T-cell Receptor Ligation Research Articles

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics.

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation. TCR microclusters (MC) are submicron-sized molecular condensates and basic signaling units that form immediately after TCR ligation. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. This CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

Differential Impact of CD43 and CD28 on T-Cell Differentiation Depending on the Order of Engagement with the TCR.

The combination of signals from the T-cell receptor (TCR) and co-stimulatory molecules triggers transcriptional programs that lead to proliferation, cytokine secretion, and effector functions. We compared the impact of engaging the TCR with CD28 and/or CD43 at different time points relative to TCR engagement on T-cell function. TCR and CD43 simultaneous engagement resulted in higher CD69 and PD-1 expression levels than in TCR and CD28-stimulated cells, with a cytokine signature of mostly effector, inflammatory, and regulatory cytokines, while TCR and CD28-activated cells secreted all categories of cytokines, including stimulatory cytokines. Furthermore, the timing of CD43 engagement relative to TCR ligation, and to a lesser degree that of CD28, resulted in distinct patterns of expression of cytokines, chemokines, and growth factors. Complete cell activation was observed when CD28 or CD43 were engaged simultaneously with or before the TCR, but ligating the TCR before CD43 or CD28 failed to complete a cell activation program regarding cytokine secretion. As the order in which CD43 or CD28 and the TCR were engaged resulted in different combinations of cytokines that shape distinct T-cell immune programs, we analyzed their upstream sequences to assess whether the combinations of cytokines were associated with different sets of regulatory elements. We found that the order in which the TCR and CD28 or CD43 are engaged predicts the recruitment of specific sets of chromatin remodelers and TFSS, which ultimately regulate T-cell polarization and plasticity. Our data underscore that the combination of co-stimulatory molecules and the time when they are engaged relative to the TCR can change the cell differentiation program.

The cellular microenvironment regulates CX3CR1 expression on CD8+ Tcells and the maintenance of CX3CR1+ CD8+ Tcells.

Expression levels of the chemokine receptor CX3CR1 serve as high-resolution marker delineating functionally distinct antigen-experienced T-cell states. The factors that influence CX3CR1 expression in Tcells are, however, incompletely understood. Here, we show that in vitro priming of naïve CD8+ Tcells failed to robustly induce CX3CR1, which highlights the shortcomings of in vitro priming settings in recapitulating in vivo T-cell differentiation. Nevertheless, in vivo generated memory CD8+ Tcells maintained CX3CR1 expression during culture. This allowed us to investigate whether T-cell receptor ligation, cell death, and CX3CL1 binding influence CX3CR1 expression. T-cell receptor stimulation led to downregulation of CX3CR1. Without stimulation, CX3CR1+ CD8+ Tcells had a selective survival disadvantage, which was enhanced by factors released from necrotic but not apoptotic cells. Exposure to CX3CL1 did not rescue their survival and resulted in a dose-dependent loss of CX3CR1 surface expression. At physiological concentrations of CX3CL1, CX3CR1 surface expression was only minimally reduced, which did not hamper the interpretability of T-cell differentiation states delineated by CX3CR1. Our data further support the broad utility of CX3CR1 surface levels as T-cell differentiation marker and identify factors that influence CX3CR1 expression and the maintenance of CX3CR1 expressing CD8+ Tcells.

A method for in vitropolarization of pathogenic ST2-expressing memory Th2 cells

Abstract IL-33 and its receptor subunit ST2 (IL1RL1) have been linked to type 2 eosinophilic asthma in multiple human and mouse studies. In particular, recent clinical trials demonstrate that monoclonal antibodies targeting IL-33/ST2 are beneficial to subsets of asthma patients. ST2 +memory Th2 cells (mTh2) are important effector cells in IL-33-mediated type 2 eosinophilic asthma. Herein we describe a unique method for polarizing a subset of pathogenic ST2 +mTh2 (ST2 +mTh2p) cells from naïve mouse CD4 T cells in vitro, which does not require multi-round activation/resting or co-culturing with antigen-presenting cells as reported in prior studies. The ST2 +mTh2p cells proliferate and produce large amounts of type 2 cytokines upon IL-33 treatment [without involving T-cell receptor (TCR) ligation]. IL-33 stimulation induces metabolic reprogramming of ST2 +mTh2p, as indicated by the increased mitochondrial respiration and glycolytic rate in Seahorse assays as well as upregulated key metabolites and metabolic genes involved in these two bioenergetic pathways. Interestingly, IL-33 stimulation also increases the metabolites and the expression of key enzymes for the production of polyamines (the important regulators of cell proliferation). Moreover, ST2 +mTh2p cells are highly pathogenic, as they are capable of propagating and producing type 2 cytokines in the lung, resulting in strong eosinophilic inflammation induced by intranasal IL-33 administration alone after adoptive transfer to Rag2−/−IL2r−/−mice (lacking both innate and adaptive lymphocytes). In summary, the current study provides useful tools to investigate IL-33/ST2-regulated molecular mechanisms and immunological functions of ST2 +mTh2p cells both in vitroand in vivo. R0 1HL144497

