Antigen-specific Memory CD8 Research Articles

GSK-3β in Dendritic Cells Exerts Opposite Functions in Regulating Cross-Priming and Memory CD8 T Cell Responses Independent of β-Catenin.

GSK-3β plays a critical role in regulating the Wnt/β-catenin signaling pathway, and manipulating GSK-3β in dendritic cells (DCs) has been shown to improve the antitumor efficacy of DC vaccines. Since the inhibition of GSK-3β leads to the activation of β-catenin, we hypothesize that blocking GSK-3β in DCs negatively regulates DC-mediated CD8 T cell immunity and antitumor immunity. Using CD11c-GSK-3β-/- conditional knockout mice in which GSK-3β is genetically deleted in CD11c-expressing DCs, we surprisingly found that the deletion of GSK-3β in DCs resulted in increased antitumor immunity, which contradicted our initial expectation of reduced antitumor immunity due to the presumed upregulation of β-catenin in DCs. Indeed, we found by both Western blot and flow cytometry that the deletion of GSK-3β in DCs did not lead to augmented expression of β-catenin protein, suggesting that GSK-3β exerts its function independent of β-catenin. Supporting this notion, our single-cell RNA sequencing (scRNA-seq) analysis revealed that GSK-3β-deficient DCs exhibited distinct gene expression patterns with minimally overlapping differentially expressed genes (DEGs) compared to DCs with activated β-catenin. This suggests that the deletion of GSK-3β in DCs is unlikely to lead to upregulation of β-catenin at the transcriptional level. Consistent with enhanced antitumor immunity, we also found that CD11c-GSK-3β-/- mice exhibited significantly augmented cross-priming of antigen-specific CD8 T cells following DC-targeted vaccines. We further found that the deletion of GSK-3β in DCs completely abrogated memory CD8 T cell responses, suggesting that GSK-3β in DCs also plays a negative role in regulating the differentiation and/or maintenance of memory CD8 T cells. scRNA-seq analysis further revealed that although the deletion of GSK-3β in DCs positively regulated transcriptional programs for effector differentiation and function of primed antigen-specific CD8 T cells in CD11c-GSK-3β-/- mice during the priming phase, it resulted in significantly reduced antigen-specific memory CD8 T cells, consistent with diminished memory responses. Taken together, our data demonstrate that GSK-3β in DCs has opposite functions in regulating cross-priming and memory CD8 T cell responses, and GSK-3β exerts its functions independent of its regulation of β-catenin. These novel insights suggest that targeting GSK-3β in cancer immunotherapies must consider its dual role in CD8 T cell responses.

Generation of antigen-specific memory CD4 T cells by heterologous immunization enhances the magnitude of the germinal center response upon influenza infection.

Current influenza vaccine strategies have yet to overcome significant obstacles, including rapid antigenic drift of seasonal influenza viruses, in generating efficacious long-term humoral immunity. Due to the necessity of germinal center formation in generating long-lived high affinity antibodies, the germinal center has increasingly become a target for the development of novel or improvement of less-efficacious vaccines. However, there remains a major gap in current influenza research to effectively target T follicular helper cells during vaccination to alter the germinal center reaction. In this study, we used a heterologous infection or immunization priming strategy to seed an antigen-specific memory CD4+ T cell pool prior to influenza infection in mice to evaluate the effect of recalled memory T follicular helper cells in increased help to influenza-specific primary B cells and enhanced generation of neutralizing antibodies. We found that heterologous priming with intranasal infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular immunization with adjuvanted recombinant LCMV glycoprotein induced increased antigen-specific effector CD4+ T and B cellular responses following infection with a recombinant influenza strain that expresses LCMV glycoprotein. Heterologously primed mice had increased expansion of secondary Th1 and Tfh cell subsets, including increased CD4+ TRM cells in the lung. However, the early enhancement of the germinal center cellular response following influenza infection did not impact influenza-specific antibody generation or B cell repertoires compared to primary influenza infection. Overall, our study suggests that while heterologous infection or immunization priming of CD4+ T cells is able to enhance the early germinal center reaction, further studies to understand how to target the germinal center and CD4+ T cells specifically to increase long-lived antiviral humoral immunity are needed.

