T-cell Priming Research Articles

Direct antigen presentation is the canonical pathway of cytomegalovirus CD8 T-cell priming regulated by balanced immune evasion ensuring a strong antiviral response.

CD8 T cells are important antiviral effectors in the adaptive immune response to cytomegaloviruses (CMV). Naïve CD8 T cells can be primed by professional antigen-presenting cells (pAPCs) alternatively by "direct antigen presentation" or "antigen cross-presentation". In the case of direct antigen presentation, viral proteins are expressed in infected pAPCs and enter the classical MHC class-I (MHC-I) pathway of antigen processing and presentation of antigenic peptides. In the alternative pathway of antigen cross-presentation, viral antigenic material derived from infected cells of principally any cell type is taken up by uninfected pAPCs and eventually also fed into the MHC class-I pathway. A fundamental difference, which can be used to distinguish between these two mechanisms, is the fact that viral immune evasion proteins that interfere with the cell surface trafficking of peptide-loaded MHC-I (pMHC-I) complexes are absent in cross-presenting uninfected pAPCs. Murine cytomegalovirus (mCMV) models designed to disrupt either of the two presentation pathways revealed that both are possible in principle and can substitute each other. Overall, however, the majority of evidence has led to current opinion favoring cross-presentation as the canonical pathway. To study priming in the normal host genetically competent in both antigen presentation pathways, we took the novel approach of enhancing or inhibiting direct antigen presentation by using recombinant viruses lacking or overexpressing a key mCMV immune evasion protein. Against any prediction, the strongest CD8 T-cell response was elicited under the condition of intermediate direct antigen presentation, as it exists for wild-type virus, whereas the extremes of enhanced or inhibited direct antigen presentation resulted in an identical and weaker response. Our findings are explained by direct antigen presentation combined with a negative feedback regulation exerted by the newly primed antiviral effector CD8 T cells. This insight sheds a completely new light on the acquisition of viral immune evasion genes during virus-host co-evolution.

Biomaterial-assisted local oxygenation safeguards the prostimulatory phenotype and functions of human dendritic cells in hypoxia.

Dendritic cells (DCs), professional antigen-presenting cells, function as sentinels of the immune system. DCs initiate and fine-tune adaptive immune responses by presenting antigenic peptides to B and T lymphocytes to mount an effective immune response against cancer and pathogens. However, hypoxia, a condition characterized by low oxygen (O2) tension in different tissues, significantly impacts DC functions, including antigen uptake, activation and maturation, migration, as well as T-cell priming and proliferation. In this study, we employed O2-releasing biomaterials (O2-cryogels) to study the effect of localized O2 supply on human DC phenotype and functions. Our results indicate that O2-cryogels effectively mitigate DC exposure to hypoxia under hypoxic conditions. Additionally, O2-cryogels counteract hypoxia-induced inhibition of antigen uptake and migratory activity in DCs through O2 release and hyaluronic acid (HA) mediated mechanisms. Furthermore, O2-cryogels preserve and restore DC maturation and co-stimulation markers, including HLA-DR, CD86, and CD40, along with the secretion of proinflammatory cytokines in hypoxic conditions. Finally, our findings demonstrate that the supplemental O2 released from the cryogels preserves DC-mediated T-cell priming, ultimately leading to the activation and proliferation of allogeneic CD3+ T cells. This work emphasizes the potential of local oxygenation as a powerful immunomodulatory agent to improve DC activation and functions in hypoxia, offering new approaches for cancer and infectious disease treatments.

