T-cell Induction Research Articles

Integrated Imaging Probe and Bispecific Antibody Development Enables In Vivo Targeting of Glypican-3–Expressing Hepatocellular Carcinoma

Abstract Glypican-3 (GPC3) is a proteoglycan with high sensitivity and specificity for hepatocellular carcinoma (HCC). We describe the integrated development and validation of a GPC3-targeting optical imaging probe and T cell–redirecting antibody (TRAB) as a theranostic strategy for the detection and treatment of HCC. A novel TRAB targeting GPC3 on HCC tumor cells and the CD3 T-cell receptor as well as a distinct GPC3-specific optical imaging probe were developed from a short peptide. The efficacy of GPC3/CD3 TRAB was evaluated in vitro using IFNγ release and calcein-AM assays. Patient-derived xenografts were used to assess the in vivo efficacy of GPC3/CD3 TRAB and the GPC3 imaging probe for the detection of GPC3+ HCC. GPC3/CD3 TRAB caused a dose-dependent escalation in IFNγ release from inactive peripheral blood T cells (P = 0.001) and higher tumor-cell lysis (P = 0.01) compared with controls in vitro. Intratumorally injected GPC3/CD3 TRAB resulted in significant prolongation of tumor doubling time in the GPC3+ tumors, with an associated reduction of tumor fluorescent signal from the HiLyte 488–conjugated GPC3-specific peptide on optical imaging. These data demonstrate that HCC cell targeting using a GPC3/CD3 TRAB derived from a small peptide enabled effective T-cell activation and induction of a cytotoxic response toward GPC3+ HCC tumor cells both in vitro and in vivo. GPC3-specific optical imaging enabled the detection of the GPC3+ HCC cells and noninvasive monitoring of tumor response to adoptive immunotherapy. The integrated development of a targeted therapeutic and molecular imaging probe provides a promising paradigm for the development of cancer theranostics.

Features of subset composition and functional activity of blood lymphocytes in tick-borne infections of different etiologies

Aim. To perform a comparative assessment of subset composition and functional activity of peripheral blood lymphocytes in patients with tick-borne encephalitis (TBE) and ixodid tick-borne borreliosis (ITBB) in the acute phase of the disease.Materials and methods. The study involved 22 patients with febrile and meningeal TBE, 15 patients with ITBB with and without erythema, and 11 healthy controls. Subset composition of blood lymphocytes was determined by flow cytometry. The blast transformation assay was applied to assess lymphocyte proliferation. Cytokine-producing activity of cells was studied in 24-hour incubated mononuclear cell cultures. Cytokine concentrations (interleukin (IL)-2, IL-4, IL-10, interferon (IFN)γ) were determined in the supernatants by the enzyme-linked immunosorbent assay (ELISA).Results. Patients with TBE demonstrated an increase in the proportion of helper – inducer T-cells, a pronounced decrease in the proportion and absolute count of cytotoxic T cells, and low T lymphocyte count compared to the control values. The study in ITBB patients revealed an increase in the helper – inducer T-cell count and the proportion of NK-cells, a decrease in the cytotoxic T cell count, and the T lymphocyte count comparable to normal values. The most significant decrease in the levels of phytohemagglutinin-induced lymphocyte proliferation was found in patients with TBE. Patients of both groups showed a decrease in IL-2 secretion in the mononuclear cell culture, a rise in IL-4 and IL-10 production, and IFNγ production levels comparable to control values.Conclusion. The study of TBE patients revealed relative lymphocytopenia with changes in the subset composition of lymphocytes characterized by an increase in the proportion of helper – inducer T-cells and a decrease in the absolute cytotoxic T lymphocyte count. Patients with ITBB demonstrated an increase in the proportion of NK-cells and a more pronounced imbalance in the T-helper / cytotoxic T lymphocyte ratio. Changes in the functional phenotype of lymphocytes, regardless of the etiology of tick-borne infection, were characterized by reduced proliferative reserve, low IL-2 secretion, increased IL-4 and IL-10 production, and depressed reactivity of lymphocytes with respect to IFNγ secretion.

A phase 3, randomized, double-blind, multicenter, placebo-controlled study of S-588410, a five-peptide cancer vaccine as an adjuvant therapy after curative resection in patients with esophageal squamous cell carcinoma.

