Virus-specific CD8 Research Articles

Characterization of CD8+ T cells in immune-privileged organs of ZIKV-infected Ifnar1-/- mice.

Zika virus (ZIKV) infection caused neurological complications and male infertility, leading to the accumulation of antigen-specific immune cells in immune-privileged organs (IPOs). Thus, it is important to understand the immunological responses to ZIKV in IPOs. We extensively investigated the ZIKV-specific T cell immunity in IPOs in Ifnar1-/- mice, based on an immunodominant epitope E294-302 tetramer. The distinct kinetics and functions of virus-specific CD8+ T cells infiltrated into different IPOs were characterized, with late elevation in the brain and spinal cord. Single epitope E294-302-specific T cells can account for 20-60% of the total CD8+ T cells in the brain, spinal cord, and testicle and persist for at least 90 days in the brain and spinal cord. The E294-302-specific TCRαβs within the IPOs are featured with the majority of clonotypes utilizing TRAV9N-3 paired with diverse TRBV chains, but with distinct αβ paired clonotypes in 7 and 30 days post-infection. Specific chemokine receptors, Ccr2 and Ccr5, were selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of virus-specific CD8+ T cells after infection. Overall, this study adds to the understanding of virus-specific CD8+ T cell responses for controlling and clearing ZIKV infection in IPOs.IMPORTANCEThe immune-privileged organs (IPOs), such as the central nervous system and testicles, presented pathogenicity and inflammation after Zika virus (ZIKV) infection with infiltrated CD8+ T cells. Our data show that CD8+ T cells keep up with virus increases and decreases in immune-privileged organs. Furthermore, our study provides the first ex vivo comparative analyses of the composition and diversity related to TCRα/β clonotypes across anatomical sites and ZIKV infection phases. We show that the vast majority of TCRα/β clonotypes in tissues utilize TRAV9N-3 with conservation. Specific chemokine expression, including Ccr2 and Ccr5, was found to be selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of the virus-specific CD8+ T cells after the infection. Our study adds insights into the anti-viral immunological characterization and chemotaxis mechanism of virus-specific CD8+ T cells after ZIKV infection in different IPOs.

Combination of oligo-fractionated irradiation with nivolumab can induce immune modulation in gastric cancer

BackgroundTumor-associated antigen (TAA)-specific CD8(+) T cells are essential for nivolumab therapy, and irradiation has been reported to have the potential to generate and activate TAA-specific CD8(+) T cells. However, mechanistic...

PLGA micro/nanoparticle vaccination elicits non-tumor antigen specific resident memory CD8+ T cell protection from hepatocellular carcinoma.

Together, tumor and virus-specific tissue-resident CD8+ memory T cells (TRMs) of hepatocellular carcinoma (HCC) patients with Hepatitis B virus (HBV) infection can provide rapid frontline immune surveillance. The quantity and activity of CD8+ TRMs were correlated with the relapse-free survival of patients with improved health. However, HBV-specific CD8+ TRMs have a more exhausted phenotype and respond more actively under anti-PDL1 or PD1 treatment of HBV+HCC patients. Vaccination strategies that induce a strong and sustained CD8+ TRMs response are quite promising. Herein, a biodegradable poly(D,L-lactide-co-glycolide) microsphere and nanosphere particle (PLGA N.M.P) delivery system co-assembled by anti-PD1 antibodies (aPD1) and loaded with ovalbumin (OVA-aPD1 N.M.P) was fabricated and characterized for size (200 nm and 1 μm diameter), charge (-15 mV), and loading efficiencies of OVA (238 μg mg-1 particles) and aPD1 (40 μg mg-1 particles). OVA-aPD1 N.M.P could stimulate the maturation of BMDCs and enhance the antigen uptake and presentation by 2-fold compared to free OVA. The nanoparticles also induced the activation of macrophages (RAW 264.7) to produce a high level of cytokines, including TNF-α, IL-6 and IL-10. In vivo stimulation of mice using OVA-aPD1 N.M.P robustly enhanced IFN-γ-producing-CD8+ T cell infiltration in tumor tissues and the secretion of IgG and IgG2a/IgG1 antibodies. OVA-aPD1 N.M.P delivered OVA to increase the activation and proliferation of OVA-specific CD8+ TRMs, and its combination with anti-PD1 antibodies promoted complete tumor rejection by the reversal of tumor-infiltrating CD8+ T cell exhaustion. Thus, PLGA N.M.P could induce a strong CD8+ TRMs response, further highlighting its therapeutic potential in enhancing an antitumor immune response.

