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Immunotherapy for cancer treatment requires the activation of cytotoxic effector lymphocytes. Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize the MHC class I–like molecule MR1. MAIT cells play an important role in the immune response against microbial infections and can directly kill tumor cells. Although MAIT cells can be expanded ex vivo, this method is time-consuming, expensive, and requires allogenic feeder layers. To overcome the limitations of conventional dendritic cell–based vaccines and ex vivo expansion of human T cells, an artificial APC (aAPC) approach to expand antitumor effector cells has several advantages. In this study, we explored an efficient in vitro method to amplify MR1-specific MAIT cells from human peripheral blood using aAPCs made by coating cell-sized latex beads with an Ag-loaded MR1 tetramer complex and anti-CD28 Ab. We further elucidated the cytotoxic potential of such expanded MAIT cells against three human glioblastoma multiforme (GBM) cell lines to explore their potential use as a novel immunotherapeutic tool, as the mostly lethal GBM poorly responds to conventional chemotherapy. When aAPCs were compared with the standard allogenic feeder layer–based approach for MAIT cell expansion, they were significantly more effective. Our results indicate that the aAPC-expanded MAIT cells remained functional, retained their original phenotype, secreted proinflammatory cytokines, and showed cytotoxicity against the GBM cell lines. Hence, MAIT cells have the potential to be a novel tool in immunotherapy approaches for the treatment of human GBM.

Introduction Gut microbiota has been reported to contribute to obesity and the pathology of obesity‐related diseases but the underlying mechanisms are largely unknown. Mucosal‐associated invariant T (MAIT) cells are a unique subpopulation of T cells characterized by the expression of a semi‐invariant T cell receptor (TCR) α chain (Vα19 in mice; Vα7.2 in humans). The expansion and maturation of MAIT cells require the gut microbiota and antigen‐presenting molecule MR1, suggesting that MAIT cells may play a unique role in bridging gut microbiota, obesity, and obesity‐associated inflammation. Methods The levels of human MAIT cells from obese patients, as well as mouse MAIT cells from obese mouse models, were determined by flow cytometry. By comparing to controls, we analyzed the change of MAIT cells in obese subjects. Results We found obese patients had fewer circulating MAIT cells than healthy‐weight donors and the difference was more distinct in male patients. Consistently, male mice (but not female mice) have shown reduced MAIT cells in the liver and adipose tissue after a 10‐week Western diet compared to mice on a control diet. We also explored the possibility of utilizing high‐throughput technology (i.e., quantitative polymerase chain reaction [qPCR]), other than flow cytometry, to determine the expression levels of the invariant TCR of human MAIT cells. But a minimal correlation (R 2 = 0.23, p = .11) was observed between qPCR and flow cytometry data. Conclusion Our study suggests that there is a sex discrepancy in the impact of obesity on MAIT cells: MAIT cells in male (but not female) humans and male mice are reduced by obesity., It has been previously reported that MAIT cells are reduced in obese patients. We also found fewer MAIT cells in both preclinical obese mouse models and obese patients. Moreover, our data suggest there is a sex discrepancy that the reduction of MAIT cells caused by obesity is only observed in males but not females in both preclinical mouse models and human subjects.

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site–specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell–based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

An imbalance in gut homeostasis results in local and systemic pathogenesis. It is still not well-understood how the immune system interacts with the gut microbiome and maintains a delicate balance. Here, we utilized a mouse model in which STAT3 expression is deleted in CD11c+ (i.e., dendritic) cells (STAT3 cKO); these mice developed an ulcerative colitis-like disease, colon carcinoma and myelodysplastic syndrome-like disease. Circulating IgE levels in STAT3 cKO mice were significantly elevated. The gut microbiome was indispensable for the observed pathogenesis, as treatment with broad-spectrum antibiotics or cross-fostering STAT3 cKO pups with mothers harboring a different microbiome prevented disease development. Gut microbiome analyses suggested that decreased commensal bacteria and increased pathogenic bacteria most likely contributed to disease. Our data suggest that STAT3 controls the manifestation of inflammation in the gut caused by the microbiome. Therefore, we conclude that a deficiency of STAT3 in DCs is sufficient to trigger uncontrolled inflammation and the development of inflammatory bowel disease.

