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T helper 17 (TH17) cells located at the Peyer’s patch (PP) inductive site and at the lamina propria effector site of the intestinal immune system are responsive to both pathogenic and commensal bacteria. Their plasticity to convert into follicular helper T (TFH) cells has been proposed to be central to gut immunoglobulin A (IgA) responses. Here, we used an IL-17A fate reporter mouse and an MHC-II tetramer to analyze antigen-specific CD4+T cell subsets and isolate them for single-cell RNA sequencing after oral immunization with cholera toxin and ovalbumin. We found a TFH-dominated response with only rare antigen-specific TH17 cells (FHand TH17 cells, arguing against TH17 plasticity as a major contributor to TFHdifferentiation. Two mouse models of TH17 deficiency confirmed that gut IgA responses to oral immunization do not require TH17 cells, withCD4CreRorcfl/flmice exhibiting normal germinal centers in PP and unperturbed total IgA production in the intestine.

T helper 17 (T

Vaccination of neonates and young infants is hampered by the relative immaturity of their immune systems and the lack of safe and efficacious vaccine adjuvants. Immaturity of the follicular dendritic cells (FDCs), in particular, appears to play a critical role for the inability to stimulate immune responses. Using the CD21mT/mG mouse model we found that at 7 days of life, FDCs exhibited a mature phenotype only in the Peyer´s patches (PP), but our unique adjuvant, CTA1-DD, effectively matured FDCs also in peripheral lymph nodes following systemic, as well as mucosal immunizations. This was a direct effect of complement receptor 2-binding to the FDC and a CTA1-enzyme-dependent enhancing effect on gene transcription, among which CR2, IL-6, ICAM-1, IL-1β, and CXCL13 encoding genes were upregulated. This way we achieved FDC maturation, increased germinal center B-cell- and Tfh responses, and enhanced specific antibody levels close to adult magnitudes. Oral priming immunization of neonates against influenza infection with CTA1-3M2e-DD effectively promoted anti-M2e-immunity and significantly reduced morbidity against a live virus challenge infection. To the best of our knowledge, this is the first study to demonstrate direct effects of an adjuvant on FDC gene transcriptional functions and the subsequent enhancement of neonatal immune responses.

Bacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti‐tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro‐inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow‐derived dendritic cells. Co‐immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma‐bearing mice through Th‐1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti‐PD‐1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti‐tumour responses using immunotherapeutic bacterial SyBV.

Background Checkpoint inhibitors work only in cancers that host inflammatory cells, and ‘cold’ tumors normally do not respond. Therefore, making ‘cold’ tumors ‘hot’ is required to increase the response rate to immunooncology therapies in general. Bacteria and bacterial products have been utilized for cancer immunotherapy for more than 100 years, but currently no such treatment is available because of the severe side effects that are observed. In this study, we produced artificial outer membrane vesicles (aOMVs) from Escherichia coli outer membrane, and injected them together with cancer tissue-derived exosomes to booster an immune response to the malignancy. Methods Outer membranes were obtained from E. coli by chemical means, followed by ionic stress and applied mild energy to generate aOMVs. The yield and purity of aOMVs were analyzed by nanoparticle tracking analysis and transmission electron microscopy. The protein and RNA contents were examined by label-free quantitative mass spectrometry and bioanalyzer. Inflammation was evaluated in macrophage cell line (RAW 264.7) and mice in vivo, and bone marrow-derived dendritic cells were used to assess the immunomodulatory functions of the aOMVs. For the study of antitumor activity, mice were subcutaneously inoculated with B16F10 cells and then subcutaneously immunized with aOMVs and melanoma exosomes five times at 3-day intervals. Also, anti-mouse PD-1 antibody was intraperitoneally injected into mice 1 day prior to immunization to investigate the effects of combination therapy. To elucidate the immunogenic mechanism, blood and spleen were obtained for antibody titer and splenocyte function study. Results Bacterial aOMVs presented nanosized spherical shape with closed membranes and exhibited high yield and purity with very few cytosolic components. These aOMVs do not cause pro-inflammatory cytokine responses in RAW 264.7 cells and mice in vivo, despite high exposure levels. The aOMVs could be taken up by dendritic cells to stimulate cytokine and maturation marker expression. Co-immunization with aOMVs and melanoma tissue-derived exosomes elicited tumor regression in melanoma-bearing mice through Th-1 type T cell immunity and anti-tumor exosome IgG antibody production. Also, the immunotherapeutic effect of aOMVs was synergistically enhanced by anti-PD-1 inhibitor. Conclusions Bacterial aOMVs can be produced in a large quantities with high purity, but are ‘detoxified’ compared to naturally released OMVs. The non-toxic aOMVs are powerful adjuvants for eliciting specific anti-tumor response, suggesting that aOMVs may be novel bacterial vesicle-mimetics clinically applicable as cancer treatment.