S-588410, a cancer peptide vaccine (CPV), comprises five HLA-A*24:02-restricted peptides from five cancer-testis antigens. In a phase 2 study, S-588410 was well-tolerated and exhibited antitumor efficacy in patients with urothelial cancer. Therefore, we aimed to evaluate the efficacy, immune response, and safety of S-588410 in patients with completely resected esophageal squamous cell carcinoma (ESCC). This phase 3 study involved patients with HLA-A*24:02-positive and lymph node metastasis-positive ESCC who received neoadjuvant therapy followed by curative resection. After randomization, patients were administered S-588410 and placebo (both emulsified with Montanide™ ISA 51VG) subcutaneously. The primary endpoint was relapse-free survival (RFS). The secondary endpoints were overall survival (OS), cytotoxic T-lymphocyte (CTL) induction, and safety. Statistical significance was tested using the one-sided weighted log-rank test with the Fleming-Harrington class of weights. A total of 276 patients were randomized (N = 138/group). The median RFS was 84.3 and 84.1weeks in the S-588410 and placebo groups, respectively (P = 0.8156), whereas the median OS was 236.3weeks and not reached, respectively (P = 0.6533). CTL induction was observed in 132/134 (98.5%) patients who received S-588410 within 12weeks. Injection site reactions (137/140 patients [97.9%]) were the most frequent treatment-emergent adverse events in the S-588410 group. Prolonged survival was observed in S-588410-treated patients with upper thoracic ESCC, grade 3 injection-site reactions, or high CTL intensity. S-588410 induced immune response and had acceptable safety but failed to reach the primary endpoint. A high CTL induction rate and intensity may be critical for prolonging survival during future CPV development.

Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD-1 antibody for BRAF-mutated melanoma.

Programmed death 1 (PD-1)/programmed death-ligand 1 inhibitors are commonly used to treat various cancers, including melanoma. However, their efficacy as monotherapy is limited, and combination immunotherapies are being explored to improve outcomes. In this study, we investigated a combination immunotherapy involving an anti-PD-1 antibody that blocks the major adaptive immune-resistant mechanisms, a BRAF inhibitor that inhibits melanoma cell proliferation, and multiple primary immune-resistant mechanisms, such as cancer cell-derived immunosuppressive cytokines, and a Toll-like receptor 7 agonist that enhances innate immune responses that promote antitumor T-cell induction and functions. Using a xenogeneic nude mouse model implanted with human BRAF-mutated melanoma, a BRAF inhibitor vemurafenib was found to restore T-cell-stimulatory activity in conventional dendritic cells by reducing immunosuppressive cytokines, including interleukin 6, produced by human melanoma. Additionally, intravenous administration of the Toll-like receptor 7 agonist DSR6434 enhanced tumor growth inhibition by vemurafenib through stimulating the plasmacytoid dendritic cells/interferon-α/natural killer cell pathways and augmenting the T-cell-stimulatory activity of conventional dendritic cells. In a syngeneic mouse model implanted with murine BRAF-mutated melanoma, the vemurafenib and DSR6434 combination synergistically augmented the induction of melanoma antigen gp100-specific T cells and inhibited tumor growth. Notably, only triplet therapy with vemurafenib, DSR6434, and the anti-PD-1 antibody resulted in complete regression of SIY antigen-transduced BRAF-mutated melanoma in a CD8 T-cell-dependent manner. These findings indicate that a triple-combination strategy targeting adaptive and primary resistant mechanisms while enhancing innate immune responses that promote tumor-specific T cells may be crucial for effective tumor eradication.

T-cell immunity induced and reshaped by an anti-HPV immuno-oncotherapeutic lentiviral vector

We recently developed an immuno-oncotherapy against human papillomavirus (HPV)-induced tumors based on a lentiviral vector encoding the Early E6 and E7 oncoproteins of HPV16 and HPV18 genotypes, namely “Lenti-HPV-07”. The robust and long-lasting anti-tumor efficacy of Lenti-HPV-07 is dependent on CD8+ T-cell induction and remodeling of the tumor microenvironment. Here, we first established that anti-vector immunity induced by Lenti-HPV-07 prime has no impact on the efficacy of a homologous boost to amplify anti-HPV T-cell immunity. To longitudinally monitor the evolution of the T-cell repertoire generated after the prime, homologous or heterologous boost with Lenti-HPV-07, we tracked T-cell clonotypes by deep sequencing of T-Cell Receptor (TCR) variable β and α chain mRNA, applied to whole peripheral blood cells (PBL) and a T cell population specific of an immunodominant E7HPV16 epitope. We observed a hyper-expansion of clonotypes post prime, accompanied by increased frequencies of HPV-07-specific T cells. Additionally, there was a notable diversification of clonotypes post boost in whole PBL, but not in the E7HPV16-specific T cells. We then demonstrated that the effector functions of such Lenti-HPV-07-induced T cells synergize with anti-checkpoint inhibitory treatments by systemic administration of anti-TIM3 or anti-NKG2A monoclonal antibodies. While Lenti-HPV-07 is about to enter a Phase I/IIa clinical trial, these results will help better elucidate its mode of action in immunotherapy against established HPV-mediated malignancies.