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes includingGGTA1,CMAH,β4galNT2,SLA-I α chain,andβ2-microglobulinthat are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti-tumor, and anti-microbial immune responses, and are activated by glycolipids presented by the MHC class I-like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)-dependent iNKT cell activation is not well-known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d-mediated Ag presentation in APCs, but also contributes to CD1d-independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d-independent, but IL-12-dependent manner, we found the virus-induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild-type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL-12 receptors. As with a VV infection, an IL-12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d-mediated Ag presentation and contributes to IL-12-induced iNKT cell activation and loss during viral infections.

Background Glioblastoma is the most common adult primary brain tumor with near-universal fatality. Major histocompatibility complex (MHC) class I molecules are important mediators of CD8 activation and can be downregulated by cancer cells to escape immune surveillance. MR1 is a nonclassical MHC-I-like molecule responsible for the activation of a subset of T cells. Although high levels of MR1 expression should enhance cancer cell recognition, various tumors demonstrate MR1 overexpression with unknown implications. Here, we study the role of MR1 in glioma. Methods Using multi-omics data from the Cancer Genome Atlas (TCGA), we studied MR1 expression patterns and its impact on survival for various solid tumors. In glioma specifically, we validated MR1 expression by histology, elucidate transcriptomic profiles of MR1 high versus low gliomas. To understand MR1 expression, we analyzed the methylation status of the MR1 gene and MR1 gene-related transcription factor (TF) expression. Results MR1 is overexpressed in all grades of glioma and many other solid cancers. However, only in glioma, MR1 overexpression correlated with poor overall survival and demonstrated global dysregulation of many immune-related genes in an MR1-dependent manner. MR1 overexpression correlated with decreased MR1 gene methylation and upregulation of predicted MR1 promoter binding TFs, implying MR1 gene methylation might regulate MR1 expression in glioma. Conclusions Our in silico analysis shows that MR1 expression is a predictor of clinical outcome in glioma patients and is potentially regulated at the epigenetic level, resulting in immune-related genes dysregulation. These findings need to be validated using independent in vitro and in vivo functional studies.

Background Glioblastoma (GBM) is a deadly brain malignancy with a dismal prognosis. While immunotherapy holds great promise for GBM treatment, most have failed due to a suppressive tumor microenvironment (TME). Antigen heterogeneity and adenosine signaling are two immunosuppressive mechanisms in GBM. The CD73-adenosine axis plays a multifaceted role in GBM pathogenesis and drives the dysfunction of NK cells in GBM TME.1,3 Our NKG2D-chimeric antigen receptor (CAR)-natural killer (NK) cells have shown anti-tumor activity when combined with CD73 blockade in vivo.2 To further extend the potency of these cells against GBM and address antigen heterogeneity in GBM, we combined the local blockade of CD73 with multi-antigen-targeting engineered NK cells. In order to improve treatment assessment, PET/MR imaging was employed to enable detailed, non-invasive assessment of tumor progression. Imaging assessment of adoptively-transferred CAR- NK cells was also developed to determine the fate of NK cell delivery to the tumor site over time. Methods We generated multifunctional engineered NK (E-NK) cells that express an anti-CD73 scFv, which is cleavable by GBM-associated proteases, an NKG2D-CAR, as well as a GD2 CAR, which can actively target the GD2 antigen overexpressed on GBM (Figure 1A). For E-NK cell radiolabeling, zirconium-89 (89Zr, ½ life = 78 Hr) radiotracer was attached covalently to the E-NK cell surface via conjugation with DFO-Bz-NCS in a range of doses from 50–600 µCi. Results An optimal balance between labeling efficiency and cell viability was attained at 120 µCi 89Zr resulting in 39% labeling efficiency and 46% cell viability over for 48 hours. After labeling, the NK cells maintained their in vitro killing activity against GBM cells (figure 1B). The 89Zr labeled E-NK cells were administered intravenously in mice containing intracranial GBM10 tumors at week 5 post-implant. PET imaging was performed at 1 and 2 days later and gamma imaging ex vivo at 4 days. Free 89Zr was visible diffusely throughout the body with low levels in the brain. The majority of 89Zr labeled E-NK cell groups localized to the lungs with detectable activity elsewhere in various organs (figure 1C and 1D). Conclusions We generated multifunctional E-NK cells which showed the improved killing of GBM cells using novel targeting approaches, including the blockade of CD73-mediated adenosinergic signaling. We also optimized E-NK cell radiolabeling with 89Zr for GB10 therapy in vitro and in vivo fate mapping against a xenograft of patient-derived GBM. Acknowledgements We gratefully acknowledge the Walther Oncology Embedding Program, Indiana University Simon Cancer Center, and In Vivo Therapeutics Core. References Wang J, Matosevic S. NT5E/CD73 as correlative factor of patient survival and natural killer cell infiltration in glioblastoma. J Clin Med 2019;8(10):1526. Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunother Cancer 2018;6(1):136. Yan A, Joachims ML, Thompson LF, Miller AD, Canoll PD, Bynoe MS. CD73 promotes glioblastoma pathogenesis and enhances its chemoresistance via A2B adenosine receptor signaling. J Neurosci 2019;39(22):4387. Flink J, Muzi M, Peck M, Krohn K. Multimodality brain tumor imaging: mr imaging, PET, and PET/MR imaging. J Nucl 2015;5(10):1554–1561.

