SummaryUsing granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 we have established dendritic cell (DC) lines from blood mononuclear cells that maintain the antigen capturing and processing capacity characteristic of immature dendritic cells in vivo. These cells have typical dendritic morphology, express high levels of major histocompatibility complex (MHC) class I and class II molecules, CD1, Fcq, RII, CD40, B7, CD44, and ICAM-1, and lack CD14. Cultured DCs are highly stimulatory in mixed leukocyte reaction (MLR) and are also capable of triggering cord blood naive T cells. Most strikingly, these DCs are as efficient as antigenspecific B cells in presenting tetanus toxoid (TT) to specific T cell clones. Their efficiency of antigen presentation can be further enhanced by specific antibodies via FoR-mediated antigen uptake. Incubation of these cultured DCs with tumor necrosis factor o~ (TNF-o 0 or soluble CD40 ligand (CD40L) for 24 h results in an increased surface expression of MHC dass I and class II molecules, B7, and ICAM-1 and in the appearance of the CD44 exon 9 splice variant (CD44-vg); by contrast, Fc'yRII is markedly and sometimes completely downregulated. The functional consequences of the short contact with TNF-c~ are an increased T cell stimulatory capacity in MLR, but a 10-fold decrease in presentation of soluble TT and a 100-fold decrease in presentation of TT-immunoglobulin G complexes.

Naive T lymphocytes travel to T-cell areas of secondary lymphoid organs in search of antigen presented by dendritic cells. Once activated, they proliferate vigorously, generating effector cells that can migrate to B-cell areas or to inflamed tissues. A fraction of primed T lymphocytes persists as circulating memory cells that can confer protection and give, upon secondary challenge, a qualitatively different and quantitatively enhanced response. The nature of the cells that mediate the different facets of immunological memory remains unresolved. Here we show that expression of CCR7, a chemokine receptor that controls homing to secondary lymphoid organs, divides human memory T cells into two functionally distinct subsets. CCR7- memory cells express receptors for migration to inflamed tissues and display immediate effector function. In contrast, CCR7+ memory cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells and differentiate into CCR7- effector cells upon secondary stimulation. The CCR7+ and CCR7- T cells, which we have named central memory (TCM) and effector memory (TEM), differentiate in a step-wise fashion from naive T cells, persist for years after immunization and allow a division of labour in the memory response.

immunology letters to nature 34 asymmetric unit (molecule B). Density for the other two molecules was broken and hard to interpret. The skeleton for molecule B was used to generate a molecular envelope for NCS averaging. Initial rotation matrices describing the relative orientations of molecules A, B and C were calculated from the heavy-atom sites (mercury bound to five sites in each of the three molecules). NCS averaging with DM 25 was used to improve phases at 2.7 Å resolution, and then extend phases to 2.2 Å , the limit of the native data set. The resulting electron density map was readily interpretable (Fig. 2). A model including residues 47-351 of each of the three molecules was built using the graphics program O 26 . No electron density is seen for 24 residues at the amino terminus. The model was refined using simulated annealing and positional refinement in X-PLOR 27 , with tight NCS restraints. The model includes 669 water molecules, which were positioned by using the program ARP (V.Lamzin). An overall thermal B factor and tightly restrained individual B factors were refined, and a bulk-solvent model was incorporated. Crystallographic R values and stereochemical parameters are presented in Table 1.The structure of the Cbl-N/ZAP70 phosphopeptide complex was determined by molecular replacement with the program AmoRe 28 . Use of the complete unliganded Cbl-N structure as a search model yielded clear rotation and translation peaks, and maps phased with the appropriately positioned model revealed strong electron density for the 4H and EF-hand domains, but no interpretable density for the SH2 domain. The model was therefore broken into two fragments (residues 47-265 and residues 266-351), and the rotation and translation searches were repeated with each fragment. The 4H/EF-hand fragment yielded a solution essentially identical to that from the intact model. The SH2 domain was then positioned using translation searches conducted in the context of the appropriately positioned 4H/EF-hand fragment. After rigid-body and positional refinement using X-PLOR 27 , electron density maps calculated with the combined model revealed clear density for all domains, and readily interpretable density for the bound ZAP-70 phosphopeptide. After construction of the peptide, the structure was refined with iterative cycles of manual refitting and simulated annealing and positional refinement in X-PLOR (Table 1). Restrained individual temperature factors were refined. The model includes residues 47-351 of c-Cbl, residues 289-297 of ZAP-70, and 358 water molecules. Figure 1a was prepared with MOLSCRIPT 29 , Fig. 2 with program O 26 , and Figs 1e and 3 with GRASP 30 . Illustrations

The memory T cell pool functions as a dynamic repository of antigen-experienced T lymphocytes that accumulate over the lifetime of the individual. Recent studies indicate that memory T lymphocytes contain distinct populations of central memory (TCM) and effector memory (TEM) cells characterized by distinct homing capacity and effector function. This review addresses the heterogeneity of TCM and TEM, their differentiation stages, and the current models for their generation and maintenance in humans and mice.

SummaryWe have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T ceils. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate--dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF+ IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal-~issociated membrane protein-I, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor or, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.

Interleukin 17 (IL-17)-producing T helper cells (T(H)-17 cells) have been characterized in mice as a distinct subset of effector cells, but their identity and properties in humans remain elusive. We report here that expression of CCR6 and CCR4 together identified human memory CD4+ T cells selectively producing IL-17 and expressing mRNA encoding the human ortholog of mouse RORgammat, a transcription factor, whereas CCR6 and CXCR3 identified T(H)1 cells producing interferon-gamma and T helper cells producing both interferon-gamma and IL-17. Memory T cells specific for Candida albicans were present mainly in the CCR6+CCR4+ T(H)-17 subset, whereas memory T cells specific for Mycobacterium tuberculosis were present in CCR6+CXCR3+ T helper type 1 subset. The elicitation of IL-17 responses correlated with the capacity of C. albicans hyphae to stimulate antigen-presenting cells for the priming of T(H)-17 responses in vitro and for the production of IL-23 but not IL-12. Our results demonstrate that human T(H)-17 cells have distinct migratory capacity and antigenic specificities and establish a link between microbial products, T helper cell differentiation and homing in response to fungal antigens.

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) have been linked to host defense and autoimmune diseases. In mice, the differentiation of T(H)-17 cells requires transforming growth factor-beta and IL-6 and the transcription factor RORgammat. We report here that for human naive CD4(+) T cells, RORgammat expression and T(H)-17 polarization were induced by IL-1beta and enhanced by IL-6 but were suppressed by transforming growth factor-beta and IL-12. Monocytes and conventional dendritic cells, but not monocyte-derived dendritic cells activated by microbial stimuli, efficiently induced T(H)-17 priming, and this function correlated with antigen-presenting cell production of IL-1beta and IL-6 but not IL-12. Our results identify cytokines, antigen-presenting cells and microbial products that promote the polarization of human T(H)-17 cells and emphasize an important difference in the requirements for the differentiation of T(H)-17 cells in humans and mice.

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.