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The continuing emergence of viral pathogens and their rapid spread into heavily populated areas around the world underscore the urgency for development of highly effective vaccines to generate protective antiviral Ab responses. Many established and newly emerging viral pathogens, including HIV and Ebola viruses, are most prevalent in regions of the world in which Mycobacterium tuberculosis infection remains endemic and vaccination at birth with M. bovis bacille Calmette–Guérin (BCG) is widely used. We have investigated the potential for using CD4+ T cells arising in response to BCG as a source of help for driving Ab responses against viral vaccines. To test this approach, we designed vaccines comprised of protein immunogens fused to an immunodominant CD4+ T cell epitope of the secreted Ag 85B protein of BCG. Proof-of-concept experiments showed that the presence of BCG-specific Th cells in previously BCG-vaccinated mice had a dose-sparing effect for subsequent vaccination with fusion proteins containing the Ag 85B epitope and consistently induced isotype switching to the IgG2c subclass. Studies using an Ebola virus glycoprotein fused to the Ag 85B epitope showed that prior BCG vaccination promoted high-affinity IgG1 responses that neutralized viral infection. The design of fusion protein vaccines with the ability to recruit BCG-specific CD4+ Th cells may be a useful and broadly applicable approach to generating improved vaccines against a range of established and newly emergent viral pathogens.

During Ag priming, naive CD4+ T cells differentiate into subsets with distinct patterns of cytokine expression that dictate to a major extent their functional roles in immune responses. We identified a subset of CD4+ T cells defined by secretion of IL-3 that was induced by Ag stimulation under conditions different from those associated with previously defined functional subsets. Using mouse models of bacterial and viral infections, we showed that IL-3–secreting CD4+ T cells were generated by infection at the skin and mucosa but not by infections introduced directly into the blood. Most IL-3–producing T cells coexpressed GM-CSF and other cytokines that define multifunctionality. Generation of IL-3–secreting T cells in vitro was dependent on IL-1 family cytokines and was inhibited by cytokines that induce canonical Th1 or Th2 cells. Our results identify IL-3–secreting CD4+ T cells as a potential functional subset that arises during priming of naive T cells in specific tissue locations.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell–specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell–specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. Significance: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

The continuing emergence of viral pathogens and their rapid spread into heavily populated areas around the world underscore the urgency for development of highly effective vaccines to generate protective anti-viral antibody responses. Many established and newly emerging viral pathogens, including HIV and Ebola viruses, are most prevalent in regions of the world in which Mycobacterium tuberculosis infection remains endemic and vaccination at birth with M. bovis bacille Calmette-Guérin (BCG) is widely used. We have investigated the potential for using CD4(+) T cells arising in response to BCG as a source of help for driving antibody responses against viral vaccines. To test this approach, we designed vaccines comprised of protein immunogens fused to an immunodominant CD4(+) T cell epitope of the secreted Antigen 85B protein of BCG. Proof of concept experiments showed that the presence of BCG-specific T helper cells in previously BCG vaccinated mice had a dose sparing effect for subsequent vaccination with fusion proteins containing the Antigen 85B epitope, and consistently induced isotype switching to the IgG2c subclass. Studies using an Ebola virus glycoprotein fused to the Antigen 85B epitope showed that prior BCG vaccination promoted high affinity IgG1 responses that neutralized viral infection. The design of fusion protein vaccines with the ability to recruit BCG-specific CD4(+) helper T cells may be a useful and broadly applicable approach to generating improved vaccines against a range of established and newly emergent viral pathogens.

Effective subunit vaccines require the incorporation of adjuvants that stimulate cells of the innate immune system to generate protective adaptive immune responses. Pattern recognition receptor agonists are a growing class of potential adjuvants that can shape the character of the immune response to subunit vaccines by directing the polarization of CD4 T cell differentiation to various functional subsets. In the current study, we applied a high-throughput in vitro screen to assess murine CD4 T cell polarization by a panel of pattern recognition receptor agonists. This identified lipopeptides with TLR2 agonist activity as exceptional Th1-polarizing adjuvants. In vivo, we demonstrated that i.v. administration of TLR2 agonists with Ag in mice replicated the findings from in vitro screening by promoting strong Th1 polarization. In contrast, TLR2 agonists inhibited priming of Th1 responses when administered cutaneously in mice. This route-specific suppression was associated with infiltrating CCR2+ cells in the skin-draining lymph nodes and was not uniquely dependent on any of the well characterized subsets of dendritic cells known to reside in the skin. We further demonstrated that priming of CD4 T cells to generate Th1 effectors following immunization with the Mycobacterium bovis bacillus Calmette–Guérin (BCG) strain, a lipoprotein-rich bacterium recognized by TLR2, was dependent on the immunization route, with significantly greater Th1 responses with i.v. compared with intradermal administration of BCG. A more complete understanding of route-dependent TLR2 responses may be critical for informed design of novel subunit vaccines and for improvement of BCG and other vaccines based on live-attenuated organisms.

