Corticostriatal combinatorics: the implications of corticostriatal axonal arborizations

The complete striatal axonal arborizations of 16 juxtacellularly stained cortical pyramidal cells were analyzed. Corticostriatal neurons were located in the medial agranular or anterior cingulate cortex of rats. All axons were of the extended type and formed synaptic contacts in both the striosomal and matrix compartments as determined by counterstaining for the mu-opiate receptor. Six axonal arborizations were from collaterals of brain stem-projecting cells and the other 10 from bilaterally projecting cells with no brain stem projections. The distribution of synaptic boutons along the axons were convolved with the average dendritic tree volume of spiny projection neurons to obtain an axonal innervation volume and innervation density map for each axon. Innervation volumes varied widely, with single axons occupying between 0.4 and 14.2% of the striatum (average = 4%). The total number of boutons formed by individual axons ranged from 25 to 2,900 (average = 879). Within the innervation volume, the density of innervation was extremely sparse but inhomogeneous. The pattern of innervation resembled matrisomes, as defined by bulk labeling and functional mapping experiments, superimposed on a low background innervation. Using this sample as representative of all corticostriatal axons, the total number of corticostriatal neurons was estimated to be 17 million, about 10 times the number of striatal projection neurons.     

Predicting interactions between T cell receptors and MHC-peptide complexes

Conserved interactions between T cell receptors (TCRs) and major histocompatibility complex (MHC) proteins with bound peptide antigens are not well understood. In order to gain a better understanding of the interaction modes of human TCR variable (V) regions, we have performed a structural analysis of the TCRs bound to their MHC-peptide ligands in human, using the available structural models determined by X-ray crystallography. We identified important differences to previous studies in which such interactions were evaluated. Based on the interactions found in the actual experimental structures we developed the first rule-based approach for predicting the ability of TCR residues in the complementarity-determining region (CDR) 1, CDR2, and CDR3 loops to interact with the MHC-peptide antigen complex. Two relatively simple algorithms show good performance under cross validation.     

Use of fluorescence polarization to monitor MHC-peptide interactions in solution

We describe here fluorescence polarization-based methods to investigate class I MHC-peptide interactions in solution. Fluorescein-labelled peptides were used to determine MHC/peptide complex association and dissociation constants as well as the equilibrium binding constant (KD). Furthermore, we developed a competition assay for the determination of IC50 values of nonlabelled compounds. Both kinetic and equilibrium parameters are of prime importance for the development of immunomodulating compounds. The assays described here show a good reproducibility and require only picomolar amounts of labelled tracers. A high ratio between the experimental values obtained for bound and free labelled ligand as well as a low standard deviation, permits the detection of class I MHC ligands with low affinity. Fluorescence polarization allows the direct measurement of the ratio between free and bound labelled ligand in solution without any separation step. Thus, in combination with microtiter-plates, the time for analysis is significantly decreased to 10 s per sample. Our assays represent versatile tools for characterizing the binding of single ligands as well as for rapid screening of large numbers of compounds.     

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides. The scMHC molecule was composed of beta2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker. Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-gamma, and were able to stain specifically CTL lines. Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.     

MHC-peptide ligand interactions establish a functional threshold for antigen-specific T cell recognition.

Antigen-specific T cell recognition is dependent on the functional density of the TCR-ligand, which consists of specific MHC molecules and a specifically bound peptide. We have examined the influence of the affinity and concentration of exogenous peptide and the density of specific MHC molecules on the proliferation of a CD4+, DQA1*0501/DQB1*0201 (DQ2.1)-restricted, HSV-2-specific T cell clone. Using antigen peptide analogs with different mutations of known DQ2-anchor residues, T cell response was reduced in an peptide-affinity and - concentration specific manner. The decrease using weaker binding peptides was gradual as stimulation with a peptide with intermediate affinity yielded intermediate T cell proliferation and the poorest binding peptide induced an even weaker T cell response. MHC class II density on the APC was modified using DQ2 homo- and heterozygous B-LCLs as APCs, however this variation of MHC concentration had no effect on T cell proliferation. We interpret this as a reflection of a low threshold for activation of the T cell clone, in which peptide-MHC avidity is the over-riding determinant of the strength of ligand signal.     

