The CD8 T cell coreceptor exhibits disproportionate biological activity at extremely low binding affinities

T lymphocytes recognize peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. Recognition specificity is determined by the alphabeta T cell receptor (TCR). The T lymphocyte surface glycoproteins CD8 and CD4 enhance T cell antigen recognition by binding to MHC class I and class II molecules, respectively. Biophysical measurements have determined that equilibrium binding of the TCR with natural agonist peptide-MHC (pMHC) complexes occurs with KD values of 1-50 microm. The pMHCI/CD8 and pMHCII/CD4 interactions are significantly weaker than this (KD >100 microm), and the relative roles of TCR/pMHC and pMHC/coreceptor affinity in T cell activation remain controversial. Here, we engineer mutations in the MHCI heavy chain and beta2-microglobulin that further reduce or abolish the pMHCI/CD8 interaction to probe the significance of pMHC/coreceptor affinity in T cell activation. We demonstrate that the pMHCI/CD8 coreceptor interaction retains the vast majority of its biological activity at affinities that are reduced by over 15-fold (KD > 2 mm). In contrast to previous reports, we observe that the weak interaction between HLA A68 and CD8, which falls within this spectrum of reduced affinities, retains substantial functional activity. These findings are discussed in the context of current concepts of coreceptor dependence and the mechanism by which TCR coreceptors facilitate T cell activation.     

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.     

Quantifying the strength of ligand antagonism in TCR triggering

Triggering of the T cell receptor (TCR) may be antagonized by ligands that are slight variants of the immunogenic peptide. This paper proposes a mathematical model to quantify the strength of the antagonistic effect. The model is based on the kinetics of association and dissociation of TCR and peptide/major histocompatibility (pMHC) molecules, and incorporates TCR triggering according to a kinetic proofreading mechanism. Model analysis indicates that while the average lifetime of the TCR/pMHC complex is the basic determinant of the contribution to TCR triggering made by the ligand, the affinity of the ligand and its MHC presentation level are also important. However, these contributions depend on the kinetic limitation regime. There is a continuum of limitation regimes, at the extremes of which are found TCR limitation and MHC limitation. Both ligand affinity and TCR and pMHC densities determine whether TCR triggering is TCR limited or MHC limited. The changing importance of affinity and antigen presentation level under various kinetic limitation regimes may explain the respective roles of antagonistic and agonistic self peptides in thymic selection. Moreover, TCR down-regulation under TCR-limited conditions may allow the T cell to differentiate between the average lifetime of the TCR/pMHC complex and the presentation level of the ligand. A method for experimental differentiation between passive and active antagonistic effects is proposed which exploits the differences between TCR and MHC limitation.     

Antibody specific for the peptide.major histocompatibility complex. Is it T cell receptor-like?

Antibodies recognizing peptide bound to a major histocompatibility complex (MHC) protein usually have a higher affinity for the composite peptide.MHC (pMHC) ligand than T cell receptors (TCR) with the same specificity. Because the solvent-accessible peptide area constitutes only a small portion of the contacting pMHC surface, we hypothesized that the contribution of the MHC moiety to the TCR-pMHC complex stability is limited, ensuring a small increment of the binding energy delivered by the peptide to be distinguishable by the TCR or the peptide-specific antibody. This suggests that the gain in affinity of the antibody-pMHC interaction can be achieved through an increase in the on-rate without a significant change in the off-rate of the interaction. To test the hypothesis, we have analyzed the binding of an ovalbumin peptide (pOV8) and its variants associated with soluble H-2Kb protein to the 25-D1.16 monoclonal antibody and compared it with the binding of the same pMHC complexes to the OT-1 TCR. This comparison revealed a substantially higher on-rate of the antibody-pMHC interaction compared with the TCR-pMHC interaction. In contrast, both the antibody and the TCR-pMHC complexes exhibited comparably fast off-rates. Sequencing of the 25-D1.16 VH and VL genes showed that they have very few somatic mutations and those occur mainly in framework regions. We propose that the above features constitute a signature of the recognition of MHC-bound peptide antigens by TCR and TCR-like antibodies, which could explain why the latter are rarely produced in vivo.     

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ～1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ～1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.     

