Affinity Threshold Research Articles

Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4+ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo, with implications for vaccine design.

Uncovering antibody cross-reaction dynamics in influenza A infections.

Influenza viruses are a cause of large outbreaks and pandemics with high death tolls. A key obstacle is that flu vaccines have inconsistent performance, in the best cases up to 60% effectiveness, but it can be as low as 10%. Uncovering the hidden pathways of how antibodies (Abs) induced by one influenza strain are effective against another, cross-reaction, is a central vexation for the design of universal flu vaccines. We conceive a stochastic model that successfully represents the antibody cross-reactive data from mice infected with H3N2 influenza strains and further validation with cross-reaction data of H1N1 strains. Using a High-Performance Computing cluster, several aspects and parameters in the model were tested. Computational simulations highlight that changes in time of infection and the B-cells population are relevant, however, the affinity threshold of B-cells between consecutive infections is a necessary condition for the successful Abs cross-reaction. Our results suggest a 3-D reformulation of the current influenza antibody landscape for the representation and modeling of cross-reactive data. The full code as a testing/simulation platform is freely available here: https://github.com/systemsmedicine/Antibody_cross-reaction_dynamics. Supplementary data are available at Bioinformatics online.

Factors that influence the thymic selection of CD8αα intraepithelial lymphocytes.

Thymocytes bearing αβ T cell receptors (TCRαβ) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.

Application of individual HLA-binding prediction thresholds increases the detection of target cell surface-presented HPV16 E6- and E7-derived epitopes as a basis for therapeutic HPV vaccine design

Abstract For the development of a therapeutic vaccine against human papillomavirus (HPV), viral epitopes bona fide presented on the surface of HPV-transformed cells need to be identified. The constitutively expressed HPV oncoproteins E6 and E7 represent ideal targets for immunotherapeutic vaccination approaches. However, viral epitopes are low abundant and human leukocyte antigen (HLA)-restricted, posing a challenge for developing peptide-based vaccines. For the identification of novel epitopes, in silico HLA-binding prediction was used to preselect HPV16 E6- and E7-derived peptides for 7 major HLA-types representing 5 HLA supertypes. Based on performance evaluation of predictors, new individual binding affinity thresholds were applied. HLA-binding of synthesized predicted binders was validated in vitro in competitive cellular binding assays. Interferon-γ ELISpot assays performed with blood samples from HLA-characterized healthy donors identified memory T cell responses against HLA-binding HPV peptides. T cells of responding donors were further characterized for epitope-specific mediation of specific target cell lysis by CD8+ CTLs using a flow cytometry based cytotoxicity assay. Complementary, an immunopeptidomics approach was used to determine the surface presentation of epitopes on HPV16-transformed cancer cells. Targeted liquid chromatography-mass spectrometry was capable of detecting epitopes predicted within the new individual affinity thresholds but missed by common thresholds. Completing the identification of bona fide target cell surface-presented E6- and E7-derived T cell epitopes will provide a map of targets for the development of a therapeutic vaccine to treat HPV16-induced malignancies.

Antigen valency dictates the affinity of B cells recruited to germinal centers and the magnitude of extrafollicular B cell responses

Abstract While it is widely agreed that antigen valency is a factor in immunogenicity, how antigen valency contributes to B cell selection during antigen-specific responses is poorly understood. Here, using HIV immunogens with a defined valency ranging from 1 to 60, we investigated the role of antigen valency during different phases of the antigen-specific B cell response. We show that highly multimerized immunogens rapidly activated cognate B cells in vivo with little affinity discrimination, initially. This subsequently led to strong extrafollicular and germinal center responses where B cells within a broad affinity range were efficiently recruited. Low-valency (1–8 copy) antigens induced less diverse effector B cell responses, with limited recruitment of low-affinity B cells. The data reveal roles of antigen valency in dictating affinity thresholds required for recruitment of B cells to germinal centers or rapid extrafollicular plasma cell responses, with important implications for vaccine design.