Peptide-Loaded HLA Class I Tetramer-Associated Magnetic Bead-Based Enrichment of HBV-Specific CD8+ T Cells.

HBV-specific CD8+ T cells are only present at the low frequency during chronic infection. Thus, they are often undetectable by conventional ex vivo staining methods using peptide-loaded HLA class I tetramers. Detection sensitivity can be increased by magnetic bead-based enrichment strategies following staining with peptide-loaded HLA class I tetramers. Additionally, some downstream applications like e.g., single cell RNA sequencing of virus-specific CD8+ T cells may also require a pre-enrichment step to increase the frequency of the cells of interest. For this, peptide-loaded HLA class I tetramers-associated magnetic bead-based enrichment is also a suitable method.

Essential role for Batf3-dependent dendritic cells in regulating CD8 T-cell response during SARS-CoV-2 infection.

SARS-CoV-2 infection elicits robust CD8 T-cell responses, yet the identity of the mechanisms playing dominant roles in initiating the virus-specific CD8 T-cell responses are largely unknown. In the present study, we interrogate the contribution of the cDC1 subset to SARS-CoV-2-specific CD8 T-cell immunity. For this purpose, we used a novel murine line which combines the SARS-CoV-2 susceptible K18-hACE2 transgenic and the Batf3 deficient mice which lack the cDC1 subset. We demonstrate that in the absence of cDC1, viral-specific CD8 T-cell responses were severely impaired both in the draining lymph node as well as in the lungs, during the effector phase of SARS-CoV-2 infection. Furthermore, SARS-CoV-2 specific memory CD8 T-cells in the lungs and spleens were also significantly impacted, whereas humoral responses, as well as CD4 T-cells were not affected. Additionally, we demonstrate that the absence of cDC1 subset, and the consequent impaired CD8 T-cell responses, resulted in significant increase in SARS-CoV-2 viral load in the lungs. The conclusions of the study were further independently corroborated in an additional COVID-19 murine model consisting infection with a mouse-adapted SARS-CoV-2 virus. These results underscore a specific role for Batf3-dependent DC in regulating SARS-CoV-2 specific CD8 T-cell responses and may contribute to future vaccine design and immunization strategies.

Safety and immunogenicity of a synthetic nanoparticle-based, T cell priming peptide vaccine against dengue in healthy adults in Switzerland: a double-blind, randomized, vehicle-controlled, phase 1 study

Vaccines that minimize the risk of vaccine-induced antibody-dependent enhancement and severe dengue are needed to address the global health threat posed by dengue. This study assessed the safety and immunogenicity of a gold nanoparticle (GNP)-based, multi-valent, synthetic peptide dengue vaccine candidate (PepGNP-Dengue), designed to provide protective CD8+ T cell immunity, without inducing antibodies. In this randomized, double-blind, vehicle-controlled, phase 1 trial (NCT04935801), healthy naïve individuals aged 18-45 years recruited at the Centre for primary care and public health, Lausanne, Switzerland, were randomly assigned to receive PepGNP-Dengue or comparator (GNP without peptides [vehicle-GNP]). Randomization was stratified into four groups (low dose [LD] and high dose [HD]), allocation was double-blind from participants and investigators. Two doses were administered by intradermal microneedle injection 21 days apart. Primary outcome was safety, secondary outcome immunogenicity. Analysis was by intention-to-treat for safety, intention-to-treat and per protocol for immunogenicity. 26 participants were enrolled (August-September 2021) to receive PepGNP-Dengue (LD or HD, n=10 each) or vehicle-GNP (LD or HD, n=3 each). No vaccine-related serious adverse events occurred. Most (90%) related adverse events were mild; injection site pain and transient discoloration were most frequently reported. Injection site erythema occurred in 58% of participants. As expected, PepGNP-Dengue did not elicit anti-DENV antibodies of significance. Significant increases were observed in specific CD8+ T cells and dengue dextramer+memory cell subsets in the LD PepGNP-Dengue but not in the HD PepGNP-Dengue or vehicle-GNP groups, specifically PepGNP-activated CD137+CD69+CD8+ T cells (day 90,+0.0318%, 95% CI: 0.0088-0.1723, p=0.046), differentiated effector memory (TemRA) and central memory (Tcm) CD8+ T cells (day 35,+0.8/105 CD8+, 95% CI: 0.19-5.13, p=0.014 and +1.34/105 CD8+, 95% CI: 0.1-7.34, p=0.024, respectively). Results provide proof of concept that a synthetic nanoparticle-based peptide vaccine can successfully induce virus-specific CD8+ T cells. The favourable safety profile and cellular responses observed support further development of PepGNP-Dengue. Emergex Vaccines Holding Limited.

