Multifunctional T-cell Research Articles

Immunogenic profiling of Mycobacterium tuberculosis Rv1513 reveals its ability to switch on Th1 based immunity.

Tuberculosis (TB) is one of the infectious diseases caused by the pathogen Mycobacterium tuberculosis that continuously threatens the global human health. Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine that has been used clinically to prevent tuberculosis in recent centuries, but its limitations in preventing latent infection and reactivation of tuberculosis do not provide full protection. In this study, we selected the membrane-associated antigen Rv1513 of Mycobacterium. In order to achieve stable expression and function of the target gene, the prokaryotic expression recombinant vector pET30b-Rv1513 was constructed and expressed and purified its protein. Detection of IFN- γ levels in the peripheral blood of TB patients stimulated by whole blood interferon release assay (WBIA) and multi-microsphere flow immunofluorescence luminescence (MFCIA) revealed that the induced production of cytokines, such as IFN-γ and IL-6, was significantly higher than that in the healthy group. Rv1513 combined with adjuvant DMT (adjuvant system liposomes containing dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL), and trehalose-660-dibenzoic acid (TDB)) was used to detect serum specific antibodies, cytokine secretion from splenic suprasplenic cell supernatants, and multifunctional T-cell levels in splenocytes in immunised mice. The levels of IFN-γ, TNF-α, and IL-2 secreted by mouse splenocytes were found in the Rv1513+DMT group and the BCG+Rv1513+DMT group. The serum levels of IgG and its subclasses and the number of IFN-γ+T cells, TNF-α+T and IFN-γ+TNF-α+T cells in the induced CD4+/CD8+T cells in mice were significantly higher than those in the BCG group, and the highest levels were found in the BCG+Rv1513+DMT group. These findings suggest that Rv1513/DMT may serve as a potential subunit vaccine candidate that may be effective as a booster vaccine after the first BCG vaccination.

COVID-19 vaccinated children, adolescents, and young adults with acute lymphoblastic leukemia show spike reactive antibodies and multifunctional T-cells.

Little is known about the efficacy of COVID-19 vaccines during acute lymphoblastic leukemia therapy (ALL); data for COVID-19 vaccine immune responses in pediatric leukemia remain sparse. We conducted a single center study of patients aged 5-25 years undergoing ALL chemotherapy who received COVID-19 vaccination. Twenty-one patients were enrolled; efficacy was evaluable in 20. Twenty were vaccinated while receiving chemotherapy. Twenty received the BNT162b2 mRNA vaccine. Spike reactive antibodies (S-IgG) and/or T-cells (SRT) were detected in 16 of 20 (80%) vaccinated patients; 13 (65%) and 9 (45%) were positive for S-IgG and SRT, respectively. Six (30%) showed both spike reactive B and T-cell responses. Eleven of the 13 with S-IgG positivity were negative for anti-Nucleocapsid IgG, an antibody profile consistent with a vaccine induced immune response. All 13S-IgG+ patients showed neutralizing antibodies. SRT included CD4+ (7) and CD8+ (6) T-cells; both CD4+ and CD8+ SRT were seen in 4. SRT were multifunctional (producing multiple cytokines) in most patients (8 of 9); 4 showed SRT with triple cytokine and B-cell co-stimulatory responses, indicating a multimodal adaptive immune response. Immune responses were seen among patients vaccinated in the settings of lymphopenia (6 of 12) intensive chemotherapy (3 of 4), and Peg allergy (6 of 8). Sequencing revealed public CD4+ and CD8+ TCR sequences reactive to epitopes across the spike protein. In conclusion, COVID-19 vaccination induced B and/or T-cell responses in a majority of children and young adults undergoing ALL chemotherapy.

Characterization of the Protective Cellular Immune Response in Pigs Immunized Intradermally with the Live Attenuated African Swine Fever Virus (ASFV) Lv17/WB/Rie1.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.

