T-cell Stimulation Research Articles

A phase 1/2 dose escalation/expansion study of OR2805 alone or in combination in subjects with advanced solid tumors.

TPS2693 Background: M2-like macrophages are thought to promote an immunoinhibitory environment and prevent activation and proliferation of T cells, leading to resistance to checkpoint inhibitor (CPI) therapy. OR2805 is a fully human monoclonal antibody identified in a patient who had an exceptional response to CPI. OR2805 recognizes CD163, an immunosuppressive scavenger receptor highly expressed on M2 tumor-associated macrophages (TAMs). OR2805 treatment reprograms M2 macrophages to take on an immunostimulatory, M1-like phenotype. In macrophage/T cell coculture assays, OR2805 treatment relieved the suppressive effect of M2c macrophages as demonstrated by increased T-cell proliferation and stimulation of IFN-γ and perforin release. OR2805 restored the IFN-γ production of exhausted T cells and showed a synergistic effect on cocultures treated in combination with CPI. OR2805 monotherapy demonstrated significant anti-tumor activity in lung cancer xenograft models in NSG-SGM3 mice. Relieving the immune suppression of CD163-expressing TAMs by OR2805 to improve anti-tumor T-cell responses will be evaluated alone and in combination with CPI therapy. Methods: This phase 1/2, open-label, multicenter, dose-escalation/expansion study is assessing the safety, tolerability and preliminary activity of OR2805 alone and in combination with anti-PD-1 or chemotherapy in up to 325 adults (≥18 years) with histologically or cytologically confirmed metastatic or locally advanced solid tumors. The study is divided into 3 parts. Part A will enroll subjects with advanced solid tumors of any histology to describe the preliminary safety profile of OR2805 and determine the recommended phase 2 dose (RP2D) of OR2805 alone and in combination with an anti-PD-1 monoclonal antibody. Part B will further characterize the safety and preliminary antitumor activity of OR2805 either alone or in combination with an anti-PD-1 or chemotherapy in previously treated non-small cell lung cancer (NSCLC), melanoma and liposarcoma, as well as in previously untreated NSCLC in combination with an anti-PD-1 antibody. Part C will evaluate OR2805 monotherapy in relapsed squamous cell carcinoma of the head and neck and other tumor types with an emphasis on correlative biomarker analysis. OR2805 will be infused on Day 1 of 21-day cycles. Primary endpoints are adverse events, laboratory abnormalities, dose-limiting toxicities, and dose-level safety and activity. Secondary endpoints are objective response rates, duration of response, complete response, progression-free survival, overall survival, PK, and antidrug antibodies. Exploratory biomarkers of OR2805-mediated pharmacodynamic effects and immune stimulation will be explored. The study was opened September 2021 and is enrolling at sites in the United States with planned expansion within the US and to Europe. Clinical trial information: Clinical trial information: NCT05094804.

Depletion of CLL cells by venetoclax treatment reverses oxidative stress and impaired glycolysis in CD4 T cells

AbstractAcquired T-cell dysfunction is characteristic of chronic lymphocytic leukemia (CLL) and is associated with reduced efficacy of T cell–based therapies. A recently described feature of dysfunctional CLL-derived CD8 T cells is reduced metabolic plasticity. To what extend CD4 T cells are affected and whether CD4 T-cell metabolism and function can be restored upon clinical depletion of CLL cells are currently unknown. We address these unresolved issues by comprehensive phenotypic, metabolic, transcriptomic, and functional analysis of CD4 T cells of untreated patients with CLL and by analysis of the effects of venetoclax plus obinutuzumab on the CD4 population. Resting CD4 T cells derived from patients with CLL expressed lower levels of GLUT-1 and displayed deteriorated oxidative phosphorylation (OXPHOS) and overall reduced mitochondrial fitness. Upon T-cell stimulation, CLL T cells were unable to initiate glycolysis. Transcriptome analysis revealed that depletion of CLL cells in vitro resulted in upregulation of OXPHOS and glycolysis pathways and restored T-cell function in vitro. Analysis of CD4 T cells from patients with CLL before and after venetoclax plus obinutuzumab treatment, which led to effective clearance of CLL in blood and bone marrow, revealed recovery of T-cell activation and restoration of the switch to glycolysis, as well as improved T-cell proliferation. Collectively, these data demonstrate that CLL cells impose metabolic restrictions on CD4 T cells, which leads to reduced CD4 T-cell functionality. This trial was registered in the Netherlands Trial Registry as #NTR6043.

