Human In vitro Modeling Identifies Adjuvant Combinations that Unlock Antigen Cross-presentation and Promote T-helper 1 Development in Newborns, Adults and Elders.

Adjuvants are vaccine components that can boost the type, magnitude, breadth, and durability of an immune response. We have previously demonstrated that certain adjuvant combinations can act synergistically to enhance and shape immunogenicity including promotion of Th1 and cytotoxic T-cell development. These combinations also promoted protective immunity in vulnerable populations such as newborns. In this study, we employed combined antigen-specific human in vitro models to identify adjuvant combinations that could synergistically promote the expansion of vaccine-specific CD4+ cells, induce cross-presentation on MHC class I, resulting in antigen-specific activation of CD8+ cells, and direct the balance of immune response to favor the production of Th1-promoting cytokines. A screen of 78 adjuvant combinations identified several T cell-potentiating adjuvant combinations. Remarkably, a combination of TLR9 and STING agonists (CpG + 2,3-cGAMP) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored production of Th1-polarizing cytokines TNF and IL-12p70 over co-regulated cytokines IL-6 and IL-12p40, respectively. Phenotypic reprogramming towards cDC1-type dendritic cells by CpG + 2,3-cGAMP was also observed. Finally, we characterized the molecular mechanism of this adjuvant combination including the ability of 2,3-cGAMP to enhance DC expression of TLR9 and the dependency of antigen-presenting cell activation on the Sec22b protein important to endoplasmic reticulum-Golgi vesicle trafficking. The identification of the adjuvant combination CpG + 2,3-cGAMP may therefore prove key to the future development of vaccines against respiratory viral infections tailored for the functionally distinct immune systems of vulnerable populations such as older adults and newborns.

Both T cell priming in lymph node and CXCR3-dependent migration are the key events for predicting the response of atezolizumab.

Anti-PD-L1 antibodies benefit many cancer patients, even those with "non-inflamed tumor". Determining which patients will benefit remains an important clinical goal. In a non-inflamed tumor mouse model, we found that PD-L1 was highly expressed on antigen-presenting cells (APCs) especially on CD103+ CD11c+ dendritic cells in tumor-draining lymph nodes (dLNs), suppressing T-cell priming by APCs. In this model, anti-PD-L1 antibodies enhanced T-cell priming and increased CXCR3+ activated T-cells in dLNs, which was followed by the trafficking of T-cells to tumors in response to CXCR3 ligands. As predictive biomarker, each APCs-related gene expression (AP score) alone or T-cells trafficking-related chemokine gene expression (T score) alone were still less than perfect among the 17 mouse models examined. However a combining score of AP score and T score (AP/T score) precisely identified anti-PD-L1-sensitive tumors. In the phase 3 trial of atezolizumab vs docetaxel in advanced NSCLC patients (OAK), the AP/T score could identify atezolizumab-treated NSCLC patients who achieved significant improvement in overall survival. This biomarker concept would be a clinically valuable for prediction of anti-PD-L1 antibody efficacy.

Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity.

Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8+ T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8+ T cell responses.

Recombinant Orthopoxvirus Primes Colon Cancer for Checkpoint Inhibitor and Cross-Primes T Cells for Antitumor and Antiviral Immunity.

Although it is known that oncolytic viruses can inflame and recruit immune cells to otherwise immunosuppressed tumor microenvironments, the influence of the antiviral immune response on antitumor immunity is less clear across viral platforms and tumor types. CF33 is a recombinant orthopoxvirus backbone effective against colon cancer. We tested derivatives of CF33 with and without immune-checkpoint inhibition (anti-PD-L1) in mouse models of colon cancer. Results showed that the efficacy of CF33 backbone with J2R deletion (single-deleted) against colon cancer is not altered by additional deletion of F14.5L in vitro or in vivo CF33 infection upregulated PD-L1 expression on tumor cells and led to an increased influx of lymphocytes and macrophages in tumors. Also, the levels of active CD8+ (IFNγ+) T cells in the virus-treated tumors were higher than those in control-treated tumors. Furthermore, a combination of CF33 derivatives with anti-PD-L1 resulted in durable tumor regression and long-term survival, resistant to tumor rechallenge. Analysis of immune cells from the treated mice showed that tumor-specific T cell activation occurred more robustly in tumors treated with the virus and that T cells were more strongly activated against the virus than against tumor, in an MHC-I-dependent manner. Our findings warrant further studies on the role of cross-priming of T cells against viral and tumor antigens, in the overall success of viroimmunotherapy.