Durability and cross-reactive immune memory to SARS-CoV-2 in individuals 2 years after recovery from COVID-19: a longitudinal cohort study

SARS-CoV-2-specific adaptive immunity more than 1 year after initial infection has not been well characterised. The aim of this study was to investigate the durability and cross-reactivity of immunological memory acquired from natural infection against SARS-CoV-2 in individuals recovered from COVID-19 2 years after infection. In this longitudinal cohort study, we recruited patients who had recovered from laboratory-confirmed COVID-19 and were discharged from Jinyintan Hospital (Wuhan, China) between Jan 7 and May 29, 2020. We carried out three successive follow-ups between June 16 and Sept 3, 2020 (6 months), Dec 16, 2020, and Feb 7, 2021 (1 year), and Nov 16, 2021, and Jan 10, 2022 (2 years), in which blood samples were taken. We included participants who did not have re-infection or receive a SARS-CoV-2 vaccination (infected-unvaccinated), and participants who received one to three doses of inactivated vaccine 1-2 years after infection (infected-vaccinated). We evaluated the presence of IgG antibodies, neutralising antibodies, and memory B-cell and memory T-cell responses against the prototype strain and delta and omicron variants. In infected-unvaccinated participants, neutralising antibody titres continually declined from 6-month to 2-year follow-up visits, with a half-life of about 141·2 days. Neutralising antibody responses to omicron sublineages (BA.1, BA.1.1, BA.2, BA.4/5, BF.7, BQ.1, and XBB) were poor. Memory B-cell responses to the prototype strain were retained at 2 years and presented cross-reactivity to the delta and omicron BA.1 variants. The magnitude of interferon γ and T-cell responses to SARS-CoV-2 were not significantly different between 1 year and 2 years after infection. Multifunctional T-cell responses against SARS-CoV-2 spike protein and nucleoprotein were detected in most participants. Recognition of the BA.1 variant by memory T cells was not affected in most individuals. The antibody titres and the frequencies of memory B cells, but not memory T cells, increased in infected-vaccinated participants after they received the inactivated vaccine. This study improves the understanding of the duration of SARS-CoV-2-specific immunity without boosting, which has implications for the design of vaccination regimens and programmes. Our data suggest that memory T-cell responses primed by initial viral infection remain highly cross-reactive after 2 years. With the increasing emergence of variants, effective vaccines should be introduced to boost neutralising antibody and overall T-cell responses to newly emerged SARS-CoV-2 variants. Chinese Academy of Medical Sciences, National Natural Science Foundation of China, Fundamental Research Funds for the Central Universities for Peking Union Medical College, Beijing Natural Science Foundation, UK Medical Research Council.

An RNA-Scaffold Protein Subunit Vaccine for Nasal Immunization.

Developing recombinant proteins as nasal vaccines for inducing systemic and mucosal immunity against respiratory viruses is promising. However, additional adjuvants are required to overcome the low immunogenicity of protein antigens. Here, a self-adjuvanted protein-RNA ribonucleoprotein vaccine was developed and found to be an effective nasal vaccine in mice and the SARS-CoV-2 infection model. The vaccine consisted of spike RBD (as an antigen), nucleoprotein (as an adaptor), and ssRNA (as an adjuvant and RNA scaffold). This combination robustly induced mucosal IgA, neutralizing antibodies and activated multifunctional T-cells, while also providing sterilizing immunity against live virus challenge. In addition, high-resolution scRNA-seq analysis highlighted airway-resident immune cells profile during prime-boost immunization. The vaccine also possesses modularity (antigen/adaptor/RNA scaffold) and can be made to target other viruses. This protein-RNA ribonucleoprotein vaccine is a novel and promising approach for developing safe and potent nasal vaccines to combat respiratory virus infections.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specificTcell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall Tcell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate Tcell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory Tcells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional Tcell responses with overall Tcell responses remaining despite the identification of escape mutations.

SARS-CoV-2-specific antibody and T-cell responses 1 year after infection in people recovered from COVID-19: a longitudinal cohort study