Mutual regulation between coupled protein condensates and ordered membrane domains.

Downstream of T-cell receptor ligation, biomolecular condensates are produced via the multivalent binding between Linker for Activation of T-cells (LAT) and its effectors (Grb2 and Sos1) to facilitate T-cell activation. LAT is a transmembrane protein known to be recruited to ordered lipid domains, suggesting potential coupling between protein cytoplasmic condensates and lateral lipid-based phase-separation and organization. We report evidence demonstrating thermodynamic coupling between lipid- and protein-based phase separation. In model membranes, condensates formed on phase-separating membranes template ordered lipid domains and induce phase separation. Conversely, lipid phase separation stabilizes protein condensate formation suggesting strong coupling between lipid- and protein-based phase separation. Furthermore, experiments in T-cells show that LAT/Grb2/Sos1 condensates recruit ordered domain lipids and proteins in living cells. We observed that crosslinking of a membrane component that prefers ordered domains (i.e. the endogenous GPI-anchored protein Thy1) stabilizes lipid phase separation and templates condensate formation. Finally, we tested whether condensate-domain coupling is necessary for T-cell activation by decoupling raft domains from condensates through either mutation of the transmembrane domain to modulate its raft affinity or mutation of residues necessary for condensate formation. We found that the coupling of cytoplasmic condensate formation to lipid rafts is essential for T-cell activation. Thus, we conclude that the thermodynamic coupling between LAT/Grb2/Sos1 cytoplasmic condensates and lipid rafts can cooperatively and functionally reorganize the membrane to facilitate T-cell activation.

Change in Programmed Death-1 And Inducible Costimulator Expression in Patients with Acute Myeloid Leukemia Following Chemotherapy and Its Cytogenetic Abnormalities

Background: Programmed death-1 (PD-1) and inducible costimulator (ICOS) are immune checkpoint receptors participating in tumor immune evasion, which counters the activation signal provided through the T-cell receptor ligation. This study aimed to investigate the relationship between the expression of PD-1 and ICOS on mononuclear cells (MNCs) isolated from the peripheral blood of acute myeloid leukemia (AML) patients and their response to induction chemotherapy. Materials and Methods: Peripheral blood samples (5cc) were collected from 56 AML patients at first diagnosis before and after the induction therapy regimen for AML. PD-1 and ICOS expression were analyzed in all patients before and after the standard induction therapy regimen. Results: The expression of PD-1 and ICOS significantly decreased (66.7 and 16.3 fold, respectively) in AML patients following chemotherapy compared to its baseline value (P=0.01 and P=0.001, respectively). The expressions of PD-1 and ICOS were significantly different between favorable and poor risk groups. Conclusions: Lower PD-1 and ICOS expressions on the surface of MNCs before induction therapy were associated with a better response to treatments. In addition, PD-1 and ICOS expression on MNCs decreased after induction therapy.

Conformational flexibility of a free and TCR-bound pMHC-I protein investigated by long-term molecular dynamics simulations

BackgroundMajor histocompatibility complexes (MHCs) play a crucial role in the cell-mediated adaptive immune response as they present antigenic peptides (p) which are recognized by host T cells through a complex formation of the T cell receptor (TCR) with pMHC. In the present study, we report on changes in conformational flexibility within a pMHC molecule upon TCR binding by looking at molecular dynamics (MD) simulations of the free and the TCR-bound pMHC-I protein of the LC13-HLA-B*44:05-pEEYLQAFTY complex.ResultsWe performed long-term MD simulations with a total simulation time of 8 µs, employing 10 independent 400 ns replicas for the free and the TCR-bound pMHC system. Upon TCR ligation, we observed a reduced dynamic flexibility in the central residues of the peptide and the MHC α1-helix, altered occurrences of hydrogen bonds between the peptide and the MHC, a reduced conformational entropy of the peptide-binding groove, as well as a decreased solvent accessible surface area.ConclusionsIn summary, our results from 8 µs MD simulations indicate a restricted conformational space of the MHC peptide-binding groove upon TCR ligation and suggest a minimum simulation time of approximately 100 ns for biomolecules of comparable complexity to draw meaningful conclusions. Given the relatively long total simulation time, our results contribute to a more detailed view on conformational flexibility properties of the investigated free and TCR-bound pMHC-I system.