Antigen-specific age-related memory CD8 T cells induce and track Alzheimer’s-like neurodegeneration

Cerebral (Aβ) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aβ/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aβ/pTau, however, appears to vary depending on the animal model. Our prior work suggested that antigen-specific memory CD8 T ("hiT") cells act upstream of Aβ/pTau after brain injury. Here, we examine whether hiT cells influence sporadic AD-like pathophysiology upstream of Aβ/pTau. Examining neuropathology, gene expression, and behavior in our hiT mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. We identify an age-related factor acting upstream of Aβ/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD.

Immune Profiling and Responses of Smoldering Multiple Myeloma Patients Treated in a Phase Ib Study of Pvx-410 Vaccine Targeting XBP1/CD138/CS1 Antigens, and Citarinostat, a Histone Deacetylase Inhibitor (HDACi) with and without Lenalidomide

BACKGROUND AND SIGNIFICANCE Immunoparesis plays an important role in the progression of smoldering multiple myeloma (SMM). Therapies that augment the immune response may prevent or slow the progression to symptomatic multiple myeloma. We have previously shown that PVX-410, a multi-peptide cancer vaccine (OncoPep., Inc.) targeting XBP1/CD138/CS1, is immunogenic in SMM, as a single therapy and in combination with lenalidomide. Here, we investigated whether co-administration of PVX-410 with an oral HDAC inhibitor, citarinostat, +/- lenalidomide enhances vaccine specific immune response in patients with SMM. STUDY DESIGN AND METHODS This 2-cohort phase 1b multicenter study accrued 16 adults (≥18 years) with HLA-A2 + SMM and high risk of progression to active MM. Patients received citarinostat in combination with PVX-410 (double combination cohort) and with PVX-410 and lenalidomide (triple combination cohort). PVX-410 was given as six doses of 0.8 mg biweekly (weeks 0, 2, 4, 6, 8, and 10) via subcutaneous injection followed by a single dose of PVX-410 during follow-up visits at month 1, 2, 3, 6, and 9; 3 cycles of citarinostat (180 mg QD PO for 21 days every 28 days) and lenalidomide (25 mg on Days 1-21 every 28 days) were given to patients in the double and triple combination cohorts respectively in conjunction with the initial 6 doses of PVX-410. Patients were followed post-treatment for 12 months. Peripheral blood (PB) and bone marrow (BM) samples at baseline and month one after treatment were analyzed by single-cell RNA sequencing. T-cell receptor and B-cell receptor sequencing were also performed to investigate T cell and B-cell clonal expansion before and after treatment. Flow cytometric analyses of anti-MM functionality of the XBP1/CD138/CS1-specific CD8 + cytotoxic T lymphocytes (CTL) was performed at various timepoints from baseline up to 12-month post-treatment. The development of antigen-specific memory CD8 + CTL through upregulation of 41BB and production of Th1-type of cytokines (IFN-g, IL-2, TNF-a) were evaluated by flow cytometry. Deep whole genome sequencing was performed on subsets of CD138 + cells from BM isolates to corroborate our findings. RESULTS We screened 16 SMM patients; 15 (94%) received treatment (7 with the double and 8 with the triple combination). By the end of the 12-month post-treatment follow-up period no patients progressed to symptomatic myeloma in the double combination cohort; one patient in the triple combination cohort withdrew by month 3 for disease progression. Most patients receiving the double combination had stable disease (SD) as their best clinical response (n=5), one had partial response (PR) and one minimal response (MR). Triple combination therapy resulted in better clinical response including PR (n=2), MR (n=4) and SD (n=2). Single-cell transcriptomic analyses of 21 PB samples from 13 patients and 15 BM samples from 11 patients at baseline and month one after treatment is in progress. Immune monitoring studies on 125 samples from 15 different patients at various timepoints from baseline up to 12-month post-treatment are ongoing. Fourteen patients (100%) had at least one treatment-related adverse event (trAEs), mostly grade 1-2 in severity. The most common trAEs among patients receiving double vs triple combination were fatigue (71% vs 63%), injection site reactions (43% vs 50%), neutropenia (57% vs 38%), anemia (29% vs 38%), and diarrhea (0% vs 50%). There was one grade 3 trAE: specifically, a thromboembolic event in one patient who was receiving lenalidomide and despite aspirin prophylaxis, but who fully recovered. DISCUSSION The combination of PVX-410 with citarinostat, with and without lenalidomide is generally safe in this patient population. The objective clinical response was relatively modest given the short duration of therapy: longer follow up is needed to assess the impact on progression to active myeloma. PB and marrow transcriptome profiling along with the immunogenicity studies are in progress and will be presented. We will evaluate whether PB mononuclear cells have the potential to replace BM as a surrogate for genetic and immunological surveillance in SMM, as we anticipate that further understanding of the immune alterations among patients with SMM and the response to immunomodulatory therapy may provide insight on early intervention and prevention of progression to symptomatic disease.

