Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab.

The mechanisms by which immune checkpoint blockade modulates tumor evolution during therapy are unclear. We assessed genomic changes in tumors from 68 patients with advanced melanoma, who progressed on ipilimumab or were ipilimumab-naive, before and after nivolumab initiation (CA209-038 study). Tumors were analyzed by whole-exome, transcriptome, and/or T cell receptor (TCR) sequencing. In responding patients, mutation and neoantigen load were reduced from baseline, and analysis of intratumoral heterogeneity during therapy demonstrated differential clonal evolution within tumors and putative selection against neoantigenic mutations on-therapy. Transcriptome analyses before and during nivolumab therapy revealed increases in distinct immune cell subsets, activation of specific transcriptional networks, and upregulation of immune checkpoint genes that were more pronounced in patients with response. Temporal changes in intratumoral TCR repertoire revealed expansion of T cell clones in the setting of neoantigen loss. Comprehensive genomic profiling data in this study provide insight into nivolumab's mechanism of action.

Nanoparticles for generating antigen-specific T cells for immunotherapy.

T cell therapy shows promise as an immunotherapy in both immunostimulatory and immunosuppressive applications. However, the forms of T cell-based therapy that are currently in the clinic, such as adoptive cell transfer and vaccines, are limited by cost, time-to-treatment, and patient variability. Nanoparticles offer a modular, universal platform to improve the efficacy of various T cell therapies as nanoparticle properties can be easily modified for enhanced cell targeting, organ targeting, and cell internalization. Nanoparticles can enhance or even replace endogenous cells during each step of generating an antigen-specific T cell response - from antigen presentation and T cell activation to T cell maintenance. In this review, we discuss the unique applications of nanoparticles for antigen-specific T cell therapy, focusing on nanoparticles as vaccines (to activate endogenous antigen presenting cells (APCs)), as artificial Antigen Presenting Cells (aAPCs, to directly activate T cells), and as drug delivery vehicles (to support activated T cells).

Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults.

BACKGROUND: Cytomegalovirus (CMV) infection leads to effector memory CD8+ T cell expansion and is associated with immune dysfunction in older adults. However, the molecular alterations of CMV-specific CD8+ T cells in CMV infected healthy young and middle-aged adults has not been fully characterized. RESULTS: We compared CD8+ T cells specific for a CMV epitope (pp65495-503, NLV) and an influenza A virus (IAV) epitope (M158-66, GIL) from the same young and middle-aged healthy adults with serum positive for anti-CMV IgG. Compared to the IAV-specific CD8+ T cells, CMV-specific CD8+ T cells contained more differentiated effector memory (TEM and TEMRA) cells. Isolated CMV-specific central memory (TCM) but not naïve (TN) cells had a significant reduced activation-induced expansion in vitro compared to their IAV-specific counterparts. Furthermore, we found that CD70 expression was reduced in CMV-specific CD28+CD8+ TCM and that CD70+ TCM had better expansion in vitro than did CD70- TCM. Mechanistically, we showed that CD70 directly enhanced MAPK phosphorylation and CMV-specific CD8+ TCM cells had a reduced MAPK signaling upon activation. Lastly, we showed that age did not exacerbate reduced CD70 expression in CMV- specific CD8+ TCM cells. CONCLUSION: Our findings showed that CMV infection causes mild expansion of CMV-NLV-specific CD8+ T cells, reduced CD70 expression and signaling, and proliferation of CMV-NLV-specific CD8+ TCM cells in young and middle-aged healthy adults and revealed an age-independent and CMV infection-specific impact on CD8+ memory T cells.

Anti-CD20 therapy depletes activated myelin-specific CD8+ T cells in multiple sclerosis.

CD8+ T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential autoantigenic targets, prior studies of myelin-reactive CD8+ T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8+ T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8+ T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8+ T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8+ T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8+ T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8+ T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20+ CD8+ T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8+ T cells in MS, indicates these cells may be attractive targets in MS therapy.

Adaptive Nanoparticle Platforms for High Throughput Expansion and Detection of Antigen-Specific T cells.

T cells are critical players in disease; yet, their antigen-specificity has been difficult to identify, as current techniques are limited in terms of sensitivity, throughput, or ease of use. To address these challenges, we increased the throughput and translatability of magnetic nanoparticle-based artificial antigen presenting cells (aAPCs) to enrich and expand (E+E) murine or human antigen-specific T cells. We streamlined enrichment, expansion, and aAPC production processes by enriching CD8+ T cells directly from unpurified immune cells, increasing parallel processing capacity of aAPCs in a 96-well plate format, and designing an adaptive aAPC that enables multiplexed aAPC construction for E+E and detection. We applied these adaptive platforms to process and detect CD8+ T cells specific for rare cancer neoantigens, commensal bacterial cross-reactive epitopes, and human viral and melanoma antigens. These innovations dramatically increase the multiplexing ability and decrease the barrier to adopt for investigating antigen-specific T cell responses.