Role of the CDR1 region of the TCR {beta} chain in the binding to purified MHC-peptide complex

Single alanine substitutions were introduced into the CDR1 regionof the ß chain of a K^d-restricted TCR. Mutants andwild-type TCR were attached to the chain of the CD3 complexand expressed at the surface of a rat basophil cell line. Transfectantswere tested for the binding of purified soluble K^d-peptlde complexes.With this experimental system, accessory molecules are unlikelyto play a major role and the contribution of each residue tothe interaction can be addressed. Results show that all positionsin the CDR1 region are involved in the binding to the K^d-peptidecomplex but at varying degrees. These effects are discussedin relation to a molecular model of the TCR. Comparison of theseresults with previous data obtained in a T cell hybridoma systemsuggests the existence of a threshold in the TCR affinity necessaryfor mature T cell activation.     

T cells distinguish MHC-peptide complexes formed in separate vesicles and edited by H2-DM

The peptide spanning residues 48-61 of hen egg white lysozyme (HEL) presented by I-A(k) gives rise to two T cell populations, referred to as type A and B, that distinguish the complex generated intracellularly upon processing of HEL from that formed with exogenous peptide. Here, we ascribe this difference to recognition of distinct conformers of the complex and show that formation of the two complexes results from antigen processing in different intracellular compartments and is dependent upon H2-DM. While the type A complex preferentially formed in a lysosome-like late vesicle, the type B complex failed to form in this compartment; this distinction was abolished in antigen-presenting cells lacking DM. Experiments in vitro indicated that H2-DM acts directly on the complex to eliminate the type B conformation. We conclude that different antigen-processing pathways generate distinct MHC-peptide conformers, priming T cells with distinct specificity that may play unique roles in immunity.     

ICAM-1 enhances MHC-peptide activation of CD8(+) T cells without an organized immunological synapse

In addition to the TCR-ligand interaction, other receptor-ligand pairs, such as LFA-1 and ICAM-1, play a major role in the activation of T cells. Recent studies of T cell activation suggest a coordinated movement of LFA-1 and ICAM-1 in forming a defined zone in the immunological synapse. It is unclear from these studies whether the organized molecular geometry of the immunological synapse is necessary for ICAM-1 enhancement of T cell activation. In this report, we demonstrate that ICAM-1 can enhance the activation of CD8(+) T cells by MHC-peptide in the absence of an organized immunologic synapse. Therefore, although the molecular organization of the immunologic synapse may amplify stimuli, it is not an absolute requirement for either CD8(+) T cell activation or the ICAM-1 enhancement of TCR activation.     

The relationship of MHC-peptide binding and T cell activation probed using chemically defined MHC class II oligomers

A series of novel chemically defined soluble oligomers of the human MHC class II protein HLA-DR1 was constructed to probe the molecular requirements for initiation of T cell activation. MHC dimers, trimers, and tetramers stimulated T cells, as measured by upregulation of the activation markers CD69 and CD25, and by internalization of activated T cell receptor subunits. Monomeric MHC-peptide complexes engaged T cell receptors but did not induce activation. For a given amount of receptor engagement, the extent of activation was equivalent for each of the oligomers and correlated with the number of T cell receptor cross-links induced. These results suggest that formation or rearrangement of a T cell receptor dimer is necessary and sufficient for initiation of T cell signaling.     

Human CD8 co-receptor is strictly involved in MHC-peptide tetramer-TCR binding and T cell activation.

Although there has been extensive analysis on the capacity of MHC-peptide tetramers to bind antigen-specific TCR, there have been comparatively few studies regarding the role of the CD4 and CD8 co-receptors in binding and activation by these multimeric molecules. Here, we start from the observation that different antibodies against human CD8 exert opposite effects on MHC-peptide tetramer binding to the TCR: tetramer staining was enhanced by OKT8 antibody, while it was blocked with SK1 antibody. We used these different anti-CD8 antibodies to modulate CD8 function during tetramer staining of Melan-A/MART1-specific CTL clones. We show that CD8 action could be variably modulated during all the phases of interaction, indicating that CD8 participates in both the initial association of the TCR with MHC-peptide tetramers and the stability of this interaction. While the blocking effect of anti-CD8 antibodies was mostly exerted during the initial binding of the TCR with MHC-peptide tetramers, the enhancing effect was exerted by augmenting the duration of this interaction. Blocking anti-CD8 antibodies were also capable of preventing tetramer-mediated T cell activation. The possibility of variably affecting MHC-peptide tetramer binding and T cell activation using anti-CD8 antibodies confirms the critical role exerted by the CD8 co-receptor in this interaction and supports the notion that TCR engagement by MHC-peptide ligands typically involves CD8.     