THEMIS functions as a cytoplasmic adaptor and nuclear transcription factor in developing thymocytes and mature T cells

Abstract THEMIS functions in the cytoplasm as an adaptor molecule which dampens TCR signaling. During thymic development, THEMIS sets the affinity threshold for activation, enabling the selection of conventional T cells in response to low affinity antigens. Although these events occur in the cytoplasm, THEMIS possess a nuclear localization signal (NLS) and a significant amount of THEMIS resides in the nucleus. To investigate the importance of THEMIS in the nucleus, we deleted the Themis NLS to force THEMIS in the cytoplasm. Surprisingly, T cell development was impaired similar to the Themis null mice, suggesting that THEMIS may have important roles in the nucleus as well. Alternatively, we established Themis mutant mice, in which THEMIS localized exclusively in the nucleus or where THEMIS was constantly present in both compartments. Normal conventional T cells were absent in both mutant mice. Since lack of THEMIS or the constant presence of THEMIS in the cytoplasm or nucleus or both, all resulted in aberrant T cell development it indicated that the translocation of THEMIS to or/and from the nucleus is essential for its function. To examine the role of THEMIS in T cells in the periphery, we generated mutant mice where THEMIS is expressed normal during thymic development but altered post-thymically. Adoptive transfer of naïve CD4 T cells with altered THEMIS expression into RAG deficient host induced enhanced sever colitis and furthermore, we observed that forced localization of THEMIS in the nucleus redirects the polarization of CD4 T cells toward the pathogenic TH17 helper lineage. These findings identify a dual function for THEMIS as a cytoplasmic adaptor during TCR signaling and a nuclear factor directing gene expression in thymocytes and mature T cells.

Affinity threshold conversion: the progressive differentiation checkpoint at which the TCRα/CD3δ signaling axis controls thymic positive selection

Abstract Upon positive selection of conventional αβ T cells, CD4+ CD8+ double-positive (DP) cells transition to single-positive (SP) and reduce their responsiveness to weak peptide/MHC ligands (affinity threshold conversion, ATC). We report that ATC actually begins prior to the completion of positive selection in the DP stage, and this checkpoint is where the TCRα/CD3δ signaling axis controls positive selection. We dissected ATC in the OT1 TCR transgenic system in conjunction with several different genes whose null mutations block thymic development at DP stage: β2m, CD3δ, and/or TCRα-Connecting Peptide Motif (αCPM). In the absence of MHC class I (β2m0), CD3δ or αCPM mutants were blocked at pre-selection DP stage and displayed immature, high-sensitivity to weak TCR ligands. In contrast, when MHC class I was present (β2m+), CD3δ or αCPM mutants were blocked at DP stage but underwent partial ATC; they began positive selection but did not complete it. Upon exogenous TCR engagement of partial-ATC thymocytes, the proximal signaling protein interaction network showed overall weakened activity, notably decreasing SOS1 participation linked to the downstream Ras/Raf/ERK selection pathway. Provision of high affinity peptide/MHC in FTOC rescued completion of positive selection for partial-ATC mutant thymocytes. Finally, comparing nontransgenic, polyclonal B6 vs. CD3δ0 DP thymocytes, although most cells showed equal maturation state, a subset of cells in B6 progressed in maturation relative to CD3δ0. We propose the existence of an intermediate checkpoint inside DP stage, in which ATC already begins to reduce responsiveness to selection ligands, yet further TCR signaling is required to complete positive selection.

Monovalent IgG4 autoantibodies require self-antigen driven affinity maturation to acquire pathogenic capacity