Background Despite aggressive treatments and care, the median survival for GBM patients is 14.6 months, which has only modestly improved over the past several decades, highlighting the need for new therapeutic approaches. NK cells, innate cytotoxic effectors, are showing potential for cancer immunotherapy for GBM.1–3 However, tumor antigen heterogeneity and a severely immunosuppressive tumor microenvironment (TME) have rendered GBM highly resistant to most single antigen-based NK monotherapies.4–6 Methods To overcome these challenges, our solution has been to develop a first multifunctional immunotherapy for GBM based on genetically-engineered NK cells bearing multiple simultaneous anti-tumor functions, including local tumor responsiveness and the ability to avoid antigen escape. The activity of these lentivirally-transduced multi-functional NK (E-pNK) cells were evaluated against patient-derived GBM cells both in vitro and in vivo. Results We have designed and synthesized a multifunctional CAR construct that expresses an anti-CD73 scFv which is cleavable by GBM-associated proteases, and a dual CAR redirected against ligands for NKG2D and GBM-associated GD2 receptors (figure 1A-B). We have isolated primary NK cells (figure 1C) and genetically manipulated them to express NKG2D, anti-GD2 scFv and anti-CD73 scFv (figure 1D-E). E-pNK cells showed a significantly higher in vitro antitumor activity towards GBM43 targets, patient-derived GBM cells, including increased percentage of tumor killing, degranulation and IFN-γ production (figure 1F-G). E-pNK cells lacking the anti-CD73 scFv following uPA treatment displayed significantly decreased killing ability of target GBM43 cells after co-culture at E/T ratios of 2.5 and 5 for 4 h (figure 1H). In addition, after treatment with cleaved anti-CD73 scFv, GBM43 cells showed a significantly reduced ability to produce adenosine due to the inhibition of CD73 enzyme activity (figure 1I). Furthermore, E-pNK cells showed potent anti-GBM activity in subcutaneously GBM43 xenografts (figure 1J-L). In vivo-adoptively transferred E-pNK cells also showed superior intratumoral infiltration into GBM43 tumors when analyzed by IHC (data not shown). Conclusions We have generated E-pNK cells showing improved antitumor activity against GBM through increased resistance to the immunosuppressive TME via adenosinergic CD73 blockade and the simultaneous ability to specifically target GBM cells via dual CARs. Based on these results, we are currently building the orthotopic GBM mouse model to further evaluate their in vivo therapeutic effects. Acknowledgements This work is supported, in part, by a Ralph W. and Grace M. Showalter Research Trust award and a Walther Cancer Foundation Embedding Grant. Ethics Approval Primary human NK (pNK) cells used in this study were obtained using Purdue University’s Institutional Review Board (IRB)-approved consent forms (IRB-approved protocol #1804020540). References Sedgwick AJ, Ghazanfari N, Constantinescu P, Mantamadiotis T, Barrow AD. The role of NK cells and innate lymphoid cells in brain cancer. Front Immunol 2020;11:01549. Burger MC, Zhang CC, Harter PN, Romanski A, Strassheimer F, Senft C, et al. CAR-engineered NK cells for the treatment of glioblastoma: turning innate effectors into precision tools for cancer immunotherapy. Front Immunol 2019;10:2683. Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunother Cancer 2018;6:136. Lim M, Xia YX, Bettegowda C, Weller M. Current state of immunotherapy for glioblastoma. Nat Rev Clin Oncol 2018;15:422-442. Lynes JP, Nwankwo AK, Sur HP, Sanchez VE, Sarpong KA, Ariyo OI, et al. Biomarkers for immunotherapy for treatment of glioblastoma. J Immunother Cancer 2020;8:e000348. Wang J, Matosevic S. NT5E/CD73 as Correlative factor of patient survival and natural killer cell infiltration in glioblastoma. J Clin Med 2019;8:1526.

