1 tumors along with the ability of the vaccine to reduce infiltration of T regulatory cells and myeloid-derived suppressor cells. Moreover, rapamycin completely abolished vaccine-induced cytotoxic T-cell responses and therapeutic activity. Taken together, our results demonstrate the powerful effects of mTOR inhibition in abolishing T-cell-mediated antitumor immune responses essential for the therapeutic efficacy of cancer vaccines.