This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4+ T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4+ T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza.

Our understanding of how class-switch recombination (CSR) to IgA occurs in the gut is still incomplete. Earlier studies have indicated that Tregs are important for IgA CSR and these cells were thought to transform into follicular helper T cells (Tfh), responsible for germinal center formation in the Peyer's patches (PP). Following adoptive transfer of T-cell receptor-transgenic (TCR-Tg) CD4 T cells into nude mice, we unexpectedly found that oral immunization did not require an adjuvant to induce strong gut IgA and systemic IgG responses, suggesting an altered regulatory environment in the PP. After sorting of splenic TCR-Tg CD4 T cells into CD25

Adjuvanted whole inactivated virus (WIV) influenza vaccines show promise as broadly protective influenza vaccine candidates. Using WIV as basis we assessed the relative efficacy of different adjuvants by carrying out a head-to-head comparison of the liposome-based adjuvants CAF01 and CAF09 and the protein-based adjuvants CTA1-DD and CTA1-3M2e-DD and evaluated whether one or more of the adjuvants could induce broadly protective immunity. Mice were immunized with WIV prepared from A/Puerto Rico/8/34 (H1N1) virus intramuscularly with or without CAF01 or intranasally with or without CAF09, CTA1-DD, or CTA1-3M2e-DD, followed by challenge with homologous, heterologous or heterosubtypic virus. In general, intranasal immunizations were significantly more effective than intramuscular immunizations in inducing virus-specific serum-IgG, mucosal-IgA, and splenic IFNγ-producing CD4 T cells. Intranasal immunizations with adjuvanted vaccines afforded strong cross-protection with milder clinical symptoms and better control of virus load in lungs. Mechanistic studies indicated that non-neutralizing IgG antibodies and CD4 T cells were responsible for the improved cross-protection while IgA antibodies were dispensable. The role of CD4 T cells was particularly pronounced for CTA1-3M2e-DD adjuvanted vaccine as evidenced by CD4 T cell-dependent reduction of lung virus titers and clinical symptoms. Thus, intranasally administered WIV in combination with effective mucosal adjuvants appears to be a promising broadly protective influenza vaccine candidate.

