Taking T cell priming down a Notch: signaling through Notch receptors enhances T cell sensitivity to antigen.

Notch receptors are widely expressed and have recognized functions in thymocytes and mature T cells. In this issue, Laky et al. (2015) show that Notch interactions with Delta-like ligand 4 (DLL4) amplify priming of naive T cells.

Disseminated Coccidioidomycosis Treated with Interferon-γ and Dupilumab.

We describe a case of life-threatening disseminated coccidioidomycosis in a previously healthy child. Like most patients with disseminated coccidioidomycosis, this child had no genomic evidence of any known, rare immune disease. However, comprehensive immunologic testing showed exaggerated production of interleukin-4 and reduced production of interferon-γ. Supplementation of antifungal agents with interferon-γ treatment slowed disease progression, and the addition of interleukin-4 and interleukin-13 blockade with dupilumab resulted in rapid resolution of the patient's clinical symptoms. This report shows that blocking of type 2 immune responses can treat infection. This immunomodulatory approach could be used to enhance immune clearance of refractory fungal, mycobacterial, and viral infections. (Supported by the Jeffrey Modell Foundation and the National Institutes of Health.).

Progressive B Cell Loss in Revertant X-SCID.

We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20 years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversion mutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID. One interesting feature was a progressive loss of B cells over 20 years. CyTOF (cytometry time of flight) analysis of bone marrow offered a potential explanation of the B cell failure, with expansions of progenitor populations that suggest a developmental block. Another interesting feature was that the patient bore extensive granulomatous disease and skin cancers that contained T cells, despite severe T cell lymphopenia in the blood. Finally, the patient had a few hundred T cells on presentation but his TCRs comprised a very limited repertoire, supporting the important conclusion that repertoire size trumps numbers of T cells.

Augmentation of T-Cell Activation by Oscillatory Forces and Engineered Antigen-Presenting Cells.

Activation of T cells by antigen presenting cells (APCs) initiates their proliferation, cytokine production, and killing of infected or cancerous cells. We and others have shown that T-cell receptors require mechanical forces for triggering, and these forces arise during the interaction of T cells with APCs. Efficient activation of T cells in vitro is necessary for clinical applications. In this paper, we studied the impact of combining mechanical, oscillatory movements provided by an orbital shaker with soft, biocompatible, artificial APCs (aAPCs) of various sizes and amounts of antigen. We showed that these aAPCs allow for testing the strength of signal delivered to T cells, and enabled us to confirm that that absolute amounts of antigen engaged by the T cell are more important for activation than the density of antigen. We also found that when our aAPCs interact with T cells in the context of an oscillatory mechanoenvironment, they roughly double antigenic signal strength, compared to conventional, static culture. Combining these effects, our aAPCs significantly outperformed the commonly used Dynabeads. We finally demonstrated that tuning the signal strength down to a submaximal "sweet spot" allows for robust expansion of induced regulatory T cells. In conclusion, augmenting engineered aAPCs with mechanical forces offers a novel approach for tuning of T-cell activation and differentiation.

CD28-CD80 interactions control regulatory T cell motility and immunological synapse formation.

Regulatory T cells (Tregs) are essential for tolerance to self and environmental Ags, acting in part by downmodulating costimulatory molecules on the surface of dendritic cells (DCs) and altering naive CD4 T cell-DC interactions. In this study, we show that Tregs form stable conjugates with DCs before, but not after, they decrease surface expression of the costimulatory molecule CD80 on the DCs. We use supported planar bilayers to show that Tregs dramatically slow down but maintain a highly polarized and motile phenotype after recognizing Ag in the absence of costimulation. These motile cells are characterized by distinct accumulations of LFA-1-ICAM-1 in the lamella and TCR-MHC in the uropod, consistent with a motile immunological synapse or "kinapse." However, in the presence of high, but not low, concentrations of CD80, Tregs form stationary, symmetrical synapses. Using blocking Abs, we show that, whereas CTLA-4 is required for CD80 downmodulation, CD28-CD80 interactions are critical for modulating Treg motility in the presence of Ag. Taken together, these results support the hypothesis that Tregs are tuned to alter their motility depending on costimulatory signals.

Enhancing tumor-infiltrating T cells with an exclusive fuel source.

