A CD64/FcγRI-mediated mechanism hijacks PD-1 from PD-L1/2 interaction and enhances anti-PD-1 functional recovery of exhausted T cells.

Therapeutic monoclonal antibodies (mAb) targeting the immune checkpoint inhibitor programmed cell death protein 1 (PD-1) have achieved considerable clinical success in anti-cancer therapy through relieving T cell exhaustion. Blockade of PD-1 interaction with its ligands PD-L1 and PD-L2 is an important determinant in promoting the functional recovery of exhausted T cells. Here, we show that anti-PD-1 mAbs act through an alternative mechanism leading to the downregulation of PD-1 surface expression on memory CD4+ and CD8+ T cells. PD-1 receptor downregulation is a distinct process from receptor endocytosis and occurs in a CD14+ monocyte dependent manner with the CD64/Fcγ receptor I acting as the primary factor for this T cell extrinsic process. Importantly, downregulation of surface PD-1 strongly enhances antigen-specific functional recovery of exhausted PD-1+CD8+ T cells. Our study demonstrates a novel mechanism for reducing cell surface levels of PD-1 and limiting the inhibitory targeting by PD-L1/2 and thereby enhancing the efficacy of anti-PD-1 Ab in restoring T cell functionality.

Intradermal skin test with mRNA vaccines as a surrogate marker of T cell immunity in immunocompromised patients.

OBJECTIVES: Intradermal skin test (IDT) with mRNA vaccines may represent a simple, reliable, and affordable tool to measure T cell response in immunocompromised patients who failed to mount serological responses following vaccination with mRNA covid-19 vaccines. METHODS: We compared anti-SARS-CoV-2 antibodies and cellular responses in vaccinated immunocompromised patients (n = 58), healthy seronegative naive controls (NC, n = 8), and healthy seropositive vaccinated controls (VC, n = 32) by Luminex, spike-induced IFN-γ Elispot and an IDT. A skin biopsy 24 h after IDT and single-cell RNAseq was performed in three vaccinated volunteers. RESULTS: Twenty-five percent of seronegative NC had a positive Elispot (2/8) and IDT (1/4), compared to 95% (20/21) and 93% (28/30) in seropositive VC, respectively. Single-cell RNAseq data in the skin of VC showed a predominant mixed population of effector helper and cytotoxic T cells. The TCR repertoire revealed 18/1064 clonotypes with known specificities against SARS-CoV-2, among which six were spike-specific. Seronegative immunocompromised patients with positive Elispot and IDT were in 83% (5/6) treated with B cell-depleting reagents, while those with negative IDT were all transplant recipients. CONCLUSIONS: Our results indicate that delayed local reaction to IDT reflects vaccine-induced T-cell immunity opening new perspectives to monitor seronegative patients and elderly populations with waning immunity.

Cytokine storm complicated by cardiogenic shock induced by anti-HER2 therapies.

Cytokine storm induced by anti-human epidermal growth factor receptor-2 (HER2) therapies has not been reported. We report a patient with breast cancer treated with trastuzumab/pertuzumab who developed severe biventricular dysfunction and cardiogenic shock (CS) 6 months after starting double anti-HER2 therapy. The CS was accompanied by severe systemic inflammation, and cardiac MRI (cMRI) showed structural changes typical of myocardial inflammation. The immuno-inflammatory profile showed significantly increased levels of activation of the complement system, proinflammatory cytokines (IL-1ß, IL-6, IL-18, IL-17A, TNF-alpha) with increased activity of classical monocytic, T helper 17 cells (Th17), CD4 T and effector memory CD8 T subsets, whereas NK cell activation was not observed. The data suggest an important role for monocytes as initiators of this FcγR-dependent antibody-dependent cytotoxicity, leading to the overactivation of an adaptive T cell response, in which Th17 cells may act in synergy with T helper 1 cells (Th1) to drive the severe cytokine release syndrome. After discontinuation of trastuzumab/pertuzumab, hypercytokinemia and complement activity normalized along with clinical recovery. Cardiac function returned to baseline within 2 months of initial presentation, together with a resolution of the myocardial inflammation on MRI.

Neutrophil Activation and Immune Thrombosis Profiles Persist in Convalescent COVID-19.