Autophagy regulates long-term cross-presentation by murine dendritic cells.

Autophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endolysosomal compartments and thereby sustain MHCI antigen cross-presentation to CD8+ T cells. In the current study, we investigated the role of autophagy in long-term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5-/- DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross-presentation. This augmented antigen cross-presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long-term MHCI cross-presentation.

Combination of Photodynamic Therapy and a Flagellin-Adjuvanted Cancer Vaccine Potentiated the Anti-PD-1-Mediated Melanoma Suppression.

Immune checkpoint inhibitors become a standard therapy for malignant melanoma. As immune checkpoint inhibitor monotherapies proved to have limited efficacy in significant portion of patients, it is envisaged that combination with other therapeutic modalities may improve clinical outcomes. We investigated the effect of combining photodynamic therapy (PDT) and TLR5 agonist flagellin-adjuvanted tumor-specific peptide vaccination (FlaB-Vax) on the promotion of PD-1 blockade-mediated melanoma suppression using a mouse B16-F10 implantation model. Using a bilateral mouse melanoma cancer model, we evaluated the potentiation of PD-1 blockade by the combination of peritumoral FlaB-Vax delivery and PDT tumor ablation. A photosensitizing agent, pheophorbide A (PhA), was used for laser-triggered photodynamic destruction of the primary tumor. The effect of combination therapy in conjunction with PD-1 blockade was evaluated for tumor growth and survival. The effector cytokines that promote the activation of CD8+ T cells and antigen-presenting cells in tumor tissue and tumor-draining lymph nodes (TDLNs) were also assayed. PDT and FlaB-Vax combination therapy induced efficacious systemic antitumor immune responses for local and abscopal tumor control, with a significant increase in tumor-infiltrating effector memory CD8+ T cells and systemic IFNγ secretion. The combination of PDT and FlaB-Vax also enhanced the infiltration of tumor antigen-reactive CD8+ T cells and the accumulation of migratory CXCL10-secreting CD103+ dendritic cells (DCs) presumably contributing to tumor antigen cross-presentation in the tumor microenvironment (TME). The CD8+ T-cell-dependent therapeutic benefits of PDT combined with FlaB-Vax was significantly enhanced by a PD-1-targeting checkpoint inhibitor therapy. Conclusively, the combination of FlaB-Vax with PDT-mediated tumor ablation would serve a safe and feasible combinatorial therapy for enhancing PD-1 blockade treatment of malignant melanoma.

Development of Spherical Nucleic Acids for Prostate Cancer Immunotherapy.

Although the strategy of therapeutic vaccination for the treatment of prostate cancer has advanced to and is available in the clinic (Sipuleucel-T), the efficacy of such therapy remains limited. Here, we develop Immunostimulatory Spherical Nucleic Acid (IS-SNA) nanostructures comprised of CpG oligonucleotides as adjuvant and prostate cancer peptide antigens, and evaluate their antitumor efficacy in syngeneic mouse models of prostate cancer. IS-SNAs with the specific structural feature of presenting both antigen and adjuvant CpG on the surface (hybridized model (HM) SNAs) induce stronger cytotoxic T lymphocyte (CTL) mediated antigen-specific killing of target cells than that for IS-SNAs with CpG on the surface and antigen encapsulated within the core (encapsulated model (EM) SNAs). Mechanistically, HM SNAs increase the co-delivery of CpG and antigen to dendritic cells over that for EM SNAs or admixtures of linear CpG and peptide, thereby improving cross-priming of antitumor CD8+ T cells. As a result, vaccination with HM SNAs leads to more effective antitumor immune responses in two prostate cancer models. These data demonstrate the importance of the structural positioning of peptide antigens together with adjuvants within IS-SNAs to the efficacy of IS-SNA-based cancer immunotherapy.

Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity.

Cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumor immune responses. Yet, key steps involved in trafficking of antigens taken up by DCs remain incompletely understood. Here, we screen 700 US Food and Drug Administration (FDA)-approved drugs and identify 37 enhancers of antigen import from endolysosomes into the cytosol. To reveal their mechanism of action, we generate proteomic organellar maps of control and drug-treated DCs (focusing on two compounds, prazosin and tamoxifen). By combining organellar mapping, quantitative proteomics, and microscopy, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release. Enhancing antigen import facilitates cross-presentation of soluble and cell-associated antigens. Systemic administration of prazosin leads to reduced growth of MC38 tumors and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumor immune responses and efficacy of checkpoint blockers.

Induction of CML-specific immune response through cross-presentation triggered by CTP-mediated BCR-ABL-derived peptides.

Although targeted therapy using tyrosine kinase inhibitors (TKIs) has made remarkable progress in treating chronic myeloid leukemia (CML), this disease remains largely incurable, warranting further investigation of new therapeutic strategies. BCR-ABL is a highly specific tumor antigen in CML and provides an attractive opportunity for vaccination therapy. Exogenous antigens must be presented on MHC class I molecules-via a process termed cross-presentation-to activate specific cytotoxic T lymphocyte response. The relative efficiency of cross-presentation is determined in part by the ability of dendritic cells (DCs) to internalize and present antigens. Here, we present a novel tool that uses cytoplasmic transduction peptide (CTP) to facilitate the internalization of antigens by DCs in an endocytosis-independent manner, which greatly enhances the efficiency of antigen presentation, thereby inducing stronger cytotoxic activity to ensure the elimination of CML cells. The data suggest that CTP-fused CML-specific peptides can be applied in vaccination therapies for CML patients.

Ex vivo IL-15 replenishment augments bone marrow precursor cell-mediated adaptive immunity via PI3K-Akt pathway.

This study tested the hypothesis that PI3K-Akt activity contributes to the superior immune function of IL-15-administrated bone marrow precursor cells (BMPC). Our previous studies revealed that PI3K-Akt play vital role in dendritic cells (DCs) cross-presentation and DC-based CTL priming. Despite the fact that IL-15 serves multiple functions in its therapeutic potential for the induction and maintenance of T cell response, the exact role of PI3K-Akt in IL-15 increased adaptive immunity is still poorly understood. In this study, we demonstrated that ex vivo IL-15 administration increased BMPC capability of antigen uptake and the expression of costimulatory molecules (such as CD80 and 4-1BB(CD137) ligand [4-1BBL]) and MHC class I molecule via PI3K-Akt pathway. Importantly, PI3K-Akt activity was not only necessary for IL-15 augmented BMPC cross-presentation and CTL priming, but also facilitated IL-15 increased therapeutic potential of the cytolytic capacity and maintenance of BMPC-activated T cells. Thus, these data suggested that PI3K-Akt activity contribute to the superior immune function of IL-15-administrated BMPC and thereby might be therapeutic potential for adaptive immunity.

A Highly Active Form of XCL1/Lymphotactin Functions as an Effective Adjuvant to Recruit Cross-Presenting Dendritic Cells for Induction of Effector and Memory CD8+ T Cells.

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8+ T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to XCR1+ DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8+ T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1+CD103+ DCs in the injection site, and most of the accumulated XCR1+CD103+ DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1+CD103+ DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8+ T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8+ T cell responses to OVA, poly (I:C) poorly recruited XCR1+CD103+ DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8+ T cells.

Wear Particle-induced Priming of the NLRP3 Inflammasome Depends on Adherent Pathogen-associated Molecular Patterns and Their Cognate Toll-like Receptors: An In Vitro Study.