Analysis of Ag-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4+ T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4+ T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from bacillus Calmette-Guérin–vaccinated mice and show that high-quality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4+ CD154+ cells was distinct from that of CD154− cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

During Ag priming, naive CD4+ T cells differentiate into subsets with distinct patterns of cytokine expression that dictate to a major extent their functional roles in immune responses. We identified a subset of CD4+ T cells defined by secretion of IL-3 that was induced by Ag stimulation under conditions different from those associated with previously defined functional subsets. Using mouse models of bacterial and viral infections, we showed that IL-3–secreting CD4+ T cells were generated by infection at the skin and mucosa but not by infections introduced directly into the blood. Most IL-3–producing T cells coexpressed GM-CSF and other cytokines that define multifunctionality. Generation of IL-3–secreting T cells in vitro was dependent on IL-1 family cytokines and was inhibited by cytokines that induce canonical Th1 or Th2 cells. Our results identify IL-3–secreting CD4+ T cells as a potential functional subset that arises during priming of naive T cells in specific tissue locations.

Analysis of antigen-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. While several methods exist for identifying antigen-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC-II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live, antigen-specific CD4+ T cells but this approach remains underexplored, and to our knowledge has not previously been applied in mycobacteria-infected animals. Here we show that CD154 expression identifies adoptively transferred or endogenous antigen-specific CD4+ T cells induced by Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination. We confirmed that antigen-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from BCG-vaccinated mice, and show that high quality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of the CD4+ CD154+ cells was distinct from that of CD154− cells, and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells characterized by the production of interleukin-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live, antigen-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination., This work was supported by the National Multiple Sclerosis Society (Grant RG 4046-A6) and the National Institutes of Health (Grant R21 NS079144-01, Neuropathology Training Grant T32 NS007098, and Cellular and Molecular Biology and Genetics Training Grant T32 GM007491).

Chagasic cardiomyopathy caused by Trypanosoma cruzi is a major health concern in Latin America and among immigrant populations in non-endemic areas. T. cruzi has a high affinity for host lipoproteins and uses the low density lipoprotein receptor (LDLr) for invasion. Herein, we report that T. cruzi infection is associated with an accumulation of LDL and cholesterol in tissues in both acute and chronic murine Chagas disease. Similar findings were observed in tissue samples from a human case of Chagasic cardiomyopathy. T. cruzi infection of cultured cells displayed increased invasion with increasing cholesterol levels in the medium. Studies of infected host cells demonstrated alterations in their cholesterol regulation. T. cruzi invasion/infection via LDLr appears to be involved in changes in intracellular cholesterol homeostasis. The observed changes in intracellular lipids and associated oxidative stress due to these elevated lipids may contribute to the development of Chagasic cardiomyopathy.

Listeria monocytogenes (Lm) is a gram-positive bacterium associated with gastrointestinal infections. Initially, infects epithelial cells in the small intestine, then migrates to other organs where it is taken up mainly by phagocytic cells. Once phagocytized, Lm may then be processed in the phagosome to generate peptides presented on MHC Class II for activation of Lm-specific CD4+ T-cell responses. Lm can also escape the phagosome, leading to secretion of protein antigens into the cytosol. These are processed and presented by MHC Class I molecules to CD8+ T-cells, generating strong protective immunity that can clear the infection and prevent subsequent reinfection. Lm has been exploited for its use as a vaccine vector, particularly for the delivery of tumor-associated antigens. Our previous work showed that the efficacy of Lm as an anti-umor therapeutic vaccine could be significantly improved by incorporation of synthetic glycolipid activators of CD1d-restricted invariant natural killer T-cells (iNKT-cells) into the bacteria. Using an attenuated Lm strain expressing the tumor-associated antigen Mage-B, this approach gave improvements in prevention of metastasis in the 4T1 breast carcinoma model in BALB/c mice, and also gave modest reductions in the size of primary tumors. Currently, our efforts are directed at further improving this approach to more effectively target primary tumors, and also at validating the effects in a more humanized experimental models. To this end, we are using two models: 1. human CD1d knock-in (hCD1dKI) mouse line in which the mouse CD1d protein has been replaced by human CD1d, and 2. VaKI mouse, which is a new model made by our lab that is a hCD1dKI mouse that has as a transgene the α-chain of the human iNKT TCR (Vα24Jα18) and is deficient of endogenous murine iNKT-cells due to a murine Jα18 KO. These mice models have diminished percentages of iNKT-cells closely resembling what is seen in most humans, and thus mount iNKT-cell responses that are comparable to those seen in humans. We found that incorporation of iNKT-cell activating glycolipids into Lm leads to improved iNKT-cell-dependent immune responses in our models, and induces synergistic antitumor immune mechanisms against implanted primary syngeneic tumors (B16 F10 and MC38). Besides, administration of iNKT-cell activating glycolipids in association with Lm did not induce anergy or exhaustion of the responding cells, thus enabling repeated treatment to boost antitumor immune responses. Citation Format: Noemi Alejandra Saavedra-Avila, Shalu Sharma, Christopher T. Johndrow, Tony Ng, Claudia Gravekamp, Steven A. Porcelli. Immunotherapy against cancer using iNKT ligands in combination with an attenuated Listeria monocytogenes in humanized mouse models [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 A151.