Abstract PO5-24-12: IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells

Abstract Research Objectives and Rationale: Previously, RARγ agonism has been shown to play an essential role in CD8 T-cell-mediated immunity to infectious pathogens. However, we found no previous reports on effects of RARγ agonists on in vivo tumor growth of triple negative or Her2+ breast cancers. Here we explored whether RARγ could play a critical role in T-cell-mediated immunity in these breast cancers, using potent highly selective novel RARγ agonist compounds. Experimental methods and results: We synthesized and screened compounds for selective transactivation of RAR γ in reporter assays. Three highly potent compounds, demonstrating highly selective RARγ transactivation at less than one nanomolar concentrations, were selected for further evaluation of potential anti-cancer activities in vitro. None of the three evaluated compounds demonstrated significant inhibitory effects on in vitro growth of EMT-6 triple negative breast cancer cells at concentrations of 20-100 millimolar, indicating that the three compounds have insignificant direct inhibitory effects on EMT-6 cancer cell growth in vitro. We then examined the effects of one of the compounds in vivo in a syngeneic mouse model of EMT-6 triple negative breast cancer. Treatment with the most potent of the three compounds, IRX5010, demonstrated dose response inhibition of in vivo growth of EMT-6 tumors. Inhibition of tumor growth at day 17 relative to vehicle was 59% and 69% respectively in mice treated with IRX5010 at 10 or 25 mg/kg/day. A cohort treated with 50 mg/kg/day was sacrificed early on day 12 due to excessive weight loss and deaths of some of the animals. This cohort experienced a 73% inhibition of tumor growth relative to vehicle. Gross necropsies of animals in the 25 and 50 mg/kg/day dose groups did not show observable pathologic findings in any major organ. Flow cytometry was performed on freshly excised tumors on the day of termination of the in-life portion of the study in the 10 and 25 mg/kg/day dose groups. Tumors from mice treated with IRX5010 had increased intratumoral numbers of Total CD45+ cells; Total CD3+, Total CD4+, and Total CD8+ T-cells per mg of tumor tissue, relative to vehicle controls. IRX5010 treated mice also had increased intratumoral CD4+IL-17+ T-helper cells; and increased CD4+CD62L- and CD8+CD62L- T-effector memory T-cells per mg of tumor tissue relative to vehicle-treated control mice. We also conducted an experiment of treatment with IRX5010 in mice transplanted with human immune cells (to provide a human immune system) to examine its effects on xenografted human Her2+ JIMT-1 cancer cells. In this model, treatment with IRX5010 at 10 mg/kg/day resulted in a 17% inhibition of tumor growth relative to vehicle control over 26 days of treatment. Flow cytometric analysis showed that tumors from the IRX5010 treated mice contained increased numbers per mg of tumor tissue of intratumoral human CD8+ T-cells, most of which were CD8+ CD7- CD45RA+ terminally differentiated effector memory phenotype T-cells. Conclusions: Treatment with IRX5010, a potent and highly selective RARγ nuclear receptor agonist compound demonstrated substantial treatment effects relative to vehicle control on inhibition of growth of murine triple negative breast cancer in vivo, despite having essentially no growth inhibitory effect on these cells in vitro. Treatment of xenografted human Her2+ tumors in mice with humanized immune systems resulted in a moderate inhibition of Her2+ breast cancer growth relative to vehicle controls. In both models, treatment with IRX5010 induced tumor infiltrating T-lymphocytes of largely effector memory phenotypes, demonstrating an effect on the tumor immune microenvironment. These data identify RARγ and RARγ agonists as a new treatment target and novel treatment modality for breast cancers, especially for the highly unmet need for new and better treatments for triple negative breast cancer. Citation Format: Martin Sanders, Mary Topalovski, Vidyasagar Vuligonda. IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-12.

Artificial Extracellular Vesicles Generated from T Cells Using Different Induction Techniques.

Cell therapy is at the forefront of biomedicine in oncology and regenerative medicine. However, there are still significant challenges to their wider clinical application such as limited efficacy, side effects, and logistical difficulties. One of the potential approaches that could overcome these problems is based on extracellular vesicles (EVs) as a cell-free therapy modality. One of the major obstacles in the translation of EVs into practice is their low yield of production, which is insufficient to achieve therapeutic amounts. Here, we evaluated two primary approaches of artificial vesicle induction in primary T cells and the SupT1 cell line-cytochalasin B as a chemical inducer and ultrasonication as a physical inducer. We found that both methods are capable of producing artificial vesicles, but cytochalasin B induction leads to vesicle yield compared to natural secretion, while ultrasonication leads to a three-fold increase in particle yield. Cytochalasin B induces the formation of vesicles full of cytoplasmic compartments without nuclear fraction, while ultrasonication induces the formation of particles rich in membranes and membrane-related components such as CD3 or HLAII proteins. The most effective approach for T-cell induction in terms of the number of vesicles seems to be the combination of anti-CD3/CD28 antibody activation with ultrasonication, which leads to a seven-fold yield increase in particles with a high content of functionally important proteins (CD3, granzyme B, and HLA II).

Induced regulatory T cells as immunotherapy in allotransplantation and autoimmunity: challenges and opportunities.