BackgroundThe memory immune response is crucial for preventing reinfection or reducing disease severity. However, the robustness and functionality of the humoral and T-cell response to SARS-CoV-2 remains unknown 12 months after initial infection. The aim of this study is to investigate the durability and functionality of the humoral and T-cell response to the original SARS-CoV-2 strain and variants in recovered patients 12 months after infection.MethodsIn this longitudinal cohort study, we recruited participants who had recovered from COVID-19 and who were discharged from the Wuhan Research Center for Communicable Disease Diagnosis and Treatment at the Chinese Academy of Medical Sciences, Wuhan, China, between Jan 7 and May 29, 2020. Patients received a follow-up visit between Dec 16, 2020, and Jan 27, 2021. We evaluated the presence of IgM, IgA, and IgG antibodies against the SARS-CoV-2 nucleoprotein, Spike protein, and the receptor-binding domain 12 months after initial infection, using ELISA. Neutralising antibodies against the original SARS-CoV-2 strain, and the D614G, beta (B.1.351), and delta (B.1.617.2) variants were analysed using a microneutralisation assay in a subset of plasma samples. We analysed the magnitude and breadth of the SARS-CoV-2-specific memory T-cell responses using the interferon γ (IFNγ) enzyme-linked immune absorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) assay. The antibody response and T-cell response (ie, IFN-γ, interleukin-2 [IL-2], and tumour necrosis factor α [TNFα]) were analysed by age and disease severity. Antibody titres were also analysed according to sequelae symptoms.FindingsWe enrolled 1096 patients, including 289 (26·4%) patients with moderate initial disease, 734 (67·0%) with severe initial disease, and 73 (6·7%) with critical initial disease. Paired plasma samples were collected from 141 patients during the follow-up visits for the microneutralisation assay. PBMCs were collected from 92 of 141 individuals at the 12-month follow-up visit, of which 80 were analysed by ELISpot and 92 by ICS assay to detect the SARS-CoV-2-specific memory T-cell responses. N-IgG (899 [82·0%]), S-IgG (1043 [95·2%]), RBD-IgG (1032 [94·2%]), and neutralising (115 [81·6%] of 141) antibodies were detectable 12 months after initial infection in most individuals. Neutralising antibodies remained stable 6 and 12 months after initial infection in most individuals younger than 60 years. Multifunctional T-cell responses were detected for all SARS-CoV-2 viral proteins tested. There was no difference in the magnitude of T-cell responses or cytokine profiles in individuals with different symptom severity. Moreover, we evaluated both antibody and T-cell responses to the D614G, beta, and delta viral strains. The degree of reduced in-vitro neutralising antibody responses to the D614G and delta variants, but not to the beta variant, was associated with the neutralising antibody titres after SARS-CoV-2 infection. We also found poor neutralising antibody responses to the beta variant; 83 (72·2%) of 115 patients showed no response at all. Moreover, the neutralising antibody titre reduction of the recovered patient plasma against the delta variant was similar to that of the D614G variant and lower than that of the beta variant. By contrast, T-cell responses were cross-reactive to the beta variant in most individuals. Importantly, T-cell responses could be detected in all individuals who had lost the neutralising antibody response to SARS-CoV-2 12 months after the initial infection.InterpretationSARS-CoV-2-specific neutralising antibody and T-cell responses were retained 12 months after initial infection. Neutralising antibodies to the D614G, beta, and delta viral strains were reduced compared with those for the original strain, and were diminished in general. Memory T-cell responses to the original strain were not disrupted by new variants. This study suggests that cross-reactive SARS-CoV-2-specific T-cell responses could be particularly important in the protection against severe disease caused by variants of concern whereas neutralising antibody responses seem to reduce over time.FundingChinese Academy of Medical Sciences, National Natural Science Foundation, and UK Medical Research Council.

Adjuvants Differentially Modulate the Immunogenicity of Lassa Virus Glycoprotein Subunits in Mice.

Lassa Fever (LF) is an acute viral hemorrhagic fever caused by Lassa virus (LASV) that is primarily transmitted through contact with wild rodents in West Africa. Although several advanced vaccine candidates are progressing through clinical trials, some effective vaccines are virally vectored and thus require a stringent cold-chain, making distribution to rural and resource-poor areas difficult. Recombinant subunit vaccines are advantageous in this aspect as they can be thermostabilized and deployed with minimal storage and transportation requirements. However, antigen dose and adjuvant formulation must be carefully selected to ensure both the appropriate humoral and cell-mediated immune responses are elicited. In this study, we examine the immunogenicity of a two-step immunoaffinity-purified recombinant LASV glycoprotein (GP) with five clinical- and preclinical-grade adjuvants. Swiss Webster mice immunized intramuscularly with 2 or 3 doses of each vaccine formulation showed complete seroconversion and maximal GP-specific antibody response after two immunizations. Formulations with GPI-0100, LiteVax, Montanide™ ISA 51, and Montanide™ ISA 720 induced both IgG1 and IgG2 antibodies suggesting a balanced Th1/Th2 response, whereas formulation of LASV GP with Alhydrogel elicited a IgG1-dominant response. Splenocytes secreting both Th1 and Th2 cytokines i.e., IFN-γ, TNF-α, IL-2, IL-4 and IL-5, were observed from mice receiving both antigen doses formulated with ISA 720, LiteVax and GPI-0100. However, robust, multifunctional T-cells were only detected in mice receiving a higher dose of LASV GP formulated with GPI-0100. Our results emphasize the importance of careful adjuvant selection and lay the immunological basis for a recombinant subunit protein LF vaccine formulation.

