CD4⁺ T cells are a critical component of the adaptive immune compartment. Each T cell expresses a clonotypic T cell receptor (TCR) that must discriminate between self and foreign peptides presented in major histocompatibility molecules (pMHC) on the surface of antigen presenting cells to direct T cell fate decisions. Information regarding TCR-pMHC interactions must then be transmitted to the TCR-associated CD3 signaling modules, which contain ITAMs that serve as signaling substrates for Src kinases. The Src kinase, Lck, is recruited to the pMHC-bound TCR-CD3 complex via association with the CD4 coreceptor that binds MHCII. It is therefore through the coordinated interactions within the TCR-CD3-pMHC-CD4 macro-complex that productive TCR signaling can occur to inform T cell activation and fate decisions. While much is known regarding the structure of the individual subunits that make up the TCR-CD3-pMHC-CD4 macro-complex, there is little information regarding how these components come together to initiate TCR signaling and determine functional outcomes. Here, we have interrogated how interaction of these individual components leads to productive T cell activation. Specifically, we interrogated the nature of TCR-MHC interactions and provide evidence that there is intrinsic specificity of the TCR for MHCII. We have also built mouse models to determine the role of TCR-CD3 interactions and TCR dimerization in the transmission of information from the TCR to the CD3 subunits following TCR-pMHC engagement. Finally, we show that both the CD4 transmembrane and extracellular domains contribute to T cell activation in vitro. Overall, this work provides insight into how the constituents of the TCR-CD3-pMHC-CD4 macro-complex interact to initiate T cell fate and function.

The mechanism of T cell recognition is the central but unsolved puzzle of adaptive immunology. The difficulties come from the multichain structure of TCR/CD3, the binate binding structure of the pMHC molecule, the diversity of the peptides presented on the APC, the critical role of coreceptor CD4/8, the communication between TCR and coreceptor CD4/8, the complex environment of interactions taking place and the binding and signaling coupled process of recognition. Most studies were using the 3D kinetic measurements or biological functional assays to address the mechanism of the T cell recognition. However, those assays are usually either lacking of physiology relevance or missing of the initial recognition signals. Here a 2D micropipette adhesion assay with high temporal resolution (-second) was used to address the in situ kinetics of molecular interaction at the membrane of live T cells. The aim of this project is to advance our understanding to the T cell recognition mechanism. The micropipette adhesion assay was firstly used to address a simple case, the resting state pMHC-CD8 interaction. In the absence of TCR-pMHC interaction, the pMHC-CD8 interaction has a very low affinity that depends on the MHC alleles and the lipid rafts of the T cell membrane where CD8 resides, but not on the peptide complexed to the MHC and whether the CD8 is an a a homodimer or an αβ heterodimer. For cognate pMHC, following the initial observation in the F5 T cell system, the binding also displays a two-step curve in the OTI T cell system. The first-step binding occurs before one second and has a very fast on-rate and off-rate (>2s ⁻¹), and the secondstep binding follows immediately but reaches a much higher level of binding. It was identified that the first-step binding is mediated by the TCR-pMHC interaction, and the second-step binding is triggered by the TCR-pMHC interaction but mediated by CD8- pMHC binding. The two-step binding is the unique property of cognate pMHC, and it can be abolished by disrupting the lipid rafts, inhibiting the Src family protein tyrosine kinases (PTK) or protein tyrosine phosphatase (PTP). The finding of two-step binding identifies a CD8-dependent signaling amplification pathway. The data also indicated the active communication between TCR and CD8 in the antigen recognition. The crosstalk between TCR and CD8 was further dissected using two anti-CD8 antibodies 53.6.7 and CT-CD8a. 53-6.7 can significantly enhance the binding of pMHC to the T cell. Although the enhancement is directly mediated by MHC-CD8 interaction, the enhancing role of this antibody is TCR dependent. Blocking the TCR-pMHC interaction on OTI T cell or expressing CD8 alone on a hybridoma abolished the enhancement. The enhancement is also dependent on the integrity of lipid rafts and the normal function of PTP. In contrast, the antibody CT-CD8 can inhibit the binding of pMHC to the T cells and interfere with the TCR-pMHC interaction. The enhancing or inhibitory role of these two anti-CD8 antibodies is reversely correlated with the affinities of TCR-pMHC interactions. Only 53-6.7, but not CT-CD8 antibody, can phosphorylate and activate Lck. The data demonstrated a dual way crosstalk between TCR and CD8, and indicated the importance of cooperation of TCR and CD8 in antigen recognition. In the physiology condition, the TCR must accurately and efficiently recognize the cognate peptide from thousands of surrounding endogenous peptides. There is an argument regarding whether the endogenous peptides plays a role in helping the TCR recognition. Our results demonstrated that the nonstimulatory peptides can significantly enhance the T cell recognition sensitivity. In the presence of nonstimulatory peptide, the TCR can efficiently detect a single antigenic pMHC. The enhancement of recognition is due to the CD8 binding to the nonstimulatory pMHC. Blocking the CD8 binding can paralyze the enhancement. In contrast, it was found that the presence of antagonist can inhibit the binding of agonist pMHC to the T cells, and the inhibition occurs in the initial recognition step. Based on the data, an "amplification and competition" model was proposed to explain the molecular mechanism of the enhancement and inhibition function of the nonstimulatory and antagonist peptides in the T cell recognition, respectively.