A replicating LCMV-based vaccine for the treatment of solid tumors

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T-cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of LCMV-based vectors to break tolerance and induce effective antigen-specific CD8+ T-cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T-cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T-cell priming, which renders previously “cold” tumors open to immune infiltration and reprograms the tumor microenvironment to “hot”. Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells

A long-lived latent reservoir of HIV-1-infected CD4 Tcells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 Tcells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory Tcells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

Association Of PTPN22 Gene Polymorphisms in Patients with Graves’ Disease in Iranian Papulation

Background: Graves’ disease (GD) is an autoimmune disease that is associated with increased thyroid gland irritation and, consequently, hyperthyroidism. Autoimmune diseases are common in the general population and are influenced by genetic and environmental factors. PTPN22, which was reported as a susceptible locus for GD in several populations, acts as a negative regulator for activation of primary T-cells, and LYP polymorphism could potentially increase susceptibility to Graves' disease, which may play a role in other autoimmune conditions as well. In this study, we investigated the association of several PTPN22 single nucleotide polymorphisms (SNPs) with Graves patients.
 Methods: After DNA extraction from peripheral blood cells, SNP Genotyping was performed through real-time PCR with allelic discrimination TaqMan genotyping assays (ABI Applied Biosystems, 7300 Real-Time PCR System, USA) based on manufacturer protocols. The frequencies of alleles and genotypes of PTPN22 SNPs (rs12760457, rs2476601, rs1310182, and rs1217414) were recorded.
 Results: In our study, the rs1310182 was significantly more frequent in patients with GD than in healthy individuals. While the C allele of rs1310182 was 1.78 times more frequent in GD patients (95%CI: 1.18- 2.69, P=0.005), the T allele was more frequent in healthy subjects (OR=0.56, 95% CI: 0.37-0.84, P=0.005). In addition, the CC genotype of this SNP was 1.86 times more common in patients (P=0.05). No significant differences were observed between the other SNPs of this gene in case and control.
 Conclusion: The results demonstrate that one SNP (rs1310182) of the PTPN22 gene is associated with susceptibility to GD in an Iranian population. Further studies, including functional analyses, are required.

Circular mRNA-based TCR-T offers a safe and effective therapeutic strategy for treatment of cytomegalovirus infection

Circular mRNA (cmRNA) is particular useful due to its high resistance to degradation by exonucleases, resulting in greater stability and protein expression compared to linear mRNA. Tcell receptor (TCR)-engineered Tcells (TCR-T) represent a promising means of treating viral infections and cancer. This study aimed to evaluate the feasibility and efficacy of cmRNA in antigen-specific-TCR discovery and TCR-T therapy. Using human cytomegalovirus (CMV) pp65 antigen as a model, we found that the expansion of pp65-responsive Tcells was induced more effectively by monocyte-derived dendritic cells transfected with pp65-encoding cmRNA compared with linear mRNA. Subsequently, we developed cmRNA-transduced pp65-TCR-T (cm-pp65-TCR-T) that specifically targets the CMV-pp65 epitope. Our results showed that pp65-TCR could be expressed on primary Tcells for more than 7days. Moreover, both invitro killing and invivo CDX models demonstrated that cm-pp65-TCR-T cells specifically and persistently kill pp65-and HLA-expressing tumor cells, significantly prolonging the survival of mice. Collectively, our results demonstrated that cmRNA can be used as a more effective technical approach for antigen-specific TCR isolation and identification, and cm-pp65-TCR-T may provide a safe, non-viral, non-integrated therapeutic approach for controlling CMV infection, particularly in patients who have undergone allogeneic hematopoietic stem cell transplantation.

Immunoneoadjuvant therapy with immune checkpoint inhibitors of gastric cancer: an emerging exemplification : Immunoneoadjuvant therapy of gastric cancer.

Advances in immune checkpoint inhibitors (ICIs) have enabled more effective treatment for individuals with various types of solid tumors. Given the improved survival benefit and acceptable safety profile of ICIs in advanced gastric cancer, there is plenty of interest in the use of ICIs in the neoadjuvant setting with curative intent. Theoretically, immunoneoadjuvant with ICIs could boost the levels of endogenous tumor antigen present in the tumor to enhance T-cell priming and further enhance systemic immunity. This systemic immune response may improve the detection and elimination of the disseminated micrometastatic tumors beyond the resected tumor, which are sources of postsurgical relapse. Numerous clinical studies have begun to explore the application of ICIs in neoadjuvant treatment of gastric cancer. This article reviews the progress in the use of ICI monotherapy and in combination with alternative therapies for the treatment of gastric cancer to aid in the development of gastric cancer immunoneoadjuvant therapy and improve the overall therapeutic benefit.