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking have rendered glioblastoma (GBM) highly resistant to therapy. As a result, GBM immunotherapies have failed to demonstrate sustained clinical improvements in patient overall survival (OS). To overcome these obstacles, here we describe a novel, sophisticated combinatorial platform for GBM: the first multifunctional immunotherapy based on genetically-engineered, human NK cells bearing multiple anti-tumor functions, including local tumor responsiveness, that addresses key drivers of GBM resistance to therapy: antigen escape, poor immune cell homing, and immunometabolic reprogramming of immune responses. We engineered dual-specific CAR-NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally-released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site specific activity in the tissue and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells, but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising new NK cell-based combinatorial strategy that can target multiple clinically-recognized mechanisms of GBM progression simultaneously.

In the naïve mouse brain, microglia and astrocytes are the most abundant immune cells; however, there is a complexity of other immune cells present including monocytes, neutrophils, and lymphocytic cells, such as natural killer (NK) cells, T cells, and B cells. In Alzheimer’s disease (AD), there is high inflammation, reactive microglia, and astrocytes, leaky blood–brain barrier, the buildup of amyloid-beta (Aβ) plaques, and neurofibrillary tangles which attract infiltrating peripheral immune cells that are interacting with the resident microglia. Limited studies have analyzed how these infiltrating immune cells contribute to the neuropathology of AD and even fewer have analyzed their interactions with the resident microglia. Understanding the complexity and dynamics of how these immune cells interact in AD will be important for identifying new and novel therapeutic targets. Thus, this review will focus on discussing our current understanding of how macrophages, neutrophils, NK cells, T cells, and B cells, alongside astrocytes, are altered in AD and what this means for the disorder, as well as how these cells are affected relative to the resident microglia.

Cell signaling pathways regulate much in the life of a cell: from shuttling cargo through intracellular compartments and onto the cell surface, how it should respond to stress, protecting itself from harm (environmental insults or infections), to ultimately, death by apoptosis. These signaling pathways are important for various aspects of the immune response as well. However, not much is known in terms of the participation of cell signaling pathways in Ag presentation, a necessary first step in the activation of innate and adaptive T cells. In this brief review, I discuss the known signaling molecules (and pathways) that regulate how Ags are presented to T cells and the mechanism(s), if identified. Studies in this area have important implications in vaccine development and new treatment paradigms against infectious diseases, autoimmunity, and cancer.

Background/aim Invariant natural killer T-cells (iNKT) stimulated by CD1d-binding glycolipids have been shown to exert antitumor effects by a number of studies in a mouse model. Breast cancer is a devastating disease, with different types of breast cancer recurring locally or distant as metastatic/advanced disease following initial treatment. The aim of this study was to examine the tumoricidal effect of a CD1d-binding glycolipid, called 7DW8-5, against a highly invasive human breast cancer cell line both in vitro and in vivo. Materials and methods Parental MDA-MB-231 cells and MDA-MB-231 cells transduced with human CD1d were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by loading with glycolipids. After co-culturing with human iNKT cells, the cells were permeabilized and stained with Alexa Flour 647-conjugated antibody to active caspase-3, and analyzed using a BD LSR II. For the in vivo tumoricidal effect, MDA-MB-231 cells transduced with human CD1d and luciferase genes were injected into the mammary fat pad of female NOD/SCID/IL2rγnull (NSG) mice, followed by the injection of human iNKT cells with or without 7DW8-5, and the levels of luminescence were analyzed with whole-body imaging. Results Human iNKT cells could kill CD1d-expressing human breast cancer cells in vitro in the presence of 7DW8-5, but not α-GalCer. As for in vivo, the adoptive transfer of human iNKT cells into tumor-challenged NSG mice significantly inhibited the growth of CD1d+ MDA-MB-231 human breast cancer cells in the presence of 7DW8-5. Conclusion CD1d-binding, glycolipid-based iNKT-cell therapy is suggested as a potent and effective treatment against breast cancer in humans.