Intramuscular Immunization with a Liposomal Multi-Epitope Chimeric Protein Induces Strong Cellular Immune Responses against Visceral Leishmaniasis.

Control of the intracellular parasite Leishmania (L.) requires the activation of strong type 1 cellular immune responses. Towards this goal, in the present study, a multiepitope chimeric protein named LiChimera was encapsulated into cationic liposomes and its protective efficacy against experimental visceral leishmaniasis was investigated. Liposomal LiChimera conferred significant protection against L. infantum as evidenced by the significantly reduced parasite loads in the spleen and liver. Protection detected in Lipo:LiChimera-immunized mice was dependent on the differentiation of long-lasting cellular immune responses and particularly the induction of antigen-specific multifunctional memory CD4+ TH1 and CD8+ T cells that persisted during infection, as evidenced by the persistent high production of IFN-γ and IL-2 and proliferation activity. Notably, protected mice were also characterized by significantly low numbers of non-regulatory CD4+ T cells able to co-produce IFN-γ and IL-10, an important population for disease establishment, as compared to non-immunized control group. Collectively, these results demonstrate that cationic liposomes containing LiChimera can be considered an effective candidate vaccine against visceral leishmaniasis.

Vaccine antigen archiving by lymphatic endothelial cells boosts archived antigen-specific CD8 T cell memory responses during a pathogenic challenge

Abstract Numerous studies have shown viral antigens can persist in lymph nodes after the resolution of the infection. We demonstrated that lymphatic endothelial cells (LEC), which comprise the lymphatic vasculature necessary for antigen drainage from the tissue, are the predominant cell type required for the persistence of both vaccine and viral antigens within the lymph node. We termed this process antigen archiving due to the ability of LEC to actively archive antigens to which an immune response has occurred. This process involves antigen acquisition, retention, and exchange of the antigen between LECs and dendritic cells (DC), resulting in the presentation of the archived antigens to archived antigen-specific CD8 memory T cells. In this study, we evaluated the effect of LEC archived antigen on archived antigen-specific memory T cells during a pathogenic challenge. We predicted that a pathogenic challenge would result in the release of the archived antigen and subsequent archived antigen presentation. Following a pathogenic challenge, we found a significant increase in archived antigen-specific endogenous or transferred memory CD8 T cells, compared to bystander activation seen by memory T cells of an unrelated specificity. The phenotype of the archived antigen-specific memory CD8 T cells was more effector-like and led to a significant increase in protection against Listeria monocytogenes (LM)-expressing the archived antigen, in the local area. In conclusion, these findings suggest that archived antigen may be released during a pathogenic challenge. As such, the archived antigen may be acquired by migratory DCs as they traffic through the lymphatics in order to present the archived antigen to memory CD8 T cells acting as an effective boost. NIH R01 AI121209

Method of manufacturing CAF®09 liposomes affects immune responses induced by adjuvanted subunit proteins