Separating T Cell Targeting Components onto Magnetically Clustered Nanoparticles Boosts Activation.

T cell activation requires the coordination of a variety of signaling molecules including T cell receptor-specific signals and costimulatory signals. Altering the composition and distribution of costimulatory molecules during stimulation greatly affects T cell functionality for applications such as adoptive cell therapy (ACT), but the large diversity in these molecules complicates these studies. Here, we develop and validate a reductionist T cell activation platform that enables streamlined customization of stimulatory conditions. This platform is useful for the optimization of ACT protocols as well as the more general study of immune T cell activation. Rather than decorating particles with both signal 1 antigen and signal 2 costimulus, we use distinct, monospecific, paramagnetic nanoparticles, which are then clustered on the cell surface by a magnetic field. This allows for rapid synthesis and characterization of a small number of single-signal nanoparticles which can be systematically combined to explore and optimize T cell activation. By increasing cognate T cell enrichment and incorporating additional costimulatory molecules using this platform, we find significantly higher frequencies and numbers of cognate T cells stimulated from an endogenous population. The magnetic field-induced association of separate particles thus provides a tool for optimizing T cell activation for adoptive immunotherapy and other immunological studies.

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.

Linking form to function: Biophysical aspects of artificial antigen presenting cell design.

Artificial antigen presenting cells (aAPCs) are engineered platforms for T cell activation and expansion, synthesized by coupling T cell activating proteins to the surface of cell lines or biocompatible particles. They can serve both as model systems to study the basic aspects of T cell signaling and translationally as novel approaches for either active or adoptive immunotherapy. Historically, these reductionist systems have not been designed to mimic the temporally and spatially complex interactions observed during endogenous T cell-APC contact, which include receptor organization at both micro- and nanoscales and dynamic changes in cell and membrane morphologies. Here, we review how particle size and shape, as well as heterogenous distribution of T cell activating proteins on the particle surface, are critical aspects of aAPC design. In doing so, we demonstrate how insights derived from endogenous T cell activation can be applied to optimize aAPC, and in turn how aAPC platforms can be used to better understand endogenous T cell stimulation. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Telomere Length as an Indicator of the Robustness of B- and T-Cell Response to Influenza in Older Adults.

BACKGROUND: Telomeres provide a key mechanism for protecting the integrity of chromosomes and their attrition after cell division and during aging are evident in lymphocytes. However, the significance of telomere shortening in age-associated decline of immune function is unknown. METHODS: We selected 22 HLA-A2-positive healthy older adults who have relatively short or long telomere lengths to compare their antibody response against the influenza vaccine, and their CD8(+) T-cell response against an influenza antigen. RESULTS: B cells from individuals with a robust antibody response to the influenza vaccine had significantly longer telomeres than those with a poor antibody response. Monocyte-derived antigen-presenting cells of both short and long telomere groups induced similar expansions of influenza M1-specific CD8(+) T cells. Vaccination did not increase M1-specific CD8(+) T cells in blood, but M1-specific CD8(+) T cells from the long telomere group exhibited significantly greater expansion in vitro than those from the short telomere group. Finally, M1-specific CD8(+) T cells that underwent more expansions had significantly longer telomeres than cells with fewer divisions. CONCLUSIONS: Telomere length is positively associated with a robust lymphocyte response, and telomere attrition may contribute to the age-associated decline of adaptive immunity.

Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation.

Non-spherical nanodimensional artificial antigen presenting cells (naAPCs) offer the potential to systemically induce an effective antigen-specific immune response. In this report it is shown biodegradable ellipsoidal naAPCs mimic the T-Cell/APC interaction better than equivalent spherical naAPCs. In addition, it is demonstrated ellipsoidal naAPCs offer reduced non-specific cellular uptake and a superior pharmacokinetic profile compared to spherical naAPCs.

Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer.