Checkpoint inhibitors work only in cancers that host inflammatory cells, and “cold” tumors normally do not respond. Therefore, making “cold” tumors “hot” is required to increase the response rate to immunooncology therapies in general. Bacteria and bacterial products have been utilized for cancer immunotherapy for more than 100 years, but currently no such treatment is available because of the severe side effects that are observed. In this study, we produced artificial outer membrane vesicles (aOMVs) from Escherichia coli outer membrane, and injected them together with cancer tissue-derived exosomes to booster an immune response to the malignancy. Firstly, outer membranes were obtained from E. coli by chemical means, followed by ionic stress and applied mild energy to generate aOMVs. The yield and purity of aOMVs were analyzed by nanoparticle tracking analysis and transmission electron microscopy. The protein and RNA contents were examined by label-free quantitative mass spectrometry and bioanalyzer. Inflammation was evaluated in macrophage cell line (RAW 264.7) and mice in vivo, and bone marrow-derived dendritic cells were used to assess the immunomodulatory functions of the aOMVs. For the study of antitumor activity, mice were subcutaneously inoculated with B16F10 cells and then subcutaneously immunized with aOMVs and melanoma exosomes five times at 3-day intervals. Also, anti-mouse PD-1 antibody was intraperitoneally injected into mice 1 day prior to immunization to investigate the effects of combination therapy. To elucidate the immunogenic mechanism, blood and spleen were obtained for antibody titer and splenocyte function study. As a result, bacterial aOMVs presented nanosized spherical shape with closed membranes and exhibited high yield and purity with very few cytosolic components. These aOMVs do not cause pro-inflammatory cytokine responses in RAW 264.7 cells and mice in vivo, despite high exposure levels. The aOMVs could be taken up by dendritic cells to stimulate cytokine and maturation marker expression. Co-immunization with aOMVs and melanoma tissue-derived exosomes elicited tumor regression in melanoma-bearing mice through Th-1 type T cell immunity and anti-tumor exosome IgG antibody production. Also, the immunotherapeutic effect of aOMVs was synergistically enhanced by anti-PD-1 inhibitor. Collectively, bacterial aOMVs can be produced in a large quantities with high purity, but are “detoxified" compared to naturally released OMVs. The non-toxic aOMVs are powerful adjuvants for eliciting specific anti-tumor response, suggesting that aOMVs may be novel bacterial vesicle-mimetics clinically applicable as cancer treatment. Citation Format: Kyong-su Park, Kristina Svennerholm, Rossella Crescitelli, Cecilia Lässer, Inta Gribonika, Jan Lötvall. Synergistic cancer immunotherapy using tumor tissue derived exosomes and artificially produced bacterial outer membrane vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-095.

Gut Th17 cells are present at the Peyer’s patch inductive site as well as at the lamina propria effector site of the intestinal immune system. These cells are responsive to pathogens as well as to the commensal microbiota and they appear to be critical for gut homeostasis. While they have been associated with immune protection against pathogens they are also known to drive autoimmune inflammation in the gut. Hence, plasticity is a hallmark of gut Th17 cells and pro- as well as anti-inflammatory functions can be expected. This places Th17 cells at center stage for immune regulation of the gut immune system. However, their role for gut IgA responses to oral immunization awaits to be better defined, although earlier studies suggested that Th17 cells play a central role as their plasticity can convert them into follicular helper T (Tfh) cells that stimulate IgA class-switching and drive gut IgA responses. Here we used an IL-17 fate-reporter mouse and a cholera toxin B-subunit (CTB)-specific tetramer to isolate CTB-specific CD4 T cell subsets following oral immunization with CT. We found no evidence of CTB-specific Th17 development in the PP, rather a clear dominance of Tfh cells was observed. Single-cell RNAseq analysis revealed a massive clonotypic dominance of Tfh cells, but no Th17 or regulatory T cells (Tregs) were found among the CTB-tetramer-specific CD4 T cells. While 3 clusters of Tfh cells were found only one cluster hosted the CTB-specific CD4 T cells. An adoptive transfer of rorgt-deficient CD4 T cells into nu/nu recipient mice exhibited unperturbed gut IgA responses to oral CT immunization, clearly demonstrating that Th17 cells are, indeed, dispensible for gut IgA responses following oral immunization.

