The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation.

SUMMARY The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4+ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1+CXCR5+ CD4+ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV+ individuals.

T cell sensitivity to antigen is intrinsically regulated during maturation to ensure proper development of immunity and tolerance, but how this is accomplished remains elusive. Here we show that increasing miR-181a expression in mature T cells augments the sensitivity to peptide antigens, while inhibiting miR-181a expression in the immature T cells reduces sensitivity and impairs both positive and negative selection. Moreover, quantitative regulation of T cell sensitivity by miR-181a enables mature T cells to recognize antagonists-the inhibitory peptide antigens-as agonists. These effects are in part achieved by the downregulation of multiple phosphatases, which leads to elevated steady-state levels of phosphorylated intermediates and a reduction of the T cell receptor signaling threshold. Importantly, higher miR-181a expression correlates with greater T cell sensitivity in immature T cells, suggesting that miR-181a acts as an intrinsic antigen sensitivity "rheostat" during T cell development.

SUMMARY In vitro cancer cultures, including 3-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated Patient-Derived Organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immunooncology investigations within the TME and facilitate personalized immunotherapy testing.

SUMMARY There is considerable heterogeneity in immunological parameters between individuals, but its sources are largely unknown. To assess the relative contribution of heritable versus non-heritable factors, we have performed a systems-level analysis of 210 healthy twins between 8–82 years of age. We measured 204 different parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated (> 50% of variance) and 58% almost completely determined (> 80% of variance) by non-heritable influences. In addition, some of these parameters become more variable with age, suggesting the cumulative influence of environmental exposure. Similarly, the serological responses to seasonal influenza vaccination are also determined largely by non-heritable factors, likely due to repeated exposure to different strains. Lastly, in MZ twins discordant for cytomegalovirus infection, over half of all parameters are affected. These results highlight the largely reactive and adaptive nature of the immune system in healthy individuals.

The activation of T cells through interaction of their T-cell receptors with antigenic peptide bound to major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a crucial step in adaptive immunity. Here we use three-dimensional fluorescence microscopy to visualize individual peptide-I-E(k) class II MHC complexes labelled with the phycobiliprotein phycoerythrin in an effort to characterize T-cell sensitivity and the requirements for forming an immunological synapse in single cells. We show that T cells expressing the CD4 antigen respond with transient calcium signalling to even a single agonist peptide-MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present. This sensitivity is highly dependent on CD4, because blocking this molecule with antibodies renders T cells unable to detect less than about 30 ligands.

The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptidemajor histocompatibility complex (pMHC) molecules on other cells1. If productive, these interactions promote the formation of an immunological synapse2,3. Here we show that synaptic TCR-pMHC binding dynamics differ significantly from TCR-pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCRpMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4-12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR-pMHC complexes, indicating that the TCR binds pMHC independently of CD4. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. (refs 4-6). Nevertheless, T cells are highly specific and sensitive for antigen, able to detect even a single antigenic pMHC complex among structurally similar yet non-stimulatory pMHCs7,8. However, although this is useful for comparative purposes, these measurements do not account for the many constraints and special features of the synaptic environment and might not reflect what occurs in situ. In particular, the restricted intercellular volume should greatly accelerate the association rate and enhance serial engagement9-11. Favourable molecular alignment of TCR and MHC, as well as any molecular pre-clustering, could also drastically affect binding12-14. Although the volume effect can be approximated, the influence of the others is essentially unknown. Author ContributionsWe have therefore devised a method to measure synaptic TCR binding to pMHC directly, using single-molecule microscopy and FRET between a donor fluorophore on the TCR and an acceptor fluorophore on the peptide bound to a MHC molecule.We used T-cell blasts from two different TCR transgenic T-cell lines, 2B4 and 5c.c7, which are specific for the same moth cytochrome c peptide (MCC 88-103) bound to the class II MHC molecule IE k . To permit the use of highly sensitive and rapid total internal reflection (TIRF) microscopy, we used a modified planar lipid bilayer system as a surrogate antigenpresenting cell surface, presenting IE k complexes, ICAM-1 adhesion molecules and B7-1 co-stimulatory polypeptides (see Methods).Structural analysis of an Fab fragment derived f...

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dualcolor fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.A principal function of most T cells is to recognize foreign antigens on other cell surfaces, with the specificity determined by the T cell antigen receptor (TCR). Once the TCR has engaged its ligand and the signaling cascade has been initiated through the associated cluster of differentiation 3 (CD3) subunits, the kinase Zap70 transmits the signal to the adaptor Lat (linker for activation of T cells; A001392). Within seconds, actin-dependent signaling microclusters are formed in the periphery of the contact site between the T cell and antigenpresenting cell, and these microclusters then migrate to the center of the interface and form the immunological synapse [1][2][3][4][5][6] . Many signaling molecules, including the TCR and Lat, colocalize in the microclusters during early T cell activation [3][4][5][6] . On the basis of electron-microscopy studies, varying degrees of preclustering of plasma membrane molecules, including the TCR and Lat, have been proposed 7-9 . However, there are concerns about potential artifacts with this approach (for example, from fixation, gold particle staining and/or drying). Additionally, fluorescence microscopy and biochemical technologies suggest the coexistence of monomeric and multivalent TCR-CD3 complexes 9,10 . It is not clear to what extent these multivalent complexes exist or if they form through protein-protein interactions or localization in membrane domains. In the case of biochemical approaches, cell lysis itself could introduce additional artifacts. To overcome such limitations and to gain insight into the organization of plasma membrane proteins in situ, we analyzed the organization of the TCR and Lat in quiescent and activated native T cell plasma membrane sheets and live T cells. We achieved this with a combination of three techniques: a modified high-speed version of photoactivated localization microscopy (hsPALM) 11-13 , dual-color fluorescence cross-correlation spectroscopy (dcFCCS) [14][15][16] and transmission electron microscopy (TEM) 7,8,17,18 . NIH Public AccessWe show that both the TCR and Lat were preclustered i...