Production of antibodies can last for a lifetime, through mechanisms that remain poorly understood. Here, we show that human memory B lymphocytes proliferate and differentiate into plasma cells in response to polyclonal stimuli, such as bystander T cell help and CpG DNA. Furthermore, plasma cells secreting antibodies to recall antigens are produced in vivo at levels proportional to the frequency of specific memory B cells, even several years after antigenic stimulation. Although antigen boosting leads to a transient increase in specific antibody levels, ongoing polyclonal activation of memory B cells offers a means to maintain serological memory for a human lifetime.

Because ethical restrictions limit in vivo studies of the human hemato-lymphoid system, substitute human to small animal xenotransplantation models have been employed. Existing models, however, sustain only limited development and maintenance of human lymphoid cells and rarely produce immune responses. Here we show that intrahepatic injection of CD34+ human cord blood cells into conditioned newborn Rag2-/-gammac-/- mice leads to de novo development of B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of functional immune responses. This provides a valuable model to study development and function of the human adaptive immune system in vivo.

Passive serotherapy can confer immediate protection against microbial infection, but methods to rapidly generate human neutralizing monoclonal antibodies are not yet available. We have developed an improved method for Epstein-Barr virus transformation of human B cells. We used this method to analyze the memory repertoire of a patient who recovered from severe acute respiratory syndrome coronavirus (SARS-CoV) infection and to isolate monoclonal antibodies specific for different viral proteins, including 35 antibodies with in vitro neutralizing activity ranging from 10(-8)M to 10(-11)M. One such antibody confers protection in vivo in a mouse model of SARS-CoV infection. These results show that it is possible to interrogate the memory repertoire of immune donors to rapidly and efficiently isolate neutralizing antibodies that have been selected in the course of natural infection.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

CD11b+/Gr‐1+ myeloid‐derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8+ T‐cells. Two major MDSC subsets were recently shown to play an equal role in MDSC‐induced immune dysfunctions: monocytic‐ and granulocytic‐like. We isolated three fractions of MDSC, i.e. CD11b+/Gr‐1high, CD11b+/Gr‐1int, and CD11b+/Gr‐1low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b+/Gr‐1int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8+ T‐cell activation, while CD11b+/Gr‐1high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b+/Gr‐1int but not CD11b+/Gr‐1high cells were also immunosuppressive in vivo following adoptive transfer. CD11b+/Gr‐1low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM‐CSF was necessary to induce preferential expansion of both CD11b+/Gr‐1int and CD11b+/Gr‐1low subsets in the spleen of tumor‐bearing mice and mediate tumor‐induced tolerance whereas G‐CSF, which preferentially expanded CD11b+/Gr‐1high cells, did not create such immunosuppressive environment. GM‐CSF also acted on granulocyte–macrophage progenitors in the bone marrow inducing local expansion of CD11b+/Gr‐1low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor‐released GM‐CSF.

Molecular characterization of the severe acute respiratory syndrome coronavirus has revealed genetic diversity among isolates. The spike (S) glycoprotein, the major target for vaccine and immune therapy, shows up to 17 substitutions in its 1,255-aa sequence; however, the biologic significance of these changes is unknown. Here, the functional effects of S mutations have been determined by analyzing their affinity for a viral receptor, human angiotensin-converting enzyme 2 (hACE-2), and their sensitivity to Ab neutralization with viral pseudotypes. Although minor differences among eight strains transmitted during human outbreaks in early 2003 were found, substantial functional changes were detected in S derived from a case in late 2003 from Guangdong province [S(GD03T0013)] and from two palm civets, S(SZ3) and S(SZ16). S(GD03T0013) depended less on the hACE-2 receptor and was markedly resistant to Ab inhibition. Unexpectedly, Abs that neutralized most human S glycoproteins enhanced entry mediated by the civet virus S glycoproteins. The mechanism of enhancement involved the interaction of Abs with conformational epitopes in the hACE-2-binding domain. Finally, improved immunogens and mAbs that minimize this complication have been defined. These data show that the entry of severe acute respiratory syndrome coronaviruses can be enhanced by Abs, and they underscore the need to address the evolving diversity of this newly emerged virus for vaccines and immune therapies.angiotensin-converting enzyme 2 ͉ enhancement ͉ immunoglobulin G ͉ pseudovirus

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared their phenotype with that of MALT1 knockout animals (Malt1−/−). Malt1PD/PD mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10–producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1−/− animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1PD/PD mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1PD/PD animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1PD/PD animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.