Regulatory T cells play a crucial role in the homeostasis of the immune response. Regulatory T cells are mainly generated in the thymus and are characterized by the expression of Foxp3, which is considered the regulatory T-cell master transcription factor. In addition, regulatory T cells can be induced from naive CD4+ T cells to express Foxp3 under specific conditions both in vivo (peripheral regulatory T cells) and in vitro (induced regulatory T cells). Both subsets of thymic regulatory T cells and peripheral regulatory T cells are necessary for the establishment of immune tolerance to self and non-self antigens. Although it has been postulated that induced regulatory T cells may be less stable compared to regulatory T cells, mainly due to epigenetic differences, accumulating evidence in animal models shows that induced regulatory T cells are stable in vivo and can be used for the treatment of inflammatory disorders, including autoimmune diseases and allogeneic transplant rejection. In this review, we describe the biological characteristics of induced regulatory T cells, as well as the key factors involved in induced regulatory T-cell transcriptional, metabolic, and epigenetic regulation, and discuss recent advances for de novo generation of stable regulatory T cells and their use as immunotherapeutic tools in different experimental models. Moreover, we discuss the challenges and considerations for the application of induced regulatory T cells in clinical trials and describe the new approaches proposed to achieve in vivo stability, including functional or metabolic reprogramming and epigenetic editing.

Abstract CT141: mRNA-4157 (V940) individualized neoantigen therapy (INT) + pembrolizumab (pembro) in advanced unresectable HPV- head & neck carcinoma (HNSCC): Clinical & translational analysis

Abstract Introduction: In HPV- HNSCC, limited durable clinical responses to PD1/PD-L1 blockade may be due to diminished effector cytolytic activity and clonal diversity.1-4 mRNA-4157 is a novel mRNA-based INT that encodes up to 34 neoantigens inducing specific antitumor T-cell activation; durable clinical responses with pembro have been shown in melanoma.5 We assessed INT + pembro in unresectable, metastatic HPV- HNSCC in the Phase 1 mRNA-4157-P101/KEYNOTE-603 study (NCT03313778). Methods: Part C of this study enrolled ≥18-year-old patients (pts) with CPI-naïve, recurrent/metastatic HPV- HNSCC. Eligible pts received 200 mg pembro Q3W IV during a 6-week lead-in, then combined with 1 mg INT ≤9 doses Q3W IM, and until disease progression, unacceptable toxicity, or ≤35 total cycles of pembro. Objectives included safety, tolerability and preliminary clinical activity. Peripheral blood was collected longitudinally for both immunogenicity via IFN-γ ELISpot and ctDNA analyses. Next generation sequencing was performed on baseline tumor biopsies. Results: A total of 28 enrolled pts received pembro; 22 received INT + pembro. Most adverse events (AEs) related to INT were grade 1-2; most common were influenza-like illness, injection site pain, and pyrexia. No grade 4/5 AEs were related to INT. Three pts had grade 3 events (pyrexia, increased lipase, and decreased lymphocyte count) attributed to both INT and pembro. Six pts discontinued prior to INT (death/disease progression/deterioration). Response rate for INT + pembro was 27.3% (6/22; 95% CI, 10.7-50.2); disease control rate was 63.6% (14/22; 95% CI, 40.7-82.8) per RECIST V1.1 with best overall response of 2 (9.1%) complete, 4 (18.2%) partial response, and 8 (36.4%) stable disease at data cut-off (May 2023); with median follow-up of 38.4 weeks (range, 7.7-198.3). Median (95% CI) PFS and OS were 15.0 weeks (11.6-38.6) and 107.1 weeks (42.7-NE), respectively. Sustained de novo T-cell induction to targeted neoantigens was observed in 5/5 pts with available samples. A decrease in ctDNA levels post-baseline was observed in 3/4 responders (8 available samples). Differential gene expression from pretreatment tumor samples suggests increased inflamed T-cell-associated gene expression (Benjamini-Hochberg-adjusted descriptive p<0.05, for hallmark inflammatory response) in INT + pembro responders. Conclusions: INT + pembro had a manageable safety profile, showed evidence for activation of immune responses, and induced preliminary clinical responses in pts with HPV- HNSCC. Further testing of INT + pembro in a randomized setting may be warranted.

