Sparsely PEGylated poly(beta-amino ester) polyplexes enhance antigen specific T-cell response of a bivalent SARS-CoV-2 DNA vaccine.

DNA technology has emerged as a promising route to accelerated manufacture of sequence agnostic vaccines. For activity, DNA vaccines must be protected and delivered to the correct antigen presenting cells. However, the physicochemical properties of the vector must be carefully tuned to enhance interaction with immune cells and generate sufficient immune response for disease protection. In this study, we have engineered a range of polymer-based nanocarriers based on the poly(beta-amino ester) (PBAE) polycation platform to investigate the role that surface poly(ethylene glycol) (PEG) density has on pDNA encapsulation, formulation properties and gene transfectability both in vitro and in vivo. We achieved this by synthesising a non-PEGylated and PEGylated PBAE and produced formulations containing these PBAEs, and mixed polyplexes to tune surface PEG density. All polymers and co-formulations produced small polyplex nanoparticles with almost complete encapsulation of the cargo in all cases. Despite high gene transfection in HEK293T cells, only the fully PEGylated and mixed formulations displayed significantly higher expression of the reporter gene than the negative control in dendritic cells. Further in vivo studies with a bivalent SARS-CoV-2 pDNA vaccine revealed that only the mixed formulation led to strong antigen specific T-cell responses, however this did not translate into the presence of serum antibodies indicating the need for further studies into improving immunisation with polymer delivery systems.

Cancer vaccines: from an immunology perspective.

The concept of a therapeutic cancer vaccine to activate anti-tumour immunity pre-dates innovations in checkpoint blockade immunotherapies. However, vaccination strategies have yet to show the hoped-for successes in patients, and unanswered questions regarding the underlying immunological mechanisms behind cancer vaccines have hampered translation to clinical practice. Recent advances in our understanding of the potential of tumour mutational burden and neo-antigen-reactive T cells for response to immunotherapy have re-ignited enthusiasm for cancer vaccination strategies, coupled with the development of novel mRNA-based vaccines following successes in prevention of COVID-19. Here we summarise current developments in cancer vaccines and discuss how advances in our comprehension of the cellular interplay in immunotherapy-responsive tumours may inform better design of therapeutic cancer vaccines, with a focus on the role of dendritic cells as the orchestrators of anti-tumour immunity. The increasing number of clinical trials and research being funnelled into cancer vaccines has demonstrated the 'proof-of-principle', supporting the hypothesis that therapeutic vaccines have potential as an immuno-oncology agent. For efficacious and safe cancer vaccines to be developed, better understanding of the underpinning immunological mechanisms is paramount.

Metabolic adaption of mucosal macrophages: Is metabolism a driver of persistence across tissues?

Macrophages play essential roles in tissue homeostasis, defense, and repair. Their functions are highly tissue-specific, and when damage and inflammation stimulate repopulation by circulating monocytes, the incoming monocytes rapidly acquire the same, tissue-specific functions as the previous, resident macrophages. Several environmental factors are thought to guide the functional differentiation of recruited monocytes, including metabolic pressures imposed by the fuel sources available in each tissue. Here we discuss whether such a model of metabolic determinism can be applied to macrophage differentiation across barrier sites, from the lung to the skin. We suggest an alternative model, in which metabolic phenotype is a consequence of macrophage longevity rather than an early driver of tissue-specific adaption.

Localized immune surveillance of primary melanoma in the skin deciphered through executable modeling.

While skin is a site of active immune surveillance, primary melanomas often escape detection. Here, we have developed an in silico model to determine the local cross-talk between melanomas and Langerhans cells (LCs), the primary antigen-presenting cells at the site of melanoma development. The model predicts that melanomas fail to activate LC migration to lymph nodes until tumors reach a critical size, which is determined by a positive TNF-α feedback loop within melanomas, in line with our observations of murine tumors. In silico drug screening, supported by subsequent experimental testing, shows that treatment of primary tumors with MAPK pathway inhibitors may further prevent LC migration. In addition, our in silico model predicts treatment combinations that bypass LC dysfunction. In conclusion, our combined approach of in silico and in vivo studies suggests a molecular mechanism that explains how early melanomas develop under the radar of immune surveillance by LC.

Which interventions improve HPV vaccination uptake and intention in children, adolescents and young adults? An umbrella review.