Early enterovirus translation deficits extend viral RNA replication and elicit sustained MDA5-directed innate signaling.

Multiple pattern recognition receptors sense vRNAs and initiate downstream innate signaling: endosomal Toll-like receptors (TLRs) 3, 7, and 8 and cytoplasmic RIG-I-like receptors (RLRs) RIG-I, and MDA5. They engage distinct signaling scaffolds: mitochondrial antiviral signaling protein (RLR), MyD88, and TLR-adaptor interacting with SLC15A4 on the lysosome (TLR7 and TLR8) and toll/IL-1R domain-containing adaptor inducing IFN (TLR3). By virtue of their unusual vRNA structure and direct host cell entry path, the innate response to EVs uniquely is orchestrated by MDA5. We reported that PVSRIPO's profound attenuation and loss of cytopathogenicity triggers MDA5-directed polar TBK1-IRF3 signaling that generates priming of polyfunctional antitumor CD8+ T-cell responses and durable antitumor surveillance in vivo. Here we unraveled EV-host relations that control suppression of host type-I IFN responses and show that PVSRIPO's deficient immediate host eIF4G cleavage generates unopposed MDA5-directed downstream signaling cascades resulting in sustained type-I IFN release.

Lactate activates the mitochondrial electron transport chain independently of its metabolism

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary Tcells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors

Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. We established an invivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity. Melanoma Research Alliance, Deutsche Forschungsgemeinschaft, German Cancer Aid, Wilhelm Sander Foundation, Novartis Foundation.

CD8 + T-cell priming is quantitatively but not qualitatively impaired in people with HIV-1 on antiretroviral therapy.

The induction of de novo CD8 + T-cell responses is essential for protective antiviral immunity, but this process is often impaired in people with HIV-1 (PWH). We investigated the extent to which the immune competence of naive CD8 + T cells, a key determinant of priming efficacy, could be preserved or restored in PWH via long-term antiretroviral therapy (ART). We used flow cytometry, molecular analyses of gene transcription and telomere length, and a fully validated priming assay to characterize naive CD8 + T cells ex vivo and evaluate the induction of antigen-specific effector/memory CD8 + T cells in vitro , comparing age-matched healthy uninfected donors (HUDs), PWH on ART, and natural HIV-1 controllers (HICs). We found that naive CD8 + T cells were numerically reduced and exhibited a trend toward shorter telomere lengths in PWH on ART compared with HUDs and HICs. These features associated with impaired priming efficacy. However, we also found that naive CD8 + T cells were fully equipped proliferatively and transcriptionally in PWH on ART, enabling the generation of antigen-specific effector/memory CD8 + T cells with functional and phenotypic attributes comparable to those primed from HUDs. Our data suggest that naive CD8 + T cells in PWH on ART are intrinsically capable of generating functionally and phenotypically intact effector/memory CD8 + T cells in response to antigen, despite evidence of senescence and an overall numerical reduction that compromises priming efficacy relative to HUDs and HICs.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

CRISPR-Cas9 genome editing has enabled advanced Tcell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human Tcells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor Tcells. Tcells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered Tcells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for Tcell engineering that mitigates this genotoxicity in the clinic.