Decreased binding of peptides-MHC class I (pMHC) multimeric complexes to CD8 affects their binding avidity for the TCR but does not significantly impact on pMHC/TCR dissociation rate

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.     

The 3A6-TCR/superagonist/HLA-DR2a complex shows similar interface and reduced flexibility compared to the complex with self-peptide

T-cell receptor (TCR) recognition of the myelin basic protein (MBP) peptide presented by major histocompatibility complex (MHC) protein HLA-DR2a, one of the MHC class II alleles associated with multiple sclerosis, is highly variable. Interactions in the trimolecular complex between the TCR of the MBP83-99-specific T cell clone 3A6 with the MBP-peptide/HLA-DR2a (abbreviated TCR/pMHC) lead to substantially different proliferative responses when comparing the wild-type decapeptide MBP90-99 and a superagonist peptide, which differs mainly in the residues that point toward the TCR. Here, we investigate the influence of the peptide sequence on the interface and intrinsic plasticity of the TCR/pMHC trimolecular and pMHC bimolecular complexes by molecular dynamics simulations. The intermolecular contacts at the TCR/pMHC interface are similar for the complexes with the superagonist and the MBP self-peptide. The orientation angle between TCR and pMHC fluctuates less in the complex with the superagonist peptide. Thus, the higher structural stability of the TCR/pMHC tripartite complex with the superagonist peptide, rather than a major difference in binding mode with respect to the self-peptide, seems to be responsible for the stronger proliferative response.     

The space and time frames of T cell activation at the immunological synapse

The selective recognition of antigenic peptides by T cells requires the spatio/temporal integration of a panoply of molecular triggers. The space frame of T cell antigen receptors (TCR) interaction with peptide/MHC complexes (pMHC) displayed by antigen presenting cells is delineated by the micrometer-scale area of the immunological synapse. The time frame of T cell stimulation is governed by a series of short TCR-pMHC interactions that are integrated into sustained signaling leading to productive activation. We discuss here how approaching antigen recognition from the time and space angles is key to the comprehension of the puzzling process of T cell activation.     

Attenuated T Cell Responses to a High-Potency Ligand In Vivo

αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4⁺ T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.     

The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.     

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.     

Functional evidence that conserved TCR CDR alpha 3 loop docking governs the cross-recognition of closely related peptide:class I complexes.

The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2K(b) and H-2K(bm8). Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2-4 and the neighboring MHC side chains. We found that CTL clones sharing TCR beta-chains exhibited disparate recognition patterns, whereas those with drastically different TCRbeta-chains but sharing identical TCRalpha CDR3 loops displayed identical functional specificity. This suggested that the CDRalpha3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:K(b) crystal structure. Importantly, these results indicate a remarkable conservation in CDRalpha3 positioning, and, therefore, in docking of diverse TCRalphabeta heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.     

Kinetics and mechanics of two-dimensional interactions between T cell receptors and different activating ligands

Adaptive immune responses are driven by interactions between T cell antigen receptors (TCRs) and complexes of peptide antigens (p) bound to Major Histocompatibility Complex proteins (MHC) on the surface of antigen-presenting cells. Many experiments support the hypothesis that T cell response is quantitatively and qualitatively dependent on the so-called strength of TCR/pMHC association. Most available data are correlations between binding parameters measured in solution (three-dimensional) and pMHC activation potency, suggesting that full lymphocyte activation required a minimal lifetime for TCR/pMHC interaction. However, recent reports suggest important discrepancies between the binding properties of ligand-receptor couples measured in solution (three-dimensional) and those measured using surface-bound molecules (two-dimensional). Other reports suggest that bond mechanical strength may be important in addition to kinetic parameters. Here, we used a laminar flow chamber to monitor at the single molecule level the two-dimensional interaction between a recombinant human TCR and eight pMHCs with variable potency. We found that 1), two-dimensional dissociation rates were comparable to three-dimensional parameters previously obtained with the same molecules; 2), no significant correlation was found between association rates and activating potency of pMHCs; 3), bond mechanical strength was partly independent of bond lifetime; and 4), a suitable combination of bond lifetime and bond strength displayed optimal correlation with activation efficiency. These results suggest possible refinements of contemporary models of signal generation by T cell receptors. In conclusion, we reported, for the first time to our knowledge, the two-dimensional binding properties of eight TCR/pMHC couples in a cell-free system with single bond resolution.     