Abstract 6554: Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Repression of the MHC-I and reduced tumor infiltration of cytotoxic T-lymphocytes is often associated with immunotherapy resistance in SCLC. Lurbinectedin is FDA-approved as a second-line treatment for SCLC and we previously showed lurbinectedin as an effective therapeutic strategy in SCLC preclinical models. There are ongoing clinical trials combining immune checkpoint blockade with lurbinectedin in ES-SCLC. However, there is no insight into the effect of lurbinectedin on the immune microenvironment in SCLC. In this study, we evaluated the effect of lurbinectedin on the immune microenvironment and the anti-tumor effect with or without PD-L1 blockade. Results: To determine the effect of lurbinectedin in SCLC models, we treated immunocompetent flank RPP (Rb1, Trp53, and p130) and RPM (Rb1, Trp53, and MYCT58A) tumor-bearing mice with lurbinectedin (0.2 mg/kg, 1/7days) and/or anti-PD-L1 (300µg, 1/7days) for 3 weeks. Mice treated with anti-PD-L1 alone showed no anti-tumor response, and single-agent lurbinectedin caused a delay in tumor growth. However, 6 of the 10 mice treated with the combination of lurbinectedin and anti-PD-L1, had a complete response (100% reduction) and the other 4 had 90% tumor regression. Tumors were resected and analyzed by multicolor flow cytometry for changes in tumor-infiltrating lymphocytes (TILs). Furthermore, analysis of immune cells infiltrating into the tumors post- treatment showed significant induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells in the combination treatment arm. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased, while immunosuppressive M2 type macrophages and MDSC cells were decreased. These effects are consistent with data showing that lurbinectedin treatment leads to an activation of the cGAS/STING pathway, type I/II interferons (IFNα/β), and pro-inflammatory chemokines (CCL5, CXCL10) in tumors. Interestingly, lurbinectedin treatment led to significant upregulation of mRNA and surface expression of MHC class-I genes (HLA-A/B/C) in vitro and in vivo. Finally, bulk RNA sequencing as well as RT-PCR of pre- and post-lurbinectedin treatment demonstrated an increase in DAMPs associated with immunogenic cell death. Conclusion: We provide the first mechanistic insight into the lurbinectedin-induced multimodal immune modulation in SCLC leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Since lurbinectedin is already approved as a second-line agent in SCLC, our preclinical data provide a strong rationale for combining this regimen with inhibitors of the PD-L1 pathway and also highlight the immune subsets that the combination targets. Citation Format: Subhamoy Chakraborty, Yazhini Mahendravarman, Utsav Sen, Avisek Banerjee, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6554.

The Promise of Complement Therapeutics in Solid Organ Transplantation.

Transplantation is the ideal therapy for end-stage organ failure, but outcomes for all transplant organs are suboptimal, underscoring the need to develop novel approaches to improve graft survival and function. The complement system, traditionally considered a component of innate immunity, is now known to broadly control inflammation and crucially contribute to induction and function of adaptive T-cell and B-cell immune responses, including those induced by alloantigens. Interest of pharmaceutical industries in complement therapeutics for nontransplant indications and the understanding that the complement system contributes to solid organ transplantation injury through multiple mechanisms raise the possibility that targeting specific complement components could improve transplant outcomes and patient health. Here, we provide an overview of complement biology and review the roles and mechanisms through which the complement system is pathogenically linked to solid organ transplant injury. We then discuss how this knowledge has been translated into novel therapeutic strategies to improve organ transplant outcomes and identify areas for future investigation. Although the clinical application of complement-targeted therapies in transplantation remains in its infancy, the increasing availability of new agents in this arena provides a rich environment for potentially transformative translational transplant research.

Intradermal Inoculation of Inactivated Foot-and-Mouth Disease Vaccine Induced Effective Immune Responses Comparable to Conventional Intramuscular Injection in Pigs.

All pigs in the Republic of Korea are given the foot-and-mouth disease virus (FMDV) vaccine intramuscularly (IM) as part of the country's vaccination policy. However, the IM administration of the FMDV vaccine to pig results in residual vaccine components in the muscle and undesirable changes in muscle and soft tissues, causing economic losses in swine production. In this study, we evaluated whether intradermal (ID) vaccination could be proposed as an alternative to IM administration. ID vaccination (0.2 mL on each side of the neck muscle) and IM vaccination (2 mL on each side of the neck muscle) were performed twice, separated by 14 days, using a commercial FMD vaccine in specific-pathogen-free pigs. We observed growth performance, gross and microscopic lesions at the inoculation site, FMDV-specific antibodies, and neutralizing antibodies for 35 days after vaccination. Side effects on the skin grossly appeared following ID administration, but most were reduced within two weeks. All ID-vaccinated pigs showed inflammatory lesions limited to the dermis, but IM-vaccinated pigs had abnormal undesirable changes and pus in the muscle. ID-vaccinated pigs performed comparably to IM-vaccinated pigs in terms of growth, FMD virus-specific antibodies, protection capability against FMDV, and T-cell induction. This study demonstrated that the ID inoculation of the inactivated FMD vaccine induced immune responses comparable to an IM injection at 1/10 of the inoculation dose and that the inoculation lesion was limited to the dermis, effectively protecting against the formation of abnormal undesirable changes in muscle and soft tissues.