BACKGROUND: Orthopaedic wear particles activate the NLRP3 inflammasome to produce active interleukin 1ß (IL1ß). However, the NLRP3 inflammasome must be primed before it can be activated, and it is unknown whether wear particles induce priming. Toll-like receptors (TLRs) are thought to mediate particle bioactivity. It remains controversial whether pathogen-associated molecular patterns (PAMPs) and/or alarmins are responsible for TLR activation by wear particles. QUESTIONS/PURPOSES: (1) Does priming of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? (2) Does priming of the NLRP3 inflammasome by wear particles depend on TLRs and TIRAP/Mal? (3) Does priming of the NLRP3 inflammasome by wear particles depend on cognate TLRs? (4) Does activation of the NLRP3 inflammasome by wear particles depend on adherent PAMPs? METHODS: Immortalized murine macrophages were stimulated by as-received titanium particles with adherent bacterial debris, endotoxin-free titanium particles, or titanium particles with adherent ultrapure lipopolysaccharide. To study priming, NLRP3 and IL1ß mRNA and IL1ß protein levels were assessed in wild-type, TLR4, TLR2, and TIRAP/Mal macrophages. To study activation, IL1ß protein secretion was assessed in wild-type macrophages preprimed with ultrapure lipopolysaccharide. RESULTS: Compared with titanium particles with adherent bacterial debris, endotoxin-free titanium particles induced 86% less NLRP3 mRNA (0.05 ± 0.03 versus 0.35 ± 0.01 NLRP3/GAPDH, p < 0.001) and 91% less IL1ß mRNA (0.02 ± 0.01 versus 0.22 ± 0.03 IL1ß/GAPDH, p < 0.001). ProIL1ß protein level was robustly increased in wild-type macrophages stimulated by particles with adherent PAMPs but was not detectably produced in macrophages stimulated by endotoxin-free particles. Adherence of ultrapure lipopolysaccharide to endotoxin-free particles reconstituted stimulation of NLRP3 and IL1ß mRNA. Particles with adherent bacterial debris induced 79% less NLRP3 mRNA (0.09 ± 0.004 versus 0.43 ± 0.13 NLRP3/GAPDH, p < 0.001) and 40% less IL1ß mRNA (0.09 ± 0.04 versus 0.15 ± 0.03 IL1ß/GAPDH, p = 0.005) in TLR4 macrophages than in wild-type. Similarly, those particles induced 49% less NLRP3 mRNA (0.22 ± 0.10 versus 0.43 ± 0.13 NLRP3/GAPDH, p = 0.004) and 47% less IL1ß mRNA (0.08 ± 0.02 versus 0.15 ± 0.03 IL1ß/GAPDH, p = 0.012) in TIRAP/Mal macrophages than in wild-type. Particles with adherent ultrapure lipopolysaccharide induced 96% less NLRP3 mRNA (0.012 ± 0.001 versus 0.27 ± 0.05 NLRP3/GAPDH, p = 0.003) and 91% less IL1ß mRNA (0.03 ± 0.01 versus 0.34 ± 0.07 IL1ß/GAPDH, p < 0.001) expression in TLR4 macrophages than in wild-type. In contrast, those particles did not induce less NLRP3 and IL1ß mRNA in TLR2 macrophages. IL1ß protein secretion was equivalently induced by particles with adherent bacterial debris or by endotoxin-free particles in a time-dependent manner in wild-type macrophages. For example, particles with adherent bacterial debris induced 99% ± 2% of maximal IL1ß secretion after 12 hours, whereas endotoxin-free particles induced 92% ± 11% (p > 0.5). CONCLUSIONS: This cell culture study showed that adherent PAMPs are required for priming of the NLRP3 inflammasome by wear particles and this process is dependent on their cognate TLRs and TIRAP/Mal. In contrast, activation of the NLRP3 inflammasome by titanium particles is not dependent on adherent PAMPs. Animal and implant retrieval studies are needed to determine whether wear particles have similar effects on the NLRP3 inflammasome in vivo. CLINICAL RELEVANCE: Our findings, together with recent findings that aseptic loosening associates with polymorphisms in the TIRAP/Mal locus, support that adherent PAMPs may contribute to aseptic loosening in patients undergoing arthroplasty.

