Effects of a local auxiliary protein on the two-dimensional affinity of a TCR-peptide MHC interaction.

The affinity of T-cell receptors (TCRs) for major histocompatibility complex molecules (MHCs) presenting cognate antigens likely determines whether T cells initiate immune responses, or not. There exist few measurements of two-dimensional (2D) TCR-MHC interactions, and the effect of auxiliary proteins on binding is unexplored. Here, Jurkat T-cells expressing the MHC molecule HLA-DQ8-glia-α1 and the ligand of an adhesion protein (rat CD2) were allowed to bind supported lipid bilayers (SLBs) presenting fluorescently labelled L3-12 TCR and rat CD2, allowing measurements of binding unconfounded by cell signaling effects or co-receptor binding. The 2D Kd for L3-12 TCR binding to HLA-DQ8-glia-α1, of 14±5 molecules/µm2 (mean±s.d.), was only marginally influenced by including CD2 up to ∼200 bound molecules/µm2 but higher CD2 densities reduced the affinity up to 1.9-fold. Cell-SLB contact size increased steadily with ligand density without affecting binding for contacts at up to ∼20% of total cell area, but beyond this lamellipodia appeared, giving an apparent increase in bound receptors of up to 50%. Our findings show how parameters other than the specific protein-protein interaction can influence binding behavior at cell-cell contacts.

Single-cell measurements of two-dimensional binding affinity across cell contacts.

The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/Kd), and the spread in affinities within the cell population was low, Kd = 4.9 ± 0.9 molecules/µm2 (mean ± SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/µm2, after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D Kd within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.

HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma.

BACKGROUND: Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours. METHODS: We studied the expression of APM components and HLA-I, as well as HLA-I tapasin-dependency profiles in glioblastoma tissues and corresponding cell lines. RESULTS: Tapasin displayed the strongest correlation to HLA-I heavy chain but also clustered with ß2-microglobulin, transporter associated with antigen processing (TAP) and LMP. Moreover, tapasin also correlated to survival of glioblastoma patients. Some APM components, for example, TAP1/TAP2 and LMP2/LMP7, showed variable but coordinated expression, whereas ERAP1/ERAP2 displayed an imbalanced expression pattern. Furthermore, analysis of HLA-I profiles revealed variable tapasin dependence of HLA-I allomorphs in glioblastoma patients. CONCLUSIONS: Expression of APM proteins is highly variable between glioblastomas. Tapasin stands out as the APM component strongest correlated to HLA-I expression and we proved that HLA-I profiles in glioblastoma patients include tapasin-dependent allomorphs. The level of tapasin was also correlated with patient survival time. Our results support the need for individualisation of immunotherapy protocols.

Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics.

Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact. A few important examples of these are: protein density, membrane fluctuations, force on the bond and the use of auxiliary binding molecules. The development of model membranes, and in particular supported lipid bilayers (SLBs), has made it possible to simplify the studied contact to analyze these effects and to measure 2D binding kinetics of individual protein-protein interactions. We will in this review give an overview of, and discuss, how different SLB systems have been used for this and compare different methods to measure binding kinetics in cell-SLB contacts. Typically, the SLB is functionalized with fluorescently labelled ligands whose interaction with the corresponding receptor on a binding cell can be detected. This interaction can either be studied 1) by an accumulation of ligands in the cell-SLB contact, whose magnitude depends on the density of the proteins and binding affinity of the interaction, or 2) by tracking single ligands in the SLB, which upon interaction with a receptor result in a change of motion of the diffusing ligand. The advantages and disadvantages of other methods measuring 2D binding kinetics will also be discussed and compared to the fluorescence-based methods. Although binding kinetic measurements in cell-SLB contacts have provided novel information on how ligands interact with receptors in vivo the number of these measurements is still limited. This is influenced by the complexity of the system as well as the required experimental time. Moreover, the outcome can vary significantly between studies, highlighting the necessity for continued development of methods to study 2D binding kinetics with higher precision and ease.

Calcium Signaling in T Cells Is Induced by Binding to Nickel-Chelating Lipids in Supported Lipid Bilayers.