BACKGROUND: Human papillomavirus (HPV) vaccination offers protection against the virus responsible for cervical, oropharyngeal, anal, vulval and penile cancers. However, there is considerable variation across, and even within, countries as to how HPV vaccination is offered and accepted. This review aimed to identify what interventions exist to promote uptake and how effective they are. METHODS: We conducted an umbrella review using the JBI (Joanna Briggs Institute) methodology to evaluate routine or catch-up interventions to increase HPV vaccination uptake and/or intention for children aged 9 years and older, adolescents and young adults up to 26. Comprehensive searches for English language quantitative systematic reviews, published between January 2011 and July 2021, were conducted across five databases. After reviewing titles and abstract, relevant papers were independently assessed in detail. MAIN RESULTS: From 1046 records identified, 10 articles were included in the review. They reported on 95 randomised controlled trials, 28 quasi-experimental studies, 14 cohort studies, 6 non-randomised pretest/post-test studies with control groups, 5 single-group pretest/post-test studies, 1 single-group post-test study and 1 randomised longitudinal study. Some interventions promoted change at the individual, community or organisational level, while others used a multicomponent approach. Face-to-face presentations, printed information and supplementing both strategies with additional components appear effective at increasing vaccination intention, while reminders and multicomponent strategies, especially ones that include some intervention aimed at provider level, appear effective at increasing vaccination uptake. Interventions that did not lead to an improvement in HPV vaccination intention or uptake varied in design and impacts were inconsistent across children/adolescents, young adults or parents. CONCLUSION: The evidence suggests that there is no single solution to increasing vaccination uptake and that different approaches may be better suited to certain populations. However, generalisations are limited by poor reporting and a paucity of studies beyond the USA. Further high-quality studies, therefore, are needed to understand how best to increase HPV vaccination uptake in different target populations.

Loss of T cell tolerance in the skin following immunopathology is linked to failed restoration of the dermal niche by recruited macrophages.

T cell pathology in the skin leads to monocyte influx, but we have little understanding of the fate of recruited cells within the diseased niche, or the long-term impact on cutaneous immune homeostasis. By combining a murine model of acute graft-versus-host disease (aGVHD) with analysis of patient samples, we demonstrate that pathology initiates dermis-specific macrophage differentiation and show that aGVHD-primed macrophages continue to dominate the dermal compartment at the relative expense of quiescent MHCIIint cells. Exposure of the altered dermal niche to topical haptens after disease resolution results in hyper-activation of regulatory T cells (Treg), but local breakdown in tolerance. Disease-imprinted macrophages express increased IL-1ß and are predicted to elicit altered TNF superfamily interactions with cutaneous Treg, and we demonstrate the direct loss of T cell regulation within the resolved skin. Thus, T cell pathology leaves an immunological scar in the skin marked by failure to re-set immune homeostasis.

Graft-versus-host disease reduces lymph node display of tissue-restricted self-antigens and promotes autoimmunity.

Acute graft-versus-host disease (GVHD) is initially triggered by alloreactive T cells, which damage peripheral tissues and lymphoid organs. Subsequent transition to chronic GVHD involves the emergence of autoimmunity, although the underlying mechanisms driving this process are unclear. Here, we tested the hypothesis that acute GVHD blocks peripheral tolerance of autoreactive T cells by impairing lymph node (LN) display of peripheral tissue-restricted antigens (PTAs). At the initiation of GVHD, LN fibroblastic reticular cells (FRCs) rapidly reduced expression of genes regulated by DEAF1, an autoimmune regulator-like transcription factor required for intranodal expression of PTAs. Subsequently, GVHD led to the selective elimination of the FRC population, and blocked the repair pathways required for its regeneration. We used a transgenic mouse model to show that the loss of presentation of an intestinal PTA by FRCs during GVHD resulted in the activation of autoaggressive T cells and gut injury. Finally, we show that FRCs normally expressed a unique PTA gene signature that was highly enriched for genes expressed in the target organs affected by chronic GVHD. In conclusion, acute GVHD damages and prevents repair of the FRC network, thus disabling an essential platform for purging autoreactive T cells from the repertoire.

A wave of monocytes is recruited to replenish the long-term Langerhans cell network after immune injury.