Peripheral T-Cell Priming and Micrometastatic Disease Control with Metastasis-Directed Therapy: Multidimensional Immunogenomic Profiling of Oligometastatic Prostate Cancer in the EXTEND Trial

Comprehensive metastasis-directed therapy (MDT) for oligometastatic prostate cancer extended progression-free survival (PFS) and time to new lesion formation in the intermittent hormone therapy (HT) basket of EXTEND. To better understand the mechanism of MDT benefit, we pooled the intermittent and continuous HT baskets of EXTEND and tested the hypothesis that adding MDT to HT would program systemic T-cells to control micrometastatic disease. A total of 174 men were randomized to HT with or without MDT to up to 5 sites of metastases. HT was given for 6 months (intermittent basket, n = 87) or indefinitely (continuous basket, n = 87). Peripheral blood samples were drawn at enrollment, at the end of MDT, at 3 months follow-up (3 mo F/U), and at progression and then analyzed by flow cytometry, T-cell receptor (TCR)-β CDR3 variable region sequencing, multiplex cytokine profiling, and next-generation circulating tumor DNA (ctDNA) sequencing. TCR clonal expansion was determined using a published betabinomial model. Repertoire changes were assessed by Morisita's index, and dominant TCR repertoire motifs were characterized with ImmunoMap. Associations between blood markers and PFS were evaluated with Cox regression adjusted hazard ratios (aHR) accounting for randomization arm and stratifying for intermittent vs continuous HT. Randomization to MDT+HT was associated with T-cell activation, proliferation, and clonal expansion. This response was first observed at end-MDT as upregulated expression of T-cell activation and inhibition markers (i.e., ICOS, Tim-3, and LAG-3) and increases in highly proliferative CD4+ and CD8+ Ki67hi T-cells (all P<0.05). TCR sequencing of 7,678,911 T-cells revealed that MDT+HT was associated with TCR clonal expansion, remodeling of the TCR repertoire, and changes in dominant TCR motifs at end-MDT and 3 mo F/U (all P<0.05). Observed T-cell priming could be driven by signaling networks of canonical T-cell stimulatory cytokines (IL-2, IL-12, and IL-15), which were upregulated at end-MDT and persisted at 3 mo F/U (all P<0.05). This modulation of T-cell phenotype, clonotype, and cytokine concentrations was not observed in the HT-monotherapy arm. At end-MDT, systemic T-cell responses were associated with improved PFS, most notably CD8+ T-cell expression of LAG-3 (aHR 0.22, 95% CI 0.03-0.91) and high TCR clonal expansion (aHR 0.13, 95% CI 0.02-0.52). High ctDNA burden at end-MDT correlated with worse PFS (aHR 1.41, 95% CI 1.04-2.54), as did CD8+ T-cell expression of inhibitory receptor TIGIT at 3 mo F/U (aHR 1.03, 95% CI 1.01-1.06). The addition of MDT to HT induced systemic T-cell activation and expansion, which was not observed in the HT-only arm. This systemic immune response was independently associated with improved PFS. In addition to cytoreduction of macroscopic disease, MDT-induced immune education may be an important complementary mechanism of micrometastatic control in oligometastatic prostate cancer.

Potentiating Local and Abscopal Antitumor Efficacy through Radiation with FAP-CD40 DARPin and Anti-PD1 Therapy