Development and preclinical evaluation of virus‐like particle vaccine against COVID‐19 infection

BackgroundVaccines that incorporate multiple SARS‐CoV‐2 antigens can further broaden the breadth of virus‐specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS‐CoV‐2 VLP vaccine that incorporates the four structural proteins of SARS‐CoV‐2.MethodsVLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable‐resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine.ResultsTransiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS‐CoV‐2. Alum adsorbed, K3‐CpG ODN‐adjuvanted VLPs elicited high titer anti‐S, anti‐RBD, anti‐N IgG, triggered multifunctional Th1‐biased T‐cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals.ConclusionThese data suggest that VLPs expressing all four structural protein antigens of SARS‐CoV‐2 are immunogenic and can protect animals from developing COVID‐19 infection following vaccination.

Phase I study of a multitargeted recombinant Ad5 PSA/MUC-1/brachyury-based immunotherapy vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC)

BackgroundAntitumor vaccines targeting tumor-associated antigens (TAAs) can generate antitumor immune response. A novel vaccine platform using adenovirus 5 (Ad5) vectors [E1–, E2b–] targeting three TAAs—prostate-specific antigen (PSA), brachyury, and MUC-1—has...

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine.

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4+ and CD8+ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4+ and CD8+ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.

In vitro 4-1BB stimulation promotes expansion of CD8+ tumor-infiltrating lymphocytes from various sarcoma subtypes.

Tumor-specific tumor-infiltrating lymphocytes (TILs) can be in vitro expanded and have the ability to induce complete and durable tumor regression in some patients with melanoma following adoptive cell therapy (ACT). In this preclinical study, we investigated the feasibility of expanding TIL from sarcomas, as well as performing functional in vitro analyses on these. TILs were expanded in vitro by the use of IL2 stimulation with or without the addition of 4-1BB and CD3 antibodies. Phenotypical and functional analyses were mainly performed by flow cytometry. TILs were expanded from 25 of 28 (89%) tumor samples from patients with 9 different sarcoma subtypes. TILs were predominantly αβ T-cells of effector memory subtype with CD4+ dominance. In particular, CD8+ TIL highly expressed LAG3 and to a lesser degree PD-1 and BTLA. In total, 10 of 20 TIL cultures demonstrated in vitro recognition of autologous tumor. In some cases, the fraction of tumor-reactive T cells was more than 20%. 4-1BB stimulation augmented expansion kinetics and favored CD8+ occurrence. In conclusion, TIL expansion from sarcoma is feasible and expanded TILs highly express LAG3 and comprise multifunctional tumor-reactive T-cells.

Tumor ablation plus co-administration of CpG and saponin adjuvants affects IL-1 production and multifunctional T cell numbers in tumor draining lymph nodes

BackgroundTumor ablation techniques, like cryoablation, are successfully used in the clinic to treat tumors. The tumor debris remaining in situ after ablation is a major antigen depot, including neoantigens, which...

T-cells producing multiple combinations of IFNγ, TNF and IL10 are associated with mild forms of dengue infection.