Invariant natural killer T (iNKT) cells recognize glycolipid antigens presented by CD1d, an antigen presenting protein structurally similar to MHC class I. Stimulation of iNKT cells by glycolipid antigens can induce strong immune responses in vivo, with rapid production of a wide variety of cytokines including those classically associated with either T helper type 1 (Th1) or type 2 (Th2) responses. Alterations in the lipid tails or other portions of CD1d-presented glycolipid ligands can bias the iNKT response towards production of predominantly Th1 or Th2 associated cytokines. However, the mechanism accounting for this structure-activity relationship remains controversial. The Th1-biasing glycolipids have been found to consistently form complexes with CD1d that preferentially localize to plasma membrane cholesterol rich microdomains (lipid rafts), whereas CD1d complexes formed with Th2-biasing ligands are excluded from these microdomains. Here we show that neutralization of endosomal pH enhanced localization of CD1d complexes containing Th2-biasing glycolipids to plasma membrane lipid rafts of antigen presenting cells (APC). Transfer of APCs presenting these "stabilized" CD1d/αGC complexes into mice resulted in immune responses with a more prominent Th1-like bias, characterized by increased NK cell transactivation and interferon-γ production. These findings support a model in which low endosomal pH controls stability and lipid raft localization of CD1d-glycolipid complexes to regulate the outcome of iNKT cell mediated responses.

Novel effective vaccines are key to solving many global health problems. Past efforts to rationally design vaccines that elicit strong cell-mediated responses have largely failed. Vaccine adjuvants, particularly toll-like receptor (TLR) agonists, are a promising tool to shape immune responses by polarizing CD4 T cells to distinct fates. Use of these adjuvants to design novel vaccines requires the full characterization of their effects on CD4 T cell differentiation, particularly in regard to the route of vaccination. In a large scale in vitro screen of vaccine adjuvants, we demonstrated that Pam3CSK4, a synthetic mimic of bacterial lipoproteins with TLR1/2 agonist activity, strongly polarized CD4 T cells to the Th1 fate (IFN-γ+, T-bet+) supporting cell-mediated responses. We adapted a vaccination system to study CD4 T cell polarization in vivo using ovalbumin-specific transgenic CD4 T cells. This in vivo system showed a route-specific effect, with strong Th1 responses from intravenous immunization with ovalbumin plus Pam3CSK4, but a lack of Th1 responses with immunization through more clinically relevant cutaneous routes. Interestingly, Pam3CSK4 in combination with a known Th1 polarizing adjuvant, CpG ODN, showed little to no Th1 response suggesting a dominant suppressive mechanism. Furthermore, suppression of Th1 differentiation occurred in response to both TLR1/2 and TLR6/2 agonists. Using a variety of cell-ablative mouse models we were able to reverse suppression of Th1 responses and identify specific candidates of innate immune cells mediating suppression during vaccination with TLR2 agonists. Current work focuses on mechanisms of suppression by TLR2 agonists in the skin and relevance to improving vaccine design.

Invariant Natural Killer T cells (iNKT) are innate like T lymphocytes that recognize glycolipid antigens bound to CD1d molecule expressed by antigen presenting cells (APCs). iNKT cells modulate many aspects of immune responses and hence are targeted in many immunotherapeutic strategies. However, the prototypical iNKT agonist α-galactosylceramide (α-GC) and its synthetic analogues have limitations that include but are not limited to toxicity and iNKT anergy. Here, we report the development of an iNKT activating tumor targeted tertiary complex composed of a CD1d-antitumor ScFv antibody fusion protein covalently coupled to novel benzophenone linked α-GC analogs (BPGCs). Covalent complexes have dramatically improved stability and higher avidity to iNKT TCR and hence up to ten times lower effective dosage of the complex can stimulate strong immune response. Notably, these complexes directly activate iNKT cells bypassing the need for APCs. Hallmarks of covalent complex mediated iNKT activation include strong and sustained IFN-y secretion with no extensive iNKT proliferation or upregulation of co-inhibitory markers such as PD-1, even after multiple treatments as opposed to free α-GC and non-covalent complexes. In vitro, anti Her2 ScFv.CD1d.BPGC complexes lead to iNKT mediated targeted killing of MC38 cells overexpressing human Her2. Preliminary antitumor immunotherapy experiments in mice have shown that one third of the covalent complex treated mice show substantial reduction in tumor growth, including apparent eradication of tumors in some cases. Altogether, our approach enables a strong iNKT-based immune activation without the need for APCs and provides long lived and localized NKT activation in the tumor microenvironment.