The ability to induce antigen-specific CD4+ and CD8+T-cell responses is one of the fundamental requirements when developing new efficacious vaccines against challenging infectious diseases and cancer. However, no adjuvants are currently approved for human subunit vaccines that induce T-cell immunity. Here, we incorporated a Toll-like receptor 4 agonist, i.e., the ionizable lipidoid L5N12, in the liposomal cationic adjuvant formulation 09 (CAF®09), and found that modified CAF®09 liposomes possess preserved adjuvant function as compared to unmodified CAF®09. CAF®09 consists of the cationic lipid dimethyldioctadecylammonium (DDA), monomycoloyl glycerol analogue 1 (MMG-1), and polyinosinic:polycytidylic acid [poly(I:C)]. By using the microfluidic mixing technology for liposome preparation, we gradually replaced DDA with L5N12, while keeping the molar ratios of MMG-1 and poly(I:C) constant. We found that this type of modification resulted in colloidally stable liposomes, which were significantly smaller and displayed reduced surface charge as compared to unmodified CAF®09, prepared by using the conventional thin film method. We showed that incorporation of L5N12 decreases the membrane rigidity of CAF®09 liposomes. Furthermore, vaccination with antigen adjuvanted with L5N12-modified CAF®09 or antigen adjuvanted with unmodified CAF®09, respectively, induced comparable antigen-specific serum antibody titers. We found that antigen adjuvanted with L5N12-modified CAF®09 induced antigen-specific effector and memory CD4+ and CD8+T-cell responses in the spleen comparable to those induced when unmodified CAF®09 was used as adjuvant. However, incorporating L5N12 did not have a synergistic immunopotentiating effect on the antibody and T-cell responses induced by CAF®09. Moreover, vaccination with antigen adjuvanted with unmodified CAF®09, which was manufactured by using microfluidic mixing, induced significantly lower antigen-specific CD4+ and CD8+T-cell responses than vaccination with antigen adjuvanted with unmodified CAF®09, which was prepared by using the thin film method. These results show that the method of manufacturing affects CAF®09 liposome adjuvanted antigen-specific immune responses, which should be taken into consideration when evaluating immunogenicity of subunit protein vaccines.

Induction of antigen specific intrahepatic CD8+ T cell responses by a secreted heat shock protein based gp96-Ig-PfCA malaria vaccine.

A highly efficacious and durable vaccine against malaria is an essential tool for global malaria eradication. One of the promising strategies to develop such a vaccine is to induce robust CD8+ T cell mediated immunity against malaria liver-stage parasites. Here we describe a novel malaria vaccine platform based on a secreted form of the heat shock protein, gp96-immunoglobulin, (gp96-Ig) to induce malaria antigen specific, memory CD8+ T cells. Gp96-Ig acts as an adjuvant to activate antigen presenting cells (APCs) and chaperone peptides/antigens to APCs for cross presentation to CD8+ T cells. Our study shows that vaccination of mice and rhesus monkeys with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum CSP and AMA1 (PfCA) vaccine candidate antigens, induces liver-infiltrating, antigen specific, memory CD8+ T cell responses. The majority of the intrahepatic CSP and AMA1 specific CD8+ T cells expressed CD69 and CXCR3, the hallmark of tissue resident memory T cells (Trm). Also, we found intrahepatic, antigen-specific memory CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses in the liver. Our novel gp96-Ig malaria vaccine strategy represents a unique approach to induce liver-homing, antigen-specific CD8+ T cells critical for Plasmodium liver-stage protection.

A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus.

Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources. We optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients. In conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level.

Comparable humoral and cellular immunity against Omicron variant BA.4/5 of once-boosted BA.1/2 convalescents and twice-boosted COVID-19-naïve individuals.

The fourth vaccination dose confers additional protective immunity against severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) infection in individuals with no prior coronavirus disease-19 (COVID-19). However, its immunological benefit against currently circulating BA.4/5 is unclear in individuals who have received a booster shot and been infected with Omicron variant BA.1/2. We analyzed immune responses in whom had been boosted once and did not have COVID-19 (n = 16), boosted once and had COVID-19 when BA.1/2 was dominant in Korea (Hybrid-6M group, n = 27), and boosted twice and did not have COVID-19 (Vx4 group, n = 15). Antibody binding activities against RBDo BA.1 and RBDo BA.4/5 , antigen-specific memory CD4+ and CD8+ T-cell responses against BA.4/5, and B-cell responses against SARS-CoV-2 wild-type did not differ statistically between the Hybrid-6M and Vx4 groups. The humoral and cellular immune responses of the Hybrid-6M group against BA.4/5 were comparable to those of the Vx4 group. Individuals who had been boosted and had an Omicron infection in early 2022 may not have high priority for an additional vaccination.