A dense population of embryo-derived Langerhans cells (eLCs) is maintained within the sealed epidermis without contribution from circulating cells. When this network is perturbed by transient exposure to ultraviolet light, short-term LCs are temporarily reconstituted from an initial wave of monocytes but thought to be superseded by more permanent repopulation with undefined LC precursors. However, the extent to which this process is relevant to immunopathological processes that damage LC population integrity is not known. Using a model of allogeneic hematopoietic stem cell transplantation, where alloreactive T cells directly target eLCs, we have asked whether and how the original LC network is ultimately restored. We find that donor monocytes, but not dendritic cells, are the precursors of long-term LCs in this context. Destruction of eLCs leads to recruitment of a wave of monocytes that engraft in the epidermis and undergo a sequential pathway of differentiation via transcriptionally distinct EpCAM+ precursors. Monocyte-derived LCs acquire the capacity of self-renewal, and proliferation in the epidermis matched that of steady-state eLCs. However, we identified a bottleneck in the differentiation and survival of epidermal monocytes, which, together with the slow rate of renewal of mature LCs, limits repair of the network. Furthermore, replenishment of the LC network leads to constitutive entry of cells into the epidermal compartment. Thus, immune injury triggers functional adaptation of mechanisms used to maintain tissue-resident macrophages at other sites, but this process is highly inefficient in the skin.

Unraveling the Mechanisms of Cutaneous Graft-Versus-Host Disease.

The skin is the most common target organ affected by graft-versus-host disease (GVHD), with severity and response to therapy representing important predictors of patient survival. Although many of the initiating events in GVHD pathogenesis have been defined, less is known about why treatment resistance occurs or why there is often a permanent failure to restore tissue homeostasis. Emerging data suggest that the unique immune microenvironment in the skin is responsible for defining location- and context-specific mechanisms of injury that are distinct from those involved in other target organs. In this review, we address recent advances in our understanding of GVHD biology in the skin and outline the new research themes that will ultimately enable design of precision therapies.

Tumor-Resident Dendritic Cells and Macrophages Modulate the Accumulation of TCR-Engineered T Cells in Melanoma.

Ongoing clinical trials explore T cell receptor (TCR) gene therapy as a treatment option for cancer, but responses in solid tumors are hampered by the immunosuppressive microenvironment. The production of TCR gene-engineered T cells requires full T cell activation in vitro, and it is currently unknown whether in vivo interactions with conventional dendritic cells (cDCs) regulate the accumulation and function of engineered T cells in tumors. Using the B16 melanoma model and the inducible depletion of CD11c+ cells in CD11c.diphtheria toxin receptor (DTR) mice, we analyzed the interaction between tumor-resident cDCs and engineered T cells expressing the melanoma-specific TRP-2 TCR. We found that depletion of CD11c+ cells triggered the recruitment of cross-presenting cDC1 into the tumor and enhanced the accumulation of TCR-engineered T cells. We show that the recruited tumor cDCs present melanoma tumor antigen, leading to enhanced activation of TCR-engineered T cells. In addition, detailed analysis of the tumor myeloid compartment revealed that the depletion of a population of DT-sensitive macrophages can contribute to the accumulation of tumor-infiltrating T cells. Together, these data suggest that the relative frequency of tumor-resident cDCs and macrophages may impact the therapeutic efficacy of TCR gene therapy in solid tumors.

Peripheral tissues reprogram CD8+ T cells for pathogenicity during graft-versus-host disease.

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic stem cell transplantation induced by the influx of donor-derived effector T cells (TE) into peripheral tissues. Current treatment strategies rely on targeting systemic T cells; however, the precise location and nature of instructions that program TE to become pathogenic and trigger injury are unknown. We therefore used weighted gene coexpression network analysis to construct an unbiased spatial map of TE differentiation during the evolution of GVHD and identified wide variation in effector programs in mice and humans according to location. Idiosyncrasy of effector programming in affected organs did not result from variation in T cell receptor repertoire or the selection of optimally activated TE. Instead, TE were reprogrammed by tissue-autonomous mechanisms in target organs for site-specific proinflammatory functions that were highly divergent from those primed in lymph nodes. In the skin, we combined the correlation-based network with a module-based differential expression analysis and showed that Langerhans cells provided in situ instructions for a Notch-dependent T cell gene cluster critical for triggering local injury. Thus, the principal determinant of TE pathogenicity in GVHD is the final destination, highlighting the need for target organ-specific approaches to block immunopathology while avoiding global immune suppression.

Redirection to the bone marrow improves T cell persistence and antitumor functions.

A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.

Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.

The immunosuppressive activity of mesenchymal stromal cells (MSCs) is well documented. However, the therapeutic benefit is completely unpredictable, thus raising concerns about MSC efficacy. One of the affecting factors is the unresolved conundrum that, despite being immunosuppressive, MSCs are undetectable after administration. Therefore, understanding the fate of infused MSCs could help predict clinical responses. Using a murine model of graft-versus-host disease (GvHD), we demonstrate that MSCs are actively induced to undergo perforin-dependent apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. When examining patients with GvHD who received MSCs, we found a striking parallel, whereby only those with high cytotoxic activity against MSCs responded to MSC infusion, whereas those with low activity did not. The need for recipient cytotoxic cell activity could be replaced by the infusion of apoptotic MSCs generated ex vivo. After infusion, recipient phagocytes engulf apoptotic MSCs and produce indoleamine 2,3-dioxygenase, which is ultimately necessary for effecting immunosuppression. Therefore, we propose the innovative concept that patients should be stratified for MSC treatment according to their ability to kill MSCs or that all patients could be treated with ex vivo apoptotic MSCs.