Myeloid derived suppressor cells potentiate virus-specific memory CD8+ T cell response

How therapeutically administered myeloid derived suppressor cells (MDSCs) modulate differentiation of virus-specific CD8+ T cell during virus infection was investigated. In vitro generated MDSCs from bone marrow precursors inhibited the expansion of stimulated CD8+ T cells but the effector cells in the recipients of MDSCs showed preferential memory transition during Influenza A virus (IAV) or an α− (Herpes Simplex Virus) as well as a γ- (murine herpesvirus 68) herpesvirus infection. Memory CD8+ T cells thus generated constituted a heterogenous population with a large fraction showing effector memory (CD62LloCCR7-) phenotype. Such cells could be efficiently recalled in the rechallenged animals and controlled the secondary infection better. Memory potentiating effects of MDSCs occurred irrespective of the clonality of the responding CD8+ T cells as well as the nature of infecting viruses. Compared to the MDSCs recipients, effector cells of MDSCs recipients showed higher expression of molecules known to drive cellular survival (IL-7R, Bcl2) and memory formation (Tcf7, Id3, eomesodermin). Therapeutically administered MDSCs not only mitigated the tissue damaging response during a resolving IAV infection but also promoted the differentiation of functional memory CD8+ T cells. Therefore, MDSCs therapy could be useful in managing virus-induced immunopathological reactions without compromising immunological memory.

Prior viral infection primes cross-reactive CD8+ T cells that respond to mouse heart allografts.

Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed. Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8+ T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens. Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8+ T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer+ CD8+ T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8+ T cells that were GFP+ was <1%. These data show that a significant percentage of viral-specific memory CD8+ T cells expressed T cell receptors that also recognized alloantigens in vivo. Notably, the frequency of cross-reactive CD8+ T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity. In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.

Identification and functional characteristic of SARS-CoV-2-specific T lymphocytes in COVID-19 patients up to 16 months after disease onset

In cases of respiratory viral infection, along with innate immunity mechanisms, the adaptive immune system plays a crucial role in the body’s defence. The efficiency of its cellular component is crucial for pathogen elimination. T cell response is detected in almost all cases of COVID-19, being among the key factors of the virus control and resistance to infection, including re-infection. So far, however, many aspects of cellular immune response to SARS-CoV-2 over one year or later after infection remain unclear. The aim of this study was to investigate the dynamics of laboratory parameters of post-infection cellular immunity to SARS-CoV-2 within 16 months from the symptoms’ onset.Fifteen healthy volunteers and 87 COVID-19 patients were included into the study. The patients were divided into 3 groups depending on the time elapsed from the onset of the first symptoms to the time when blood samples were collected (from 14 to 500 days). For all samples, the number of S- and N-specific T lymphocytes and the cytokines secreting profiles were determined. Also, the Phenograph automatic clustering algorithm was used to discern different functional groups of the cells.Approximately 1 in 5 × 103 peripheral blood mononuclear cells was specific for SARS-CoV-2 S-protein, and 1 in 104 was specific for N-protein. Since the first weeks of infection, the number of specific CD8+ cells was significantly higher in COVID-19 patients, as compared with the group of healthy volunteers. As the postinfection period increased, the number of virus-specific CD4+ and CD8+ cells gradually decreased, but remained significantly higher than in control group. Among CD4+ cell population, the proportion of IFNγ-IL- 2-TNFα+ cells decreased and the ratio of IFNγ+IL-2-TNFα- cells increases. During first weeks of the disease, CD8+ lymphocytes are represented predominantly by IFNγ+IL-2-TNFα- cells and IFNγ-IL-2-TNFα+ cells by the end of the observation period. The clustering results showed that, in the early post-infection period, virusspecific T lymphocytes were mostly presented by populations of IFNγ- and TNFα-producing CD4+ effector memory cells. Meanwhile, in later time period, the most common populations were TNFα-producing CD8+ TEMRA and IFNγ-producing CD8+ central memory T lymphocytes.T cell adaptive immunity plays an important role in the control and elimination of viral infections. In this study, we demonstrated that robust cellular immunity against SARS-CoV-2 is present in the vast majority of patients from the first weeks up to 16 months after the onset of the first symptoms of COVID-19. The immune memory to SARS-CoV-2 is provided by production of central and effector memory T cells, and the data on their time dynamics during the study period allow us to hope for a longer duration of cellular immune memory to SARS-CoV-2.