Loss of the Nuclear Receptor Pparγ Primes T Effector (TEFF) Differentiation of Antigen-Specific CD8+ T Cells

Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to the nuclear hormone superfamily, with three distinct types identified: PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). PPARs possess a critical role in the regulation of lipid metabolism, and thus dictate the differentiation and fate of immune cells. PPARγ is a lipid-activated transcription factor mainly known to mediate anti-inflammatory effects on both innate and adaptive immune system. Synthetic ligands for activating PPARγ ameliorate disease outcomes in various models of inflammation and autoimmunity. The role of PPARγ in the immune system has been mainly studied in macrophages, where it is known to mediate M2 polarization. In the present study we investigated how PPARγ affects the differentiation and function of antigen-specific effector (TEFF) and memory (TM) CD8+ T cells using OTI TCR transgenic mice and OTI mice with T cell-specific PPARγ deletion (OTI/PPARγf/f-CD4-cre; thereafter named OTI/PPARγ-KO). Antigen-specific TEFF were generated in vitro by stimulation of OTI and OTI/PPARγ-KO T cells with Ova257-264 followed by culture with IL-2, whereas TM were generated by stimulation with Ova257-264 followed by culture with IL-15. Under both TEFF and TM conditions in vitro, antigen-specific stimulation of OTI/PPARγ-KO cells resulted in significantly greater proliferation and expansion relative to OTI control. Additionally, OTI/PPARγ-KO TM cells cultured with IL-15 co-expressed significantly higher CD44+/CD62L+ fractions indicating a greater expansion of central memory cells. Consistent with these, metabolomics analysis showed that under both culture conditions, PPARγ-deficient OTI cells had increased levels of glycolysis intermediates but also TCA metabolites consistent with a program of metabolically active cells with high levels of glycolysis and OXPHOS. RNA-sequencing studies showed that upon TEFF differentiation in vitro, OTI/PPARγ-KO T cells displayed a differential gene expression program compared to OTI controls. Pathway enrichment analysis revealed that PPARγ-deficient OTI cells were significantly enriched for genes related to T cell activation and proliferation, integrin activation, cytokine production, ribosomal function and biogenesis, RNA processing and protein translation. During antigen-specific stimulation in vivo performed after adoptive transfer of OTI/PPARγ-KO and OTI cells into syngeneic recipients followed by immunization with Ova-expressing Listeria monocytogenes (Lm-OVA), PPARγ deletion resulted in a rapid and enhanced TEFF cell activation and expansion as determined by the higher mitotic index, higher enrichment of Dextramer-positive antigen-specific T cells, and the higher expression of activation markers including KLRG1, CD44 and CD69, but also CTLA-4, PD-1, Lag-3 and Tim-3. These cells also developed higher numbers of TNF-α and IFN-γ-producing T cells, as well as ROR-γt expressing T cells consistent with Tc17 differentiation. In vivo stimulated antigen-specific PPARγ-deficient and control OTI T cells showed no numerical differences in TM differentiation or expansion. However, upon rechallenge in vitro, PPARγ-deficient TM cells exhibited improved capacity for TEFF differentiation with higher release of inflammatory cytokines and expression of CD69, PD-1 and CTLA-4. Our findings uncover a new role of PPARγ ablation in programming CD8+ T responses by regulating metabolism-mediated cues, which imprint T cell differentiation and long-term effector capacity.

BCGΔBCG1419c increased memory CD8+ T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG in a model of chronic tuberculosis

Previously, we reported that a hygromycin resistant version of the BCGΔBCG1419c vaccine candidate reduced tuberculosis (TB) disease in BALB/c, C57BL/6, and B6D2F1 mice infected with Mycobacterium tuberculosis (Mtb) H37Rv. Here, the second-generation version of BCGΔBCG1419c (based on BCG Pasteur ATCC 35734, without antibiotic resistance markers, and a complete deletion of BCG1419c) was compared to its parental BCG for immunogenicity and protective efficacy against the Mtb clinical isolate M2 in C57BL/6 mice. Both BCG and BCGΔBCG1419c induced production of IFN-γ, TNF-α, and/or IL-2 by effector memory (CD44+CD62L−), PPD-specific, CD4+ T cells, and only BCGΔBCG1419c increased effector memory, PPD-specific CD8+ T cell responses in the lungs and spleens compared with unvaccinated mice before challenge. BCGΔBCG1419c increased levels of central memory (CD62L+CD44+) T CD4+ and CD8+ cells compared to those of BCG-vaccinated mice. Both BCG strains elicited Th1-biased antigen-specific polyfunctional effector memory CD4+/CD8+ T cell responses at 10 weeks post-infection, and both vaccines controlled Mtb M2 growth in the lung and spleen. Only BCGΔBCG1419c significantly ameliorated pulmonary inflammation and decreased neutrophil infiltration into the lung compared to BCG-vaccinated and unvaccinated mice. Both BCG strains reduced pulmonary TNF-α, IFN-γ, and IL-10 levels. Taken together, BCGΔBCG1419c increased memory CD8+T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG.