It is unknown whether brain astrocytes and microglia have the capacity to present microbial antigens via the innate immune MR1/MAIT cell axis. We have detected MAIT cells in the normal mouse brain and found that both astrocytes and microglia are MR1(+). When we stimulated brain astrocytes and microglia with E. coli, and then co-cultured them with MAIT cells, MR1 surface expression was upregulated and MAIT cells were activated in an antigen-dependent manner. Considering the association of MAIT cells with inflammatory conditions, including those in the CNS, the MR1/MAIT cell axis could be a novel therapeutic target in neuroinflammatory disorders.

Many reviews on the CD1d/NKT cell axis focus on the ability of CD1d-restricted NKT cells to serve as effector cells in a variety of disorders, be they infectious diseases, cancer or autoimmunity. In contrast, here, we discuss the ways that viruses, bacteria and tumor cells can evade the CD1d/NKT cell axis. As a result, these disease states have a better chance to establish a foothold and potentially cause problems for the subsequent adaptive immune response, as the host tries to rid itself of infections or tumors.

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT) cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair) host antitumor immunity. We have previously shown that CD1d-mediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1+/- mice. Nf1+/- mice were found to have similar levels of NKT cells as wildtype (WT) littermates. Interestingly, however, reduced CD1d expression was observed in Nf1+/- mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1+/- mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1+/- mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1+/- mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.

Cytokines that regulate the immune response signal through the Janus kinase / signal transducer and activation of transcription (JAK/STAT) pathway, but whether this pathway can regulate CD1d-mediated lipid antigen presentation to natural killer T (NKT) cells is unknown. Here, we found that STAT3 promotes antigen presentation by CD1d. Antigen-presenting cells (APCs) in which STAT3 expression was inhibited exhibited markedly reduced endogenous lipid antigen presentation to NKT cells without an impact on exogenous lipid antigen presentation by CD1d. Consistent with this observation, in APCs where STAT3 was knocked down, dramatically decreased levels of UDP glucose ceramide glucosyltransferase (UGCG), an enzyme involved in the first step of glycosphingolipid biosynthesis, were observed. Impaired lipid antigen presentation was reversed by ectopic expression of UGCG in STAT3-silenced CD1d(+) APCs. Hence, by controlling a fundamental step in CD1d-mediated lipid antigen presentation, STAT3 signalling promotes innate immune responses driven by CD1d.

Summary CD1d-mediated lipid antigen presentation activates a subset of innate immune lymphocytes called invariant natural killer T (NKT) cells that, by virtue of their potent cytokine production, bridge the innate and adaptive immune systems. Transforming growth factor (TGF-β) is a known immune modulator that can activate the mitogen-activated protein kinase p38; we have previously shown that p38 is a negative regulator of CD1d-mediated antigen presentation. Several studies implicate a role for TGF-β in the activation of p38. Therefore, we hypothesized that TGF-β would impair antigen presentation by CD1d. Indeed, a dose-dependent decrease in CD1d-mediated antigen presentation and impairment of lipid antigen processing was observed in response to TGF-β treatment. However, it was found that this inhibition was not through p38 activation. Instead, Smads 2, 3 and 4, downstream elements of the TGF-β canonical signalling pathway, contributed to the observed effects. In marked contrast to that observed with CD1d, TGF-β was found to enhance MHC class II-mediated antigen presentation. Overall, these results suggest that the canonical TGF-β/Smad pathway negatively regulates an important arm of the host's innate immune responses – CD1d-mediated lipid antigen presentation to NKT cells.

Mucosal‐associated invariant T (MAIT) cells are conserved T cells that express a semi‐invariant T‐cell receptor (Vα7.2 in humans and Vα19 in mice). The development of MAIT cells requires the antigen‐presenting MHC‐related protein 1 (MR1), as well as commensal bacteria. The mechanisms that regulate the functional expression of MR1 molecules and their loading with bacterial antigen in antigen‐presenting cells are largely unknown. We have found that treating B cells with the Toll‐like receptor 9 (TLR9) agonist CpG increases MR1 surface expression. Interestingly, activation of TLR9 by CpG‐A (but not CpG‐B) enhances MR1 surface expression. This is limited to B cells and not other types of cells such as monocytes, T or natural killer cells. Knocking‐down TLR9 expression by short hairpin RNA reduces MR1 surface expression and MR1‐mediated bacterial antigen presentation. CpG‐A triggers early endosomal TLR9 activation, whereas CpG‐B is responsible for late endosomal/lysosomal activation of TLR9. Consistently, blocking endoplasmic reticulum to Golgi protein transport, rather than lysosomal acidification, suppressed MR1 antigen presentation. Overall, our results indicate that early endosomal TLR9 activation is important for MR1‐mediated bacterial antigen presentation.