Multifunctional interleukin 10 (IL10)+ Th1 cells have been implicated in favorable evolution of many infectious diseases, promoting an efficacious immune response while limiting immunopathology. Here, we investigated the presence of multifunctional CD4+ and CD8+ T-cells that expressed interferon gamma (IFNγ), IL10 and tumor necrosis factor (TNF), or its combinations during dengue infection. Peripheral blood mononuclear cells (PBMCs) from outpatients with dengue (mild dengue forms) and hospitalized patients (or patients with dengue with warning signs and severe dengue) were cultured in the presence of envelope (ENV) or NS3 peptide libraries of DENV during critical (hospitalization period) and convalescence phases. The production of IFNγ, IL10 and TNF by CD4+ and CD8+ T-cells was assessed by flow cytometry. Our data show that patients with mild dengue, when compared with patients with dengue with warning signs and severe dengue, presented higher frequencies of multifunctional T-cells like NS3-specific IFNγ/IL10-producing CD4+ T-cells in critical phase and NS3- and ENV-specific CD8+ T-cells producing IFNγ/IL10. In addition, NS3-specific CD8+ T-cells producing high levels of IFNγ/TNF and IFNγ/TNF/IL10 were also observed in the mild dengue group. We observed that multifunctional T-cells produced higher levels of cytokines as measured by intracellular content when compared with single producer T-cells. Importantly, multifunctional CD4+ and CD8+ T-cells producing IFNγ, TNF and IL10 simultaneously displayed positive correlation with platelet levels, suggesting a protective role of this population. The presence of IL10+ Th1 and IL10+ Tc1 multifunctional cells was associated with mild dengue presentation, suggesting that these cells play a role in clinical evolution of dengue infection.

Elite athletes on regular training show more pronounced induction of vaccine-specific T-cells and antibodies after tetravalent influenza vaccination than controls

Compliance of elite athletes with vaccination recommendations is low mainly based on concerns about side-effects and perceived poor vaccine efficacy due to continued physical training. We therefore employed seasonal influenza vaccination to investigate the effect of regular physical training on vaccine-induced cellular and humoral immunity in elite athletes and controls.Lymphocyte subpopulations and vaccine-specific T-cells were quantified and functionally characterized from 45 athletes and 25 controls before, and 1, 2 and 26 weeks after vaccination. Moreover, influenza-specific antibodies and their neutralizing function were quantified.Both groups showed a significant increase in vaccine-reactive CD4 T-cell levels which peaked one week after vaccination (p < 0.0001). The increase was significantly more pronounced in athletes (4.1-fold) compared to controls (2.3-fold; p = 0.0007). The cytokine profile changed from multifunctional T-cells co-producing IFNγ, IL-2 and TNFα to cells with restricted cytokine expression. This change in functionality was associated with a significant increase in CTLA-4 expression (p < 0.0001), which again was more pronounced in athletes. Likewise, the increase in neutralizing antibodies was stronger in athletes (p = 0.004 for H1N1; p = 0.032 for H3N2).In conclusion, both groups mounted a strong vaccine-specific cellular and humoral immunity after standard vaccination. The more pronounced increase in specific T-cells and neutralizing antibodies indicates that high frequency and intensity of training enhance vaccine-responses in elite athletes.

Prospective Assessment of Cytomegalovirus Immunity in High-Risk Donor-Seropositive/Recipient-Seronegative Liver Transplant Recipients Receiving Either Preemptive Therapy or Antiviral Prophylaxis.

The differential impact of preemptive therapy (PET) and antiviral prophylaxis (AP) on development of cytomegalovirus (CMV)-specific neutralizing antibody (nAb) and T-cell responses have not previously been directly compared in high-risk donor-seropositive/recipient-seronegative (D+R-) organ transplant recipients. We prospectively assessed T-cell and nAb responses 3 months after transplantation in cohorts of high-risk D+R- liver transplant recipients who received either PET (n = 15) or AP (n = 25) and a control group of CMV-seropositive transplant recipients (R+) (AP; n = 24). CMV phosphoprotein 65 (pp65)- and immediate early protein 1-specific multifunctional T-cell responses were determined by means of intracellular cytokine staining and nAbs against BADrUL131-Y4 CMV in adult retinal pigment epithelial cell line-19 human epithelial cells; nAbs were detected in 8 of 12 (67%) in the PET group, none of 17 in the AP group, and 20 of 22 (91%) in the R+ group. Multifunctional CD8 and CD4 T-cell responses to pp65 were generally similar between PET and R+ groups, and lower for the AP group; multifunctional CD4 responses were similar across all groups. Among D+R- liver transplant recipients, PET was associated with the development of greater nAb and multifunctional CD8 T-cell responses compared with AP, providing a potential mechanism to explain the relative protection against late-onset disease with PET. Future studies are needed to define specific immune parameters predictive of late-onset CMV disease with AP.