Overall avidity declines in TCR repertoires during latent CMV but not EBV infection.

The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. To address these issues, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCRαβ clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. The parameters involved during the long-term persistence of virus-specific T cell clonotypes were further evaluated by gene expression profiling, phenotype and functional analyses. Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. These findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data further suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.

Immunotherapy of cytomegalovirus infection by low-dose adoptive transfer of antiviral CD8 T cells relies on substantial post-transfer expansion of central memory cells but not effector-memory cells.

Cytomegaloviruses (CMVs) are host species-specific in their replication. It is a hallmark of all CMVs that productive primary infection is controlled by concerted innate and adaptive immune responses in the immunocompetent host. As a result, the infection usually passes without overt clinical symptoms and develops into latent infection, referred to as "latency". During latency, the virus is maintained in a non-replicative state from which it can reactivate to productive infection under conditions of waning immune surveillance. In contrast, infection of an immunocompromised host causes CMV disease with viral multiple-organ histopathology resulting in organ failure. Primary or reactivated CMV infection of hematopoietic cell transplantation (HCT) recipients in a "window of risk" between therapeutic hemato-ablative leukemia therapy and immune system reconstitution remains a clinical challenge. Studies in the mouse model of experimental HCT and infection with murine CMV (mCMV), followed by clinical trials in HCT patients with human CMV (hCMV) reactivation, have revealed a protective function of virus-specific CD8 T cells upon adoptive cell transfer (AT). Memory CD8 T cells derived from latently infected hosts are a favored source for immunotherapy by AT. Strikingly low numbers of these cells were found to prevent CMV disease, suggesting either an immediate effector function of few transferred cells or a clonal expansion generating high numbers of effector cells. In the murine model, the memory population consists of resting central memory T cells (TCM), as well as of conventional effector-memory T cells (cTEM) and inflationary effector-memory T cells (iTEM). iTEM increase in numbers over time in the latently infected host, a phenomenon known as 'memory inflation' (MI). They thus appeared to be a promising source for use in immunotherapy. However, we show here that iTEM contribute little to the control of infection after AT, which relies almost entirely on superior proliferative potential of TCM.

Antibody-mediated delivery of viral epitopes to redirect EBV-specific CD8+ T-cell immunity towards cancer cells.

Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo.

C-Myc uses Cul4b to preserve genome integrity and promote antiviral CD8+ T cell immunity

During infection, virus-specific CD8+ T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8+ T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8+ T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8+ T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8+ T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.

Virus-Specific CD8+ Stem Memory T Lymphocytes Display Enhanced Anti-Virus Responses