Antigen-specific CD8+ T-cell tolerance, induced by myeloid-derived suppressor cells (MDSCs), is one of the main mechanisms of tumor escape. Using in vivo models, we show here that MDSCs directly disrupt the binding of specific peptide-major histocompatibility complex (pMHC) dimers to CD8-expressing T cells through nitration of tyrosines in a T-cell receptor (TCR)-CD8 complex. This process makes CD8-expressing T cells unable to bind pMHC and to respond to the specific peptide, although they retain their ability to respond to nonspecific stimulation. Nitration of TCR-CD8 is induced by MDSCs through hyperproduction of reactive oxygen species and peroxynitrite during direct cell-cell contact. Molecular modeling suggests specific sites of nitration that might affect the conformational flexibility of TCR-CD8 and its interaction with pMHC. These data identify a previously unknown mechanism of T-cell tolerance in cancer that is also pertinent to many pathological conditions associated with accumulation of MDSCs.

Nanoscale artificial antigen presenting cells for T cell immunotherapy.

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. FROM THE CLINICAL EDITOR: Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.

Artificial Antigen-Presenting Cell Fabrication for Murine T Cell Expansion.

Antigen-presenting cells (APCs), such as dendritic cells and macrophages, have a unique ability to survey the body and present information to T cells via peptide-loaded major histocompatibility complexes (signal 1). This presentation, along with a co-stimulatory signal (signal 2), leads to activation and subsequent expansion of T cells. This process can be harnessed and utilized for therapeutic applications, but the use of patient-derived APCs can be complex and inefficient. Alternatively, artificial APCs (aAPCs) provide a simplified method to achieve T cell activation by presenting the two necessary stimulatory signals. This protocol describes the utilization of magnetic nanoparticles and stimulatory proteins to create aAPCs that can be employed for activating and expanding antigen-specific T cells for both basic and translational immunology and immunotherapy studies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Protein and particle modification for aAPC fabrication Basic Protocol 2: aAPC validation by immunolabeling of conjugated protein Support Protocol 1: Quantification of aAPC stock concentration Basic Protocol 3: Determination of aAPC usage for murine CD8+ T cell activation Support Protocol 2: Isolation of murine CD8+ T cells.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

Dynamic regulation of functionally distinct virus-specific T cells.

The functional capacities of CD8(+) T cells important for virus clearance are influenced by interactions with antigen presenting cells (APCs) and CD4(+) T cells during initial selection, subsequent expansion, and development of memory. Recently, investigators have shown that polyfunctional T cells correlate best with long-term protection, however, it is still unknown how to stimulate T cells to achieve these responses. To study this, we examined the phenotypes and functions of CD8(+) T cells specific for two different virus antigens stimulated ex vivo using either autologous monocyte-derived dendritic cells (moDCs) or HLA-A2-Ig-based artificial APCs (aAPCs). Although similar numbers of influenza virus and measles virus tetramer-positive cells were generated by stimulation with peptide-loaded moDCs and aAPCs, T cell function, assessed by expression of IL-2, IFN-gamma, TNF-alpha, MIP1beta, and CD107a, showed that aAPC-generated CD8(+) T cells were multifunctional, whereas moDC-generated cells were mostly monofunctional. aAPC-generated cells also produced more of each cytokine per cell than CD8(+) T cells generated with moDCs. These phenotypes were not fixed, as changing the culture conditions of expanding T cells from aAPCs to moDCs, and moDCs to aAPCs, reversed the phenotypes. We conclude that CD8(+) T cells are heterogeneous in their functionality and that this is dependent, in a dynamic way, on the stimulating APC. These studies will lead to understanding the factors that influence induction of optimal CD8(+) T cell function.

Shape matters: Biodegradable anisotropic nanoparticle artificial antigen presenting cells for cancer immunotherapy.

Artificial antigen presenting cells are biomimetic particles that recapitulate the signals presented by natural antigen presenting cells in order to stimulate T cells in an antigen-specific manner using an acellular platform. We have engineered an enhanced nanoscale biodegradable artificial antigen presenting cell by modulating particle shape to achieve a nanoparticle geometry that allows for increased radius of curvature and surface area for T cell contact. The non-spherical nanoparticle artificial antigen presenting cells developed here have reduced nonspecific uptake and improved circulation time compared both to spherical nanoparticles and to traditional microparticle technologies. Additionally, the anisotropic nanoparticle artificial antigen presenting cells efficiently engage with and activate T cells, ultimately leading to a marked anti-tumor effect in a mouse melanoma model that their spherical counterparts were unable to achieve. STATEMENT OF SIGNIFICANCE: Artificial antigen presenting cells (aAPC) can activate antigen-specific CD8+ T cells but have largely been limited to microparticle-based platforms and ex vivo T cell expansion. Although more amenable to in vivo use, nanoscale aAPC have traditionally been ineffective due to limited surface area available for T cell interaction. In this work, we engineered non-spherical biodegradable nanoscale aAPC to investigate the role of particle geometry and develop a translatable platform for T cell activation. The non-spherical aAPC developed here have increased surface area and a flatter surface for T cell engagement and, therefore, can more effectively stimulate antigen-specific T cells, resulting in anti-tumor efficacy in a mouse melanoma model.

Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity.

Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles.

Modulation of MHC binding by lateral association of TCR and coreceptor.

The structure of a T cell receptor (TCR) and its affinity for cognate antigen are fixed, but T cells regulate binding sensitivity through changes in lateral membrane organization. TCR microclusters formed upon antigen engagement participate in downstream signaling. Microclusters are also found 3-4 days after activation, leading to enhanced antigen binding upon rechallenge. However, others have found an almost complete loss of antigen binding four days after T cell activation, when TCR clusters are present. To resolve these contradictory results, we compared binding of soluble MHC-Ig dimers by transgenic T cells stimulated with a high (100 µM) or low (100 fM) dose of cognate antigen. Cells activated by a high dose of peptide bound sixfold lower amounts of CD8-dependent ligand K(b)-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand L(d)-QL9 equally well. Consistent with the differences between binding of CD8-dependent and CD8-independent peptide/MHC, Förster resonance energy transfer (FRET) measurements of molecular proximity reported little nanoscale association of TCR with CD8 (16 FRET units) compared to their association on cells stimulated by low antigen dose (62 FRET units). Loss of binding induced by changes in lateral organization of TCR and CD8 may serve as a regulatory mechanism to avoid excessive inflammation and immunopathology in response to aggressive infection.

Inhibition of Fas ligand in NOD mice unmasks a protective role for IL-10 against insulitis development.

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of pancreatic insulin-producing ß cells by autoreactive T cells early in life. Despite daily insulin injections, patients typically develop cardiovascular and other complications; and intensive efforts are being directed toward identifying therapeutic targets to prevent the disease without directly impinging on the host defense. Fas ligand (FasL) is one potential target. Fas-FasL interactions primarily regulate T-cell homeostasis, not activation. Nevertheless, spontaneous gene mutation of Fas (called lpr mutation) or FasL (called the gld mutation) prevents autoimmune diabetes in nonobese diabetic (NOD) mice, the widely used model for T1D. Furthermore, although homozygous gld mutations cause age-dependent lymphoproliferation, limiting the gld mutation to one allele (NOD-gld/+) or treating NOD-wild-type mice with FasL-neutralizing monoclonal antibody completely prevents the disease development without causing lymphoproliferation or immune suppression. Herein, we show that the heterozygous gld mutation inhibits the accumulation of diabetogenic T cells in the pancreas, without interfering with their proliferation and expansion in the draining pancreatic lymph nodes. Pancreata from NOD-gld/+ mice contained B cells that expressed CD5 and produced IL-10, which was critical for maintenance of the disease resistance because its neutralization with an IL-10 receptor-blocking monoclonal antibody allowed accumulation of CD4 T cells in the pancreas and led to insulitis development. The results provide novel insights into the pathogenesis of T1D that could have important therapeutic implications.

Wnt activation promotes memory T cell polyfunctionality via epigenetic regulator PRMT1.

T cell polyfunctionality is a hallmark of protective immunity against pathogens and cancer, yet the molecular mechanism governing it remains mostly elusive. We found that canonical Wnt agonists inhibited human memory CD8+ T cell differentiation while simultaneously promoting the generation of highly polyfunctional cells. Downstream effects of Wnt activation persisted after removal of the drug, and T cells remained polyfunctional following subsequent cell division, indicating the effect is epigenetically regulated. Wnt activation induced a gene expression pattern that is enriched with stem cell-specific gene signatures and upregulation of protein arginine methyltransferase 1 (PRMT1), a known epigenetic regulator. PRMT1+CD8+ T cells are associated with enhanced polyfunctionality, especially the ability to produce IL-2. In contrast, inhibition of PRMT1 ameliorated the effects of Wnt on polyfunctionality. Chromatin immunoprecipitation revealed that H4R3me2a, a permissive transcription marker mediated by PRMT1, increased at the IL-2 promoter loci following Wnt activation. In vivo, Wnt-treated T cells exhibited superior polyfunctionality and persistence. When applied to cytomegalovirus (CMV) donor-seropositive, recipient-seronegative patients (D+/R-) lung transplant patient samples, Wnt activation enhanced CMV-specific T cell polyfunctionality, which is important in controlling CMV diseases. These findings reveal a molecular mechanism governing T cell polyfunctionality and identify PRMT1 as a potential target for T cell immunotherapy.