Dendritic Cells Cross-Present Immunogenic Lentivector-Encoded Antigen from Transduced Cells to Prime Functional T Cell Immunity.

Recombinant lentiviral vectors (LVs) are highly effective vaccination vehicles that elicit protective T cell immunity in disease models. Dendritic cells (DCs) acquire antigen at sites of vaccination and migrate to draining lymph nodes, where they prime vaccine-specific T cells. The potency with which LVs activate CD8+ T cell immunity has been attributed to the transduction of DCs at the immunization site and durable presentation of LV-encoded antigens. However, it is not known how LV-encoded antigens continue to be presented to T cells once directly transduced DCs have turned over. Here, we report that LV-encoded antigen is efficiently cross-presented by DCs in vitro. We have further exploited the temporal depletion of DCs in the murine CD11c.DTR (diphtheria toxin receptor) model to demonstrate that repopulating DCs that were absent at the time of immunization cross-present LV-encoded antigen to T cells in vivo. Indirect presentation of antigen from transduced cells by DCs is sufficient to prime functional effector T cells that control tumor growth. These data suggest that DCs cross-present immunogenic antigen from LV-transduced cells, thereby facilitating prolonged activation of T cells in the absence of circulating LV particles. These are findings that may impact on the future design of LV vaccination strategies.

Depletion of CD11c⁺ cells in the CD11c.DTR model drives expansion of unique CD64⁺ Ly6C⁺ monocytes that are poised to release TNF-α.

Dendritic cells (DCs) play a vital role in innate and adaptive immunities. Inducible depletion of CD11c(+) DCs engineered to express a high-affinity diphtheria toxin receptor has been a powerful tool to dissect DC function in vivo. However, despite reports showing that loss of DCs induces transient monocytosis, the monocyte population that emerges and the potential impact of monocytes on studies of DC function have not been investigated. We found that depletion of CD11c(+) cells from CD11c.DTR mice induced the expansion of a variant CD64(+) Ly6C(+) monocyte population in the spleen and blood that was distinct from conventional monocytes. Expansion of CD64(+) Ly6C(+) monocytes was independent of mobilization from the BM via CCR2 but required the cytokine, G-CSF. Indeed, this population was also expanded upon exposure to exogenous G-CSF in the absence of DC depletion. CD64(+) Ly6C(+) monocytes were characterized by upregulation of innate signaling apparatus despite the absence of inflammation, and an increased capacity to produce TNF-α following LPS stimulation. Thus, depletion of CD11c(+) cells induces expansion of a unique CD64(+) Ly6C(+) monocyte population poised to synthesize TNF-α. This finding will require consideration in experiments using depletion strategies to test the role of CD11c(+) DCs in immunity.

G-CSF mobilizes CD34+ regulatory monocytes that inhibit graft-versus-host disease.

Granulocyte colony-stimulating factor (G-CSF) is routinely used to collect peripheral blood stem cells (PBSCs) from healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT). We show that, in both humans and mice, G-CSF mobilizes a subset of CD34(+) cells with mature monocyte features. These cells, which are phenotypically and functionally conserved in mice and humans, are transcriptionally distinct from myeloid and monocytic precursors but similar to mature monocytes and endowed with immunosuppressive properties. In response to interferon-γ released by activated T cells, these cells produce nitric oxide, which induces allogeneic T cell death both in vitro and in vivo. These apoptotic T cells are engulfed by macrophages that release transforming growth factor-ß and promote regulatory T cell expansion. Indeed, the fraction of CD34(+) monocytes in peripheral blood CD34(+) cells inversely correlates with the incidence of acute graft-versus-host disease (GVHD) in humans. Therefore, G-CSF-mobilized cells are an attractive candidate population to be expanded ex vivo for cellular therapy against GVHD.

OX40- and CD27-mediated costimulation synergizes with anti-PD-L1 blockade by forcing exhausted CD8+ T cells to exit quiescence.