Virus specific memory CD8+ T cells can be divided into three subsets, stem memory (T SCM), central (T CM) and effector (T EM), but their lineage relationships and their ability to persist and confer protective immunity against virus infection are not well understood. Our results show that virus-specific CD8+T SCM have a greater capacity than T CM and T EM to persist in vivo and in vitro and are more efficient in mediating protective immunity against CMV infection and EBV infection because of their increased self-renew potential whether in transcriptome or in epigenome. We firstly expanded virus-specific T cells containing CD8+TSCM and CD8+TCM cells in vitro by stimulating healthy human PBMCs with cytokines and CMV (pp65 and IE1) and EBV (LMP2a and EBNA1) virus peptides. Then we fully evaluated the phenotypic and functional differences of diverse virus-specific memory T subsets and found that CD8+TSCM cells are a subset of low exhaustion, high migration and high secretion of IL-2. However, the expression of IFN-γand TNF-a and CD107a were lower compared with TEM cells, although comparable to CD8+TCM cells. We then evaluated the self-renewal and differentiation capacity of CD8+TSCM cells, compared with TCM or TEM cells. The results showed after stimulated with a-CD3/CD2/CD28 for 5 days, that CD8+TSCM cells possess around 50% of CD45RA+CD62L+ subpopulation cells. Meanwhile, CD8+TSCM cells constantly keep a higher expression of CD45RA+ cells than TCM and TEM cells in different proliferation generations. Notably, around 20% of TCM cells,5% of TEM cells and 10% of TEFF cells were found, suggesting that TSCM cells can reconstitute the whole memory T subsets. By contrast, the TCM cells mainly retained a central memory phenotype or differentiated into effector memory T cells and the TEM cells maintained its original phenotype. We also used the stem cell index to evaluate the stemness of different subgroups, and the results confirmed that the stemness evaluation of TSCM cells were significantly higher than TCM and TEM subsets. In vivo rein-fusion of different virus-specific T cell into irradidated NPG mice also showed that CD8+TSCM cells in vivo can differentiate into all memory T subsets in vivo while TCM cells mainly retained TEM and TCM subgroups at 4W post T cell infusion in mouse liver,lung and spleen(Figure 1A-G). To explore whether the antiviral ability of TSCM cells lasted longer than that of TCM and TEM cells. We first evaluated the apoptosis of different T cell subsets stimulated by AD169 virus and found less apoptosis rate was lower than TCM and TEM cells, suggesting persistent virus infection leads to damage to T cells and CD8+TSCM cells have a strong ability to resist virus damage, which may potentially lead to stronger and longer lasting antiviral effects. Based on this, we also evaluated the apoptosis level of virus-infected MRC-5 cells to evaluate whether CD8+TSCM cells exert a stronger anti-virus effect. The result showed that AD-169 infected MRC-5 cells co-incubated with CD8+TSCM had a higher proportion of apoptotic level than CD8+TCM cells. We also used the 5day-long-term infection model in vitro to evaluate the effect of virus infection on T cell differentiation and evaluated the change of virus copy number after long-term co-culture of different cell subsets. We found that sorted CD8+TSCM system can significantly reduce the virus copy number compared with the control group, while there is no significant difference among TCM and TEM cells (Figure1H,). Further analysis into survival ability of T cell subpopulations found that the proportion of CD3+ T cells in TSCM group were significantly higher and the CD8+TSCM group was dominated by TCM and TEM cells after 5-day stimulation but still kept about 22 percentage of TSCM phenotype. (Figure1I). In summary, CD8+ TSCM cells have a sustainable ability against CMV than TCM and TEM. To examine the in vivo activity of virus-specific CD8+TSCM cells. We used Raji-HLA*A0201-pp65-luciferase cells to construct tumor infiltration mouse model. The result showed that in contrast to control mice, we observed a reduction in bioluminescence over three weeks in mice treated with CD8+TSCM cells. Although mice treated with CD8+TCM and CD8+TEM cells retained a lower tumor size in the early two weeks post adoptive T infusion, they showed a rapid tumor progresses and event lead to shorter survival compared with CD8+TSCM group, indicating a sustained anti-CMV effect of CD8+TSCM cells.

Deep Characterization of Immune Dysfunction in Patients with Multiple Myeloma (MM) and Identification of Cellular Biomarkers for Tailored Vaccination Strategies