Selected Articles from This Issue

Interleukin-15 (IL-15) regulates the generation, persistence, and differentiation of natural killer (NK) and CD8 T cells. To expand its role in cancer immunotherapy, Chen and colleagues designed a multitumor model treated with the combination of intratumoral anti-CD40, parenteral IL-15 and anti-PD-1. Anti-CD40 given intratumorally not only reduced dosing requirements and toxicity, but it also stimulated the antitumor activity of the distal tumors, suggestive of an abscopal effect. IL-15 with intratumoral anti-CD40 was additive/synergistic, and the inclusion of the anti-PD-1 was additive to the combination. Thus, this treatment shows promise for patients with solid tumors or resistance to anti-PD-1 therapy.Cancer-associated fibroblasts (CAF) are a heterogenous cell population with a complex immunoregulatory role, which has not been well defined in head and neck squamous cell carcinoma (HNSCC). Leveraging protein activity inference tools to analyze scRNA-sequencing of HNSCC patients, pre- and post-nivolumab, Obradovic and colleagues identified distinct subpopulations of head and neck CAF (HNCAF). Enrichment of a T-cell stimulating population (tsCAF) was found to associate with treatment response and ex vivo assays confirmed this CAF subset reduces CD8 T-cell exhaustion. Analysis of an independent HNSCC cohort treated with pembrolizumab validated this tsCAF subset as a potential biomarker of response.Approximately 30% of MSI-H/dMMR tumors do not respond to immune checkpoint inhibitors (ICI), highlighting the importance of identifying predictive biomarkers or molecular profiling correlated with resistance to these agents. Here, Chida and colleagues performed transcriptomic profiling of MSI-H/dMMR gastrointestinal tumors treated with PD-1 blockade. The study revealed that nonresponders had the enrichment of epithelial mesenchymal transition, angiogenesis, hypoxia, mTORC1, KRAS, Wnt/β-catenin, TGF-β, and various metabolism-related signaling pathways, which have been associated with an immunosuppressive tumor microenvironment. Meanwhile, the IFN-γ pathway was upregulated in responders. High expression of VEGF-A might predict a negative response to ICI in this population.Acral melanoma is a rare subtype of melanoma that arises on non-sun exposed skin of the hands, feet, and nailbeds. Patients with acral melanoma have lower response rates to immunotherapy than those with nonacral cutaneous melanoma, suggesting unique microenvironmental features. In their study, Li and colleagues performed comprehensive single cell analysis of a cohort of acral melanoma specimens and found them to harbor fewer effector CD8 T cells and NK cells and a complete absence of γδ T cells compared to non-acral cutaneous melanoma. VISTA, TIGIT, and adenosine signaling were identified as targetable immune checkpoints in acral melanoma.

Effects of immune checkpoint blockade on antigen-specific CD8+ T cells for use in adoptive cellular therapy.

Adoptive T-cell therapies have been successfully used as prophylaxis or treatment for immunocompromised patients at risk of viral infections or advanced cancers. Unfortunately, for some refractory cancers, they have failed. To overcome this, checkpoint inhibitors are used to rescue immune antitumor responses. We hypothesized that in vitro checkpoint blockade during T-cell stimulation and expansion with messenger RNA (mRNA)-pulsed DCs may enhance the activity of antigen-specific T cells and improve the efficacy of adoptive cellular therapy platforms. Human peripheral blood mononuclear cells were isolated from cytomegalovirus (CMV)-seropositive donors to generate DCs. These were pulsed with CMV matrix phosphoprotein 65 (CMVpp65)-mRNA to educate T cells in coculture for 15 days. Three checkpoint blockade conditions were evaluated (anti-PD1, anti-Tim3, and anti-PD1 + Tim3). IL-2 and antibodies blockades were added every 3 days. Immunophenotyping was performed on Day 0 and Day 15. Polyfunctional antigen-specific responses were evaluated upon rechallenge with CMVpp65 peptides. CMVpp65-activated CD8+ T cells upregulate Lag3 and Tim3 (P ≤ 0.0001). Tim3 antibody blockade alone or in combination led to a significant upregulation of Lag3 expression on CD8+ pp65Tetramer+ central memory, effector memory, and terminal effector memory cells re-expressing RA (TEMRA) T cells. This latter T-cell subset uniquely maintains double-positive Tim3/Lag3 expression after checkpoint blockade. By contrast, PD1 blockade had minimal effects on Tim3 or Lag3 expression. In addition, IFN-γ secretion was reduced in T cells treated with Tim3 blockade in a dose-dependent manner (P = 0.004). In this study, we have identified a potential activating component of Tim3 and linkage between Tim3 and Lag3 signaling upon blocking the Tim3 axis during T-cell-antigen-presenting cell interactions that should be considered when targeting immune checkpoints for clinical use.