Although the advent of Immune Checkpoint Inhibitors (ICI) has changed the facet of oncology, about 70% of patients develop resistance. CD40 is a potent costimulatory molecule that drives dendritic cells (DCs) and tumor-associated macrophages (TAMs) to prime T-cells. Clinically, agonistic CD40 antibodies have demonstrated antitumor activity, but dose-limiting toxicities have impaired efficacy. Radiation (RT), especially at higher doses, activates DCs and TAMs, but also upregulates Fibroblast Activation Protein (FAP) in the tumor microenvironment (TME). The upregulated FAP expression can be harnessed for the design of targeted drug delivery to the TME. The FAP-CD40 DARPin is a molecule that utilizes FAP and CD40 binding domains to selectively cross-link and activate CD40 in the tumor, thus avoiding systemic CD40 activation. This study aimed to evaluate if RT combined with a murine FAP-CD40 (mFAP-CD40) and ICI could improve survival and local and abscopal responses in a pre-clinical lung adenocarcinoma model. Human FAPxCD40 (MP0317) is being evaluated in Phase I trials. The 344SQ-P tumor cells were bilaterally injected into the right and left hind legs of 129Sv/Ev mice to establish primary and secondary tumors respectively. When primary tumors reached ∼7mm in diameter, they were irradiated with 12 Gy x 3 fractions, while secondary tumors were monitored. mFAP-CD40 (5mg/kg) was injected intraperitoneally at 2, 7, and 11 days after the last fraction of RT. A backbone of α-PD1 ICI was given twice per week starting with RT for 5 shots total. Mice were euthanized when tumors reached 14mm in diameter. Lungs were collected at experimental endpoints, fixed, and enumerated for metastases. For FAP detection in the TME, we first conducted an IHC analysis on 344SQ-P tumors harvested 11 days post RT. Although 5 Gy x 3 was superior to 12 Gy x 3 in upregulating FAP expression compared to unirradiated controls (P = 0.0088), we chose the 12 Gy x 3 dose for subsequent experiments due to its superiority in releasing tumor antigens, priming T-cells, and promoting abscopal responses. In our bilaterally established murine groups, we have recorded the following median survival days: Control = 23; RT = 26; RT+α-PD1 = 33; mFAP-CD40+α-PD1 = 23; and RT+mFAP-CD40+α-PD1 (Triple therapy) = 44 days. These results were further buttressed by the difference in average tumor growth observed between groups; whereby there was a significant delay in tumor growth of both primary (P<0.0001) and secondary tumors (P<0.0001) of the Triple therapy group compared to either RT+α-PD1 or mFAP-CD40+α-PD1 dual therapies. In addition, the Triple therapy cohort had a significantly lower count of lung metastases vs. control (P = 0.0007) and vs. mFAP-CD40+α-PD1 (P = 0.0246) cohorts. mFAP-CD40 DARPin with RT and α-PD1 proved efficacious to control primary and secondary tumors in a murine lung carcinoma model with no detected toxicities.

Radiomics Based Assessment of Tumor Infiltrating CD8 T-Cells Predicts Induction Systemic Therapy Response in HPV+ Oropharyngeal Cancer: Exploratory Analysis of Prospective Trials

The adaptive immune system is increasingly recognized to be important for tumor control in HPV+ oropharyngeal cancer (HPV+OPC), and pre-clinical models suggest tumor specific T-cell priming occurs in draining lymph nodes. De-escalation strategies incorporating T-cell infiltration present a novel prognostic biomarker. The purpose of this study was to examine the utility of a validated radiomics model of CD8 T-cell infiltration within the framework of 2 prospective HPV+OPC response-adaptive de-escalation trials. We hypothesized that nodal radiomics scores (RS) would be correlated with response to induction systemic therapy. Patients enrolled on 2 prospective phase II response adapted de-escalation trials for low- and high-risk HPV+OPC were included in the analysis. Patients received induction chemotherapy and chemoimmunotherapy in trials 1 and 2, respectively. Primary tumors and nodes were retrospectively delineated on diagnostic CT scans before and after induction therapy to ascertain volumetric tumor burden & response. RS were generated on pre-induction scans for both primary tumors and nodes. RS and tumor response were dichotomized using a 50% threshold (high vs low, responder vs non-responder, respectively). Linear regression was used to evaluate the correlation between % volume change in tumor burden and RS. Fisher's exact test was used to evaluate concordance between RS and tumor response. A total of 87 patients (N = 47 Trial 1, N = 40 Trial 2) were evaluable. The mean post-induction therapy reduction in primary and nodal volumetry was 80.7% and 67% respectively. Primary and nodal RS were not associated with primary (T1/T2 vs T3/T4) or nodal stage (N1-N2b vs N2c-N, p > 0.10 both). Both primary and nodal RS were correlated with % volume change, r = 0.24 (p = 0.034) and r = 0.37 (p = 0.002), respectively. The mean difference in % volume change in primary and node, using the RS, was 11.13% (p = 0.015) and 17. 69% (p = 0.004). There was no association between primary tumor RS and total lesion responder status (p = 0.312). However, there was an association between high vs low nodal RS and total lesion responder status (p = 0.005). This is the first report of a validated radiomics score of CD8-T cell infiltration in HPV+OPC to predict response to systemic therapy. While radiomics scores in both the primary and nodes were associated with percent volume response, this association was stronger in nodes. Higher nodal radiomics scores were associated with improved volume reduction in total lesion burden. This effect however was not observed in the primary tumor. CD8-T cell infiltration in nodes, but not in the primary tumor, was associated with overall tumor response after systemic induction therapy in HPV+ oropharyngeal cancer.