T cells are an essential part of the adaptive immune system and protect the body from intracellular infections. The specificity with which αßTCR-bearing T cells recognize cognate antigen presented on MHC molecules is paramount to maintaining the balance between mounting effector functions against pathogens and establishing peripheral tolerance to self. The mechanism by which T cells translate qualitative differences in TCR:pMHC binding to sensitive proximal signalling events which ultimately result in specific Tcell effector responses to infected cells but not to self is mostly unknown. To address how T cell signalling responds to qualitative differences in TCR triggering by pMHC, I established a system of stimulating T cells bearing the 1G4 TCR specifically in vitro with a panel of four NY-ESO-1_156-165 peptide variant MHC tetramers. Single amino acid substitutions to the NY-ESO-1_156-165 peptide conferred a maximum 35-fold difference in the monomeric affinity for the 1G4 TCR. The system allows the highly controlled investigation of very rapid TCR proximal signalling events simultaneously and quantitatively using flow cytometry. Stimulations with pMHC tetramers showed rapid sensitive sequential signalling responses which were able to confer ligand discrimination. Very early signalling events such as CD3ζ phosphorylation showed analogue responses to the different affinity pMHC tetramers. Later signalling events including phospho-ERK showed a distinct on/off switch-like response. The amplitude of the very early analogue signalling responses determined the extent of later digital ERK signals. This indicates that a certain analogue signalling threshold must be passed to result in T cell activation. The thymocyte protein Themis has been shown proximal TCR signalling to modulate thymocyte selection thresholds. Its deletion results in profound defects in positive thymocyte selection. Themis locates to the LAT signalosome of the TCR signalling cascade via Grb2, yet its molecular function is unknown. Employing the system I established, I demonstrate that Themis-k/d cells show increased levels of CD3z-chain phosphorylation, phospho-ERK signalling and signal-induced apoptosis which was independent of the ERK signal. This shows that Themis is a global attenuator of proximal TCR signalling. We are currently investigating possible associations of Themis to proteins phosphastases such as SHP-1 which could attenuate TCR proximal protein tyrosine signalling events.

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

The 23 pairs of chromosomes comprising the human genome are intricately folded within the nucleus of each cell in a manner that promotes efficient gene regulation and cell function. Consequently, active gene rich regions are compartmentally segregated from inactive gene poor regions of the genome. To better understand the mechanisms driving compartmentalization we investigated what would occur if this system was disrupted. By digesting the genome to varying sizes and analyzing the fragmented 3D structure over time, our work revealed essential laws governing nuclear compartmentalization. At a finer resolution within compartments, chromatin forms loop structures capable of regulating gene expression. Genome wide association studies have identified numerous single nucleotide polymorphisms (SNPs) associated with the neuropsychiatric disease schizophrenia. When these SNPs are not located within a gene it is difficult to gain insight into disease pathology; however, in some cases chromatin loops may link these noncoding schizophrenia risk variants to their pathological gene targets. By generating 3D genome maps, we identified and analyzed loops of glial cells, neural progenitor cells, and neurons thereby expanding the set of genes conferring schizophrenia risk. The binding of T-cell receptors (TCRs) to foreign peptides on the surface of diseased cells triggers an immune response against the foreign invader. Utilizing available structural information of the TCR antigen interface, we developed computational methods for successful prediction of TCR-antigen binding. As this binding is a prerequisite for immune response, such improvements in binding prediction could lead to important advancements in the fields of autoimmunity and TCR design for cancer therapeutics.

T cell activation and thymic selection are thought to be determined by the binding propensity (avidity or affinity) of the T cell receptor (TCR) to its ligands. However, binding propensity quantified by previous 3D TCR–pMHC kinetics such as using tetramer staining or surface plasmon resonance (SPR) under estimate TCR–pMHC interaction due to neglecting physiological conditions. Recent studies considering membrane contribution in TCR–pMHC interaction reported 2D kinetics and force regulated bond dissociation kinetics have better prediction to biological responses in CD8+ T cells. In this study, we further tested the findings in CD4+ T cells and CD4+ CD8+ (double-positive, DP) thymocytes. We analyzed TCR–pMHC interaction for a well-characterized panel of altered peptide ligands (APLs) on multiple transgenic mouse TCR systems. Using ultrasensitive 2D mechanical assays, in situ 2D kinetic measurements show better sensitivity than the SPR 3D kinetic measurements in gauging the ligand potency and thymic selection. Furthermore, force-regulated bond lifetime of TCR–pMHC interaction amplifies the discrimination in recognition of APLs and thymic selection. When force was applied to TCR–pMHC–CD4/8 bonds, two distinct patterns emerged: agonist/negative selecting ligands formed CD4/8-dependent catch-slip bonds where lifetime first increased, reached a maximum, then decreased with increasing force, whereas antagonist/positive selecting ligands formed slip-only bonds where lifetime monotonically decreases with increasing force. Our results highlight an important role of mechanical force in ligand discrimination and suggest a new mechanism for T cell activation and thymic selection that is distinct from previous models based on 3D measurements.