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic.

The PD-L1/4-1BB Bispecific Antibody-Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner.

While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.

Neoantigens as potential vaccines in hepatocellular carcinoma

BackgroundNeoantigens, new immunogenic sequences arising from tumor mutations, have been associated with response to immunotherapy and are considered potential targets for vaccination. Hepatocellular carcinoma (HCC) is a moderately mutated tumor,...

Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1β in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Fc-null anti-PD-1 monoclonal antibodies deliver optimal checkpoint blockade in diverse immune environments

BackgroundDespite extensive clinical use, the mechanisms that lead to therapeutic resistance to anti-programmed cell-death (PD)-1 monoclonal antibodies (mAbs) remain elusive. Here, we sought to determine how interactions between the Fc...

Functional condition of the sensitized T-lymphocytes according to RBTL in children with non-rheumatic myocarditis

Summary. The increased proportion of non-rheumatic myocarditis in children has recently determined the urgency of this problem in pediatrics. It is known that non-rheumatic myocarditis in children often occurs against the background of viral or bacterial infection under conditions of altered reactivity of the immune system. There is evidence, mainly in rheumatism, that spontaneous and stimulated lymphocyte blatransformation response in such patients reflects the activity of the process, its tendency to chronicity and may serve as a criterion for the adequacy of therapy. Studies on the functional status of T lymphocytes in patients with non-rheumatic myocarditis are mainly related to experimental models. This determines the relevance of this work. The aim was to compare the functional activity of T-lymphocytes in spontaneous and phytohemagglutenin-stimulated lymphocyte blastranformation reactions in children with non-rheumatic myocarditis in acute and chronic course. Materials and methods. Under observation were 42 children aged 4 to 13 years with non-rheumatic myocarditis. A study was made of the functional activity of T-lymphocytes in the reaction of blast transformation using a radioisotope technique with thymidine-3H. Phytohemagglutenin from Reanal was used as a nonspecific stimulant. The reaction was evaluated on an SBS-2 automatic scintillation counter. The functional activity of T-lymphocytes was studied upon admission of children to the cardiology department and after the treatment. As a result of the study of the functional state of T-lymphocytes in the blast transformation reaction using the thymidine-3H radioisotope label in 52 children with acute and chronic non-rheumatic myocarditis, it was shown that during the period of advanced clinical manifestations, high spontaneous stimulation to the nonspecific phytohemagglutenin stimulator was recorded. The complex of generally accepted therapeutic agents leads to the restoration of the functional state of T-lymphocytes in children with an acute course of non-rheumatic myocarditis, and in chronic, despite the positive clinical dynamics, increased spontaneous stimulation of T-lymphocytes is still recorded, and the response to phytohemagglutenin is not fully restored in them. These data make it possible to recommend indicators of the functional activity of T-lymphocytes as criteria for recovery and determining the timing of treatment. Conclusions. The criterion for recovery and determining the duration of treatment of children with non-rheumatic myocarditis should be not only clinical and electrophysiological indicators, but also indicators of functional activity of T-lymphocytes, reflecting the sensitization of the body

Myelin Peptide-Mannan Conjugate Multiple Sclerosis Vaccines: Conjugation Efficacy and Stability of Vaccine Ingredient.