Abstract B039: Peptide-MHC-directed expansion of multifunctional antigen-responsive T-cells

Abstract Functional properties and antigen specificity of expanded T-cells are crucial for the efficacy of adoptive cell transfer-based therapies in cancer. Most current strategies involve nonspecific expansion of bulk tumor-infiltrating lymphocytes, often providing growth preference to co-infiltrated virus specific T-cells and driving an exhausted phenotype of the expanded T-cell product.A potential way to resolve this challenge, is the use of artificial antigen-presenting scaffolds providing both an antigen specific stimulation through peptide-MHC interaction and additional the required co-stimulatory and growth signals through associated stimulatory molecules and cytokines. We have designed such antigen-presenting scaffolds; build on a dextran-polysaccharide, carrying both peptide-MHC and relevant stimulatory molecules. The artificial antigen-presenting scaffolds interacts specifically with T-cells based on recognition of the peptide-MHC molecule and effectively expand and functionally stimulate specific T-cells in a peptide-MHC-directed fashion, while leaving all other T-cell specificities untouched. Results from in vitro experiments have showed that antigen specific CD8 T-cells stimulated with these artificial antigen-presenting scaffolds express a less differentiated phenotype and low PD-1 expression, associated with high proliferation potential and enhanced antitumor effect in vivo. Furthermore, this expansion strategy provides a high frequency of multifunctional antigen specific CD8 T-cells expressing IFN-γ, TNF-α, and CD107a upon target recognition and provide improved cancer cell killing over IL2-driven expansion of tumor-infiltrating lymphocytes. Furthermore, the current strategy allows for simultaneous expansion of numerous different T-cell populations, required to generate T-cell products with broad recognition profiles based on the personal cancer-antigen and mutational profile. All of these characteristics are of significant importance for in vivo tumor cell killing following adoptive transfer of expanded T-cell products. Thus, the present strategy represents an optimized method for expansion of cancer-restricted T-cells for adoptive cell therapy. Citation Format: Vibeke Mindahl Rafa, Mona Bodenhöfer, Amalie Kai Bentzen, Tripti Tamhane, Marco Donia, Inge Marie Stentoft Svane, Søren Nyboe Jakobsen, Christian Schmess, Sine Reker Hadrup. Peptide-MHC-directed expansion of multifunctional antigen-responsive T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B039.

Abstract A020: Immunomonitoring for actively personalized peptide vaccines (APVACs) during immunotherapeutic treatment of glioblastoma

Abstract Cancer immunotherapy to gliomas has so far failed to show encouraging results, as gliomas are rarely mutated and show various mechanisms of immune escape. To improve therapy to these type of cancer, the Glioma Actively Personalized Vaccine Consortium (GAPVAC) integrated a highly personalized peptide vaccine approach into glioblastoma standard of care treatment combining neoepitope and nonmutated tumor antigens to exploit the full repertoire of tumor antigens. In this phase I clinical trial fifteen patients received two different types of personalized peptide vaccines (APVAC1 and APVAC2), that were selected based on transcriptome, immunopeptidome and mutational analysis of the patient’s individual tumors. While APVAC1 vaccines were composed of nonmutated tumor antigens selected in a warehouse-based approach, APVAC2 vaccines primarily targeted neoepitopes. Both vaccines were used in combination with poly-ICLC and GM-CSF as adjuvants and demonstrated expected safety profile and outstanding Immunogenicity. Immunomonitoring of APVAC1 peptides was carried out using a combinatorial ex vivo Class I 2D multimer (2DMM) and Class II intracellular cytokine staining (ICS) assay with an outstanding sensitivity to detect even one peptide-specific cell in one million of CD4 or CD8 T-cells. Nonmutated APVAC1 class I peptides showed induction of persistent CD8 T-cell responses, mainly consisting of a highly favorable central memory phenotype (CM). Furthermore, APVAC1 class II peptides demonstrated induction of polyfunctional CD4 T-cells predominantly of a type 1 T helper cell (TH1) phenotype. Notably, an APVAC1 class II specific T-cell response was detected in tumor-infiltrating lymphocyte (TIL) fraction obtained from resection of one patient. On the other side, immune responses to APVAC2 peptides were analyzed using a pan-ICS assay including a single in vitro sensitization step to analyze a broad array of cytokines produced by CD4 T helper (TH) cells and CD8 CTLs in parallel. APVAC2 peptides showed excellent immunogenicity and induced potent and multifunctional CD4 T-cell responses, mostly of a TH1 phenotype that often concurred with CTL responses. Furthermore, the induction of APVAC1-specific CD8 memory cells, as a marker for the potency of the vaccine-induced immune responses, reversely correlated with the baseline frequencies of regulatory T-cells (Treg). Taken together, actively personalized peptide vaccines (APVACs) were highly immunogenic and induced sustained responses of a highly favorable CD4 and CD8 T-cell phenotype. The vaccination showed the expected safety profile and the approach was feasible, even in this highly individualized setting. Therefore, the APVAC vaccination approach clearly represents a step forward on the path to bring the benefit of immunotherapy to glioblastoma patients. Citation Format: Alexander Ulges, Norbert Hilf, Wolfgang Wick, Michael Platten, Pierre-Yves Dietrich, Katrin Frenzel, Arie Admon, Sjoerd S.H. van der Burg, Andreas von Deimling, Per thor Straten, Cecile Gouttefangeas, Judith R. Kroep, Francisco Martínez-Ricarte, Hideo Okada, Christian H. Ottensmeier, Berta Ponsati, Hans S. Poulsen, Stefan Stevanovic, Ghazaleh Tabatabai, Hans-Georg Rammensee, Ugur Sahin, Dominik Maurer, Regina Mendrzyk. Immunomonitoring for actively personalized peptide vaccines (APVACs) during immunotherapeutic treatment of glioblastoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A020.