Background: Infection is the leading cause of death in MM. However, the extent of immune dysfunction compared to other B-cell lymphoproliferative disorders (B-CLPD) and healthy adults remains poorly studied. Greater knowledge is needed for tailored vaccination strategies and to improve outcomes upon new pathogens or breakthrough infections. Aim: Generate an atlas of the basal immune status and its response to vaccination in MM vs B-CLPD patients and age-matched health care practitioners (HCP), using the COVID-19 mRNA vaccines as a case study. Methods: A total of 1,099 blood and serum samples were collected from 177 individuals: 28 MM, 53 B-CLPD and 96 HCP older than 50. They were studied before COVID-19 mRNA vaccination, at days 7 and 14 after the first dose, at days 7 and 62 after the second dose, as well as before and at day 17 after the booster. Immune profiling was performed using multidimensional and computational flow cytometry that systematically analyzed 56 immune cell types per sample and time point: 17 B, 30 T, 6 antigen-presenting cell (APC) and 3 granulocytic subsets. Serum levels of IgM, IgG and IgA against the receptor-binding domain (RBD) of the spike (S) glycoprotein, S glycoprotein, nucleocapsid (N) and main protease were quantified using a multiplex-microsphere-based flow cytometry assay. SARS-CoV-2-specific CD8 T cells were quantified using a dextramer panel of S, N, membrane, and ORF3 proteins. Results: When compared to HCP and/or B-CLPD, MM patients showed abnormal distribution of 17/17 B, 22/30 T, 4/6 APC and 1/3 granulocytic cell subsets prior to vaccination. The most deviated cell types in MM were naïve CD21+ B-cells, naïve CD4 T-cells, PD1- and PD1+CD127low effector memory (EM) CD8 T cells, classical monocytes and neutrophils. The B cell compartment of MM patients showed impaired response to COVID-19 mRNA vaccination. While B-CLPD patients and HCP displayed significant expansions of naïve CD21-, IgM+IgD+CD27+CD21-, IgG+CD27-CD21-, and IgG+CD27+CD21- B-cell subsets, as well as of IgA+ circulating plasma cells, such expansions were not observed in MM patients. Accordingly, anti-RBD IgM, IgA and IgG titers were significantly reduced in MM compared to B-CLPD and HCP after the second dose ( P ≤.002). Importantly, the booster increased anti-RBD IgG levels in HCP (20,184 to 186,629 IU/mL, P &amp;lt;.001) but not in MM (1,991 to 5,998 IU/mL, P =.14) and B-CLPD (33,548 to 41,288 IU/mL, P =.19) patients. The immune response of the T-cell compartment was also altered in MM patients. The significant expansion of CD127lowPD1+ and CD127+CD25+ EM CD4, naïve CD4 and CD127lowPD1+ EM CD8 subsets observed in HCP was unnoted in MM and B-CLPD patients. Accordingly, the percentage of virus-specific CD8 T cells after the second dose did not increase in MM and B-CLPD patients. The booster increased virus-specific CD8 T cells in HCP (0.08 to 0.14%, P =.02), but not in MM (0.08 to 0.09%, P =.94) and B-CLPD (0.23 to 0.16%, P =.85). Furthermore, the booster induced virus-specific CD8 T cell differentiation into an EM phenotype in HCP but not in MM and B-CLPD patients. In addition to the dysfunctional B and T cell response to vaccination, MM patients showed abnormal kinetics of APC such as intermediateclassical and non-classical SLAN- and SLAN+ monocytes, as well as of granulocytic subsets such as basophils and neutrophils. Based on differences in immune-cell distribution using HCP as a reference, we calculated an immune dysregulation longitudinal cumulative score in each individual that included 48,496 immune parameters. Compared with HCP, up to 34% MM and 17% B-CLPD patients showed high immune dysregulation scores. Among them, 75% and 33%, respectively, had low seroconversion after the second dose. We found 7/30 T and 14/17 B-cell subsets associated with poor vaccine response in MM patients, namely CD127lowPD1+CXCR5+ EM CD8 T cells, as well as CD21- and CD21+ naïve, transitional, IgM+IgD+CD27+CD21+, IgM+CD27-CD21+, and IgG+CD27-CD21+ B cells. Conclusion: We provide an atlas of the immune dysfunction in MM patients and how it affects the efficacy of vaccination strategies such as for COVID-19. The schedule of vaccine doses may thus benefit from individualization according to patients' immune status, which could act as a surrogate of host, tumor and treatment-related immune dysfunction. Accordingly, we have identified key cell types for readily monitoring of patients' immune status using routinely available flow cytometry.

An interferon-integrated mucosal vaccine provides pan-sarbecovirus protection in small animal models

A pan-sarbecovirus or pan-betacoronavirus vaccine that can prevent current and potential future beta-coronavirus infections is important for fighting possible future pandemics. Here, we report a mucosal vaccine that cross-protects small animal models from sarbecoviruses including SARS-CoV-1, SARS-CoV-2 and its variants. The vaccine comprises a live-but-defective SARS-CoV-2 virus that is envelope deficient and has the orf8 segment replaced by interferon-beta, hence named Interferon Beta Integrated SARS-CoV-2 (IBIS) vaccine. Nasal vaccination with IBIS protected mice from lethal homotypic SARS-CoV-2 infection and hamsters from co-housing-mediated transmission of homotypic virus. Moreover, IBIS provided complete protection against heterotypic sarbecoviruses, including SARS-CoV-2 Delta and Omicron variants, and SARS-CoV-1 in both mice and hamsters. Besides inducing a strong lung CD8 + T cell response, IBIS specifically heightened the activation of mucosal virus-specific CD4 + T cells compared to the interferon-null vaccine. The direct production of interferon by IBIS also suppressed virus co-infection of SARS-CoV-2 in human cells, reducing the risk of genetic recombination when using as live vaccines. Altogether, IBIS is a next-generation pan-sarbecovirus vaccine and warrants further clinical investigations.