Myelin peptide–mannan conjugates have been shown to be potential vaccines in the immunotherapy of multiple sclerosis. The conjugates are comprised from the epitope peptide and the polysaccharide mannan which transfers as a carrier the antigenic peptide to dendritic cells that process and present antigenic peptides at their surface in complex with MHC class I or class II resulting in T-cell stimulation. The conjugation of antigenic peptide with mannan occurs through the linker (Lys–Gly)5, which connects the peptide with the oxidized mannose units of mannan. This study describes novel methods for the quantification of the vaccine ingredient peptide within the conjugate, a prerequisite for approval of clinical trials in the pursuit of multiple sclerosis therapeutics. Myelin peptides, such as MOG35–55, MBP83–99, and PLP131–145 in linear or cyclic form, as altered peptide ligands or conjugated to appropriate carriers, possess immunomodulatory properties in experimental models and are potential candidates for clinical trials.

Distinct exhaustion features of T lymphocytes shape the tumor-immune microenvironment with therapeutic implication in patients with non-small-cell lung cancer

BackgroundReinvigoration of T-cell exhaustion with antibodies has shown promising efficacy in patients with non-small-cell lung cancer (NSCLC). However, the characteristics of T-cell exhaustion with regard to tumor-infiltrating lymphocytes (TILs) are...

IMMU-52. EFFECTS OF IMMUNE CHECKPOINT BLOCKADE ON ANTIGEN-SPECIFIC CD8+ T CELLS FOR USE IN ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive T-cell therapies have been successfully used as treatment for patients diagnosed with advanced cancers. Unfortunately, for some refractory cancers, they have failed. To overcome this, checkpoint inhibitors have shown to rescue immune anti-tumor responses. We hypothesized that in-vitro checkpoint blockade during T-cell stimulation and expansion with RNA-pulsed dendritic cells may enhance the activity of antigen-specific T-cells and improve the efficacy of ACT platforms. METHODS Human PBMCs were isolated from CMV seropositive donors to generate DCs and pulsed them with CMVpp65-mRNA to educate T-cells in co-culture for 15-days. We targeted pp65 antigen which is ubiquitously expressed by glioblastoma cells. Three checkpoint blockade conditions were evaluated (anti-PD1, anti-Tim3 and anti-PD1+Tim3). IL2 was added every 3 days as well as the blockade antibodies. Immunephenotyping was performed on Day-0 and Day-15. Polyfunctional antigen specific responses were evaluated upon rechallenge with CMVpp65 peptides. RESULTS CMVpp65 activated CD8+ T-cells upregulate Lag3 and Tim3 (p= < 0.0001). Tim3 blockade alone or in combination led to a significant upregulation of Lag3 expression on CD8+pp65Tetramer+ central memory, effector memory, and TEMRA T-cells. This latter T-cell subset uniquely maintain double-positive Tim3/Lag3 expression after blockade. In contrast, PD-1 blockade had minimal effects on Tim3 or Lag3 expression. In addition, IFN-g secretion was reduced in T-cells treated with Tim3 blockade in a dose-dependent manner (p= 0.004). CONCLUSION In this study, we have identified a potential activating component of Tim3 and linkage between Tim3 and Lag3 signaling upon blocking Tim3 axis during T-cell antigen presenting cell interactions that should be considered when targeting immune checkpoints for clinical use.

Antigen multimers: Specific, sensitive, precise, and multifunctional high-avidity CAR-staining reagents

Although chimeric antigen receptor (CAR) T-cell therapy has transformed cancer treatment, high-quality and universal CAR-staining reagents are urgently required to manufacture CAR T cells, predict therapy response, decipher CAR biology, and engineer new CARs. Here, we developed tetrameric and dodecameric forms of a multifunctional and extensible category of high-avidity CAR-staining reagents: antigen-multimers. Antigen-multimers detected CARs against CD19, HER2, and Tn-glycoside with significantly higher specificity, sensitivity, and precision than existing reagents. In addition to accurate CAR T-cell detection by flow cytometry, antigen-multimers also enabled ≥100-fold magnetic enrichment of rare CAR T cells, selective CAR T-cell stimulation, and high-dimensional CAR T-cell profiling by single-cell multi-omics analyses. Finally, antigen-multimers accurately captured clinical anti-CD19 CAR T cells from patients' cellular infusion products, post-infusion peripheral blood, and tumor biopsies. Antigen-multimers can be readily extended to other CAR systems by switching its antigen ligand. As such, antigen-multimers have broad clinical and research applications.