Abstract INTRODUCTION The mechanisms underlying B cell-mediated autoimmune disease and the origin of autoreactive B cells are not well understood. Human monoclonal autoantibodies (mAbs) are valuable tools for investigating both. In this study, we used mAbs derived from patients with the autoimmune disorder, myasthenia gravis (MG). A subset of patients with MG have pathogenic autoantibodies that recognize muscle-specific tyrosine kinase (MuSK). The autoantibodies of MuSK MG are predominantly of the IgG4 subclass and functionally monovalent as a result of Fab-arm exchange. OBJECTIVE To gain insight into the origin and development of these unique autoantibodies. METHODS AND RESULTS We examined MG patient-derived mAbs, their corresponding germline-encoded unmutated common ancestors (UCA) and monovalent antigen-binding fragments (Fabs) to investigate how antigen-driven affinity maturation contributes to both binding and immunopathology. Mature mAbs, their UCA counterparts and mature monovalent Fabs bound to the MuSK autoantigen and retained their pathogenic capacity. However, monovalent UCA Fabs, which still bound the autoantigen, lost their pathogenic capacity. The mature Fabs were characterized by very high affinity (sub-nanomolar) driven by a rapid on-rate and slow off-rate. However, the UCA affinity was approximately 100-fold less than the mature Fabs, which was driven by a rapid off-rate. SUMMARY/CONCLUSION These findings indicate that the autoantigen initiates the autoimmune response in MuSK MG and drives autoimmunity through the accumulation of somatic hypermutations such that monovalent IgG4 Fab-arm exchanged MG autoantibodies reach a high affinity threshold required for pathogenic capacity.

TCR Affinity for In Vivo Peptide-Induced Thymic Positive Selection Fine-Tunes TCR Responsiveness of Peripheral CD8+ T Cells.

The affinity for TCR interactions with self-peptide/MHC complexes (pMHC) in the thymus critically affects immature thymocytes that newly express TCRs. Previous fetal thymus organ culture experiments have indicated that difference in the affinity for thymic TCR/pMHC interactions not only determines thymocyte fate between positive and negative selection, but also affects Ag responsiveness of positively selected thymocytes. In the current study, we examined whether TCR/pMHC affinity during positive selection in the thymus would further affect Ag responsiveness of mature T cells in the periphery. To do so, OVA peptide variants were in vivo administered to TAP1-deficient OT-I/TCR-transgenic mice in which T cell development was otherwise arrested at CD4+CD8+ thymocytes because of the lack of self-pMHC presentation in thymic APCs. We found that a group of peptide variants induced the transient generation of OT-I CD8+ T cells in the thymus and the periphery. We also noticed that the affinity threshold for positive and negative selection detected in adult mice in vivo was higher than that measured in fetal thymus organ culture experiments in vitro. Interestingly, we further found that the affinity for positively selecting peptides proportionally affected TCR responsiveness of peripheral naive CD8+ T cells. These results indicate that in vivo administration of a peptide can promote T cell selection in the thymus and the affinity for TCR/pMHC interaction during positive selection fine-tunes Ag responsiveness of peripheral T cells.

Quantification of Uncertainty in Peptide-MHC Binding Prediction Improves High-Affinity Peptide Selection for Therapeutic Design.

The computational identification of peptides that can bind the major histocompatibility complex (MHC) with high affinity is an essential step in developing personal immunotherapies and vaccines. We introduce PUFFIN, a deep residual network-based computational approach that quantifies uncertainty in peptide-MHC affinity prediction that arises from observational noise and the lack of relevant training examples. With PUFFIN's uncertainty metrics, we define binding likelihood, the probability a peptide binds to a given MHC allele at a specified affinity threshold. Compared to affinity point estimates, we find that binding likelihood correlates better with the observed affinity and reduces false positives inhigh-affinity peptide design. When applied to examine an existing peptide vaccine, PUFFIN identifies an alternative vaccine formulation with higher binding likelihood. PUFFIN is freely available for download at http://github.com/gifford-lab/PUFFIN.

Natural Product Target Network Reveals Potential for Cancer Combination Therapies.