CD1d-restricted T (natural killer T [NKT]) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to downregulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell's cytoskeletal network and viral spreading. We have previously shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found that an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared with the wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not the ΔUL49 virus. Ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Moreover, inhibiting viral protein synthesis rescued VP22-dependent inhibition of CD1d antigen presentation. In conclusion, our findings suggest that VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.

What does it take to be successful as a tenure-track research faculty member in a School of Medicine? What are the elements necessary to run a successful laboratory? How does one find the resources and help to know what is important for promotion and tenure? Most training in graduate school or in clinical fellowships does not answer these questions. Too often, new junior tenure-track research faculty members are left to learn from the "school of hard knocks" and essentially are reinventing the wheel, which is a huge waste of time. This article describes the history of research faculty, what makes them successful, and offers suggestions on how we can help them reach their greatest potential.

CD1d molecules are MHC class I-like molecules that present lipids to a unique subpopulation of T cells called NKT cells. The cytoplasmic tail of human CD1d possesses a tyrosine-based endosomal targeting motif (YXXZ). As such, these molecules traffic through the endocytic pathway, where it is believed that they are loaded with the antigenic lipid that stimulates NKT cells. In the current study, it was found that the T322 residue in the human CD1d tail is a major signal controlling transport to the cell surface and thus its functional expression. Mimicking the phosphorylation of this residue or removal of the entire cytoplasmic tail negates its ability to regulate CD1d trafficking, resulting in lysosomal targeting and degradation. These results demonstrate an important role of a heretofore unknown signal in the cytoplasmic tail of CD1d that may have relevance to other type I integral membrane proteins that traverse through the endocytic pathway.

For over three decades, the scientific community has expressed concern over the paucity of African American, Latino and Native American researchers in the biomedical training pipeline. Concern has been expressed regarding what is forecasted as a shortage of these underrepresented minority (URM) scientists given the demographic shifts occurring worldwide and particularly in the United States. Increased access to graduate education has made a positive contribution in addressing this disparity. This article describes the multiple pathway approaches that have been employed by a school of medicine at an urban Midwest research institution to increase the number of URM students enrolled in, and graduating from, doctoral programs within basic science departments, through the combination of R25 grants and other grant programs funded by the National Institutes of Health (NIH). This article outlines the process of implementing a strong synergistic approach to the training of URM students through linkages between the NIH-funded "Bridges to the Doctorate (BRIDGES)" and "Initiative for Maximizing Graduate Student Diversity (IMGSD)" programs. The article documents the specific gains witnessed by this particular institution and identifies key components of the interventions that may prove useful for institutions seeking to increment the biomedical pipeline with scientists from diverse backgrounds.

Statins are widely used as cholesterol-lowering agents that also decrease inflammation and target enzymes essential for prenylation, an important process in the activation and intracellular transport of proteins vital for a wide variety of cellular functions. Here, we report that statins impair a critical component of the innate immune response, CD1d-mediated Ag presentation. The addition of specific intermediates in the isoprenylation pathway reversed this effect, whereas specific targeting of enzymes responsible for prenylation mimicked the inhibitory effects of statins on Ag presentation by CD1d as well as MHC class II molecules. This study demonstrates the importance of isoprenylation in the regulation of Ag presentation and suggests a mechanism by which statins reduce inflammatory responses.

Natural killer T (NKT) cells are a T-cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant T-cell receptor (TCR) α chain rearrangement (Vα14Jα18 in mice; Vα24Jα18 in humans) and are called type I NKT cells; all other NKT cells are type II. In the current study, we have analyzed the roles for these NKT-cell subsets in the host's innate antitumor response against a murine B-cell lymphoma model in vivo. In tumor-bearing mice, we found that type I NKT cells conferred protection in a CD1d-dependent manner, whereas type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice correlated with tumor progression. Myeloid cells (CD11b+Gr1+) were present in large numbers at the tumor site and in the spleen of tumor-bearing type I NKT–deficient mice, suggesting that antitumor immunosurveillance was inhibited by CD11b+Gr1+ cells. Overall, these data suggest that there are distinct roles for NKT-cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in immunotherapeutic approaches against blood cancers.