Transcriptional Immunoediting of AML Cells after Allogeneic Hematopoietic Stem Cell Transplantation

Relapse of AML after allogeneic hematopoietic stem cell transplantation (alloSCT) has been linked to reduced expression of major histocompatibility complex type II proteins (MHC-II), which appears to have a regulatory basis. To understand the mechanism of immune evasion in AML, we developed CORENODE, a computational algorithm for genome-wide transcription network decomposition. CORENODE identified the transcription factors (TFs) IRF8 and MEF2C as positive regulators and MYB and MEIS1 as negative regulators of the MHC-II expression in AML. We validated this prediction using several approaches. First, inactivation of IRF8 and MEF2C in AML cell lines by CRISPR-Cas9 resulted in decreased MHC-II expression, while inactivation of MYB and MEIS1 led to MHC-II induction. Second, decreased levels of IRF8 and MEF2C, and increased levels of MYB and MEIS1 were seen in patients with AML relapse after alloSCT, correlating with reduced expression of MHC-II. Finally, MHC-II expression in various patient-derived datasets could be accurately predicted by CORENODE from the combinatorial levels of the 4 TFs. Although immune evasion in AML has been linked to the transcriptional coactivator CIITA, CORENODE predicted that members of the TF tetrad regulate MHC-II genes independently from CIITA. Indeed, inactivation of MYB and MEIS1 in a CIITA knockout AML cell line was able to cause MHC-II induction. Beyond MHC-II, CORENODE identified a module of 58 genes that were up-regulated by IRF8 and down-regulated by MYB. The module was highly enriched for genes participating in graft-versus-leukemia (GvL) reactivity and we found it to be downregulated in 5 of 6 patients with a post-alloSCT AML relapse. Thus, the post-alloSCT changes in MHC-II expression are part of a broader transcriptional program of GvL response in AML, antagonistically regulated by MYB and IRF8. However, reduced expression of MHC-II and other GvL response genes at relapse does not correlate with the myeloid differentiation state, indicating that it is regulated independently. To understand the clonal basis of MHC-II expression loss at the time of relapse, we analyzed MHC-II expression at the single-cell level. Analysis of previously published paired scRNA data at the time of initial diagnosis and after alloSCT demonstrated a small number of cells with reduced MHC-II expression driven by altered MYB and IRF8 levels present at the time of initial diagnosis. These MHC-II-low cells appear to be selected by the immune pressure, resulting in eventual relapse. In summary, our findings reveal an adaptive transcriptional mechanism of AML immunoediting after allogenic transplantation whereby combinatorial fluctuations of TF levels under immune pressure result in selection of cells with a silenced T-cell stimulation program. [Display omitted] DisclosuresStegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding.

Treatment-Free Intervals during CD19xCD3 BiTE ® Construct-Mediated T-Cell Stimulation Induce Functional Reinvigoration and Transcriptional Reprogramming of Exhausted T Cells

Treatment-Free Intervals during CD19xCD3 BiTE ® Construct-Mediated T-Cell Stimulation Induce Functional Reinvigoration and Transcriptional Reprogramming of Exhausted T Cells

Axicabtagene Ciloleucel in Relapsed or Refractory Large B-Cell Lymphoma Patients in Complete Metabolic Response at Time of Infusion

Axicabtagene Ciloleucel in Relapsed or Refractory Large B-Cell Lymphoma Patients in Complete Metabolic Response at Time of Infusion

Dual-Antigen COVID-19 Vaccine Subcutaneous Prime Delivery With Oral Boosts Protects NHP Against SARS-CoV-2 Challenge.

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

Critically Ill Coronavirus Disease 2019 Patients Exhibit Hyperactive Cytokine Responses Associated With Effector Exhausted Senescent T Cells in Acute Infection.

COVID-19 can progress to severe pneumonia with respiratory failure and is aggravated by the deregulation of the immune system causing an excessive inflammation including the cytokine storm. We herein report that severe acutely infected patients have high levels of both type-1 and type-2 cytokines. Our results show abnormal cytokine levels upon T cell stimulation, in a non-polarized profile. Furthermore, our findings indicate that this hyperactive cytokine response is associated with a significantly increased frequency of late-differentiated T cells with particular phenotype of effector exhausted/senescent CD28 -CD57 + cells. Interestingly, we demonstrated for the first time an increased frequency of CD3 +CD4 +CD28 -CD57 + T cells with expression of programmed death 1 (PD-1), one of the hallmarks of T cell exhaustion. These findings reveal that COVID-19 is associated with acute immunodeficiency, especially within the CD4 + T cell compartment and points to possible mechanisms of loss of clonal repertoire and susceptibility to viral relapse and reinfection events.