Single-cell profiling of the antigen-specific response to BNT162b2 SARS-CoV-2 RNA vaccine

RNA-based vaccines against SARS-CoV-2 have proven critical to limiting COVID-19 disease severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. Here we identify and characterize antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 using multiple single-cell technologies for in depth analysis of longitudinal samples from a cohort of healthy participants. Mass cytometry and unbiased machine learning pinpoint an expanding, population of antigen-specific memory CD4+ and CD8+ T cells with characteristics of follicular or peripheral helper cells. B cell receptor sequencing suggest progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlate with eventual SARS-CoV-2 IgG, and a participant lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms identify an antigen-specific cellular basis of RNA vaccine-based immunity.

Archived antigen boosts CD8 T cell memory responses during an unrelated infection.

Abstract Numerous studies have shown viral antigens can persist in lymph nodes after the resolution of the infection. We showed that lymphatic endothelial cells (LECs), which comprise the lymphatic vasculature, are the predominant cell type required for the persistence of antigen within the lymph node. We termed this process antigen archiving due to the ability of LECs to actively acquire and store antigens to which an active immune response occurred. This process involves antigen acquisition, retention, and exchange of the antigen between LECs and dendritic cells, resulting in the presentation of the archived antigens to CD8 memory T cells and improving effector function. In more recent data, we demonstrated that LEC death causes the release of archived antigens during LN contraction. We found that archived antigens could be released during an unrelated viral infection, during the time frame of LEC death. This release of antigen was visualized by a significant increase in archived antigen-specific endogenous or transferred memory CD8 T cells, but not memory T cells of an unrelated specificity. The increase in archived antigen-specific CD8 T cells maintained a more effector-like phenotype and performed significantly better upon antigenic challenge with Listeria monocytogenes (LM) expressing ovalbumin. Mice that received an unrelated infection 2–3 weeks prior to antigenic challenge demonstrated improved protection against LM-ova. In conclusion, archived antigen release during a secondary unrelated infection boosts the archived antigen-specific memory CD8 T cell population. These findings bring into question whether some previous data demonstrating heterologous immunity may be a result of archived antigen release. Supported by R01 AI121209 R21 AI155929

Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination.

Antigen recognition through the T cell receptor (TCR) αβ heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naïve T cells with high specificity to expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naïve hosts. In this study, we use high-throughput sequencing of memory CD4 TCRβ repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCRβ sequence patterns into a hepatitis B epitope-specific annotation model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T cell clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination.

The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Antigen-specific memory CD4+ T cells can persist and confer rapid and efficient protection from microbial reinfection. However, the mechanisms underlying the long-term maintenance of the memory CD4+ T cell pool remain largely unknown. Here, using a mouse model of acute infection with lymphocytic choriomeningitis virus (LCMV), we found that the serine/threonine kinase complex mammalian target of rapamycin complex 2 (mTORC2) is critical for the long-term persistence of virus-specific memory CD4+ T cells. The perturbation of mTORC2 signaling at memory phase led to an enormous loss of virus-specific memory CD4+ T cells by a unique form of regulated cell death (RCD), ferroptosis. Mechanistically, mTORC2 inactivation resulted in the impaired phosphorylation of downstream AKT and GSK3β kinases, which induced aberrant mitochondrial reactive oxygen species (ROS) accumulation and ensuing ferroptosis-causative lipid peroxidation in virus-specific memory CD4+ T cells; furthermore, the disruption of this signaling cascade also inhibited glutathione peroxidase 4 (GPX4), a major scavenger of lipid peroxidation. Thus, the mTORC2-AKT-GSK3β axis functions as a key signaling hub to promote the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Abstract 12526: T Cell Response to Influenza Vaccination Remains Intact in Adults With Congenital Heart Disease Who Underwent Early Thymectomy