We have identified a unique mouse model in which oral immunization with soluble protein, without a potent mucosal adjuvant, is highly effective at stimulating strong gut IgA responses. In fact, cholera toxin (CT), which is the gold standard for oral adjuvants, did not enhance the gut IgA response to ovalbumin (OVA) in this model. As we dissected the mechanism allowing for an unadjuvanted strong gut IgA response we found that adoptively transferred OVA-peptide-specific TCR Tg DO11.10 CD4 T cells into nude Balb/c mice were sufficient to promote the gut IgA response. However, only CD4 T cells with a rearranged TCR a-chain, reactive to unknown, but possibly microbial, antigens in the gut, were effective, because SCID DO11.10 CD4 T cells failed to support a gut IgA response. Co-transfer of wild-type CD4 T cells with DO11.10 CD4 T cells into nude Balb/c hosts completely obliterated the response, but not if the CD4 T cells were isolated from IL-10−/− mice. Hence, the gut IgA response was under strict regulatory CD4 T cell control. By contrast, if CT adjuvant was admixed with OVA we completely rescued the gut IgA response following oral immunization. Thus, oral CT adjuvant primarily exerted a modulating effect on the regulatory CD4 T cell control in Peyer’s patches (PP), impacting on the balance between follicular helper and regulatory T cells. While both Th17 and CD25+ Tregs, have been claimed sufficient for supporting gut IgA responses, our model clearly suggests that the regulatory environment in PP is more complex, as will be discussed.

The gut is the largest lymphoid organ in the body. Due to intense and constant exposure to the outside world, it also functions as the most important portal of entry for many pathogens. T cell-dependent secretory immunoglobulin A (IgA) prevents pathogens from spreading to systemic tissues and, hence, oral immunization represents the most effective route for vaccination against these pathogens. The detailed mechanism of oral vaccination-induced protective IgA immunity is not fully understood. The main aim of this thesis was to investigate the role of the gut CD4 T subsets for the induction of IgA responses. By using Ovalbumin-specific TCR-Tg CD4 T cells in an adoptive transfer system and mucosal immunization with or without cholera toxin (CT) adjuvant I show that IgA induction in the Peyer's patch (PP) is regulated in a distinct two-step process, where T follicular helper cells (TFH) and thymus-derived T regulatory cells (tTreg) orchestrate the IgA induction. Effective B cell help in the germinal center (GC) is maintained by antigen-specific TFH cells, while IgA class-switch recombination (CSR) is promoted by tTregs independently of the immunizing antigen. It should be emphasized that the default response pathway activated by oral antigen administration is oral tolerance. In this doctoral thesis, I demonstrate that the suppressive pathway is regulated by IL-10. Thus, CD4 T cells upon exposer to cognate antigen in the presence of IL-10 differentiate into peripherally induced Tregs (pTreg). In the absence of IL-10 or after addition of CT adjuvant TFH differentiation is enhanced, resulting in a strong gut IgA response. CT has been reported to be the most potent oral adjuvant. Some reports suggest that CT preferentially exerts the adjuvant function via Th17 cells. The immuno-dominant part of CT is its B subunit, therefore, I used CTB-specific tetramer to monitor if CT induced T cell response is dominated by Th17 cells. Surprisingly, the CTB-specific T cell repertoire was nearly absent of Th17 lineage, however that did not prevent adjuvant’s ability to induce a strong gut IgA response. Instead, CT induced CD4 T cells were overrepresented by TFH lineage that did not derive from Th17 cells as shown by using IL-17 fate reporter mice. These observations were confirmed using single-cell RNAseq technology. Gene signature of sorted CTB-specific CD4 T cells showed an almost complete dominance of the TFH phenotype with virtually no Th17 signature. Besides, the adoptive transfer of Th17 deficient CD4 T cells (Rorc-/-) into nude host allowed for a robust gut IgA induction after oral immunization with CT. These findings argue strongly against the observations that upon CT immunization gut IgA B cell responses are driven by Th17 cells that exhibit great plasticity towards the TFH lineage. Interestingly, obtained data suggest that TFH cells in the PP do not share clonal relatedness with Th17, Th1 or Treg cells which have been a long-standing controversy in this field. Together, these findings provide a new paradigm for how gut IgA responses are regulated and which two types of CD4 T cell subsets are needed; tTregs for IgA CSR and TFH for GC formation and B cell maturation.