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

Therapeutic vaccination following early antiretroviral therapy elicits highly functional T cell responses against conserved HIV-1 regions

‘Kick and kill’ cure strategies aim to induce HIV protein expression in latently infected cells (kick), and thus trigger their elimination by cytolytic T cells (kill). In the Research in Viral Eradication of HIV Reservoirs trial (NCT02336074), people diagnosed with primary HIV infection received immediate antiretroviral therapy (ART) and were randomised 24 weeks later to either a latency-reversing agent, vorinostat, together with ChAdV63.HIVconsv and MVA.HIVconsv vaccines, or ART alone. This intervention conferred no reduction in HIV-1 reservoir size over ART alone, despite boosting virus-specific CD4+ and CD8+ T cells. The effects of the intervention were examined at the cellular level in the two trial arms using unbiased computational analysis of polyfunctional scores. This showed that the frequency and polyfunctionality of virus-specific CD4+ and CD8+ T cell populations were significantly increased over 12 weeks post-vaccination, compared to the ART-only arm. HIV-specific IL-2-secreting CD8+ T cells also expanded significantly in the intervention arm and were correlated with antiviral activity against heterologous HIV in vitro. Therapeutic vaccination during ART commenced in primary infection can induce functional T cell responses that are phenotypically similar to those of HIV controllers. Analytical therapy interruption may help determine their ability to control HIV in vivo.

Phenotype and fate of liver-resident CD8 T cells during acute and chronic hepacivirus infection.

Immune correlates of hepatitis C virus (HCV) clearance and control remain poorly defined due to the lack of an informative animal model. We recently described acute and chronic rodent HCV-like virus (RHV) infections in lab mice. Here, we developed MHC class I and class II tetramers to characterize the serial changes in RHV-specific CD8 and CD4 T cells during acute and chronic infection in C57BL/6J mice. RHV infection induced rapid expansion of T cells targeting viral structural and nonstructural proteins. After virus clearance, the virus-specific T cells transitioned from effectors to long-lived liver-resident memory T cells (TRM). The effector and memory CD8 and CD4 T cells primarily produced Th1 cytokines, IFN-γ, TNF-α, and IL-2, upon ex vivo antigen stimulation, and their phenotype and transcriptome differed significantly between the liver and spleen. Rapid clearance of RHV reinfection coincided with the proliferation of virus-specific CD8 TRM cells in the liver. Chronic RHV infection was associated with the exhaustion of CD8 T cells (Tex) and the development of severe liver diseases. Interestingly, the virus-specific CD8 Tex cells continued proliferation in the liver despite the persistent high-titer viremia and retained partial antiviral functions, as evident from their ability to degranulate and produce IFN-γ upon ex vivo antigen stimulation. Thus, RHV infection in mice provides a unique model to study the function and fate of liver-resident T cells during acute and chronic hepatotropic infection.

Usp22 Deficiency Leads to Downregulation of PD-L1 and Pathological Activation of CD8+ T Cells and Causes Immunopathology in Response to Acute LCMV Infection.

Ubiquitin-specific peptidase 22 (Usp22) cleaves ubiquitin moieties from numerous proteins, including histone H2B and transcription factors. Recently, it was reported that Usp22 acts as a negative regulator of interferon-dependent responses. In the current study, we investigated the role of Usp22 deficiency in acute viral infection with lymphocytic choriomeningitis virus (LCMV). We found that the lack of Usp22 on bone marrow-derived cells (Usp22fl/fl Vav1-Cre mice) reduced the induction of type I and II interferons. A limited type I interferon response did not influence virus replication. However, restricted expression of PD-L1 led to increased frequencies of functional virus-specific CD8+ T cells and rapid death of Usp22-deficient mice. CD8+ T cell depletion experiments revealed that accelerated CD8+ T cells were responsible for enhanced lethality in Usp22 deficient mice. In conclusion, we found that the lack of Usp22 generated a pathological CD8+ T cell response, which gave rise to severe disease in mice.