E3 ubiquitin ligase casitas B-lineage lymphoma-b modulates T-cell anergic resistance via phosphoinositide 3-kinase signaling in patients with immune thrombocytopenia

BackgroundThe E3 ubiquitin ligase casitas B-lineage lymphoma-b (CBLB) is a newly identified component of the ubiquitin-dependent protein degradation system and is considered an important negative regulator of immune cells. CBLB is essential for establishing a threshold of T-cell activation and regulating peripheral T-cell tolerance through various mechanisms. However, the involvement of CBLB in the pathogenesis of immune thrombocytopenia (ITP) is unknown. ObjectivesWe aimed to investigate the expression and role of CBLB in CD4+ T cells obtained from patients with ITP through quantitative proteomics analyses. MethodsCD4+ T cells were transfected with adenoviral vectors overexpressing CBLB to clarify the effect of CBLB on anergic induction of T cells in patients with ITP. DNA methylation levels of the CBLB promoter and 5′ untranslated region (UTR) in patient-derived CD4+ T cells were detected via MassARRAY EpiTYPER assay (Agena Bioscience). ResultsCD4+ T cells from patients with ITP showed resistance to anergic induction, highly activated phosphoinositide 3-kinase–protein kinase B (AKT) signaling, decreased CBLB expression, and 5′ UTR hypermethylation of CBLB. CBLB overexpression in T cells effectively attenuated the elevated phosphorylated protein kinase B level and resistance to anergy. Low-dose decitabine treatment led to significantly elevated levels of CBLB expression in CD4+ T cells from 7 patients showing a partial or complete response. ConclusionThese results indicate that the 5′ UTR hypermethylation of CBLB in CD4+ T cells induces resistance to T-cell anergy in ITP. Thus, the upregulation of CBLB expression by low-dose decitabine treatment may represent a potential therapeutic approach to ITP.

Sustained Liver HBsAg Loss and Clonal T- and B-Cell Expansion upon Therapeutic DNA Vaccination Require Low HBsAg Levels.

Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.

Analysis of the Impact of Body Mass Index (BMI) on the Durability of Response in Patients with Aplastic Anemia Treated with Weight-Adjusted Horse Anti-Thymocyte Globulin (hATG)

Introduction: Immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG), cyclosporine A (CsA) and Eltrombopag (Epag) has recently been established as standard of care in adult patients (pts.) with severe (sAA) or very severe (vsAA) aplastic anemia > 40-50 years or ineligible for transplant for other reasons. ATG works via immunosuppressive properties including T-cell depletion and induction of immune tolerance. Horse ATG (hATG) is applied for 4 days with 40 mg/kg/day based on the patient's current body weight. Thus, obese pts., defined by a body mass index (BMI) ≥ 30, receive higher absolute hATG dosages than non-obese pts. (BMI < 30). To date, it is unknown whether increased hATG dosages in obese pts. might have beneficial or adverse effects on AA treatment. To address the role of obesity in AA, we investigated (1) the prevalence of obesity in 334 pts. with (suspected) AA and (2) compared the overall survival (OS) and response of 89 obese and non-obese AA pts. to the treatment with hATG/CsA ±Epag. Methods: Retrospective analysis of data from pts. enrolled in the German Registry for Aplastic Anemia and Bone Marrow Failure (AA-BMF). 334 pts. with (suspected) AA and available BMI data (49% (n=164) male/ 51% (n=170) female, 83% (n=278) BMI < 30, 17% (n=56) BMI ≥ 30) were identified of which 89 pts. (43 male/ 46 female, mean age 50 ± 17 years, 75 BMI < 30/ 14 BMI ≥ 30 at hATG administration) with confirmed diagnosis of AA (4 mAA/ 49 sAA/ 18 vsAA/ 14 AA not otherwise specified/ 4 AA-PNH Overlap) and treatment with hATG/CSA were analyzed in detail. 14 pts. received hATG/CSA/Epag (all BMI < 30). Follow-up data were compiled over 35 ± 38.5 months between 2000 and 2023, whereby follow-up data of 12 pts. were incomplete. Results are given as mean ± standard deviation. Results: Analysis of the age distribution of the 343 pts. with (suspected) AA showed a mean age of 50 ±17 years with a biphasic peak at age approx. 25 and 65 years. The average BMI stratified by age (20-30y: 22.9, 31-40y: 24.5, 41-50y: 26.8, 51-60y: 26.4, 61-70y: 27.5, >70y: 27.8) corresponded to the BMI distribution of the German population surveyed by the German Federal Statistical Office. In accordance with the expected survival rates, the 89 AA pts. treated with hATG/CSA (including 14 pts. with Epag) revealed a 5-year overall survival (OS) of 93%/ 86%/ 67 % at < 40/ 40-60/ > 60 years. Regarding BMI, no significant difference was observed in 5-year OS between obese (BMI ≥ 30, 5y-OS 67%) and non-obese pts. (BMI < 30, 5y-OS 85%, p = 0.51). In pts. responding to IST, hematological response at six months after ATG was reached in 67 % (n=7/11) of the obese and 63 % (n=21/33, in 9 pts. exact timepoint not available) of the non-obese pts. (p = 0.85). The proportion of primary IST refractory pts. was numerically lower in obese (21 %, n=3/14) than in non-obese pts. (32 %, n=21/65, p=0.53). Among all 53 responders (2 pts. had incomplete follow-up to assess relapse), a significantly lower relapse rate of 9 % (n=1/11) in obese (median follow-up: 16 months, range 3 to 120 months) compared to 55 % (n=22/40) in non-obese pts. was observed (median follow-up: 19 months, range 1 to 87 months, p=0.008). Conclusion: BMI distribution of AA pts. by age is comparable to that of the general population with a higher rate of obesity in older pts.. Pts. with a BMI ≥ 30 thus receiving higher ATG dosages do appear to have comparable OS and hematological response rates compared to pts. with BMI <30. However, pending validation in a larger patient cohort, we hypothesize, based on our analysis, that a higher cumulative total ATG dose may have a beneficial impact on relapse rates in pts. with BMI ≥ 30 compared to those with BMI < 30.