PURPOSE: Following a severe COVID-19 infection, a proportion of individuals develop prolonged symptoms. We investigated the immunological dysfunction that underlies the persistence of symptoms months after the resolution of acute COVID-19. METHODS: We analyzed cytokines, cell phenotypes, SARS-CoV-2 spike-specific and neutralizing antibodies, and whole blood gene expression profiles in convalescent severe COVID-19 patients 1, 3, and 6 months following hospital discharge. RESULTS: We observed persistent abnormalities until month 6 marked by (i) high serum levels of monocyte/macrophage and endothelial activation markers, chemotaxis, and hematopoietic cytokines; (ii) a high frequency of central memory CD4+ and effector CD8+ T cells; (iii) a decrease in anti-SARS-CoV-2 spike and neutralizing antibodies; and (iv) an upregulation of genes related to platelet, neutrophil activation, erythrocytes, myeloid cell differentiation, and RUNX1 signaling. We identified a "core gene signature" associated with a history of thrombotic events, with upregulation of a set of genes involved in neutrophil activation, platelet, hematopoiesis, and blood coagulation. CONCLUSION: The lack of restoration of gene expression to a normal profile after up to 6 months of follow-up, even in asymptomatic patients who experienced severe COVID-19, signals the need to carefully extend their clinical follow-up and propose preventive measures.

Ocrelizumab Impairs the Phenotype and Function of Memory CD8+ T Cells: A 1-Year Longitudinal Study in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: Depleting CD20+ B cells is the primary mechanism by which ocrelizumab (OCRE) is efficient in persons with multiple sclerosis (pwMS). However, the exact role of OCRE on other immune cell subsets directly or indirectly remains elusive. The purpose of this study is to characterize the dynamics of peripheral immune cells of pwMS on OCRE. METHODS: We collected blood samples from 38 pwMS before OCRE onset (T0) and at 6 and 12 months (T6, T12) after initiation. To cover the immune cell diversity, using mass cytometry time of flight, we designed a 38-parameter panel to analyze B, T, and innate immune cell markers and CNS migratory markers. In parallel, viral-specific CD8+ T-cell responses were assessed by the quantification of interferon-γ secretion using the enzyme-linked immunospot assay on cytomegalovirus, Epstein-Barr virus, and influenza stimulations. RESULTS: Beside B-cell depletion, we observed a loss in memory CD8+CD20+ and central memory CD8+ T cells but not in CD4+CD20+ T cells already at T6 and T12 (p < 0.001). The loss of memory CD8+ T cells correlated with a lower CXCR3 expression (p < 0.001) and CNS-related LFA-1 integrin expression (p < 0.001) as well as a reduced antiviral cellular immune response observed at both time points (p < 0.001). Of note, we did not observe major changes in the phenotype of the other cell types studied. Seven of 38 (18.4%) patients in our cohort presented with infections while on OCRE; 4 of which were switched from dimethyl fumarate. Finally, using a mixed linear model on mass cytometry data, we demonstrated that the immunomodulation induced by previous disease-modifying therapies (DMTs) was prolonged over the period of the study. DISCUSSION: In addition to its well-known role on B cells, our data suggest that OCRE also acts on CD8+ T cells by depleting the memory compartment. These changes in CD8+ T cells may be an asset in the action of OCRE on MS course but might also contribute to explain the increased occurrence of infections in these patients. Finally, although more data are needed to confirm this observation, it suggests that clinicians should pay a special attention to an increased infection risk in pwMS switched from other DMTs to OCRE.

Patient-derived monoclonal antibody neutralizes SARS-CoV-2 Omicron variants and confers full protection in monkeys.

The SARS-CoV-2 Omicron variant has very high levels of transmission, is resistant to neutralization by authorized therapeutic human monoclonal antibodies (mAb) and is less sensitive to vaccine-mediated immunity. To provide additional therapies against Omicron, we isolated a mAb named P2G3 from a previously infected vaccinated donor and showed that it has picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other variants tested. We solved the structure of P2G3 Fab in complex with the Omicron spike using cryo-electron microscopy at 3.04 Å resolution to identify the P2G3 epitope as a Class 3 mAb that is different from mAb-binding spike epitopes reported previously. Using a SARS-CoV-2 Omicron monkey challenge model, we show that P2G3 alone, or in combination with P5C3 (a broadly active Class 1 mAb previously identified), confers complete prophylactic or therapeutic protection. Although we could select for SARS-CoV-2 mutants escaping neutralization by P2G3 or by P5C3 in vitro, they had low infectivity and 'escape' mutations are extremely rare in public sequence databases. We conclude that this combination of mAbs has potential as an anti-Omicron drug.