Targeted Pubic Neoantigen Immunotherapy Using Encapsulated Nanoparticles, Directed by Radiation

We tested a treatment combining radiotherapy with pubic neoantigen nanoparticles of CAND1 and ADGRF5-II (120 ± 56 nm) in nanocapsules detectable by computed tomography (CT) (545± 24 nm) with IFN γ or PD-L1 antibody (Ab), which released their contents upon radiation exposure. We performed two radiotherapy sessions: 1) induction of MHC-I and II in tumor cells and dendritic cells (DC) by the first round of radiation with concurrently released IFN γ from nanoparticles; 2) targeted neoantigen immunotherapy with PD-L1 blockade by the second round of radiation, elicited by CAND1-specific CD8+T-cell and ADGRF5-II-specific CD4+T-cell, related to induced MHC-I and MHC-II in session 1, respectively. For session one, nanocapsules were generated by mixing iopamiron and 15 mg IFN γ with a 1.0 mL solution containing 4.0% alginate, 3.0% hyaluronate, and 1 μg/mL P-selectin. This mixture was sprayed into 0.5 mmol/l FeCl2 supplemented with 1 µg/mL anti-VEGFR-1/2 Ab. For session two, Mixture of 110 fmol CAND1 and fmol ADGRF5-II, were encapsulated into poly lactic-co-Glycolic acid (PLGA) particle, using nanoprecitation method. Those particles were encapsulated by mixing into above alginate-hyaluronate cocktail with 400 mg PD-L1 antibody (Ab), then sprayed into 0.5 mmol/l FeCl2 supplemented with P-selectin Ab. In session one, 1 × 1010 nanocapsules were intravenously injected into BALB/c mice exhibiting primary 4TI mammary carcinoma in the left hind leg and lung metastases. Tumor accumulation was monitored by CT. Subsequently, 10 or 20 Gy 60Co γ-radiation was administered to primary tumors and lung metastasis. In session two, 1 × 1010 nanocapsules were injected i.v. and allowed to interact with P-selectin for 24 h; a further 10 or 20 Gy 60Co γ-radiation was administered to tumors and lung metastasis. In session one, CT imaged the accumulation of anti-VEGFR-1/2 nanocapsules around primary and metastatic tumors, which helped determine the timing of the first radiation. The nanocapsules released P-selectin and IFN γ in response to first radiation. P-selectin was deposited on tumor vessels. Radiation and released IFN γ elicited MHC-I and II on tumor cells and DCs. In session two, released CAND1 and ADGRF5-II from PLGA particle, which were emitted from nanocapsules upon second radiation, were loaded onto MHC-I and II. Subsequently, two kinds of tertiary complex were formed on tumor cells and DC: 1) MHC-I-CAND1-CD8+T-cell receptor (TCR); and 2) MHC-II- ADGRF5-II-CD4+TCR, which destroyed tumor cells, in collaboration with released PD-L1 Ab. Those T-cell mediated cell killing was magnified via enhanced DC-mediated T-cell priming by the two kinds of tertiary complex on DC. Those treatments resulted in EF 1.5 and 86% reduction of new metastasis formation. Our encapsulated pubic neoantigen particle will lead to quick and effective neoantigen immunotherapy.