Heat shock factor 1 drives regulatory T-cell induction to limit murine intestinal inflammation

The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of CD4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and TGFβ, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using ChIP, we demonstrate direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when WT Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T cell component.

Microbiota-Specific T Cells Contribute to Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation

Background: Allogeneic T-cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a fixed genetic system within which donor T cells interact with recipient-derived peptide-MHC complexes. However, the current model does not account for potential cognate interactions between donor T cells and microbe-derived antigen presented by the recipient. Prior studies in healthy patients have demonstrated the existence of circulating αβT cells targeting common bacterial species in the gastrointestinal (GI) tract. Whether microbe-specific T cells derived from the donor graft impact GVHD outcomes has not been studied. We tested the hypothesis that the recipient GI microbiota serve as a source of cognate antigen, contributing to clonotypic donor T-cell expansion and induction of GVHD. Methods: Functional studies utilized allogeneic stem cell transplant models in murine systems including both matched (Marilyn Tg to B6 male) and mismatched (B6 to B6D2F1) transplants as well as inducible MHCII knockout mice. Microbiota-specific transgenic T cells (MSTCs) targeting commensal flagellin (CBir1) and vendor-specific microbiome (segmented filamentous bacteria - SFB) in the context of I-A(b) were incorporated as part of the donor graft. Mechanistic studies utilized flow cytometry, cytokine stimulation and profiling, single-cell RNA sequencing, gene set enrichment analysis, NFAT-GFP T-hybridoma reporters, live-cell imaging (xCelligence RTCA eSight), and tissue analysis with RNA in-situ hybridization (ISH). Results: MSTCs lethally augmented GVHD when given as part of the donor graft in respectively colonized recipients. Taconic B6 male recipients (SFB-bearing) developed lethal GVHD when both donor Marilyn (Allo) Tg T cells and SFB T cells were introduced, but not when either transgenic T cell was introduced alone ( Figure A, p<0.0001). In contrast, Jackson B6 recipients (SFB-absent) receiving both Allo and SFB T cells did not develop lethal GVHD ( Figure A, p<0.0001), confirming functional relevance of microbe-specificity among the donor T cell pool. Lethal GVHD was also seen with addition of CBir1 T cells to the donor graft but not with addition of bulk T cells (log-rank p<0.0001). Similar results were seen in a mismatched transplant system. To determine how MSTCs promoted lethality in the context of an allogeneic (allo) response, we performed scRNA sequencing of T cells harvested in the recipient spleen at day 6 post-transplant (prior to mortality onset). Post-transplant MSTCs harbored a naïve phenotype (Sell high/CD44 low/CD69 low) when given as the primary source of donor T cells but shifted to an early activated phenotype (Sell int/CD44 low/CD69 high/TCF7 low) when Allo T cells were concurrently introduced (ANOVA q<0.01). MSTC activation was confirmed with augmented expression of IFNγ upon PMA/ionomycin stimulation observed in allo vs. syngeneic response (Wilcoxon rank-sum, p< 0.01). Introduction of Allo T cells increased the abundance of MSTCs in the mesenteric lymph nodes (day 5, 6, Wilcoxon, p<0.01). Ablation of MHCII on the GI epithelial surface using Villin-CreERT2 I-Ab flox recipients abrogated microbe-specific T cell activation in co-culture with recipient GI intraepithelial cells harvested at day 6 post-transplant vs. non-ablated control (8, 12, 24-hr NFAT-GFP intensity, p<0.001). RNA ISH probes targeting the CDR3 region of anti-microbial CBir1 T cells demonstrated an increase in MSTC infiltration at day 6 in the recipient GI lamina propria during an allo response ( Figure B) compared to a syngeneic response (# of MSTCs/terminal ileum cross section at day 6, Wilcoxon p<0.01). Conclusions: We confirm that clonotypic T cell expansion following allogeneic transplantation is not only driven by donor-recipient genetics, as reflected by major and minor histocompatibility matching, but also by differences in microbiota composition that can serve as a source of antigenic stimulation, providing a feed-forward mechanism to propagate GVHD. Furthermore, in the context of an alloreactive T cell response that damages gut integrity, microbial antigens can be presented by GI epithelia, allowing the gut to be concurrently targeted by microbiota specific T cells whose function is additionally enhanced by the alloreactive response.