Blocking the function of Stat (signal transducer and activator of transcription) proteins, which are critical for antiviral responses, has evolved as a common mechanism for pathogen immune evasion. The poxvirus-encoded phosphatase H1 is critical for viral replication, and may play an additional role in the evasion of host defense by dephosphorylating Stat1 and blocking interferon (IFN)-stimulated innate immune responses. Vaccinia virus (VACV) H1 can inhibit the phosphorylation of the transcription factor Stat1 after IFN-gamma stimulation of epithelial cells, greatly attenuating IFN-induced biological functions. In this study, we demonstrate that VACV infection is capable of inhibiting the phosphorylation of Stat1 and Stat2 after stimulation of fibroblasts or bone marrow-derived macrophages with either type I or type II IFNs, but did not inhibit the activation of Stat3 or Stat5 in either cell type. By using recombinant proteins for in vitro assays, we observe that variola virus H1 is more active than VACV H1, although it has similar selectivity for Stat targets. Differential effects of VACV infection were observed on the induction of IFN-stimulated genes, with complete inhibition of some genes by VACV infection, while others were less affected. Despite the IFN-gamma-induced expression of some genes in VACV-infected cells, IFN-gamma was unable to rescue the VACV-mediated inhibition of MHC class II antigen presentation. Moreover, VACV infection can affect the IFN-induced expression of Stat1-dependent and Stat1-independent genes, suggesting that the virus may target additional IFN-activated pathways. Thus, VACV targets multiple signaling pathways in the evasion of antiviral immune responses.

We have recently demonstrated that the p38 and ERK1/2 MAP kinases play reciprocal roles in the control of CD1d-mediated antigen presentation. Although the use of specific inhibitors for these pathways clearly had an effect, the effects were not complete, leading to speculations that additional pathways were involved. Here, we show that inhibiting protein kinase C delta (PKCdelta) substantially impairs antigen presentation by murine CD1d1 to NKT cells. This effect was accompanied by marked changes in the intracellular localization of CD1d. Expression of a dominant-negative mutant of PKCdelta in CD1d(+) cells resulted in nearly undetectable endogenous antigen presentation, substantially impaired CD1d recycling, a decrease in MAPK activation, and a decrease in the ability to present low (but not high) concentrations of alpha-galactosylceramide at the cell surface. These data strongly suggest that PKCdelta is a critical regulator of CD1d-mediated antigen presentation and is involved in multiple steps of the process.

Vaccinia virus (VV) has been most commonly used as the vaccine to protect individuals against the causative agent of smallpox (variola virus), but it also uses a number of strategies meant to evade or blunt the host's antiviral immune response. Natural killer T (NKT) cells are a subset of immunoregulatory CD1d-restricted T lymphocytes believed to bridge the innate and adaptive immune responses. It is shown here that the VV-encoded molecules, B1R and H5R, play a role in the ability of VV to inhibit CD1d-mediated antigen presentation to NKT cells. These are the first poxvirus-encoded molecules identified that can play such a role in the evasion of an important component of the innate immune response.

The ability to correlate changes in antigen‐reactive lymphocytes with disease will provide information needed to develop strategies for combating illness. One critical group of lymphocytes are the CD1‐restricted T cells. It is desirable to use CD1 molecules in an array format to query CD1‐restricted lymphocytes in humans. To investigate the feasibility of this technique, we employed mCD1d and α‐galactosylceramide to demonstrate that‐slide immobilized, CD1d‐α‐GalCer complexes capture an NKT cell hybridoma in the presence of a competitor. The success of this scheme represents the first step toward the development of CD1‐antigen arrays that could be used to profile biological samples.

The MHC class I-like CD1d glycoprotein is a member of the CD1 family of Ag-presenting molecules and is responsible for the selection of NKT cells. A number of ligands that can be presented by CD1d to NKT or other CD1d-restricted T cells have been identified. These include glycolipids from a marine sponge, bacterial glycolipids, normal endogenous glycolipids, tumor-derived phospholipids and glycolipids, and nonlipidic molecules. The presentation of many of these molecules can have immunopotentiating effects, such as serving as an adjuvant against malaria or resulting in a more rapid clearance of certain virus infections. They can also be protective in autoimmune diseases or cancer or can be deleterious. This review will highlight these ligands in a discussion of their potential use against (and role in the pathogenesis of) these diseases.