Myo9b mutations are associated with altered dendritic cell functions and increased susceptibility to autoimmune diabetes onset

The regulation of autoimmunity against pancreatic islet β cells for type 1 diabetes (T1D) onset is still unclear. NOD/ShiLtJ (NOD) mice are prone to the onset of autoimmune diabetes, but its congenic strain, ALR/Lt (ALR), is not. Here we show that dendritic cells (DC) in ALR mice have impaired migratory and T-cell priming capability. Genomic comparative analysis maps a 33-bp deletion in the ALR Myosin IXb (Myo9b) gene when compared with NOD genome; meanwhile, data from knock-in models show that this ALR Myo9b allele impairs phenotypic and functional maturation of DCs, and prevents the development and progression of spontaneous autoimmune diabetes in NOD mice. In parallel, while the ALR 33-bp deletion of Myo9b is not conserved in human, we find a MYO9B R133Q polymorphism associating with increased risk of T1D and enhanced DC function in patients with T1D. Our results thus hint that alterations in Myo9b may contribute to altered DC function and autoimmune diabetes onset.

Tumor immunogenicity dictates reliance on TCF1 in CD8+ T cells for response to immunotherapy

Stem-like CD8+ Tcells are regulated by Tcell factor 1 (TCF1) and are considered requisite for immune checkpoint blockade (ICB) response. However, recent findings indicate that reliance on TCF1+CD8+ Tcells for ICB efficacy may differ across tumor contexts. We find that TCF1 is essential for optimal priming of tumor antigen-specific CD8+ Tcells and ICB response in poorly immunogenic tumors that accumulate TOX+ dysfunctional Tcells, but is dispensable for Tcell priming and therapy response in highly immunogenic tumors that efficiently expand transitory effectors. Importantly, improving Tcell priming by vaccination or by enhancing antigen presentation on tumors rescues the defective responses of TCF1-deficient CD8+ Tcells upon ICB in poorly immunogenic tumors. Our study highlights TCF1's role during the early stages of anti-tumor CD8+ Tcell responses with important implications for guiding optimal therapeutic interventions in cancers with low TCF1+CD8+ Tcells and low-neo-antigen expression.

T-cell priming transcriptomic markers: implications of immunome heterogeneity for precision immunotherapy

Immune checkpoint blockade is effective for only a subset of cancers. Targeting T-cell priming markers (TPMs) may enhance activity, but proper application of these agents in the clinic is challenging due to immune complexity and heterogeneity. We interrogated transcriptomics of 15 TPMs (CD137, CD27, CD28, CD80, CD86, CD40, CD40LG, GITR, ICOS, ICOSLG, OX40, OX40LG, GZMB, IFNG, and TBX21) in a pan-cancer cohort (N = 514 patients, 30 types of cancer). TPM expression was analyzed for correlation with histological type, microsatellite instability high (MSI-H), tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) expression. Among 514 patients, the most common histological types were colorectal (27%), pancreatic (11%), and breast cancer (10%). No statistically significant association between histological type and TPM expression was seen. In contrast, expression of GZMB (granzyme B, a serine protease stored in activated T and NK cells that induces cancer cell apoptosis) and IFNG (activates cytotoxic T cells) were significantly higher in tumors with MSI-H, TMB ≥ 10 mutations/mb and PD-L1 ≥ 1%. PD-L1 ≥ 1% was also associated with significantly higher CD137, GITR, and ICOS expression. Patients’ tumors were classified into “Hot”, “Mixed”, or “Cold” clusters based on TPM expression using hierarchical clustering. The cold cluster showed a significantly lower proportion of tumors with PD-L1 ≥ 1%. Overall, 502 patients (98%) had individually distinct patterns of TPM expression. Diverse expression patterns of TPMs independent of histological type but correlating with other immunotherapy biomarkers (PD-L1 ≥ 1%, MSI-H and TMB ≥ 10 mutations/mb) were observed. Individualized selection of patients based on TPM immunomic profiles may potentially help with immunotherapy optimization.

Phage-assisted evolution and protein engineering yield compact, efficient prime editors

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed invivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.