A body of research demonstrates examples of in vitro and in vivo synergy between natural products and anti-neoplastic drugs for some cancers. However, the underlying biological mechanisms are still elusive. To better understand biological entities targeted by natural products and therefore provide rational evidence for future novel combination therapies for cancer treatment, we assess the targetable space of natural products using public domain compound-target information. When considering pathways from the Reactome database targeted by natural products, we found an increase in coverage of 61% (725 pathways), relative to pathways covered by FDA approved cancer drugs collected in the Cancer Targetome, a resource for evidence-based drug-target interactions. Not only is the coverage of pathways targeted by compounds increased when we include natural products, but coverage of targets within those pathways is also increased. Furthermore, we examined the distribution of cancer driver genes across pathways to assess relevance of natural products to critical cancer therapeutic space. We found 24 pathways enriched for cancer drivers that had no available cancer drug interactions at a potentially clinically relevant binding affinity threshold of < 100nM that had at least one natural product interaction at that same binding threshold. Assessment of network context highlighted the fact that natural products show target family groupings both distinct from and in common with cancer drugs, strengthening the complementary potential for natural products in the cancer therapeutic space. In conclusion, our study provides a foundation for developing novel cancer treatment with the combination of drugs and natural products.

Study of energy level alignment at weakly interacting small organic molecular thin film interfaces: The validity of classical model from inorganics

In this study, we discussed the validity of an inorganic semiconductor-based model on energy level alignment at weakly interacting substrate-organic film interfaces, in which the influence of Gaussian distributed density of states on injection barrier based on the thermal equilibrium condition after contacted with substrates was quantitatively investigated. A “Z”-like shape of the energy level alignment has been quantitatively obtained: a slope “S = 1” is clearly given, when the substrate work function is located approximately in between the threshold of ionization energy (IEth) and electron affinity (EAth); on the other hand, “S ∼ 0” is given while the substrate work function is lower than EAth or larger than IEth. Although different calculated results compared with experimental data can also be found, in which the calculated minimal injection barrier is ∼0.2 eV smaller than experimental data, this simplified practical method can be easily applied to calculate the device-related electrostatic potential inside dielectric films.

Extraction and intracellular trafficking of membrane associated antigen distinguishes human germinal center B cells from naïve B cells and memory B cells

Abstract Protective antibody responses to vaccination or infection in humans depend on the competitive selection of high-affinity germinal-center (GC) B cells into the long-lived memory B cell compartment. Selection is determined in large part by the ability of GC B cells to gather, process and present antigen to T follicular helper T cells (TFH cells). We have shown that human GC B cells are better able to distinguish their affinity for antigen as compared to naïve B cells in the gathering and internalization of antigen. Here we provide evidence that human naïve B cells and GC B cells rely on different endocytic processes for antigen internalization and trafficking. We found that the Microtubule Organizing Centers in activated GC B cells were not polarized toward the immune synapse with the antigen-containing membrane as in naïve B cells but rather dispersed large distances from the immune synapse in GC B cells. GC B cells trafficked antigen-bound BCRs from the synapse outside the sides of the cells to distal sites for internalization whereas naïve B cells internalized antigen from the immune synapse. Moreover, as compared to naïve B cells, LAMP-1+ vesicles in GC B cells were less polarized and less internalized antigen was colocalized with LAMP-1+ vesicles. We also found that the expression of SNX9 and SNX18, key components of the endocytosis machinery and modulators of clathrine mediated endocytosis, was significantly lower in GC B cells as compared to naïve B cells and SNX9 and SNX18 were poorly polarized toward the immune synapse in activated GC B cells. We propose that these differences in endocytosis processes for antigen internalization contribute the higher affinity thresholds of GC B cells.

Low HLA binding of diabetes-associated CD8+ T-cell epitopes is increased by post translational modifications