Humoral Responses Against Variants of Concern by COVID-19 mRNA Vaccines in Immunocompromised Patients.

Importance: There are limited comparative data on the durability of neutralizing antibody (nAb) responses elicited by messenger RNA (mRNA) vaccines against the SARS-CoV-2 variants of concern (VOCs) in immunocompromised patients and healthy controls. Objective: To assess the humoral responses after vaccination with BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccines. Design, Setting, and Participants: In this prospective, longitudinal monocentric comparative effectiveness study conducted at the Lausanne University Hospital, binding IgG anti-spike antibody and nAb levels were measured at 1 week, 1 month, 3 months, and 6 months after vaccination with mRNA-1273 (24.6% of participants) or BNT162b2 (75.3% of participants). Interventions: All participants received 2 doses of either mRNA-1273 or BNT162b2 vaccines 4 to 6 weeks apart. Main Outcomes and Measures: The primary outcome of the study was the persistence of nAb responses against the original, nonvariant SARS-CoV-2 (2019-nCoV) and different VOCs at 6 months after vaccination. Key secondary outcomes were associations of the type of mRNA vaccine, the underlying disease, and the treatment with the response to vaccination. Results: Among the 841 participants enrolled between January 14 and August 8, 2021, the patient population comprised 637 participants (mean [SD] age, 61.8 [13.7] years; 386 [60.6%] female), and the healthy control population comprised 204 participants (mean [SD] age, 45.9 [12.0] years; 144 [70.6%] female). There were 399 patients with solid cancers, 101 with hematologic cancers, 38 with solid organ transplants, 99 with autoimmune diseases, and 204 healthy controls. More than 15 000 nAb determinations were performed against the original, nonvariant 2019-nCoV and the Alpha, Beta, Gamma, and Delta variants. The proportions of nAbs and their titers decreased in all study groups at 6 months after vaccination, with the greatest decreases for the Beta and Delta variants. For Beta, the proportion decreased to a median (SE) of 39.2% (5.5%) in those with hematologic cancers, 44.8% (2.7%) in those with solid cancers, 23.1% (8.3%) in those with solid organ transplants, and 22.7% (4.8%) in those with autoimmune diseases compared with 52.1% (4.2%) in healthy controls. For Delta, the proportions decreased to 41.8% (5.6%) in participants with hematologic cancer, 51.9% (2.7%) in those with solid cancers, 26.9% (8.7%) in those with solid organ transplants, and 30.7% (5.3%) in those with autoimmune diseases compared with 56.9% (4.1%) healthy controls. Neutralizing antibody titers decreased 3.5- to 5-fold between month 1 and month 6, and the estimated duration of response was greater and more durable among those participants vaccinated with mRNA-1273. In participants with solid cancers, the estimated duration of nAbs against the Beta variant was 221 days with mRNA-1273 and 146 days with BNT162b2, and against the Delta variant, it was 226 days with mRNA-1273 and 161 with BNT162b2. The estimated duration of nAbs in participants with hematologic cancers was 113 and 127 days against Beta and Delta variants, respectively. Conclusions and Relevance: This comparative effectiveness study suggests that approximately half of patients with hematologic cancers and solid cancers, about 70% of patients with solid organ transplants or autoimmune diseases, and 40% of healthy controls have lost nAbs against the circulating VOCs at 6 months after vaccination. These findings may be helpful for developing the best boosting vaccination schedule especially in immunocompromised patients.

68Ga-DOTATOC PET/CT to detect immune checkpoint inhibitor-related myocarditis.

BACKGROUND: Immune checkpoint inhibitor (ICI)-related myocarditis is a rare but potentially fatal adverse event that can occur following ICI exposure. Early diagnosis and treatment are key to improve patient outcomes. Somatostatin receptor-based positron emission tomography-CT (PET/CT) showed promising results for the assessment of myocardial inflammation, yet information regarding its value for the diagnosis of ICI-related myocarditis, especially at the early stage, is limited. Thus, we investigated the value of 68Ga-DOTA(0)-Phe(1)-Tyr(3)-octreotide (68Ga-DOTATOC) PET/CT for the early detection and diagnosis of ICI-related myocarditis. METHODS: Consecutive patients with clinically suspected ICI-related myocarditis from July 2018 to February 2021 were retrospectively evaluated in this single-center study. All patients underwent imaging for the detection of ICI-related myocarditis using either cardiac magnetic resonance (CMR) imaging or 68Ga-DOTATOC PET/CT. PET/CT images were acquired 90 min after the injection of 2 MBq/kg 68Ga-DOTATOC with pathological myocardial uptake in the left ventricle (LV) suggestive of myocarditis defined using a myocardium-to-background ratio of peak standard uptake value to mean intracavitary LV standard uptake (MBRpeak) value above 1.6. Patients had a full cardiological work-up including ECG, echocardiography, serum cardiac troponin I (cTnI), cardiac troponin T and creatine kinase (CK), CK-MB. Endomyocardial biopsy and inflammatory cytokine markers were also analyzed. The detection rate of ICI-related myocarditis using 68Ga-DOTATOC PET/CT and CMR was assessed. RESULTS: A total of 11 patients had clinically suspected ICI-related myocarditis; 9 underwent 68Ga -DOTATOC PET/CT. All nine (100%) patients with 68Ga-DOTATOC PET/CT presented with pathological myocardial uptake in the LV that was suggestive of myocarditis (MBRpeak of 3.2±0.8, range 2.2-4.4). Eight patients had CMR imaging and 3/8 (38%) patients had lesions evocative of myocarditis. All PET-positive patients were previously treated with a high dose of steroids and intravenous immunoglobulin prior to PET/CT had elevated serum cTnI except for one patient for whom PET/CT was delayed several days. Interestingly, in 5/6 (83%) patients who presented with concomitant myositis, pathological uptake was seen on whole-body 68Ga-DOTATOC PET/CT images in the skeletal muscles, suggesting an additional advantage of this method to assess the full extent of the disease. In contrast, four patients with CMR imaging had negative findings despite having elevated serum cTnI levels (range 20.5-5896.1 ng/mL), thus defining possible myocarditis. Newly identified immune correlates could provide specific biomarkers for the diagnosis of ICI-related myocarditis. Most tested patients (six of seven patients) had serum increases in the inflammatory cytokine interleukin (IL)-6 and in the chemokines CXCL9, CXCL10, and CXCL13, and the mass cytometry phenotypes of immune cell populations in the blood also showed correlations with myocardial inflammation. Four of five patients with myocarditis exhibited a Th1/Th2 imbalance favoring a pronounced inflammatory Th1, Th1/Th17, and Th17 CD4 memory T-cell response. The high proportion of non-classical monocytes and significantly reduced levels of CD31 in four to five patients was also consistent with an inflammatory disease. CONCLUSION: The use of 68Ga-DOTATOC PET/CT along with immune correlates is a highly sensitive method to detect ICI-related myocarditis especially in the early stage of myocardial inflammation, as patients with elevated cTnI may present normal CMR imaging results. 68Ga-DOTATOC PET/CT is also useful for detecting concomitant myositis. These results need to be confirmed in a larger population of patients and validated against a histological gold standard if available.

Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques.

Achieving sufficient worldwide vaccination coverage against SARS-CoV-2 will require additional approaches to currently approved viral vector and mRNA vaccines. Subunit vaccines may have distinct advantages when immunizing vulnerable individuals, children and pregnant women. Here, we present a new generation of subunit vaccines targeting viral antigens to CD40-expressing antigen-presenting cells. We demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with long-term memory phenotypes, in a humanized mouse model. Additionally, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Vaccine-elicited antibodies cross-neutralize different SARS-CoV-2 variants, including D614G, B1.1.7 and to a lesser extent B1.351. Such vaccination significantly improves protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals.

Case Report: Stepwise Anti-Inflammatory and Anti-SARS-CoV-2 Effects Following Convalescent Plasma Therapy With Full Clinical Recovery.