Full eradication of pre-clinical human papillomavirus-induced tumors by a lentiviral vaccine.

Human papillomavirus (HPV) infections are the cause of all cervical and numerous oropharyngeal and anogenital cancers. The currently available HPV vaccines, which induce neutralizing antibodies, have no therapeutic effect on established tumors. Here, we developed an immuno-oncotherapy against HPV-induced tumors based on a non-integrative lentiviral vector encoding detoxified forms of the Early E6 and E7 oncoproteins of HPV16 and 18 genotypes, namely, "Lenti-HPV-07". A single intramuscular injection of Lenti-HPV-07 into mice bearing established HPV-induced tumors resulted in complete tumor eradication in 100% of the animals and was also effective against lung metastases. This effect correlated with CD8+ T-cell induction and profound remodeling of the tumor microenvironment. In the intra-tumoral infiltrates of vaccinated mice, the presence of large amounts of activated effector, resident memory, and transcription factor T cell factor-1 (TCF-1)+ "stem-like" CD8+ T cells was associated with full tumor eradication. The Lenti-HPV-07-induced immunity was long-lasting and prevented tumor growth after a late re-challenge, mimicking tumor relapse. Lenti-HPV-07 therapy synergizes with an anti-checkpoint inhibitory treatment and therefore shows promise asan immuno-oncotherapy against established HPV-mediated malignancies.

Outcome for Children and Young Adults With T-Cell ALL and Induction Failure in Contemporary Trials.

Historically, patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission at the end of induction (EOI) have had poor long-term survival. The goal of this study was to examine the efficacy of contemporary therapy, including allogeneic hematopoietic stem cell transplantation (HSCT) in first remission (CR1). Induction failure (IF) was defined as the persistence of at least 5% bone marrow (BM) lymphoblasts and/or extramedullary disease after 4-6 weeks of induction chemotherapy. Disease features and clinical outcomes were reported in 325 of 6,167 (5%) patients age 21 years and younger treated in 14 cooperative study groups between 2000 and 2018. With a median follow-up period of 6.4 years (range, 0.3-17.9 years), the 10-year overall survival (OS) was 54.7% (SE = 2.9), which is significantly higher than the 27.6% (SE = 2.9) observed in the historical cohort from 1985 to 2000. There was no significant impact of sex, age, white blood cell count, central nervous system disease status, T-cell maturity, or BM disease burden at EOI on OS. Postinduction complete remission (CR) was achieved in 93% of patients with 10-year OS of 59.6% (SE = 3.1%) and disease-free survival (DFS) of 56.3% (SE = 3.1%). Among the patients who achieved CR, 72% underwent HSCT and their 10-year DFS (with a 190-day landmark) was significantly better than nontransplanted patients (63.8% [SE = 3.6] v 45.5% [SE = 7.1]; P = .005), with OS of 66.2% (SE = 3.6) versus 50.8% (SE = 6.8); P = .10, respectively. Outcomes for patients age 21 years and younger with T-ALL and IF have improved in the contemporary treatment era with a DFS benefit among those undergoing HSCT in CR1. However, outcomes still lag considerably behind those who achieve remission at EOI, warranting investigation of new treatment approaches.

Combined intranasal and intramuscular parainfluenza 5-, simian adenovirus ChAdOx1- and poxvirus MVA-vectored vaccines induce synergistically HIV-1-specific T cells in the mucosa.

The primary goal of this work is to broaden and enhance the options for induction of protective CD8+ T cells against HIV-1 and respiratory pathogens. We explored the advantages of the parainfluenza virus 5 (PIV5) vector for delivery of pathogen-derived transgenes alone and in combination with the in-human potent regimen of simian adenovirus ChAdOx1 prime-poxvirus MVA boost delivering bi-valent mosaic of HIV-1 conserved regions designated HIVconsvX. We showed in BALB/c mice that the PIV5 vector expressing the HIVconsvX immunogens could be readily incorporated with the other two vaccine modalities into a single regimen and that for specific vector combinations, mucosal CD8+ T-cell induction was enhanced synergistically by a combination of the intranasal and intramuscular routes of administration. Encouraging safety and immunogenicity data from phase 1 human trials of ChAdOx1- and MVA-vectored vaccines for HIV-1, and PIV5-vectored vaccines for SARS-CoV-2 and respiratory syncytial virus pave the way for combining these vectors for HIV-1 and other indications in humans.