PYR-41 and Thalidomide Impair Dendritic Cell Cross-Presentation by Inhibiting Myddosome Formation and Attenuating the Endosomal Recruitments of p97 and Sec61 via NF-κB Inactivation.

PYR-41 and thalidomide have therapeutic effects on inflammation-associated diseases with side effects such as tumorigenesis. Cross-presentation allows dendritic cells (DC) to present endogenous antigen and induce protective immunity against microbe infection and tumors. But, up to now, the effects of PYR-41 and thalidomide on cross-presentation are still uncertain. In this study, we investigated the effect and mechanism of PYR-41 and thalidomide on DC cross-presentation by observing Myddosome formation, endosomal recruitment of p97 and Sec61, NF-κB activation, and cross-priming ability. We demonstrated that the inhibition of endosomal recruitment of p97 and Sec61, together with attenuated NF-κB activation and Myddosome formation, contributes to PYR-41- and thalidomide-impaired cross-presentation and thereby reverses cross-activation of T cells. These observations suggest that NF-κB signaling and p97 and Sec61 molecules are candidates for dealing with the side effects of PYR-41 and thalidomide.

Polyethyleneimine modification of aluminum hydroxide nanoparticle enhances antigen transportation and cross-presentation of dendritic cells.

BACKGROUND: The aim of this study was to explore the feasibility of delivering tumor antigens and enhancing the antigen cross-presentation of dendritic cells (DCs) by aluminum hydroxide nanoparticle with polyethyleneimine (PEI) modification (LV@HPA/PEI). MATERIALS AND METHODS: The LV@HPA nanoparticles were modified by PEI first, then the influence of LV@HPA/PEI on DCs was examined. The distinct expression of ovalbumin (OVA) protein transported into DCs by LV@HPA/PEI was observed by flow cytometry and Western blot. The biocompatibility of LV@HPA/PEI, maturity and antigen cross-presentation of DCs was observed in vitro. Tumor derived autophagosomes (DRibbles) combined with LV@HPA/PEI were loaded into DCs, and DC vaccines were used to immunize mice. The percentage of CD3+CD8+IFN-γ+ T cells in immunized mice was determined by flow cytometry. Additionally, the functional properties of the LV@HPA/PEI-DRibble-DCs vaccine were examined in vivo in PancO2 tumor-bearing mice. RESULTS: In our study, we described how LV@HPA/PEI can be a functionalized antigen delivery system with notable antigen transport effect and negligible cytotoxicity. It was found that LV@HPA/PEI could be easily internalized into DCs to assist antigen release into the cytoplasm. In addition, DCs matured gradually after loading with LV@HPA/PEI-OVA, which increased significantly the cytokine IL-12 secretion and expression of surface molecules CD80 and CD86. Interestingly, DCs loaded with LV@HPA/PEI-DRibbles could promote the activation of tumor-specific T cells both in murine and in human T cells. In the following in vivo experiments, the vaccine of LV@HPA/PEI-DRibble-DCs significantly inhibited tumor growth and improved the survival rate of the PancO2 tumor-bearing mice. CONCLUSION: We established a high-performance anti-tumor vaccine of DCs loaded with LV@ HPA/PEI nanoparticles and tumor-associated antigens in autophagosomes (DRibbles), which could serve as a therapeutic strategy in cancer immunotherapy.

HMGB1 released from intestinal epithelia damaged by cholera toxin adjuvant contributes to activation of mucosal dendritic cells and induction of intestinal cytotoxic T lymphocytes and IgA.