In these times of COVID-19 pandemic, concern has been raised about the potential effects of SARS-CoV-2 infection on immunocompromised patients, particularly on those receiving B-cell depleting agents and having therefore a severely depressed humoral response. Convalescent plasma can be a therapeutic option for these patients. Understanding the underlying mechanisms of convalescent plasma is crucial to optimize such therapeutic approach. Here, we describe a COVID-19 patient who was deeply immunosuppressed following rituximab (anti-CD20 monoclonal antibody) and concomitant chemotherapy for chronic lymphoid leukemia. His long-term severe T and B cell lymphopenia allowed to evaluate the treatment effects of convalescent plasma. Therapeutic outcome was monitored at the clinical, biological and radiological level. Moreover, anti-SARS-CoV-2 antibody titers (IgM, IgG and IgA) and neutralizing activity were assessed over time before and after plasma transfusions, alongside to SARS-CoV-2 RNA quantification and virus isolation from the upper respiratory tract. Already after the first cycle of plasma transfusion, the patient experienced rapid improvement of pneumonia, inflammation and blood cell counts, which may be related to the immunomodulatory properties of plasma. Subsequently, the cumulative increase in anti-SARS-CoV-2 neutralizing antibodies due to the three additional plasma transfusions was associated with progressive and finally complete viral clearance, resulting in full clinical recovery. In this case-report, administration of convalescent plasma revealed a stepwise effect with an initial and rapid anti-inflammatory activity followed by the progressive SARS-CoV-2 clearance. These data have potential implications for a more extended use of convalescent plasma and future monoclonal antibodies in the treatment of immunosuppressed COVID-19 patients.

Personalized Cytokine-Directed Therapy With Tocilizumab for Refractory Immune Checkpoint Inhibitor-Related Cholangiohepatitis.

INTRODUCTION: For patients with corticosteroid (CS)-refractory immune checkpoint inhibitor-related cholangiohepatitis (irCH), no consensus exists regarding treatment, and outcomes remain poor. We evaluated the possibility of personalized treatment according to the patient's cytokine profile and the immunohistopathologic assessment of the predominant immune infiltrate type of liver tissue. METHODS: NSCLCs with CS-refractory irCH were analyzed by immunohistochemistry of liver biopsy specimen, serum cytokine panel, and assessment of peripheral blood mononuclear cell immune cell monitoring by mass cytometry. RESULTS: A total of three consecutive patients with irCH were identified. We found a predominant T-cell infiltrate and an interferon-gamma or T helper 1 proinflammatory cytokine profile. Here, we report for the first time that a T-cell-targeted therapy with the interleukin (IL)-6 receptor-neutralizing antibody tocilizumab, which inhibits signaling downstream of interferon-gamma and several other Janus kinase-dependent cytokines, is an effective single cytokine-directed therapy for CS-refractory irCH. Three patients with severe, CS-refractory irCH who were treated with tocilizumab were found to have persistent clinical and biological remission. CONCLUSIONS: Dysregulation of the IL-6/T-cell axis may contribute to the pathogenesis of CS-refractory irCH. Our observations suggest that IL-6 blockade seems to have promise in the treatment of CS-refractory irCH. The results from our three patients need to be confirmed in a larger patient population.

BCG Vaccination Induces Long-Term Functional Reprogramming of Human Neutrophils.

The tuberculosis vaccine bacillus Calmette-Guérin (BCG) protects against some heterologous infections, probably via induction of non-specific innate immune memory in monocytes and natural killer (NK) cells, a process known as trained immunity. Recent studies have revealed that the induction of trained immunity is associated with a bias toward granulopoiesis in bone marrow hematopoietic progenitor cells, but it is unknown whether BCG vaccination also leads to functional reprogramming of mature neutrophils. Here, we show that BCG vaccination of healthy humans induces long-lasting changes in neutrophil phenotype, characterized by increased expression of activation markers and antimicrobial function. The enhanced function of human neutrophils persists for at least 3 months after vaccination and is associated with genome-wide epigenetic modifications in trimethylation at histone 3 lysine 4. Functional reprogramming of neutrophils by the induction of trained immunity might offer novel therapeutic strategies in clinical conditions that could benefit from modulation of neutrophil effector function.

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice.

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG+ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS+ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.

In Situ Characterization of Follicular Helper CD4 T Cells Using Multiplexed Imaging.