P063 Expression of the coinhibitory receptor TIGIT on memory CD4+ T cells is induced by TCR ligation and retinoic acid co-stimulation and is elicited during microbial colonization of the intestine

Abstract Background The coinhibitory receptor T cell immunoglobulin and ITIM domain (TIGIT) is widely expressed by immune cells, including CD4+ T cells. Previously, we showed that circulating gut homing CD38+ (CD62LnegCD4+) effector T cells are enriched for TIGIT+ cells with an immunoregulatory phenotype. In newly diagnosed inflammatory bowel disease (IBD) patients, low frequencies of regulatory TIGIT+ cells in circulating gut homing CD38+CD62LnegCD4+ effector T cells associate with more severe disease. IBD is a chronic disease characterized by aberrant CD4+ T cell responses to intestinal microbiota. Together, this led us to hypothesize that TIGIT+CD4+ T cells regulate immune responses to intestinal bacteria thereby maintaining intestinal homeostasis. Methods We studied TIGIT expression in subpopulations of murine and human intestinal CD4+ T cells. In addition, we assessed the signals required for the induction of TIGIT expression in vitro using CD4+ T cells isolated from human healthy donor peripheral blood. Results TIGIT was expressed by more than 50% of memory, but not naïve, CD4+ T cells in the intestinal lamina propria of both mouse and human. Both Foxp3+ and Foxp3neg intestinal CD4+ T cells expressed TIGIT. Intestinal TIGIT expression depended on the presence of microbial antigens, as conventionalization of germ-free mice with specific-pathogen free (SPF) microbiota dramatically increased TIGIT expression in both small intestine and colon. T cell receptor (TCR) ligation mimicked by anti-CD3 stimulation was essential to induce TIGIT expression on CD4+ T cells isolated from human peripheral blood, but CD28 co-stimulation led to reduced TIGIT expression. TIGIT expression could only be induced on antigen-experienced CD4+ memory T cells, but not naïve CD4+ T cells. Using the calcium ionophore ionomycin and the calcineurin inhibitor tacrolimus, we demonstrated that the induction of TIGIT expression upon TCR ligation depended on downstream calcium-dependent signaling. We next assessed whether retinoic acid (RA), a metabolite produced by intestinal antigen-presenting cells (APCs), can further enhance TIGIT expression. RA plays an important role in mucosal immune responses, as it promotes the development of Foxp3+ regulatory T cells and induces the expression of the gut-homing markers CD38 and α4β7. We showed that co-stimulation with RA also significantly enhanced TCR-induced TIGIT expression, as it increased both the mean fluorescence intensity (MFI) and frequencies of TIGIT+ cells in CD4+ memory T cells. Conclusion In conclusion, these data argue that TCR ligation and retinoic acid production by intestinal APCs induces TIGIT expression on CD4+ memory T cells, which in turn may contribute to regulation of immune responses against microbial antigens.

CD146 bound to LCK promotes T cell receptor signaling and antitumor immune responses in mice.

Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, we found CD146 to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the antitumor response of T cells. Together, our data reveal an LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.

Regulations of T Cell Activation by Membrane and Cytoskeleton.

Among various types of membrane proteins that are regulated by cytoskeleton, the T cell receptor (TCR) greatly benefits from these cellular machineries for its function. The T cell is activated by the ligation of TCR to its target agonist peptide. However, the binding affinity of the two is not very strong, while the T cell needs to discriminate agonist from many nonagonist peptides. Moreover, the strength and duration of the activation signaling need to be tuned for immunological functions. Many years of investigations revealed that dynamic acto-myosin cytoskeletons and plasma membranes in T cells facilitate such regulations by modulating the spatiotemporal distributions of proteins in plasma membranes and by applying mechanical loads on proteins. In these processes, protein dynamics in multiple scales are involved, ranging from collective molecular motions and macroscopic molecular organizations at the cell–cell interface to microscopic changes in distances between receptor and ligand molecules. In this review, details of how cytoskeletons and membranes regulate these processes are discussed, with the emphasis on how all these processes are coordinated to occur within a single cell system.

IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation.

Molecular design and optimization of hepatic cancer SLP76-derived PLCγ1 SH3-binding peptide with the systematic N-substitution of peptide PXXP motif.

The phospholipase Cγ1 (PLCγ1) is essential for T-cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP-containing decapeptide segment (185 QPPVPPQRPM194 , termed as SLP76185-194 peptide) of adaptor protein SLP76 following T-cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76185-194 PXXP motif with nonnatural N-substituted amino acids, as the proline is the only endogenous N-substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate-sized N-substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N-substituted peptides as well as native SLP76185-194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence-based assays. Two N-substituted peptides, SLP76185-194 (N-Leu187/N-Gln190) and SLP76185-194 (N-Thr187/N-Gln190), are designed to have high potency (Kd =0.67±0.18 and 1.7±0.3μM, respectively), with affinity improvement by, respectively, 8.5-fold and 3.4-fold relative to native peptide (Kd =5.7±1.2μM).

The Role of Adaptor Proteins in the Biology of Natural Killer T (NKT) Cells

Adaptor proteins contribute to the selection, differentiation and activation of natural killer T (NKT) cells, an innate(-like) lymphocyte population endowed with powerful immunomodulatory properties. Distinct from conventional T lymphocytes NKT cells preferentially home to the liver, undergo a thymic maturation and differentiation process and recognize glycolipid antigens presented by the MHC class I-like molecule CD1d on antigen presenting cells. NKT cells express a semi-invariant T cell receptor (TCR), which combines the Vα14-Jα18 chain with a Vβ2, Vβ7, or Vβ8 chain in mice and the Vα24 chain with the Vβ11 chain in humans. The avidity of interactions between their TCR, the presented glycolipid antigen and CD1d govern the selection and differentiation of NKT cells. Compared to TCR ligation on conventional T cells engagement of the NKT cell TCR delivers substantially stronger signals, which trigger the unique NKT cell developmental program. Furthermore, NKT cells express a panoply of primarily inhibitory NK cell receptors (NKRs) that control their self-reactivity and avoid autoimmune activation. Adaptor proteins influence NKT cell biology through the integration of TCR, NKR and/or SLAM (signaling lymphocyte-activation molecule) receptor signals or the variation of CD1d-restricted antigen presentation. TCR and NKR ligation engage the SH2 domain-containing leukocyte protein of 76kDa slp-76 whereas the SLAM associated protein SAP serves as adaptor for the SLAM receptor family. Indeed, the selection and differentiation of NKT cells selectively requires co-stimulation via SLAM receptors. Furthermore, SAP deficiency causes X-linked lymphoproliferative disease with multiple immune defects including a lack of circulating NKT cells. While a deletion of slp-76 leads to a complete loss of all peripheral T cell populations, mutations in the SH2 domain of slp-76 selectively affect NKT cell biology. Furthermore, adaptor proteins influence the expression and trafficking of CD1d in antigen presenting cells and subsequently selection and activation of NKT cells. Adaptor protein complex 3 (AP-3), for example, is required for the efficient presentation of glycolipid antigens which require internalization and processing. Thus, our review will focus on the complex contribution of adaptor proteins to the delivery of TCR, NKR and SLAM receptor signals in the unique biology of NKT cells and CD1d-restricted antigen presentation.

The CD28-B7 Family of Co-signaling Molecules.

Immune responses are controlled by the optimal balance between protective immunity and immune tolerance. T-cell receptor (TCR) signals are modulated by co-signaling molecules, which are divided into co-stimulatory and co-inhibitory molecules. By expression at the appropriate time and location, co-signaling molecules positively and negatively control T-cell differentiation and function. For example, ligation of the CD28 on T cells provides a critical secondary signal along with TCR ligation for naive T-cell activation. In contrast, co-inhibitory signaling by the CD28-B7 family is important to regulate immune homeostasis and host defense, as these signals limit the strength and duration of immune responses to prevent autoimmunity. At the same time, microorganisms or tumor cells can use these pathways to establish an immunosuppressive environment to inhibit the immune responses against themselves. Understanding these co-inhibitory pathways will support the development of new immunotherapy for the treatment of tumors and autoimmune and infectious diseases. Here, we introduce diverse molecules belonging to the members of the CD28-B7 family.

Naïve CD8+ T-Cells Engage a Versatile Metabolic Program Upon Activation in Humans and Differ Energetically From Memory CD8+ T-Cells.

Background: Characterization of the intracellular biochemical processes that regulate the generation and maintenance of effector and memory CD8+ T-cells from naïve precursors is essential for our understanding of adaptive immune responses and the development of immunotherapies. However, the metabolic determinants of antigen-driven activation and differentiation remain poorly defined, especially in humans.Methods: We used a variety of different approaches, including gene expression profiling and measurements of nutrient flux, to characterize the basal and activation-induced energetic requirements of naïve and phenotypically-defined subsets of human memory CD8+ T-cells.Findings: Profound metabolic differences were apparent as a function of differentiation status, both at rest and in response to stimulation via the T cell receptor (TCR). Of particular note, resting naïve CD8+ T cells were largely quiescent, but rapidly upregulated diverse energetic pathways after ligation of surface-expressed TCRs. Moreover, autophagy and the mechanistic target of rapamycin (mTOR)-dependent glycolytic pathway were identified as critical mediators of antigen-driven priming in the naïve CD8+ T cell pool, the efficiency of which was dampened by the presence of neutral lipids and fatty acids.Interpretation: These observations provide a metabolic roadmap of the CD8+ T-cell compartment in humans and reveal potentially selective targets for novel immunotherapies.

Integrating Experiment and Theory to Understand TCR-pMHC Dynamics.

The conformational dynamism of proteins is well established. Rather than having a single structure, proteins are more accurately described as a conformational ensemble that exists across a rugged energy landscape, where different conformational sub-states interconvert. The interaction between αβ T cell receptors (TCR) and cognate peptide-MHC (pMHC) is no exception, and is a dynamic process that involves substantial conformational change. This review focuses on technological advances that have begun to establish the role of conformational dynamics and dynamic allostery in TCR recognition of the pMHC and the early stages of signaling. We discuss how the marriage of molecular dynamics (MD) simulations with experimental techniques provides us with new ways to dissect and interpret the process of TCR ligation. Notably, application of simulation techniques lags behind other fields, but is predicted to make substantial contributions. Finally, we highlight integrated approaches that are being used to shed light on some of the key outstanding questions in the early events leading to TCR signaling.

Reprogramming of Th1 cells into regulatory T cells through rewiring of the metabolic status.

T helper type 1 (Th1) cells form one of the most stable CD4 T-cell subsets, and direct conversion of fully differentiated Th1 to regulatory T (Treg) cells has been poorly investigated. Here, we established a culture method for inducing Foxp3 from Th1 cells of mice and humans. This is achieved simply by resting Th1 cells without T-cell receptor ligation before stimulation in the presence of transforming growth factor-beta (TGF-β). We named the resulting Th1-derived Foxp3+ cells Th1reg cells. Mouse Th1reg cells showed an inducible Treg-like phenotype and suppressive ability both in vitro and in vivo. Th1reg cells could also be induced from in vivo-developed mouse Th1 cells. Unexpectedly, the resting process enabled Foxp3 expression not through epigenetic changes at the locus, but through metabolic change resulting from reduced mammalian target of rapamycin complex 1 (mTORC1) activity. mTORC1 suppressed TGF-β-induced phosphorylation of Smad2/3 in Th1 cells, which was restored in rested cells. Our study warrants future research aiming at development of immunotherapy with Th1reg cells.

Biologically Inspired Design of Nanoparticle Artificial Antigen-Presenting Cells for Immunomodulation

Particles engineered to engage and interact with cell surface ligands and to modulate cells can be harnessed to explore basic biological questions as well as to devise cellular therapies. Biology has inspired the design of these particles, such as artificial antigen-presenting cells (aAPCs) for use in immunotherapy. While much has been learned about mimicking antigen presenting cell biology, as we decrease the size of aAPCs to the nanometer scale, we need to extend biomimetic design to include considerations of T cell biology-including T-cell receptor (TCR) organization. Here we describe the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology. We show that aAPCs, larger than 300 nm, activate T cells more efficiently than smaller aAPCs, 50 nm. The 50 nm aAPCs require saturating doses or require artificial magnetic clustering to activate T cells. Increasing ligand density alone on the 50 nm aAPCs did not increase their ability to stimulate CD8+ T cells, confirming the size-dependent phenomenon. These data support the need for multireceptor ligation and activation of T-cell receptor (TCR) nanoclusters of similar sizes to 300 nm aAPCs. Quantitative analysis and modeling of a nanoparticle system provides insight into engineering constraints of aAPCs for T cell immunotherapy applications and offers a case study for other cell-modulating particles.

Radiotherapy in Combination with αOX40 and Actla-4 Immunotherapy Reverses Anergy and Prevents Development of Functional Exhaustion within Tumor-Specific CD8 T Cells

The immunosuppressive tumor microenvironment and chronic T cell stimulation that occurs in the presence of cancer results in CD8 T cell dysfunction that has been difficult to reverse with immunotherapy (IT) alone. This dysfunction can be separated into two categories of anergy and exhaustion. Anergy is a lack of proliferative response to stimulation with cytokine or antigen resulting from inadequate co-stimulation or suboptimal T cell receptor (TCR) stimulation during T cell priming. Functional exhaustion is the inability of T cells to kill or produce effector cytokines in response to stimulation. We hypothesized the addition of ionizing radiotherapy (RT) to IT with agonist αOX40 & blocking αCTLA-4 antibodies would reverse tumor associated CD8 T cell anergy and exhaustion. We utilized an established model of CD8 T cell anergy in which transgenic, OVA-specific, OT-I CD8 T cells are adoptively transferred into centrally tolerant POET-1 mice bearing OVA expressing tumors. The development of anergy/exhaustion was monitored within this T cell population and animals were treated with a single 20 Gy fraction of RT using a SARRP, αOX40/αCTLA-4, or combined IT/RT 21 days after transfer. To assess anergy/exhaustion in endogenous CD8 T cell responses CT26 colon carcinoma bearing BALB/c mice were treated with IT, RT, or combined IT/RT either 10 or 17 days after tumor implantation. The frequency/function of tumor-specific (AH1 or OT-I) CD8 T cells was monitored in blood, lymph node (LN), and tumor infiltrating lymphocytes (TIL) in these models. Reporter Nur77-GFP-OT-I CD8 T cells expressing green fluorescent protein in response to TCR ligation were used in to measure the presence/strength of TCR signaling following IT or RT. We demonstrate OT-I cells become anergic/exhausted and produce few effector cytokines 21 days after adoptive transfer. Combination therapy with IT/RT resulted in increased TCR stimulation (measured by GFP expression) and reversed anergy (measured by proliferation) within OT-I T cells in LN and TIL compared to IT or RT alone. Combination therapy was able to reverse functional exhaustion (measured by cytokine production) within LN but not TIL in this model. In CT26 tumor bearing mice combination IT/RT significantly increased (up to 4-fold) the frequency of tumor-specific endogenous AH1 CD8 TIL and prevented development of functional exhaustion within TIL when compared to IT or RT alone. Combined ablative RT and IT with αOX40/αCTLA-4 results in increased CD8 TCR signaling, reversal of T cell anergy, and can prevent or reverse functional exhaustion. These novel results suggest the addition of RT to IT can effectively reverse tumor-associated T cell dysfunction where IT alone is insufficient and provide rationale for early phase clinical trial design.

TCR Signal Quality Modulates Fate Decisions of Single CD4+ T Cells in a Probabilistic Manner

To what extent the lineage decisions of activated CD4+ Tcells are determined by the quality of Tcell receptor (TCR) ligation is incompletely understood. Here, we show that individual Tcells expressing identical TCRs take highly variable fate decisions despite binding the same ligand. We identify a mathematical model that correctly captures this probabilistic behavior and allows one to formalize changes inTCR signal quality-due to cognate versus altered peptide ligation-as changes of lineage-specific proliferation and differentiation rates. We show that recall responses also adhere to this probabilistic framework requiring recruitment of multiple memory clones to provide reliable differentiation patterns. By extending our framework to simulate hypothetical TCRs of distinct binding strength, we reconstruct primary and secondary response patterns emerging from a polyclonal TCR repertoire insilico. Collectively, these data suggest that individual Tcells harboring distinct TCRs generate overlapping primary differentiation patterns that segregate only upon repetitive immunization.