BackgroundType 1 diabetes (T1D) is thought to be an autoimmune disease driven by anti-islet antigen responses and mediated by T-cells. Recent published data suggests that T-cell reactivity to modified peptides, effectively neoantigens, may promote T1D. These findings have given more credence to the concept that T1D may not be solely an error of immune recognition but may be propagated by errors in protein processing or in modifications to endogenous peptides occurring as result of hyperglycemia, endoplasmic reticulum (ER) stress, or general beta cell dysfunction. In the current study, we hypothesized that diabetes-associated epitopes bound human leukocyte antigen (HLA) class I poorly and that post-translational modifications (PTM) to key sequences within the insulin-B chain enhanced peptide binding to HLA class I, conferring the CD8+ T-cell reactivity associated with T1D.ResultsWe first identified, through the Immune Epitope Database (IEDB; www.iedb.org), 138 published HLA class I-restricted diabetes-associated epitopes reported to elicit positive T-cell responses in humans. The peptide binding affinity for their respective restricting allele(s) was evaluated in vitro. Overall, 75% of the epitopes bound with a half maximal inhibitory concentration (IC50) of 8250 nM or better, establishing a reference affinity threshold for HLA class I-restricted diabetes epitopes. These studies demonstrated that epitopes from diabetes-associated antigens bound HLA with a lower affinity than those of microbial origin (binding threshold of 500 nM for 85% of the epitopes). Further predictions suggested that diabetes epitopes also bind HLA class I with lower affinity than epitopes associated with other autoimmune diseases. Therefore, we measured the effect of common PTM (citrullination, chlorination, deamidation, and oxidation) on HLA-A*02:01 binding of insulin-B-derived peptides, compared to native peptides. We found that these modifications increased binding for 44% of the insulin-B epitopes, but only 15% of the control peptides.ConclusionsThese results demonstrate that insulin-derived epitopes, commonly associated with T1D, generally bind HLA class I poorly, but can be subject to PTM that improve their binding capacity and may, in part, be responsible for T-cell activation in T1D and subsequent beta cell death.

An unexpected protective role of low-affinity allergen-specific IgG through the inhibitory receptor FcγRIIb

Induction of allergen-specific IgG antibodies is a critical parameter for successful allergen-specific immunotherapy. IgG antibodies can inhibit IgE-mediated mast cell activation through direct allergen neutralization or through the inhibitory receptor FcγRIIb. The affinity of IgE antibodies to the allergen has been shown to be critical for cellular activation. Here we addressed the question of affinity thresholds of allergen-specific IgG antibodies for inhibition of mast cell activation using 2 different mAbs against the major cat allergen Fel d 1 both invitro and invivo in mice. Sequences of the 2 high-affinity mAbs were back-mutated to germline, resulting in low-affinity (10-7mol/L) antibodies of the exact same specificity. Using these newly generated recombinant antibodies, we demonstrate that low-affinity antibodies are still able to inhibit mast cell activation through FcγRIIb but do not neutralize the allergen. Antibody affinity dictates the mechanism of mast cell inhibition, and IgG antibodies triggering the inhibitory FcγRIIb pathway can show a broader cross-reactivity pattern than previously thought. This indicates that allergen-specific immunotherapy generates a larger protective umbrella of inhibitory IgG antibodies than previously appreciated.

Lie algebraic discussion for affinity based information diffusion in social networks

Abstract In this paper we develop a dynamical information diffusion model which features the affinity of people with information disseminated in social networks. Four types of agents, i.e., susceptible, informed, known, and refractory ones, are involved in the system, and the affinity mechanism composing of an affinity threshold which represents the fitness of information to be propagated is incorporated. The model can be generally described by a time-inhomogeneous Markov chain, which is governed by its master (Kolmogorov) equation. Based on the Wei-Norman method, we derive analytical solutions of the model by constructing a low-dimensional Lie algebra. Numerical examples are provided to illustrate the obtained theoretical results. This study provides useful insights into the closed-form solutions of complex social dynamics models through the Lie algebra method.

Abstract LB-002: Optimizing immunogenicity prediction of neoepitopes

Abstract Epitopes that arise from a somatic mutation, so called neoepitopes, are now known to play a key role in cancer immunology and immunotherapy. Recent advances in high-throughput sequencing have made it possible to identify all mutations and thereby all potential neoepitope candidates in an individual cancer. It has however become evident that the vast majority of these neoepitope candidates do not induce a T cell response when tested in vivo or in vitro, i.e. they are not immunogenic. Especially in patients with a high mutational load, usually hundreds of potential neoepitopes are detected, highlighting the need to further narrow down this candidate list. Several studies have used different combinations of immunoinformatic tools such as MHC binding predictions to prioritize the initial set of neoepitopes candidates. The tools to use and thresholds to apply for this prioritization has so far been largely based on experience with epitope identification in other settings such as infectious disease and allergy. To establish the appropriate tools and thresholds in the cancer setting, we here curated a set of immunogenic neoepitopes from the published literature and performed detailed analyses to detect what features discriminate immunogenic neoepitopes from a background set of mutated peptides. We experimentally measured the HLA binding affinity of all curated immunogenic neoepitopes. In doing so, we aimed to identify the optimal affinity threshold to effectively identify immunogenic neoepitopes. As a next step, we sought to assess the added value of different immunoinformatics tools, including various HLA binding prediction algorithms, processing prediction, stability prediction, and immunogenicity prediction, to most effectively detect immunogenic neoepitopes. The obtained results are now going to be used to facilitate the development of more accurate prediction algorithms. Citation Format: Zeynep Kosaloglu-Yalcin, Manasa Lanka, John Sidney, Kerrie Vaughan, Jason Greenbaum, Alessandro Sette, Bjoern Peters. Optimizing immunogenicity prediction of neoepitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-002. doi:10.1158/1538-7445.AM2017-LB-002

A Chemically Inducible Helper Module for Detecting Protein-Protein Interactions with Tunable Sensitivity Based on KIPPIS.

As protein-protein interactions (PPIs) play essential roles in regulating their functional consequences in cells, methods to detect PPIs in living cells are desired for correct understanding of intracellular PPIs and pharmaceutical development therefrom. Here we demonstrate a c-kit-based PPI screening (KIPPIS) system in combination with a chemically inducible helper module for detecting PPIs in living mammalian cells. In this system, a mutant of FK506-binding protein 12 (FKBPF36V) is fused with a protein of interest and the intracellular domain of a receptor tyrosine kinase c-kit. Constitutive expression of two fusion proteins with interacting proteins of interest in interleukin-3 (IL-3)-dependent cells results in dimerization and subsequent activation of the c-kit intracellular domains, which allows cell proliferation in a culture medium devoid of IL-3. A helper ligand, a small synthetic chemical that homodimerizes FKBPF36V, assists the formation of stable complexes of the fusion proteins and serves as a tuner for sensitivity of the system. Using this system, two model PPIs were successfully detected on the basis of cell proliferation, which was featured by the helper-ligand- and PPI-dependent phosphorylation of the Src family kinases, a hallmark of the c-kit signaling. Notably, the inclusion of the helper module enabled PPI detection with tunable sensitivity. The helper-assisted KIPPIS allows us to configure various affinity thresholds by changing the concentration of the helper ligand, which may be applied to select affinity-matured variants using the advantage of cell proliferation.

CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold.

The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.

Opposing effects of actin signaling and LFA-1 on establishing the affinity threshold for inducing effector T-cell responses in mice.

Mature CD8(+) T cells use a narrow antigen affinity threshold to generate tissue-infiltrating cytotoxic effector T cells and induce autoimmune pathology, but the mechanisms that establish this antigen affinity threshold are poorly understood. Only antigens with affinities above the threshold induce stable contacts with APCs, polarization of a T cell, and asymmetric T-cell division. Previously published data indicate that LFA-1 inside-out signaling might be involved in establishing the antigen affinity threshold. Here, we show that subthreshold antigens weakly activate all major distal TCR signaling pathways. Low-affinity antigens are more dependent on LFA-1 than suprathreshold antigens. Moreover, augmenting the inside-out signaling by hyperactive Rap1 does not increase responses to the subthreshold antigens. Thus, LFA-1 signaling does not contribute to the affinity-based antigen discrimination. However, we found that subthreshold antigens do not induce actin rearrangement toward an APC, mediated by Rho-family GTPases, Cdc42, and Rac. Our data suggest that Rac and Cdc42 contribute to the establishment of the antigen affinity threshold in CD8(+) T cells by enhancing responses to high-affinity antigens, or by reducing the responses to low-affinity antigens.