Follicular helper CD4 T (Tfh) cells play an essential role in the formation of germinal centers (GCs), where mature B cells proliferate, differentiate, and provide long-term protective humoral responses. Despite the extensive phenotypic characterization and identification of human Tfh cell subsets, their spatial positioning at tissue level is not well understood. Here, we describe a quantitative multiplexed immunofluorescence approach allowing for the comprehensive in situ characterization of Tfh cells in human tonsils and lymph nodes (LNs) from individuals with angioimmunoblastic T-cell lymphoma (AITL). We have developed eight multiplexed panels comprising a spectrum of Tfh cell markers, like PD-1, CXCR5, and ICOS, along with transcription factors (Bcl6, Tbet, GATA3), to assess their expression, frequencies, spatial distribution and co-localization in a quantitative manner. Combined analysis of relevant markers revealed the presence of several Tfh cell subsets at tissue level based on the differential expression of surface receptors, nuclear factors as well as their distinct localization within the follicular areas. Interestingly, we found a considerable amount of tonsillar Tfh cells expressing high levels of the Th2 regulator GATA3. The co-expression of GATA3, CXCR5, and BCL6, points to an important role of GATA3 for the generation of effector human Tfh cells. Furthermore, our data revealed significantly different Tfh cell profile signatures between health and disease. Therefore, our imaging platform generates meaningful information for the in situ characterization of human Tfh cells and could provide the base for future studies aiming to a comprehensive understanding of Tfh cell tissue heterogeneity.

T-cell exhaustion in HIV infection.

The T-cell response is central in the adaptive immune-mediated elimination of pathogen-infected and/or cancer cells. This activated T-cell response can inflict an overwhelming degree of damage to the targeted cells, which in most instances leads to the control and elimination of foreign invaders. However, in conditions of chronic infection, persistent exposure of T cells to high levels of antigen results in a severe T-cell dysfunctional state called exhaustion. T-cell exhaustion leads to a suboptimal immune-mediated control of multiple viral infections including the human immunodeficiency virus (HIV). In this review, we will discuss the role of T-cell exhaustion in HIV disease progression, the long-term defect of T-cell function even in aviremic patients on antiretroviral therapy (ART), the role of exhaustion-specific markers in maintaining a reservoir of latently infected cells, and exploiting these markers in HIV cure strategies.

Development of a novel human phage display-derived anti-LAG3 scFv antibody targeting CD8+ T lymphocyte exhaustion.

BACKGROUND: Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. RESULTS: We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γ release by both ELISA and ELISPOT assays. CONCLUSIONS: Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment.

Tumor suppression of novel anti-PD-1 antibodies mediated through CD28 costimulatory pathway.

Classical antagonistic antibodies (Abs) targeting PD-1, such as pembrolizumab and nivolumab, act through blockade of the PD-1-PDL-1 interaction. Here, we have identified novel antagonistic anti-PD-1 Abs not blocking the PD-1-PDL-1 interaction. The nonblocking Abs recognize epitopes on PD-1 located on the opposing face of the PDL-1 interaction and overlap with a newly identified evolutionarily conserved patch. These nonblocking Abs act predominantly through the CD28 coreceptor. Importantly, a combination of blocking and nonblocking Abs synergize in the functional recovery of antigen-specific exhausted CD8 T cells. Interestingly, nonblocking anti-PD-1 Abs have equivalent antitumor activity compared with blocker Abs in two mouse tumor models, and combination therapy using both classes of Abs enhanced tumor suppression in the mouse immunogenic tumor model. The identification of the novel nonblocker anti-PD-1 Abs and their synergy with classical blocker Abs may be instrumental in potentiating immunotherapy strategies and antitumor activity.

Sensitive and frequent identification of high avidity neo-epitope specific CD8 + T cells in immunotherapy-naive ovarian cancer.

Immunotherapy directed against private tumor neo-antigens derived from non-synonymous somatic mutations is a promising strategy of personalized cancer immunotherapy. However, feasibility in low mutational load tumor types remains unknown. Comprehensive and deep analysis of circulating and tumor-infiltrating lymphocytes (TILs) for neo-epitope specific CD8+ T cells has allowed prompt identification of oligoclonal and polyfunctional such cells from most immunotherapy-naive patients with advanced epithelial ovarian cancer studied. Neo-epitope recognition is discordant between circulating T cells and TILs, and is more likely to be found among TILs, which display higher functional avidity and unique TCRs with higher predicted affinity than their blood counterparts. Our results imply that identification of neo-epitope specific CD8+ T cells is achievable even in tumors with relatively low number of somatic mutations, and neo-epitope validation in TILs extends opportunities for mutanome-based personalized immunotherapies to such tumors.