Solid tumors harbor immunosuppressive microenvironments that inhibit tumor infiltrating lymphocytes (TILs) through the voracious consumption of glucose. We sought to restore TIL function by providing them with an exclusive fuel source. The glucose disaccharide cellobiose, which is a building block of cellulose, contains a ß-1,4-glycosidic bond that cannot be hydrolyzed by animals (or their tumors), but fungal and bacterial organisms have evolved enzymes to catabolize cellobiose and use the resulting glucose. By equipping T cells with two proteins that enable import and hydrolysis of cellobiose, we demonstrate that supplementation of cellobiose during glucose withdrawal restores T cell cytokine production and cellular proliferation. Murine tumor growth is suppressed and survival is prolonged. Offering exclusive access to a natural disaccharide is a new tool that augments cancer immunotherapies. Beyond cancer, this approach could be used to answer questions about the regulation of glucose metabolism across many cell types, biological processes, and diseases.

Harnessing Biomaterials to Amplify Immunity in Aged Mice through T Memory Stem Cells.

The durability of a protective immune response generated by a vaccine depends on its ability to induce long-term T cell immunity, which tends to decline in aging populations. The longest protection appears to arise from T memory stem cells (TMSCs) that confer high expandability and effector functions when challenged. Here we engineered artificial antigen presenting cells (aAPC) with optimized size, stiffness and activation signals to induce human and mouse CD8+ TMSCs in vitro. This platform was optimized as a vaccine booster of TMSCs (Vax-T) with prolonged release of small-molecule blockade of the glycogen synthase kinase-3ß together with target antigens. By using SARS-CoV-2 antigen as a model, we show that a single injection of Vax-T induces durable antigen-specific CD8+ TMSCs in young and aged mice, and generates humoral responses at a level stronger than or similar to soluble vaccines. This Vax-T approach can boost long-term immunity to fight infectious diseases, cancer, and other diseases.

Cyclosporine-resistant, Rab27a-independent mobilization of intracellular preformed CD40 ligand mediates antigen-specific T cell help in vitro.

CD40L is critically important for the initiation and maintenance of adaptive immune responses. It is generally thought that CD40L expression in CD4(+) T cells is regulated transcriptionally and made from new mRNA following Ag recognition. However, recent studies with two-photon microscopy revealed that most cognate interactions between effector CD4(+) T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4(+) T cells store preformed CD40L (pCD40L) in lysosomal compartments and that pCD40L comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during brief encounters in vivo. However, it has not been shown that this relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. In this study, we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while de novo CD40L and cytokine expression are completely inhibited. These drugs thus provide a tool to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and that mobilization of pCD40L does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4(+) T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery.

Systemic enhancement of antitumour immunity by peritumourally implanted immunomodulatory macroporous scaffolds.

A tumour microenvironment abundant in regulatory T (Treg) cells aids solid tumours to evade clearance by effector T cells. Systemic strategies to suppress Treg cells or to augment immunity can elicit autoimmune side effects, cytokine storms and other toxicities. Here we report the design, fabrication and therapeutic performance of a biodegradable macroporous scaffold, implanted peritumourally, that releases a small-molecule inhibitor of transforming growth factor ß to suppress Treg cells, chemokines to attract effector T cells and antibodies to stimulate them. In two mouse models of aggressive tumours, the implant boosted the recruitment and activation of effector T cells into the tumour and depleted it of Treg cells, which resulted in an 'immunological abscopal effect' on distant metastases and in the establishment of long-term memory that impeded tumour recurrence. We also show that the scaffold can be used to deliver tumour-antigen-specific T cells into the tumour. Peritumourally implanted immunomodulatory scaffolds may represent a general strategy to enhance T-cell immunity and avoid the toxicities of systemic therapies.

Diversity in immunological synapse structure.

Immunological synapses (ISs) are formed at the T cell-antigen-presenting cell (APC) interface during antigen recognition, and play a central role in T-cell activation and in the delivery of effector functions. ISs were originally described as a peripheral ring of adhesion molecules surrounding a central accumulation of T-cell receptor (TCR)-peptide major histocompatibility complex (pMHC) interactions. Although the structure of these 'classical' ISs has been the subject of intense study, non-classical ISs have also been observed under a variety of conditions. Multifocal ISs, characterized by adhesion molecules dispersed among numerous small accumulations of TCR-pMHC, and motile 'immunological kinapses' have both been described. In this review, we discuss the conditions under which non-classical ISs are formed. Specifically, we explore the profound effect that the phenotypes of both T cells and APCs have on IS structure. We also comment on the role that IS structure may play in T-cell function.

Mechanosensing through YAP controls T cell activation and metabolism.

Upon immunogenic challenge, lymph nodes become mechanically stiff as immune cells activate and proliferate within their encapsulated environments, and with resolution, they reestablish a soft baseline state. Here we show that sensing these mechanical changes in the microenvironment requires the mechanosensor YAP. YAP is induced upon activation and suppresses metabolic reprogramming of effector T cells. Unlike in other cell types in which YAP promotes proliferation, YAP in T cells suppresses proliferation in a stiffness-dependent manner by directly restricting the translocation of NFAT1 into the nucleus. YAP slows T cell responses in systemic viral infections and retards effector T cells in autoimmune diabetes. Our work reveals a paradigm whereby tissue mechanics fine-tune adaptive immune responses in health and disease.

Augmenting T-cell responses to tumors by in situ nanomanufacturing.

Recent innovations in immunoregulatory treatments have demonstrated both the impressive potential and vital role of T cells in fighting cancer. These treatments come at a cost, with systemic side effects including life-threatening autoimmunity and immune dysregulation the norm. Here, we developed an approach to locally synthesize immune therapies and in this way, avoid systemic toxicity. Rather than just encapsulating cytokines, we endowed our nanoparticles with transcriptional and translational machinery to make cytokines locally, in situ, and on demand (activated by light). We demonstrated the capabilities of these particles in vitro and in vivo, in a mouse model of melanoma, and showed that tumor-infiltrating T cells were more highly activated in the context of these "microfactory" particles that make the synthetic cytokine.

Intact B-Cell Signaling and Function With Host B-Cells 47 Years After Transplantation for X-SCID.

Introduction: Severe Combined Immunodeficiency (SCID) is a life-threatening immunodeficiency caused by several pathogenic genetic variants, and it is characterized by profound defects in T-cell numbers and immune function. First performed in the late 1960's, hematopoietic stem cell transplantation remains the standard treatment for most cases of SCID. There is a growing number of post-transplant SCID patients, and it is imperative to assess the long-term outcomes of these patients. We have reported here the longest follow-up of a post-transplant SCID patient who, to our knowledge, bears the first gamma chain (γc) variant to show intact IL-21 signaling. Case Presentation: The patient presented at 5 months of age with recurrent thrush and Pneumocystis jiroveci pneumonia. In 1971, at the age of 11 months, he received an unconditioned, matched, related donor transplant comprising whole, unprocessed bone marrow. He is now 48 years old without significant illness and has never required immunoglobulin replacement. He exhibits T-dependent vaccine responses. He does suffer from chronic warts and bacterial infections that have worsened in recent years. We confirmed a known pathogenic variant in the IL2RG gene showing a hemizygous variant NM_000206.2:c.675C>A, resulting in p.Ser225Arg. His chimerism studies revealed donor T cells, host B cells, host myeloid cells, and mixed NK cells. Lymphocyte enumeration revealed normal numbers and distribution of B cells. The host B cells carry the pathogenic variant in IL2RG, but, when stimulated with IL-21, they demonstrated intact, γc-dependent signaling. Conclusions: Even with host B cells, reconstitution with donor T cells can be sufficient to allow over four decades of survival when B-cell function is intact. Our case demonstrates that satisfactory B-cell function can arise as a consequence of both intact IL-21 signaling due to a hypomorphic γc variant, and close HLA matching with the donor to allow for effective T-cell help.

T-cell activation is modulated by the 3D mechanical microenvironment.