Friends and foes of tuberculosis: modulation of protective immunity.

Protective immunity in tuberculosis (TB) is subject of debate in the TB research community, as this is key to fully understand TB pathogenesis and to develop new promising tools for TB diagnosis and prognosis as well as a more efficient TB vaccine. IFN-γ producing CD4+ T cells are key in TB control, but may not be sufficient to provide protection. Additional subsets have been identified that contribute to protection such as multifunctional and cytolytic T-cell subsets, including classical and nonclassical T cells as well as novel innate immune cell subsets resulting from trained immunity. However, to define protective immune responses against TB, the complexity of balancing TB immunity also has to be considered. In this review, insights into effector cell immunity and how this is modulated by regulatory cells, associated comorbidities and the host microbiome, is discussed. We systematically map how different suppressive immune cell subsets may affect effector cell responses at the local site of infection. We also dissect how common comorbidities such as HIV, helminths and diabetes may bias protective TB immunity towards pathogenic and regulatory responses. Finally, also the composition and diversity of the microbiome in the lung and gut could affect host TB immunity. Understanding these various aspects of the immunological balance in the human host is fundamental to prevent TB infection and disease.

The increased protection and pathology in Mycobacterium tuberculosis-infected IL-27R-alpha-deficient mice is supported by IL-17A and is associated with the IL-17A-induced expansion of multifunctional T cells.

During Mycobacterium tuberculosis (Mtb) infection, mice lacking the IL-27R exhibit lower bacterial burdens but develop an immunopathological sequelae in comparison to wild-type mice. We here show that this phenotype correlates with an enhanced recruitment of antigen-specific CCR6+ CD4+ T cells and an increased frequency of IL-17A-producing CD4+ T cells. By comparing the outcome of Mtb infection in C57BL/6, IL-27R-deficient and IL-27R/IL-17A-double deficient mice, we observed that both the increased protection and elevated immunopathology are supported by IL-17A. Whereas IL-17A neither impacts the development of Tr1 cells nor the expression of PD1 and KLRG1 on T cells in IL-27R-deficient mice during infection, it regulates the presence of multifunctional T-cells in the lungs, co-expressing IFN-γ, IL-2 and TNF. Eventually, IL-17A supports Cxcl9, Cxcl10 and Cxcl13 expression and the granulomatous response in the lungs of infected IL-27R-deficient mice. Taken together, IL-17A contributes to protection in Mtb-infected IL-27R-deficient mice probably through a chemokine-mediated recruitment and strategic positioning of multifunctional T cells in granulomas. As IL-27 limits optimal antimycobacterial protection by inhibiting IL-17A production, blocking of IL-27R-mediated signaling may represent a strategy for improving vaccination and host-directed therapy in tuberculosis. However, because IL-27 also prevents IL-17A-mediated immunopathology, such intervention has to be tightly controlled.