Introduction: Thymectomy is routine during surgery for congenital heart defects to access to the heart. T cells developed in the thymus play a key role in immunity. Individuals with thymectomy in infancy have altered T cell populations suggesting early immunosenescence. Hypothesis: Adults with Congenital Heart Disease (ACHD) who underwent thymectomy in the first year of life have an altered response to influenza vaccination due to T cell immunosenescence. Methods: We recruited ACHD with early thymectomy ≤ 1 year of age (ACHD-ET; n = 12), ACHD and no thymectomy (ACHD-NT; n = 8), and healthy controls (HC; n = 14). Peripheral blood was collected prior to influenza vaccine and 4 weeks following administration. Flow cytometry of T cell subsets and intracellular cytokine staining of CD4 + T cells was done following in vitro stimulation with influenza viral antigen. Results: Subject's mean age was 34 ± 10.6 years with no difference between the groups. At baseline, the median (IQR) frequency of naïve CD4 + T cells was 24.7% (15.9) in ACHD-ET vs. 43.6% (16.9) in HC ( P =0.01). Similarly, naïve CD8 + T cells were lower with 37.5% (25.7) in ACHD-ET vs 62.8% (22.9) in HC ( P =0.02). This also resulted in a reciprocal increase in memory CD4 + and CD8 + T Cells in the ACHD-ET group. The ACHD-NT was not significantly different than the other groups. The frequencies of influenza antigen-specific memory CD4 + T cells expressing IFN-γ and TNF-α were significantly increased in post-vaccine blood samples compared to pre-vaccine samples across all 3 groups ( P <0.05). Conclusions: ACHD-ET have a smaller population of naïve T cells, suggestive of immunosenescence. Despite this they have an equivalent cytokine response suggesting that early thymectomy does not inhibit the response to vaccination in young adulthood. Our findings support the recommendation that preventative vaccination against pathogens including influenza virus and the newly emerging SARS-CoV-2 should continue to be routinely performed in ACHD.

Enhancement of the Local CD8+ T-Cellular Immune Response to Mycobacterium tuberculosis in BCG-Primed Mice after Intranasal Administration of Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens.

BCG is the only licensed vaccine against Mycobacterium tuberculosis (M.tb) infection. Due to its intramuscular administration route, BCG is unable to induce a local protective immune response in the respiratory system. Moreover, BCG has a diminished ability to induce long-lived memory T-cells which are indispensable for antituberculosis protection. Recently we described the protective efficacy of new mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing TB10.4 and HspX proteins of M.tb within an NS1 influenza protein open reading frame. In the present work, the innate and adaptive immune response to immunization with the Flu/THSP and the immunological properties of vaccine candidate in the BCG-prime → Flu/THSP vector boost vaccination scheme are studied in mice. It was shown that the mucosal administration of Flu/THSP induces the incoming of interstitial macrophages in the lung tissue and stimulates the expression of co-stimulatory CD86 and CD83 molecules on antigen-presenting cells. The T-cellular immune response to Flu/THSP vector was mediated predominantly by the IFNγ-producing CD8+ lymphocytes. BCG-prime → Flu/THSP vector boost immunization scheme was shown to protect mice from severe lung injury caused by M.tb infection due to the enhanced T-cellular immune response, mediated by antigen-specific effector and central memory CD4+ and CD8+ T-lymphocytes.

Impairment of CD4+ T and Memory B Cell Responses but Normal Memory CD8+T-Cell Activation on Crohn's Disease after COVID-19 Vaccination: A Twin Case.

Vaccines to prevent the impact of SARS-CoV-2 are now available, including for patients with autoimmune diseases. However, there is no information about how inflammatory bowel disease (IBD) treatment could impact the cellular and humoral immune responses. This study evaluated SARS-CoV-2-specific humoral and cellular responses after vaccination with a two-dose schedule in a Crohn’s disease patient treated with Infliximab (10 mg/kg); we included comparisons with a monozygotic twin. The results showed that the Crohn’s disease’s twin (twin 2) had no antibody detection and reduced activation of CD4+ T cell responses, unlike the twin without the autoimmune disease (twin 1). Twin 2 developed antigen-specific central memory CD8+ T-cells and IFNγ production after the second dose of COVID-19 vaccination, similar to twin 1. These findings elucidated the role of T-cell immunity after COVID-19 immunization on IBD patients despite the lack of antibody production. Finally, our observation supports the consensus recommendation for IBD patients to receive COVID-19 vaccines.

Cancer immunotherapy with PI3K and PD-1 dual-blockade via optimal modulation of T cell activation signal

BackgroundImmune checkpoint blockade (ICB) induces durable clinical responses in patients with various types of cancer. However, its limited clinical efficacy requires the development of better approaches. In addition to immune...