T cells recognize mechanical forces through a variety of cellular pathways, including mechanical triggering of both the T-cell receptor (TCR) and integrin LFA-1. Here we show that T cells can recognize forces arising from the mechanical rigidity of the microenvironment. We fabricated 3D scaffold matrices with mechanical stiffness tuned to the range 4-40 kPa and engineered them to be microporous, independently of stiffness. We cultured T cells and antigen presenting cells within the matrices and studied T-cell activation by flow cytometry and live-cell imaging. We found that there was an augmentation of T-cell activation, proliferation, and migration speed in the context of mechanically stiffer 3D matrices as compared to softer materials. These results show that T cells can sense their 3D mechanical environment and alter both their potential for activation and their effector responses in different mechanical environments. A 3D scaffold of tunable stiffness and consistent microporosity offers a biomaterial advancement for both translational applications and reductionist studies on the impact of tissue microenvironmental factors on cellular behavior.

Unraveling the mechanobiology of immune cells.

Immune cells can sense and respond to biophysical cues - from dynamic forces to spatial features - during their development, activation, differentiation and expansion. These biophysical signals regulate a variety of immune cell functions such as leukocyte extravasation, macrophage polarization, T cell selection and T cell activation. Recent studies have advanced our understanding on immune responses to biophysical cues and the underlying mechanisms of mechanotransduction, which provides rational basis for the design and development of immune-modulatory therapeutics. This review discusses the recent progress in mechanosensing and mechanotransduction of immune cells, particularly monocytes/macrophages and T lymphocytes, and features new biomaterial designs and biomedical devices that translate these findings into biomedical applications.

The Actin-Capping Protein Alpha-Adducin Is Required for T-Cell Costimulation.

Alpha-adducin (Add1) is a critical component of the actin-spectrin network in erythrocytes, acting to cap the fast-growing, barbed ends of actin filaments, and recruiting spectrin to these junctions. Add1 is highly expressed in T cells, but its role in T-cell activation has not been examined. Using a conditional knockout model, we show that Add1 is necessary for complete activation of CD4+ T cells in response to low levels of antigen but is dispensable for CD8+ T cell activation and response to infection. Surprisingly, costimulatory signals through CD28 were completely abrogated in the absence of Add1. This study is the first to examine the role of actin-capping in T cells, and it reveals a previously unappreciated role for the actin cytoskeleton in regulating costimulation.

Case Study: Mechanism for Increased Follicular Helper T Cell Development in Activated PI3K Delta Syndrome.

Gain-of-function variants in p110δ, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) expressed in lymphocytes, cause activated PI3-kinase δ syndrome (APDS), a primary immunodeficiency that is characterized by recurrent infections, viremia, lymphadenopathy, and autoimmunity. The mechanism of autoimmunity in APDS has not been well-understood. Here, we show the profound skewing of peripheral CD4+ T cells to a T follicular helper (TFH) phenotype in a patient with APDS bearing a novel p110δ variant, Y524S. We also saw a diminishment of transient Foxp3 expression in activated T cells. Mechanistic studies revealed that both the new variant and a previously described, pathogenic variant (E81K) enhanced an interaction between intracellular Osteopontin and p85α. This interaction had been shown in mice to promote TFH differentiation. Our results demonstrate a new influence of PI3K on human T cell differentiation that is unrelated to its lipid-kinase activity and suggest that TFH should be monitored in APDS patients.

Cytokine Secreting Microparticles Engineer the Fate and the Effector Functions of T-Cells.

T-cell immunotherapy is a promising approach for cancer, infection, and autoimmune diseases. However, significant challenges hamper its therapeutic potential, including insufficient activation, delivery, and clonal expansion of T-cells into the tumor environment. To facilitate T-cell activation and differentiation in vitro, core-shell microparticles are developed for sustained delivery of cytokines. These particles are enriched by heparin to enable a steady release of interleukin-2 (IL-2), the major T-cell growth factor, over 10+ d. The controlled delivery of cytokines is used to steer lineage specification of cultured T-cells. This approach enables differentiation of T-cells into central memory and effector memory subsets. It is shown that the sustained release of stromal cell-derived factor 1α could accelerate T-cell migration. It is demonstrated that CD4+ T-cells could be induced to high concentrations of regulatory T-cells through controlled release of IL-2 and transforming growth factor beta. It is found that CD8+ T-cells that received IL-2 from microparticles are more likely to gain effector functions as compared with traditional administration of IL-2. Culture of T-cells within 3D scaffolds that contain IL-2-secreting microparticles enhances proliferation as compared with traditional, 2D approaches. This yield a new method to control the fate of T-cells and ultimately to new strategies for immune therapy.