Exhaustion of chronically stimulated CD8(+) T cells is a significant obstacle to immune control of chronic infections or tumors. Although coinhibitory checkpoint blockade with anti-programmed death ligand 1 (PD-L1) Ab can restore functions to exhausted T cell populations, recovery is often incomplete and dependent upon the pool size of a quiescent T-bet(high) subset that expresses lower levels of PD-1. In a model in which unhelped, HY-specific CD8(+) T cells gradually lose function following transfer to male bone marrow transplantation recipients, we have explored the effect of shifting the balance away from coinhibition and toward costimulation by combining anti-PD-L1 with agonistic Abs to the TNFR superfamily members, OX40 and CD27. Several weeks following T cell transfer, both agonistic Abs, but especially anti-CD27, demonstrated synergy with anti-PD-L1 by enhancing CD8(+) T cell proliferation and effector cytokine generation. Anti-CD27 and anti-PD-L1 synergized by downregulating the expression of multiple quiescence-related genes concomitant with a reduced frequency of T-bet(high) cells within the exhausted population. However, in the presence of persistent Ag, the CD8(+) T cell response was not sustained and the overall size of the effector cytokine-producing pool eventually contracted to levels below that of controls. Thus, CD27-mediated costimulation can synergize with coinhibitory checkpoint blockade to switch off molecular programs for quiescence in exhausted T cell populations, but at the expense of losing precursor cells required to maintain a response.

Cell-intrinsic regulation of murine dendritic cell function and survival by prereceptor amplification of glucocorticoid.

Although the inhibitory effects of therapeutic glucocorticoids (GCs) on dendritic cells (DCs) are well established, the roles of endogenous GCs in DC homeostasis are less clear. A critical element regulating endogenous GC concentrations involves local conversion of inactive substrates to active 11-hydroxyglucocorticoids, a reduction reaction catalyzed within the endoplasmic reticulum by an enzyme complex containing 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) and hexose-6-phosphate dehydrogenase (H6PDH). In this study, we found that this GC amplification pathway operates both constitutively and maximally in steady state murine DC populations and is unaffected by additional inflammatory stimuli. Under physiologic conditions, 11ßHSD1-H6PDH increases the sensitivity of plasmacytoid DCs (pDCs) to GC-induced apoptosis and restricts the survival of this population through a cell-intrinsic mechanism. Upon CpG activation, the effects of enzyme activity are overridden, with pDCs becoming resistant to GCs and fully competent to release type I interferon. CD8α(+) DCs are also highly proficient in amplifying GC levels, leading to impaired maturation following toll-like receptor-mediated signaling. Indeed, pharmacologic inhibition of 11ßHSD1 synergized with CpG to enhance specific T-cell responses following vaccination targeted to CD8α(+) DCs. In conclusion, amplification of endogenous GCs is a critical cell-autonomous mechanism for regulating the survival and functions of DCs in vivo.

PD-L1 co-stimulation contributes to ligand-induced T cell receptor down-modulation on CD8+ T cells.

T cell receptor (TCR) down-modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down-modulation. Here, we show that the interaction between programmed cell death 1 ligand 1 (PD-L1) on dendritic cells (DCs) and programmed death 1 (PD-1) on CD8 T cells contributes to ligand-induced TCR down-modulation. We provide evidence that this occurs via Casitas B-lymphoma (Cbl)-b E3 ubiquitin ligase up-regulation in CD8 T cells. Interference with PD-L1/PD-1 signalling markedly inhibits TCR down-modulation leading to hyper-activated, proliferative CD8 T cells as assessed in vitro and in vivo in an arthritis model. PD-L1 silencing accelerates anti-tumour immune responses and strongly potentiates DC anti-tumour capacities, when combined with mitogen-activated kinase (MAPK) modulators that promote DC activation.

Langerhans cells regulate cutaneous injury by licensing CD8 effector cells recruited to the skin.

Langerhans cells (LCs) are a distinct population of dendritic cells that form a contiguous network in the epidermis of the skin. Although LCs possess many of the properties of highly proficient dendritic cells, recent studies have indicated that they are not necessary to initiate cutaneous immunity. In this study, we used a tractable model of cutaneous GVHD, induced by topical application of a Toll-like receptor agonist, to explore the role of LCs in the development of tissue injury. By adapting this model to permit inducible and selective depletion of host LCs, we found that GVHD was significantly reduced when LCs were absent. However, LCs were not required either for CD8 T-cell activation within the draining lymph node or subsequent homing of effector cells to the epidermis. Instead, we found that LCs were necessary for inducing transcription of IFN-γ and other key effector molecules by donor CD8 cells in the epidermis, indicating that they license CD8 cells to induce epithelial injury. These data demonstrate a novel regulatory role for epidermal LCs during the effector phase of an inflammatory immune response in the skin.