Cholera toxin (CT) is a potent mucosal adjuvant and oral administration of ovalbumin (OVA) antigens plus CT induces OVA-specific CD8+ cytotoxic T lymphocytes (CTLs) and IgA production in intestinal mucosa. However, the mechanisms of induction of these immune responses remain unknown. Intestinal OVA-specific CD8+ CTLs were not induced by oral administration of the CT active (CTA) or CT binding (CTB) subunit as an adjuvant and CD11c+ DCs were involved in cross-priming of intestinal CTLs. CD8+CD103+CD11c+CD11b-DCs and DCIR2+CD103+CD11c+CD11b+ DCs were distributed in the intestinal lamina propria and mesenteric lymph nodes, both DC subsets expressed DEC-205, and the expression of co-stimulatory molecules such as CD80 and CD86 was enhanced in both DC subsets after oral administration of intact CT but not the CTA or CTB subunit. Intestinal DCs activated by the oral administration of OVA plus CT cross-presented OVA antigens and DCs that captured OVA antigen through DEC-205, but not DCIR2, could cross-present antigen. We found that oral administration of intact CT, but not the CTA or CTB subunit, enhanced cell death, cytoplasmic expression of high-mobility group box 1 protein (HMGB1) in epithelial cell adhesion molecule (EpCAM)+CD45- intestinal epithelial cells (IECs), and HMGB1 levels in fecal extracts. HMGB1 dose-dependently enhanced the expression of CD80 and CD86 on DCs in vitro, and intravenous or oral administration of glycyrrhizin, an HMGB1 inhibitor, significantly suppressed activation of mucosal DCs and induction of intestinal OVA-specific CTLs and IgA by oral CT administration. These results showed that oral administration of intact CT triggers epithelial cell death in the gut and the release of HMGB1 from damaged IECs, and that the released HMGB1 may mediate activation of mucosal DCs and induction of CTLs and IgA in the intestine.

Targeting the Leukemia Antigen PR1 with Immunotherapy for the Treatment of Multiple Myeloma.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies.Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples.Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivoConclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386-96. ©2018 AACR.

IRE1α Activation in Bone Marrow-Derived Dendritic Cells Modulates Innate Recognition of Melanoma Cells and Favors CD8+ T Cell Priming.

The IRE1α/XBP1s signaling pathway is an arm of the unfolded protein response (UPR) that safeguards the fidelity of the cellular proteome during endoplasmic reticulum (ER) stress, and that has also emerged as a key regulator of dendritic cell (DC) homeostasis. However, in the context of DC activation, the regulation of the IRE1α/XBP1s axis is not fully understood. In this work, we report that cell lysates generated from melanoma cell lines markedly induce XBP1s and certain members of the UPR such as the chaperone BiP in bone marrow derived DCs (BMDCs). Activation of IRE1α endonuclease upon innate recognition of melanoma cell lysates was required for amplification of proinflammatory cytokine production and was necessary for efficient cross-presentation of melanoma-associated antigens without modulating the MHC-II antigen presentation machinery. Altogether, this work provides evidence indicating that ex-vivo activation of the IRE1α/XBP1 pathway in BMDCs enhances CD8+ T cell specific responses against tumor antigens.

Adjuvants Enhancing Cross-Presentation by Dendritic Cells: The Key to More Effective Vaccines?

Over the last decades, vaccine development has advanced significantly in pursuing higher safety with less side effects. However, this is often accompanied by a reduction in vaccine immunogenicity and an increased dependency on adjuvants to enhance vaccine potency. Especially for diseases like cancer, it is important that therapeutic vaccines contain adjuvants that promote strong T cell responses. An important mode of action for such adjuvants is to prolong antigen exposure to dendritic cells (DCs) and to induce their maturation. These mature DCs are extremely effective in the activation of antigen-specific T cells, which is a pre-requisite for induction of potent and long-lasting cellular immunity. For the activation of CD8+ cytotoxic T cell responses, however, the exogenous vaccine antigens need to gain access to the endogenous MHCI presentation pathway of DCs, a process referred to as antigen cross-presentation. In this review, we will focus on recent insights in clinically relevant vaccine adjuvants that impact DC cross-presentation efficiency, including aluminum-based nanoparticles, saponin-based adjuvants, and Toll-like receptor ligands. Furthermore, we will discuss the importance of adjuvant combinations and highlight new developments in cancer vaccines. Understanding the mode of action of adjuvants in general and on antigen cross-presentation in DCs in particular will be important for the design of novel adjuvants as part of vaccines able to induce strong cellular immunity.