T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment

A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64)Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.     

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

The mammalian alpha/beta T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR-MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential.     

Epitope flexibility and dynamic footprint revealed by molecular dynamics of a pMHC-TCR complex

The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.     

TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01 + Rhesus Monkeys

Many of the factors that contribute to CD8⁺ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8⁺ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8⁺ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8⁺ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8⁺ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8⁺ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.     

Cutting edge: LFA-1 integrin-dependent T cell adhesion is regulated by both ag specificity and sensitivity

Ab stimulation of the TCR rapidly enhances the functional activity of the LFA-1 integrin. Although TCR-mediated changes in LFA-1 activity are thought to promote T cell-APC interactions, the Ag specificity and sensitivity of TCR-mediated triggering of LFA-1 is not clear. We demonstrate that peptide/MHC (pMHC) tetramers rapidly enhance LFA-1-dependent adhesion of OT-I TCR transgenic CD8(+) T cells to purified ICAM-1. Inhibition of src family tyrosine kinase or PI3K activity blocked pMHC tetramer- and anti-CD3-stimulated adhesion. These effects are highly specific because partial agonist and antagonist pMHC tetramers are unable to stimulate OT-I T cell adhesion to ICAM-1. The Ag thresholds required for T cell adhesion to ICAM-1 resemble those of early T cell activation events, because optimal LFA-1 activation occurs at tetramer concentrations that fail to induce maximal T cell proliferation. Thus, TCR signaling to LFA-1 is highly Ag specific and sensitive to low concentrations of Ag.     

The relationship between predicted peptide-MHC class II affinity and T-cell activation in a HLA-DRbeta1*0401 transgenic mouse model

The HLA-DRB1*0401 MHC class II molecule (DR4) is genetically associated with rheumatoid arthritis. It has been proposed that this MHC class II molecule participates in disease pathogenesis by presenting arthritogenic endogenous or exogenous peptides to CD4+ T cells, leading to their activation and resulting in an inflammatory response within the synovium. In order to better understand DR4 restricted T cell activation, we analyzed the candidate arthritogenic antigens type II collagen, human aggrecan, and the hepatitis B surface antigen for T-cell epitopes using a predictive model for determining peptide-DR4 affinity. We also applied this model to determine whether cross-reactive T-cell epitopes can be predicted based on known MHC-peptide-TCR interactions. Using the HLA-DR4-IE transgenic mouse, we showed that both T-cell proliferation and Th1 cytokine production (IFN-gamma) correlate with the predicted affinity of a peptide for DR4. In addition, we provide evidence that TCR recognition of a peptide-DR4 complex is highly specific in that similar antigenic peptide sequences, containing identical amino acids at TCR contact positions, do not activate the same population of T cells.     

A Novel Flow Cytometric High Throughput Assay for a Systematic Study on Molecular Mechanisms Underlying T Cell Receptor-Mediated Integrin Activation

Lymphocyte function-associated antigen 1 (LFA-1), a member of β2-integrin family, exerts multiple roles in host T cell immunity and has been identified as a useful drug-development target for inflammatory and autoimmune diseases. Applying the findings that primary resting T cells absorb nanometric membrane vesicles derived from antigen presenting cells (APC) via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide-major histocompatibility complex (MHC) complex (pMHC) and LFA-1 with its ligand, intercellular adhesion molecule-1 (ICAM-1), and that signaling cascades triggered by TCR/pMHC interaction take a part in the vesicle-absorption, we established a cell-based high throughput assay for systematic investigation, via isolation of small molecules modulating the level of vesicle-absorption, of molecular mechanisms underlying the T cell absorption of APC-derived vesicles, i.e., structural basis of TCR/pMHC and LFA-1/ICAM-1 interactions and TCR-mediated LFA-1 activation. As primary T cells along with physiological ligands expressed in biological membrane are used and also individual cells in assay samples are analyzed by flow cytometry, results obtained using the assay system hold superior physiological and therapeutic relevance as well as statistical precision.