Supported lipid bilayers (SLBs) are one of the most common cell-membrane model systems to study cell-cell interactions. Nickel-chelating lipids are frequently used to functionalize the SLB with polyhistidine-tagged ligands. We show here that these lipids by themselves can induce calcium signaling in T cells, also when having protein ligands on the SLB. This is important to avoid "false" signaling events in cell studies with SLBs, but also to better understand the molecular mechanisms involved in T-cell signaling. Jurkat T cells transfected with the non-signaling molecule rat CD48 were found to bind to ligand-free SLBs containing ≥2 wt% nickel-chelating lipids upon which calcium signaling was induced. This signaling fraction steadily increased from 24 to 60% when increasing the amount of nickel-chelating lipids from 2 to 10 wt%. Both the signaling fraction and signaling time did not change significantly compared to ligand-free SLBs when adding the CD48-ligand rat CD2 to the SLB. Blocking the SLB with bovine serum albumin reduced the signaling fraction to 11%, while preserving CD2 binding and the exclusion of the phosphatase CD45 from the cell-SLB contacts. Thus, CD45 exclusion alone was not sufficient to result in calcium signaling. In addition, more cells signaled on ligand-free SLBs with copper-chelating lipids instead of nickel-chelating lipids and the signaling was found to be predominantly via T-cell receptor (TCR) triggering. Hence, it is possible that the nickel-chelating lipids act as ligands to the cell's TCRs, an interaction that needs to be blocked to avoid unwanted cell activation.

Dimensions and Interactions of Large T-Cell Surface Proteins.

The first step of the adaptive immune response involves the interaction of T cells that express T-cell receptors (TCRs) with peptide-loaded major histocompatibility complexes expressed by antigen-presenting cells (APCs). Exactly how this leads to activation of the TCR and to downstream signaling is uncertain, however. Recent findings suggest that one of the key events is the exclusion of the large receptor-type tyrosine phosphatase CD45, from close contacts formed at sites of T-cell/APC interaction. If this is true, a full understanding of how close contact formation leads to signaling would require insights into the structures of, and interactions between, large membrane proteins like CD45 and other proteins forming the glycocalyx, such as CD43. Structural insights into the overall dimensions of these proteins using crystallographic methods are hard to obtain, and their conformations on the cell surface are also unknown. Several imaging-based optical microscopy techniques have however been developed for analyzing protein dimensions and orientation on model cell surfaces with nanometer precision. Here we review some of these methods with a focus on the use of hydrodynamic trapping, which relies on liquid flow from a micropipette to move and trap membrane-associated fluorescently labeled molecules. Important insights that have been obtained include (i) how protein flexibility and coverage might affect the effective heights of these molecules, (ii) the height of proteins on the membrane as a key parameter determining how they will distribute in cell-cell contacts, and (iii) how repulsive interactions between the extracellular parts of the proteins influences protein aggregation and distribution.

Hydrodynamic trapping measures the interaction between membrane-associated molecules.

How membrane proteins distribute and behave on the surface of cells depends on the molecules' chemical potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. Here, we present a method we call hydrodynamic trapping that can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding above a lipid membrane. The chemical potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this method to study four representative proteins, with different height-to-width ratios and molecular properties; from globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The data we obtain illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins, as well as underlining the general applicability of the method.

Role of Blimp-1 in programing Th effector cells into IL-10 producers.

Secretion of the immunosuppressive cytokine interleukin (IL) 10 by effector T cells is an essential mechanism of self-limitation during infection. However, the transcriptional regulation of IL-10 expression in proinflammatory T helper (Th) 1 cells is insufficiently understood. We report a crucial role for the transcriptional regulator Blimp-1, induced by IL-12 in a STAT4-dependent manner, in controlling IL-10 expression in Th1 cells. Blimp-1 deficiency led to excessive inflammation during Toxoplasma gondii infection with increased mortality. IL-10 production from Th1 cells was strictly dependent on Blimp-1 but was further enhanced by the synergistic function of c-Maf, a transcriptional regulator of IL-10 induced by multiple factors, such as the Notch pathway. We found Blimp-1 expression, which was also broadly induced by IL-27 in effector T cells, to be antagonized by transforming growth factor (TGF) ß. While effectively blocking IL-10 production from Th1 cells, TGF-ß shifted IL-10 regulation from a Blimp-1-dependent to a Blimp-1-independent pathway in IL-27-induced Tr1 (T regulatory 1) cells. Our findings further illustrate how IL-10 regulation in Th cells relies on several transcriptional programs that integrate various signals from the environment to fine-tune expression of this critical immunosuppressive cytokine.