BCR-ABL-specific CD4+ T-helper cells promote the priming of antigen-specific cytotoxic T cells via dendritic cells.

The advent of tyrosine kinase inhibitor (TKI) therapy markedly improved the outcome of patients with chronic-phase chronic myeloid leukemia (CML). However, the poor prognosis of patients with advanced-phase CML and the lifelong dependency on TKIs are remaining challenges; therefore, an effective therapeutic has been sought. The BCR-ABL p210 fusion protein's junction region represents a leukemia-specific neoantigen and is thus an attractive target for antigen-specific T-cell immunotherapy. BCR-ABL p210 fusion-region-specific CD4+ T-helper (Th) cells possess antileukemic potential, but their function remains unclear. In this study, we established a BCR-ABL p210 b3a2 fusion-region-specific CD4+ Th-cell clone (b3a2-specific Th clone) and examined its dendritic cell (DC)-mediated antileukemic potential. The b3a2-specific Th clone recognized the b3a2 peptide in the context of HLA-DRB1*09:01 and exhibited a Th1 profile. Activation of this clone through T-cell antigen receptor stimulation triggered DC maturation, as indicated by upregulated production of CD86 and IL-12p70 by DCs, which depended on CD40 ligation by CD40L expressed on b3a2-specific Th cells. Moreover, in the presence of HLA-A*24:02-restricted Wilms tumor 1 (WT1)235-243 peptide, DCs conditioned by b3a2-specific Th cells efficiently stimulated the primary expansion of WTI-specific cytotoxic T lymphocytes (CTLs). The expanded CTLs were cytotoxic toward WT1235-243-peptide-loaded HLA-A*24:02-positive cell lines and exerted a potent antileukemic effect in vivo. However, the b3a2-specific Th-clone-mediated antileukemic CTL responses were strongly inhibited by both TKIs and interferon-α. Our findings indicate a crucial role of b3a2-specific Th cells in leukemia antigen-specific CTL-mediated immunity and provide an experimental basis for establishing novel CML immunotherapies.

Effect of Mesenchymal Stromal Cells on T Cells in a Septic Context: Immunosuppression or Immunostimulation?

Sepsis is a complex process, including a first wave of damage partially due to the body's response to pathogens, followed by a phase of immune cell dysfunction. The efficacy of a pharmacological approach facing a rapidly evolving system implies a perfect timing of administration-this difficulty could explain the recent failure of clinical trials. Mesenchymal stromal cells (MSCs) are usually defined as immunosuppressive and their beneficial effects in preclinical models of acute sepsis have been shown to rely partly on such ability. If nonregulated, this phenotype could be harmful in the immunosuppressed context arising hours after sepsis onset. However, MSCs being environment sensitive, we hypothesized that they could reverse their immunosuppressive properties when confronted with suffering immune cells. Our objective was to evaluate the effect of human MSCs on activated human lymphocytes in an in vitro endotoxemia model. Peripheral blood mononuclear cells (PBMCs) underwent a 24-h lipopolysaccharide (LPS) intoxication and were stimulated with phytohemagglutinin (PHA) in contact with MSCs. MSCs induced a differential effect on lymphocytes depending on PBMC intoxication with LPS. Unintoxicated lymphocytes were highly proliferative with PHA and were inhibited by MSCs, whereas LPS-intoxicated lymphocytes showed a low proliferation rate, but were supported by MSCs, even when monocytes were depleted. These data, highlighting MSC plasticity in their immunomodulatory activity, pave the way for further studies investigating the mechanisms of mutual interactions between MSCs and immune cells in sepsis. Thus, MSCs might be able to fight against both early sepsis-induced hyperinflammatory response and later time points of immune dysfunction.