On the role of high- and low-abundance class II MHC-peptide complexes in the thymic positive selection of CD4(+) T cells

The role of self-peptides bound to MHC molecules in the selection of T cells in the thymus remains controversial. Here, we have tested whether a high-abundance single class II MHC-peptide complex has a dominant effect on the repertoire of CD4(+) T cells selected by low-abundance class II MHC-peptide complexes. For these studies, we have used H-2(b) mice that lack an invariant chain (Ii) (A(b)Ii(-)) and their transgenic variant (A(b)A(b)EpIi(-)) that co-expresses A(b) molecules covalently bound with a single peptide Ep(52-68). In these latter mice, close to 50% of all A(b) molecules are occupied by Ep(52-68) peptide. Although the A(b)Ep complex was abundantly expressed in the thymus under conditions excluding negative selection on bone marrow-derived cells, no striking quantitative difference between repertoires of TCR expressed on CD4(+) T cells in A(b)Ii(-) and A(b)A(b)EpIi(-) mice was noticed. Our results are consistent with the view that diverse, low-abundance self-peptides play an important role in thymic positive selection and do not support the notion that dominant, high-abundance peptides may be critical for shaping the TCR repertoire.     

Towards assessing the sympathovagal balance

Exact assessment of the autonomic nervous system’s (ANS) activity by means of heart rate variability (HRV) is a long-standing challenge. Although many techniques have been proposed to take up the challenge, none ever proposed a rationale for the approach behind the technique or a satisfying discrimination of the two activities which underlie the autonomic control of HRV. We here propose a new method, providing both an understanding of the discrimination’s nature and a framework which we believe leads to a thorough assessment of the sympathovagal balance, as a trajectory between points in a well-chosen space. The methodology assumes tools from scale invariance/covariance physics. The sympathovagal balance is obtained on a beat-to-beat basis with the dynamics portrayed through a trajectory. Furthermore, universal trajectories are sought which would comprehensively describe the effect of atropine and isoproterenol injections on systems underlying the heart pace variations. Non-invasive assessment of the respective activities of the sympathetic and parasympathetic subsystems of the ANS would be possible through cardiac autonomic measurements.     

Extended presentation of specific MHC-peptide complexes by mature dendritic cells compared to other types of antigen-presenting cells

Dendritic cells are known as the most potent antigen-presenting cells for the induction of T cell-mediated immune responses. To discriminate between the presentation of antigens and the co-stimulatory aspects of this high immunostimulatory capacity, we used recombinant antibodies with T cell receptor-like specificity to detect defined MHC-peptide complexes on living cells. Mature human dendritic cells (mDC) were compared with immature DC (iDC), monocytes, CD4(+) T lymphocytes, melanoma cells, T2 cells and B lymphoblastoid cells for their capacity to present MHC class I-restricted tumor-associated T cell epitopes and were found to display the specific peptides two to six times longer than other cells. The most short-lived peptide had an average half-life of 8.7 h on mDCvs. 3.5 h on B lymphoblastoid cells, while the most long-lived peptide had a half-life of 118.5 h vs. 20.7 h on these two cell types. The decay kinetics of specific MHC-peptide complexes on iDC were among the fastest observed. The high potency of dendritic cells to induce specific T cell responses is thus based, in addition to the expression of co-stimulatory molecules, on an extended antigenic memory, which increases the likelihood and the extent of contacts between dendritic cells and antigen-specific T cells.     

Towards cytoprotection in the peritransplant period

As a consequence of ischemia–reperfusion injury of whole organ transplants and hypoxia–anoxia of cell transplants, transplantation unavoidably triggers adverse, cytodestructive inflammation within the allograft. Interventions that dampen adverse inflammation may limit the extent and duration of this injury, and preserve tissue function. Moreover, these interventions should create a milieu that guides many donor-activated T cells into a tissue-protective phenotype, thus promoting graft acceptance or even tolerance. Hence, it is useful, maybe crucial, to identify the measures that minimize deleterious consequences of acute and chronic inflammation upon allograft. Several therapies that inhibit activity of certain proinflammatory cytokines or expression of tissue “danger signals”, while sustaining or potentially enhancing the expression of tissue-intrinsic anti-inflammatory and cytoprotective genes, are awaiting clinical trials or are already approved for the treatment of immuno-inflammatory disorders. If applied in the peritransplant period, such cytoprotective regimens may increase the pool of donor organs suitable for transplantation, reduce the overall requirements for maintenance immunosuppression and perhaps foster transplant tolerance.