Exhausted PD-1hi T-Cells Are Enriched in Secondary Lymphoid Organs of CLL Patients

Chronic lymphocytic leukemia (CLL) is associated with increased numbers of T-cells that exhibit substantial alteration in composition and function. But their role in CLL pathogenesis remains largely elusive. On the one side, T-cells provide proliferation and pro-survival signals to CLL cells, but on the other side, they show features of activation and exhaustion, including overexpression of inhibitory molecules, like PD-1, suggesting their involvement in anti-tumoral activity. Since CLL T-cells retain the ability to produce cytokines and cytotoxic molecules, they were termed pseudo-exhausted. Results of previous studies were mostly based on blood samples of CLL patients, allowing only limited conclusions as interactions of immune cells and CLL cells mainly occur in lymphoid organs.In the present study, we aimed at deciphering differences in phenotype, differentiation and functional status of T-cells in paired peripheral blood (PB) and lymph node (LN) samples of CLL patients, particularly focusing on T-cell exhaustion. We applied 12-color flow cytometry and ex vivo stimulation of primary CLL T-cells followed by intracellular cytokine and cytotoxic molecule detection. In addition, we quantified transcription factors that are known to regulate T-cell differentiation by intracellular flow cytometry.In line with published data, we detected an increased proportion of CD45RO- CCR7- CD8+ effector T-cells as well as increased numbers of PD-1+ CD8+ T-cells in PB of CLL patients (n=22), compared to age-matched healthy individuals (n=19), and a positive correlation of these numbers with disease load. Despite of their enhanced expression of PD-1 and other inhibitory receptors, PB CD8+ T-cells from CLL patients exhibited increased production of granzyme B (GzmB), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in comparison to cells from age-matched healthy donors, indicating disease-associated activation of PB CD8+ T-cells with intact functional capacity.Subsequently, we compared CD8+ T-cells from paired LN and PB samples of CLL patients (n=7). This analysis revealed that LNs were enriched in CD45RO+ CCR7- effector memory T-cells (TEM cells) and contained significantly smaller numbers of terminally differentiated effector memory cells re-expressing CD45RA (TEMRA cells). Moreover, LN CD8+ T-cells strongly expressed the activation marker CD69, yet presented with higher levels of PD-1 compared to PB CD8+ T-cells. In line with high PD-1 expression, LN CD8+ T-cells exhibited a significantly lower production of IFNγ and TNFα, and a substantially reduced cytotoxic capability as indicated by lower GzmB production compared to PB T-cells. TEM cells in CLL patients' LNs exhibited different levels of PD-1 expression, and we observed a significantly higher percentage of cells with high PD-1 expression (PD-1hi) in LNs compared to PB, which was mainly composed of cells with intermediate PD-1 expression (PD-1int), (Figure 1). Comparison of PD-1hi and PD-1int subsets in LNs showed that PD-1hi cells expressed higher levels of CD69 compared to PD-1int cells, but were substantially inferior in GzmB production. Furthermore, PD-1hi cells expressed significantly higher levels of EOMES, a transcription factor that is known to drive terminal differentiation and exhaustion of T-cells in chronic infections. In contrast, PD-1int cells showed higher expression of the transcription factors T-BET and TCF7, which mediate long-term endurance of T-cells in chronic situations.In summary, this study shows that exhausted T-cells are enriched in LNs rather than PB of CLL patients. Our data suggests that the increased leukemia-associated activation of CD8+ T-cells in patients' LNs drives the accumulation of PD-1hi exhausted T-cells. A better understanding of these processes will be essential for the development of novel immunotherapeutic approaches for CLL.Figure 1:(A) CD8+ T-cells from paired peripheral blood (PB) and lymph node (LN) samples of CLL patients (n=7) were analyzed by flow cytometry for the expression of PD-1 (representative of seven samples). (B) The relative abundance of PD-1low, PD-1int and PD-1hi subsets within the CD45RO+ CCR7- CD8+ effector memory (EM) population in PB and LN was quantified. [Display omitted] DisclosuresStilgenbauer:AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding.