MHC Class Research Articles

Serum multiplex analysis of checkpoint proteins in association with immune-related adverse events in patients with melanoma receiving adjuvant anti-PD-1 therapy.

9588 Background: Immune checkpoint inhibitors have significantly improved melanoma (MEL) patient (PT) outcomes. Immune-related adverse events (irAEs) may cause significant morbidity, rarely including death. Soluble immune checkpoints have been identified as biomarkers of autoimmune activity and potential off-target pathology. Understanding their relationship to pathologic outcomes may assist in the identification of PTs at highest risk of irAEs. Methods: PTs with resectable stage IIB-IV MEL who received adjuvant anti-programmed-death-1 (PD-1) therapy were identified from the University of Pittsburgh’s MEL Center biospecimen repository (N=273). MILLIPLEX Human Immuno-Oncology Checkpoint Protein Panel 2 was used to study serum levels of immunomodulatory proteins. Mean pretreatment serum levels in PTs with and without irAEs were compared using a two-sample t-test after performing Box-Cox transformation for non-normally distributed data. Results: Sera from 43 PTs were analyzed. The cohort included 28 (65.1%) male, mean age at diagnosis (62), including MEL stage IIIA = 7 (16.3%), IIIB = 16(37.2%) and IIIC = 20(46.5%). The most common irAEs (n=24, 55.8%) were thyroiditis (n=9, 20.9%), dermatitis (n=8, 18.6%), and adrenal insufficiency (n=9, 21.0%). Multiple irAEs were observed amongst 22 (51.2%) PTs with ≥3 irAEs and 6 (13.9%) with 5 irAEs. Visceral irAEs included Hepatitis (n=3, 7.0%), pneumonitis (n=2, 4.7%), and colitis (n=5, 11.6%). Seven of 8 patients with visceral irAEs required corticosteroids. Siglec-7 (mean 23.4 vs 3.51 pg/mL, p=0.018) and siglec-9 (mean 77.0 vs 33.1 pg/mL, p=0.049) were significantly elevated in the PTs who developed any irAE. After stratifying for each individual irAE, PTs with thyroiditis had elevated Nectin-4 (mean 2785.5 vs 494.7 pg/mL, p<0.001) and serum arginase (mean 2437.5 vs 1383.1 pg/mL, p= 0.046) was elevated in PTs who developed colitis. PTs with hepatitis demonstrated increased galectin-3 (mean 10103.9 vs 9915.3 pg/mL, p=0.045) and decreased levels of CD40L (mean 2869.2 vs 9496.9 pg/mL, p=0.033). In PTs with dermatitis B7-H3 (mean 2503.3 vs 732.4 pg/mL, p=0.009) and MHC class I polypeptide-related sequence B (mean 1598.0 vs 259.1 pg/mL, p=0.048) were elevated. Conclusions: We have demonstrated unique serum checkpoint protein profiles related to specific irAEs in melanoma patients receiving adjuvant anti-PD1. Further studies are warranted to confirm and validate the utility of these irAE predictors during adjuvant anti-PD-1.

Influenza Virus-Derived CD8 T Cell Epitopes: Implications for the Development of Universal Influenza Vaccines.

The influenza virus poses a global health burden. Currently, an annual vaccine is used to reduce influenza virus-associated morbidity and mortality. Most influenza vaccines have been developed to elicit neutralizing Abs against influenza virus. These Abs primarily target immunodominant epitopes derived from hemagglutinin (HA) or neuraminidase (NA) of the influenza virus incorporated in vaccines. However, HA and NA are highly variable proteins that are prone to antigenic changes, which can reduce vaccine efficacy. Therefore, it is essential to develop universal vaccines that target immunodominant epitopes derived from conserved regions of the influenza virus, enabling cross-protection among different virus variants. The internal proteins of the influenza virus serve as ideal targets for universal vaccines. These internal proteins are presented by MHC class I molecules on Ag-presenting cells, such as dendritic cells, and recognized by CD8 T cells, which elicit CD8 T cell responses, reducing the likelihood of disease and influenza viral spread by inducing virus-infected cell apoptosis. In this review, we highlight the importance of CD8 T cell-mediated immunity against influenza viruses and that of viral epitopes for developing CD8 T cell-based influenza vaccines.

A multi-ethnic reference panel to impute HLA classical and non-classical class I alleles in admixed samples: Testing imputation accuracy in an admixed sample from Brazil.

The MHC class I region contains crucial genes for the innate and adaptive immune response, playing a key role in susceptibility to many autoimmune and infectious diseases. Genome-wide association studies have identified numerous disease-associated SNPs within this region. However, these associations do not fully capture the immune-biological relevance of specific HLA alleles. HLA imputation techniques may leverage available SNP arrays by predicting allele genotypes based on the linkage disequilibrium between SNPs and specific HLA alleles. Successful imputation requires diverse and large reference panels, especially for admixed populations. This study employed a bioinformatics approach to call SNPs and HLA alleles in multi-ethnic samples from the 1000 genomes (1KG) dataset and admixed individuals from Brazil (SABE), utilising 30X whole-genome sequencing data. Using HIBAG, we created three reference panels: 1KG (n = 2504), SABE (n = 1171), and the full model (n = 3675) encompassing all samples. In extensive cross-validation of these reference panels, the multi-ethnic 1KG reference exhibited overall superior performance than the reference with only Brazilian samples. However, the best results were achieved with the full model. Additionally, we expanded the scope of imputation by developing reference panels for non-classical, MICA, MICB and HLA-H genes, previously unavailable for multi-ethnic populations. Validation in an independent Brazilian dataset showcased the superiority of our reference panels over the Michigan Imputation Server, particularly in predicting HLA-B alleles among Brazilians. Our investigations underscored the need to enhance or adapt reference panels to encompass the target population's genetic diversity, emphasising the significance of multiethnic references for accurate imputation across different populations.

Unraveling the immunologic vulnerabilities of diffuse hemispheric glioma, H3 G34-mutant.

2082 Background: Recent insights into the molecular basis of diffuse hemispheric glioma, H3 G34-mutant (DHG-G34), an incurable high-grade glioma, have opened new therapeutic possibilities. The oncohistone mutation in DHG-G34 impairs the epigenetic regulator SETD2, which normally orchestrates DNA-mismatch-repair and negatively regulates the transcriptional silencing activity of the Polycomb Repressive Complex 2 (PRC2). In DHG-G34, a mismatch-repair-deficiency phenotype is predicted, but an anti-tumor immune response is notably absent in clinical observations, which we posit is linked to the aberrant transcriptional repression by PRC2. Here, we investigate DHG-G34 immunosuppression mechanisms in a large real-world multi-omics cohort and human cell lines. Methods: Clinical, molecular, and immunologic characteristics of a DHG-G34 cohort, sequenced (DNA and RNA) at Caris Life Sciences (Phoenix, AZ), are contrasted against diffuse midline glioma (DMG) and pediatric low-grade glioma (LGG). A significant difference between genomic subgroups was defined as fold-change > 1.2. Statistical significance was determined using chi-square, Fishers-exact, and Mann Whitney U tests with corrections for multiple hypothesis testing (q < 0.05).Hazard ratio (HR) was calculated using the Cox proportional hazards model. KNS-42 DHG-G34 cell line was exposed in triplicate to 1 µM valemetostat (an EZH1/2 inhibitor) or DMSO in vitro. Flow cytometry and RNA-sequencing were utilized to determine differential immunologic characteristics. Results: 29 DHG-G34, 51 DMG, and 52 LGG were identified in Caris database. Median OS among DHG-G34 was 15.4 mo., similar to DMG (HR = 1.1, 95% CI: 0.70-1.73, p = 0.686). Most frequent alterations in DHG-G34 were TP53 (93.1%), ATRX (69.0%), PDGFRA (31.0%). Tumor mutational burden (TMB) was high, >=10mt/MB, in 10% of DHG-G34 and 0/51 in DMG and 0/52 LGG (p < 0.01, q > 0.05), but immune fractions including dendritic cells, T cells, macrophages, microglia, and antigen-presenting genes were decreased (fold-change: 0.19 – 0.83, q < 0.05). In KNS-42, PRC2 inhibition upregulated MHC class 1 (p < 0.001). Of 402 KNS-42 missense variants, transcription of 43 were upregulated following EHZ2 inhibition, and 4 were downregulated (q < 0.05). Among immunostimulatory genes, P2RX7, which engages T-cells and stimulates an inflammatory response, was downregulated in patient samples (q < 0.001) and upregulated in KNS-42 following EZH2 inhibition (q < 0.001). Conclusions: DHG-G34 has an increased propensity for high TMB but remains immunologically cold. Our pilot experiment suggests inhibiting PRC2’s unique role in DHG-G34 may sensitize DHG-G34 to an immune response by inducing MHC class 1 expression and transcriptional readthrough of mutated transcripts. Understanding whether treatment induces TMB in DHG-G34 may affect timing of an immunotherapeutic intervention.

INSIGHT-005: A new stratum of the explorative, open-labeled, phase I INSIGHT study to evaluate the feasibility and safety, as well as preliminary efficacy, of subcutaneous injections with IMP321 (eftilagimod alpha) in combination with PD-L1 inhibitor (avelumab) for metastatic or unresectable locally advanced urothelial carcinoma (UC)—IKF-s614.

TPS4620 Background: UCs are the 6th most common malignancies in the Western world being localized in the upper (5-10%) or the lower (90-95%) urinary tract. Metastatic UC (mUC), which accounts for 5% of all cases, is associated with a dismal prognosis and prompt progression. So far, the 1st line therapy for mUC encompassed platinum-based combination regimens. Recent data indicate beneficial patient treatment with immune checkpoint inhibitors. Thus, avelumab was approved for maintenance therapy after progression-free platinum-based chemotherapy for locally advanced (LA) or mUC. Further immune checkpoint inhibitors (e.g. pembrolizumab, atezolizumab, nivolumab) achieved approval or showed promising results for treatment of different patient populations. Eftilagimod alpha (efti) is a soluble LAG-3 fusion protein and a MHC class II agonist activating APCs followed by CD8 T-cell activation. The combination of efti with PD-1/PD-L1 blockade is not yet available and is proposed to enhance efficacy. Based on previous results from the INSIGHT trial and change in treatment landscape we hypothesize that combining avelumab and efti will display clinically relevant efficacy in unresectable LA UC or mUC subgroups with acceptable toxicity. Methods: INSIGHT-005 is a new stratum within the investigator-initiated INSIGHT phase I platform trial ongoing at multiple sites (n=9) in Germany. Patients with unresectable LA UC or mUC will receive efti in combination with avelumab. 30 patients will be enrolled in 3 subgroups: I) Previously untreated, eligible for platinum-based therapy, with PD-L1 CPS≥10; II) Previously untreated, not-eligible for platinum-based therapy, irrespective of the PD-L1 status; III) Suffered disease progression after platinum-based chemotherapy for metastatic disease and did not receive avelumab maintenance therapy, irrespective of the PD-L1 status. Enrolled patients will receive avelumab 800 mg i.v. and efti 30 mg s.c. on the same day Q2W for a maximum of 24 cycles. Tumor evaluation will be performed via CT or MRI every 8 weeks. The primary endpoint of this study is to explore feasibility, safety, and preliminary efficacy of efti when added to avelumab in unresectable LA UC or mUC. Secondary endpoints include safety and efficacy parameters as defined by objective response, time to and duration of response and PFS according to RECIST 1.1, OS and biomarker analyses. First patient was enrolled on 2023-11-29. Currently, recruitment is ongoing. ClinicalTrials.gov ID: NCT03252938 Clinical trial information: NCT03252938 .

The impact of MICB mismatches in unrelated haematopoietic stem cell transplantation.

MICA polymorphisms have been associated with increased incidence of acute GvHD and adverse outcome in allogeneic haematopoietic stem cell transplantation (HSCT). MICB is another expressed member of MHC class I-related chain genes and its impact on HSCT outcome is yet to be fully defined. We typed a large cohort of patients and donors for MICB polymorphisms and investigated the impact of MICB matching on outcome after unrelated HSCT. 69.2% of the patients were 10/10 human leukocyte antigen (HLA) matched and 30.8% were 9/10 HLA matched. MICB typing was performed using a short amplicon-based NGS typing assay on the Illumina MiSeq platform. Differences in proteins were considered as mismatches. MICA polymorphisms were identified as possible confounder and were therefore included as parameter in the multivariate analyses. Due to the strong linkage disequilibrium with the classical HLA-genes, sub-stratification for HLA matching status was necessary, and no effect of MICB mismatches was seen in the 10/10 HLA matched group when compared to the MICB matched cases. However, in the 9/10 HLA matched group, MICB mismatched cases showed significantly worse disease free survival (DFS), GvHD and relapse free survival (GRFS) compared to the MICB matched cases (DFS: HR 1.24, p = 0.011; GRFS: HR 1.26, p = 0.002). MICA mismatches had no impact on any outcome parameter. According to our findings, effects previously attributed to MICA differences may have been confounded by MICB polymorphisms. We show that MICB differences contribute a small but relevant effect in 9/10 HLA-matched transplantations, which in turn highlights the possible usefulness of MICB typing in donor selection among similarly suitable 9/10 matched donors, especially when HLA-B mismatches have to be accepted.

Unveiling a novel NKG2A inhibitor based on monoclonal antibody to HLAE-peptide complex.

e14677 Background: HLAE is a non-classical MHC class I molecule complexed with the VMAPRTLFL nonapeptide, derived from the leader sequence of HLAG (HLAEpHLAG). HLAEpHLAG is overexpressed across a wide type of tumors prominently on Multiple Myeloma (MM). HLAEpHLAG complex is the ligand to CD94-NKG2A in the immune system which negatively regulates the activation of natural killer (NK) cells as well as NKG2A positive T cells. Harnessing of the HLAE-NKG2A axis by the cancer cells leads to evasion from the immune system attack resulting in cancer progression. Currently, several Immune Checkpoint Inhibitors (ICIs) target the NKG2A receptor which has a low specificity to cancer and high toxicity to the patient. Engineering a new ICI that targets the HLAEpHLAG complex can potentially yield improved outcomes in cancer therapy. Methods: Using the hybridoma system, a new mouse monoclonal antibody (mAb) called 4D7 that targets the HLAEpHLAG complex was developed. mAb 4D7 affinity was assessed in vitro with recombinant proteins and a panel of cell lines that expressed zero to high levels of HLAE. The functional impacts of the mAb 4D7 was examined in a system where various MM cell lines were separately co-cultured with primary NK (pNK) cells. pNK were isolated from the peripheral blood of 5 healthy donors, and their activity was examined in the presence of mAb 4D7 by CD107a degranulation assay. Also, the functional impacts of the mAb 4D7 on allogenic pNK and MM blast cells, isolated from the bone marrow of 6 MM patients were assessed. Results: mAb 4D7 was determined to be an IgG-kappa isotype showing a high specific binding to recombinant HLAEpHLAG. Cell lines with overexpression of membranal HLAEpHLAG: RPMI8226, U266, 721.221 HLAE, and 721.221 HLAG showed positive staining with the mAb 4D7. While cell lines with low to zero expression of HLA-E like 721.221 WT showed low or negative staining with the mAb 4D7. The degranulation of NKG2A+ pNK subset cells against MM target cells increased in presence of mAb 4D7. However, adding mAb 4D7 to NKG2A -pNK subset cells co-cultured with MM target cells did not affect the pNK activity. Similar results were obtained when the target and effector cells were isolated from the bone marrow of active treatment-naive MM patients. Conclusions: MAb 4D7 demonstrates a remarkably high affinity for binding to the HLAEpHLAG complex, ensuring the safety of its application. The ability of 4D7 mAb to effectively block the HLAEpHLAG complex and counteract the inhibition signal in NK cells. mAb 4D7 is a promising monoclonal antibody with substantial potential to serve as an ICI for clinical use in MM patients.

Tumor-specific MHC class II upregulation associated with response to anti-PD-L1 therapy in patients with urothelial cancer.

Tumor-specific MHC class II upregulation associated with response to anti-PD-L1 therapy in patients with urothelial cancer.

Correlation of allele-specific HLA loss of heterozygosity in patients with cancer with driver mutations.

e14658 Background: While immunotherapies have revolutionized cancer treatment, the limited predictability of response and resistance mechanisms present challenges to the development of novel therapeutics for solid tumors. Many of these use a sensitive and specific T cell receptor (TCR)-HLA:peptide interaction as a mechanism of action and can be impacted by HLA loss of heterozygosity (LOH). Allele-agnostic HLA LOH analyses assessing loss of any MHC class I allele are relevant for HLA-agnostic approaches such as immune checkpoint inhibitors; however, context-specific studies remain limited. For example, adoptive TCR T cell therapies and bi-specific TCR T cell engagers target neoantigen peptides derived from oncogenic driver mutations, such as those of KRAS and TP53, presented by a specific HLA allele. Thus, allele-agnostic analyses may overestimate the frequency and relevance of HLA LOH for allele-specific modalities. Methods: A real-world comprehensive genomic profiling dataset consisting of 78,418 cases (1), germline heterozygous for one or more HLA class I locus, was analyzed for HLA LOH in the context of frequent driver mutations (KRAS G12C, G12D, G12V and TP53 R175H), the corresponding HLA alleles (A*02:01, A*03:01, and A*11:01), and within tumor subtypes. Results: Allele-agnostic HLA LOH was observed in 16.6% of all samples and varied between indications. Notably, allele-specific loss of HLAs A*02:01, A*03:01, and A*11:01 across all samples was much lower than allele-agnostic loss (6.3%, 6.6%, and 6.3% cases, respectively). Although, a trend for increased allele-specific HLA LOH was observed when analyzed by driver mutation, the frequency remained low. Specifically, across all cases, A*02:01, A*03:01, and A*11:01 allele-specific LOH ranged between 5.9% - 9.7% in relation to KRAS G12C, G12D, and G12V compared to 5.9% - 6.2% for wild-type (WT). Moreover, it did not achieve statistical significance for any HLA-KRAS mutation pairs in colorectal (CRC), non-small cell lung (NSCLC), and pancreatic (PDAC) cancers, except for A*11:01 and KRAS G12C in NSCLC, which was lower in mutated cases (5.3% for G12C vs 10.1% for WT KRAS, p=0.045). Both allele-agnostic and A*02:01-specific HLA LOH in TP53 WT samples were lower than average. The TP53 R175H mutation was associated with limited increase in HLA-A*02:01 LOH (from 4.4% for WT to 8.1% for TP53 R175H) and varied between indications, e.g. no significant increase was observed in R175H-positive breast cancer and NSCLC cases contrary to CRC and PDAC. Conclusions: Based on this real-world dataset, over 90% of cases retain the TCR-targeted allele and, therefore, preserve the molecular complex necessary for HLA/driver mutation-based therapies. Notably, the database is enriched for advanced cancer patients. Further studies should test correlations with treatments and whether tumors in earlier lines of treatment have an even lower rate of HLA LOH. 1. Montesion et al., PMID: 33127846.

AIPAC-003: A randomized, double-blind, placebo-controlled phase 3 trial testing eftilagimod alpha (soluble LAG-3) in patients with HER2-neg/low metastatic breast cancer receiving paclitaxel, following an open-label dose optimization.

TPS1125 Background: Eftilagimod alpha (efti), a soluble LAG-3 protein, acts as an MHC class II agonist that enhances immunity by mediating antigen presenting cell and CD8 T-cell activation. Data from a randomized, phase 2b trial of efti plus paclitaxel compared to placebo plus paclitaxel in patients (pts) with HR+ HER2– metastatic breast cancer (MBC) (AIPAC; NCT02614833) showed sustained pharmacodynamic activity that was linked to improved overall survival (OS) in the efti arm. AIPAC-003 is a randomized, double-blind, placebo-controlled phase 3 trial testing efti plus paclitaxel in HER2-neg/low MBC pts, following an initial open-label dose optimization lead-in (DOL) component to determine the optimal biological dose (OBD) of efti in this combination. Methods: The DOL phase will determine the OBD of efti prior to start of the P3 part of the study. Enrolment for the DOL began in November 2023 with the aim to recruit 66 pts. Primary objectives in the DOL are: safety and tolerability of 90 mg vs 30 mg efti and defining the OBD of efti in combination with weekly paclitaxel. Determination of the OBD will be based on the totality of safety and tolerability data together with overall response rate (ORR) and pharmacodynamic marker (CD8+ T cells, absolute lymphocyte count) data. Secondary objectives in the DOL include efficacy, QoL and PK profile always of 30 mg efti vs. 90 mg on top of weekly paclitaxel. In the Phase 3 component pts will be randomized to receive either paclitaxel + efti or paclitaxel + placebo in a double-blinded fashion. The primary endpoint (EP) for the proposed Phase 3 is OS and the exact patient population will be defined after determination of the OBD. Key secondary EPs include progression free survival and ORR by RECIST 1.1, quality of life and safety. Pts will receive paclitaxel (80 mg/m2 I.V. on D1, 8 and 15 in a 4-week cycle), followed by efti or placebo (DOL: 30 or 90 mg efti; Phase 3: OBD of efti or placebo) S.C. on D1 and 15 in a 4-week cycle for up to 12 months. Imaging is done every 8 weeks and assessed by the investigator. in the DOL blood samples are taken from 0 to 96 hours in cycle 1 and 4 to determine PK / PD profiles. PK / PD parameters are assessed by a central lab. Key inclusion criteria: Pts with either a) HR+ and HER2-neg/low and endocrine therapy-resistant MBC or b) TNBC not eligible for anti-PD-1-based therapy. Pts must have measurable disease, ECOG PS 0-1 and no prior chemotherapy for metastatic disease. Key exclusion criteria: Pts if HR+ who are not considered endocrine resistant and/or have received < 1 line of therapy in the metastatic setting. Pts who are considered TNBC and are eligible for anti-PD-(L)1 containing therapy. Clinical trial information: NCT05747794 .

A phase I pilot study of personalized neoantigen peptide-based vaccine in combination with pembrolizumab in advanced solid tumors (PNeoVCA).

TPS2700 Background: Though immunotherapy with anti-PD-1 immune checkpoint inhibitors (ICIs) has led to improvements in clinical outcomes in patients with cancer, when used as monotherapy in the advanced setting, only 18-25% of patients respond to ICI. Notably, ICI frequently lacks anti-tumor activity in immune-cold cancers where T cell priming is missing. Recent breakthroughs in identifying personal neoantigens via a comprehensive analysis of cancer sequencing data have brought increased attention to neoantigen cancer vaccines. Personalized cancer vaccines targeting neoantigens have shown early promise. While neoantigens have recently been investigated in some cancer types, the current neoantigen prediction algorithms have often focused on the MHC class-I subtype, single nucleotide mutations (SNM), small insertions and deletions (INDEL). We recently developed an informatics workflow, REAL-neo, for the identification, quality control (QC), and prioritization of both class-I and class-II human leukocyte antigen (HLA) bound neoantigens that arise from somatic SNM, INDEL, aberrant RNA splicing, and gene fusions, generating much more potent neoantigen candidates. Furthermore, we demonstrated robust T-cell response and prolonged survival by combining ICI and cancer vaccines in preclinical models. This single-arm phase I clinical trial is in progress to assess the safety, feasibility, and immunogenicity of personalized neoantigen vaccines in combination with pembrolizumab in patients with advanced solid cancers. Methods: The vaccine consists of up to 20 peptides (15-30 mer) comprising patient-specific neoantigens identified from tumor DNA and mRNA sequencing data using REAL-neo and delivered with GM-CSF as an adjuvant. The first cohort of three patients will be treated at dose level 1 consisting of 4 or 5 peptides at 300 mcg/peptide and GM-CSF 125 mcg per injection site in each of four limbs. After Cohort 1 is deemed safe, the study will expand to cohort 2, when patients will be enrolled at the same dose level for vaccine plus Pembrolizumab 200 mg i.v. Neoantigen vaccine will be given via subcutaneous injection on days 1, 4, 8, 15, and 21 (cohorts 1 and 2) and weeks 5 and 8 (booster dose for cohort 2 only). Cohort 1 has been completed without dose-limiting toxicity (DLT). Enrollment in Cohort 2 began in January 2024. AEs are assessed according to CTCAE v5. and safety findings are reviewed by data safety medical board (DSMB). Key eligibility criteria include 1) histologically confirmed locally advanced or metastatic solid malignancies and 2) cancer progression after at least one line of the standard-of-care (SOC) systemic treatment. For cohort 2, patients must be eligible to receive pembrolizumab per SOC or the treating physician’s judgment. Clinical trial information: NCT05269381 .

Cloning, Expression, and Bioinformatics Modeling of Human Papillomavirus Type 52 L1/L2 Chimeric Protein in Escherichia coli BL21 (DE3)

Human papillomavirus (HPV) L1 major capsid protein generates a highly immunogenic virus like particles (VLPs), which have been used as the main component of its prophylactic vaccine. However, the neutralizing antibodies against L1 VLPs are mostly type specific and may not be effective to prevent infection from different strains of HPV. On the other hand, HPV L2 minor capsid protein has low antigenic variation, thus can induce cross-neutralization. This study aims to obtain HPV 52 L1/L2 chimeric protein, which is designed based on HPV type 52 as one of the most circulated high-risk types in Indonesia, to develop a broad-spectrum HPV vaccine. Substitution of HPV 52 H4 helix L1 region with an HPV 52 L2 epitope was carried out using overlap extension PCR. HPV 52 L1/L2 chimeric gene was constructed into pET-SUMO expression vector and expressed in Escherichia coli BL21 (DE3). Bioinformatics modeling suggested that L2 epitope was located inside of the loop region in monomer form, and on the contrary, it was located outside of the pentamer surface. Furthermore, B cell and T cell epitopes predictions were conducted using Immune Epitope Database (IEDB) analysis. The B cell epitopes prediction revealed eleven potential epitopes, whereas the T cell epitopes prediction showed seven potential epitopes for each MHC class I and MHC class II. This study showed that HPV 52 L1/L2 chimeric protein has the potential to induce cross-neutralizing antibodies and can be developed as a promising candidate for a new HPV vaccine.

Augmentation of tumor expression of HLA-DR, CXCL9, and CXCL10 may improve olfactory neuroblastoma immunotherapeutic responses

BackgroundOlfactory neuroblastoma is a rare malignancy of the anterior skull base typically treated with surgery and adjuvant radiation. Although outcomes are fair for low-grade disease, patients with high-grade, recurrent, or metastatic disease oftentimes respond poorly to standard treatment methods. We hypothesized that an in-depth evaluation of the olfactory neuroblastoma tumor immune microenvironment would identify mechanisms of immune evasion in high-grade olfactory neuroblastoma as well as rational targetable mechanisms for future translational immunotherapeutic approaches.MethodsMultispectral immunofluorescence and RNAScope evaluation of the tumor immune microenvironment was performed on forty-seven clinically annotated olfactory neuroblastoma samples. A retrospective chart review was performed and clinical correlations assessed.ResultsA significant T cell infiltration was noted in olfactory neuroblastoma samples with a stromal predilection, presence of myeloid-derived suppressor cells, and sparse natural killer cells. A striking decrease was observed in MHC-I expression in high-grade olfactory neuroblastoma compared to low-grade disease, representing a mechanism of immune evasion in high-grade disease. Mechanistically, the immune effector stromal predilection appears driven by low tumor cell MHC class II (HLA-DR), CXCL9, and CXCL10 expression as those tumors with increased tumor cell expression of each of these mediators correlated with significant increases in T cell infiltration.ConclusionThese data suggest that immunotherapeutic strategies that augment tumor cell expression of MHC class II, CXCL9, and CXCL10 may improve parenchymal trafficking of immune effector cells in olfactory neuroblastoma and augment immunotherapeutic responses.

The Improved Antigen Uptake and Presentation of Dendritic Cells Using Cell-Penetrating D-octaarginine-Linked PNVA-co-AA as a Novel Dendritic Cell-Based Vaccine.

Cancer immunotherapy using antigen-pulsed dendritic cells can induce strong cellular immune responses by priming cytotoxic T lymphocytes. In this study, we pulsed tumor cell lysates with VP-R8, a cell-penetrating D-octaarginine-linked co-polymer of N-vinylacetamide and acrylic acid (PNVA-co-AA), into the DC2.4 murine dendritic cell line to improve antigen uptake and then determined the anti-tumor effect in tumor-bearing mice. DC2.4 cells were pulsed with the cell lysate of EL4, a murine lymphoma cell line, and VP-R8 to generate the DC2.4 vaccine. For the in vivo study, DC2.4 cells pulsed with EL4 lysate and VP-R8 were subcutaneously injected into the inguinal lymph node to investigate the anti-tumor effect against EL4 and EL4-specific T cell immune responses. VP-R8 significantly improved antigen uptake into DC2.4 compared to conventional keyhole limpet hemocyanin (p < 0.05). The expression of MHC class I, MHC class II, and CD86 in DC2.4 cells significantly increased after pulsing tumor lysates with VP-R8 compared to other treatments (p < 0.05). The intra-lymph node injection of DC2.4 pulsed with both VP-R8 and EL4 lysate significantly decreased tumor growth compared to DC2.4 pulsed with KLH and lysates (p < 0.05) and induced tumor-infiltrating CD8T cells. The DC2.4 vaccine also remarkably increased the population of IFN-gamma-producing T cells and CTL activity against EL4 cells. In conclusion, we demonstrated that VP-R8 markedly enhances the efficiency of dendritic cell-based vaccines in priming robust anti-tumor immunity, suggesting its potential as a beneficial additive for dendritic cell-based immunotherapy.

Validation of novel conditional ligands and large-scale detection of antigen-specific T cells for H-2Dd and H-2Kd

The UV-mediated peptide exchange has enabled the generation of multiple different MHC multimer specificities in parallel, surpassing tedious individual refolding of MHC molecules with peptide ligands. Murine models are acknowledged as an effective tool for preclinical research to advance our understanding of immunological mechanisms, with the potential translatability of key learnings from mouse models to the clinic. The common inbred mouse strain BALB/c is frequently used in immunological research. However, for the BALB/c histocompatibility (H)-2 alleles availability of conditional ligand has been limited. To overcome this challenge, we design and experimentally validate conditional ligands restricted to murine MHC class I alleles H2Dd and H2Kd. In addition, we demonstrate the ability of the three H2d molecules and two additional C57BL/6 H2b molecules folded in-house with conditional ligands to generate fluorescently labeled peptide-H2 tetramers that allow staining of antigen-specific CD8+ T cells in splenocyte samples. Finally, we generate large peptide-H-2 multimer libraries with a DNA-barcode labeling system for high-throughput interrogation of CD8+ T cell specificity in murine splenocyte samples. Consequently, the described techniques will contribute to our understanding of the antigen-specific CD8+ T cell repertoire in murine preclinical models of various diseases.

Memory CD8 T cells are vulnerable to chronic IFN-γ signals but not to CD4 T cell deficiency in MHCII-deficient mice

The mechanisms by which the number of memory CD8 T cells is stably maintained remains incompletely understood. It has been postulated that maintaining them requires help from CD4 T cells, because adoptively transferred memory CD8 T cells persist poorly in MHC class II (MHCII)-deficient mice. Here we show that chronic interferon-γ signals, not CD4 T cell-deficiency, are responsible for their attrition in MHCII-deficient environments. Excess IFN-γ is produced primarily by endogenous colonic CD8 T cells in MHCII-deficient mice. IFN-γ neutralization restores the number of memory CD8 T cells in MHCII-deficient mice, whereas repeated IFN-γ administration or transduction of a gain-of-function STAT1 mutant reduces their number in wild-type mice. CD127high memory cells proliferate actively in response to IFN-γ signals, but are more susceptible to attrition than CD127low terminally differentiated effector memory cells. Furthermore, single-cell RNA-sequencing of memory CD8 T cells reveals proliferating cells that resemble short-lived, terminal effector cells and documents global downregulation of gene signatures of long-lived memory cells in MHCII-deficient environments. We propose that chronic IFN-γ signals deplete memory CD8 T cells by compromising their long-term survival and by diverting self-renewing CD127high cells toward terminal differentiation.

Death receptors 4/5 mediate tumour sensitivity to natural killer cell-mediated cytotoxicity in mismatch repair deficient colorectal cancer.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.

Cutting Edge: LAG3 Dimerization Is Required for TCR/CD3 Interaction and Inhibition of Antitumor Immunity.

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor that plays a critical role in controlling T cell tolerance and autoimmunity and is a major immunotherapeutic target. LAG3 is expressed on the cell surface as a homodimer but the functional relevance of this is unknown. In this study, we show that the association between the TCR/CD3 complex and a murine LAG3 mutant that cannot dimerize is perturbed in CD8+ T cells. We also show that LAG3 dimerization is required for optimal inhibitory function in a B16-gp100 tumor model. Finally, we demonstrate that a therapeutic LAG3 Ab, C9B7W, which does not block LAG3 interaction with its cognate ligand MHC class II, disrupts LAG3 dimerization and its association with the TCR/CD3 complex. These studies highlight the functional importance of LAG3 dimerization and offer additional approaches to therapeutically target LAG3.

Designing a multi-epitope vaccine against Shigella dysenteriae using immuno-informatics approach.

Shigella dysenteriae has been recognized as the second most prevalent pathogen associated with diarrhea that contains blood, contributing to 12.9% of reported cases, and it is additionally responsible for approximately 200,000 deaths each year. Currently, there is no S. dysenteriae licensed vaccine. Multidrug resistance in all Shigella spp. is a growing concern. Current vaccines, such as O-polysaccharide (OPS) conjugates, are in clinical trials but are ineffective in children but protective in adults. Thus, innovative treatments and vaccines are needed to combat antibiotic resistance. In this study, we used immuno-informatics to design a new multiepitope vaccine and identified S. dysenteriae strain SD197's membrane protein targets using in-silico methods. The target protein was prioritized using membrane protein topology analysis to find membrane proteins. B and T-cell epitopes were predicted for vaccine formulation. The epitopes were shortlisted based on an IC50 value <50, antigenicity, allergenicity, and a toxicity analysis. In the final vaccine construct, a total of 8 B-cell epitopes, 12 MHC Class I epitopes, and 7 MHC Class II epitopes were identified for the Lipopolysaccharide export system permease protein LptF. Additionally, 17 MHC Class I epitopes and 14 MHC Class II epitopes were predicted for the Lipoprotein-releasing ABC transporter permease subunit LolE. These epitopes were selected and linked via KK, AAY, and GGGS linkers, respectively. To enhance the immunogenic response, RGD (arginine-glycine-aspartate) adjuvant was incorporated into the final vaccine construct. The refined vaccine structure exhibits a Ramachandran score of 91.5% and demonstrates stable interaction with TLR4. Normal Mode Analysis (NMA) reveals low eigenvalues (3.925996e-07), indicating steady and flexible molecular mobility of docked complexes. Codon optimization was carried out in an effective microbial expression system of the Escherichia coli K12 strain using the recombinant plasmid pET-28a (+). Finally, the entire in-silico analysis suggests that the suggested vaccine may induce a significant immune response against S. dysenteriae, making it a promising option for additional experimental trials.

Omicron B.A 2: Vaccine Construction with MHC Class-I, MHC Class-II by using Bioinformatics Tools

Omicron B.A 2: Vaccine Construction with MHC Class-I, MHC Class-II by using Bioinformatics Tools