NK T (NKT) cells are an important component of the innate immune system and recognize the MHC class I-like CD1d molecule. NKT cells possess significant immunoregulatory activity due to their rapid secretion of large quantities of pro- and anti-inflammatory cytokines following CD1d-dependent stimulation. Because the innate immune system is programmed to respond to a multitude of diverse stimuli and must be able to quickly differentiate between pathogenic and endogenous signals, we hypothesized that, apart from stimulation via the TCR (e.g., CD1d-dependent activation), there must be multiple activation pathways that can be triggered through other cell surface receptors on NKT cells. Therefore, we analyzed the ability of CD44, a structurally diverse cell surface receptor expressed on most cells, to stimulate murine NKT cells, compared with conventional T cells. Stimulation of CD44 through Ab cross-linking or binding to its natural ligands hyaluronan and osteopontin induced NKT cells to secrete cytokines, up-regulate activation markers, undergo morphological changes, and resist activation-induced cell death, whereas conventional T cells only exhibited changes in morphology and protection from activation-induced cell death. This CD44-specific stimulation of NKT cells correlated with their ability to bind hyaluronan. Thus, fundamental differences in CD44 function between these lymphocyte subsets suggest an important biological role for CD44 in the innate immune response.

A critical component of the host’s innate immune response involves lipid Ag presentation by CD1d molecules to NK T cells. In this study we used murine CD1d1-transfected L (L-CD1) cells to study the effect of viruses on CD1d-mediated Ag presentation to NKT cells and found that an infection with vesicular stomatitis and vaccinia (but not lymphocytic choriomeningitis) virus inhibited murine CD1d1-mediated Ag presentation. This was under the reciprocal control of the MAPKs, p38 and ERK, and was due to changes in the intracellular trafficking of CD1d1. The reciprocal regulation of CD1d1-mediated Ag presentation by MAPK suggests that the targeting of these pathways is a novel means of immune evasion by viruses.

SummaryExtracellular antigens are internalized and processed before binding MHC class II molecules within endosomal and lysosomal compartments of professional antigen presenting cells (APC) for subsequent presentation to T cells. Yet select cytoplasmic peptides derived from autoantigens also intersect and bind class II molecules via an unknown mechanism. In human B lymphoblasts, inhibition of the peptide transporter associated with antigen processing (TAP) failed to alter class II-restricted cytoplasmic epitope presentation. By contrast, decreased display of cytoplasmic epitopes via class II molecules was observed in cells with diminished expression of the lysosome-associated membrane protein-2 (Lamp-2). Overexpression of Lamp-2 isoform A (Lamp-2a), an established component of chaperone-mediated autophagy, enhanced cytoplasmic autoantigen presentation. Manipulating APC expression of heat shock cognate protein 70 (hsc70), a cofactor for Lamp-2a, also altered cytoplasmic class II peptide presentation. These results demonstrate a novel role for the lysosomal Lamp-2a-hsc70 complex in promoting immunological recognition and antigen presentation.

CD1d is an MHC class I-like surface molecule that presents endogenous glycoplipid antigens. The effect of HIV infection on CD1d surface expression has not yet been reported. FACS analysis revealed significantly lower levels of CD1d on CD14 + monocytes from HIV-infected subjects compared to HIV-infected subjects on HAART and healthy controls. CD1d expression correlated inversely with viral load in infected individuals. CD1d surface expression on human cell lines was downregulated after infection with M-tropic HIV, T-tropic HIV, or after exposure to HIV gp120 in vitro. These data suggest that CD1d-mediated responses are altered during HIV infection and may thus contribute to the global immunodeficiency seen in these patients.

The primary screening of hybridoma clones secreting monoclonal antibodies (MAbs) requires the testing of a large number of hybridoma culture supernatants within a short time and is very labor-intensive. In addition, the type of antigen and its location in the cell have to be considered when selecting the appropriate screening procedure, but relatively few reagents are available for analyzing these molecules. We have developed an intracellular and cell surface ELISA technique for screening hybridoma supernatants that hastens the screening procedure of a large number of clones in a short period of time, as the supernatants of fused cells grown in 96-well plates are used directly in the assay. This novel screening technique is rapid, sensitive, specific, and applicable to MAbs specific for a wide variety of intracellular and/or cell surface proteins.

CD11b, CD11c, and F4/80 are normally used to define dendritic cell and/or macrophage populations. In this study, the expression of all three markers was observed on CD8(+) T cells following infection of mice with several distinct viruses. Using lymphocytic choriomeningitis virus as a model virus, it was found that relatively more CD11b(+)CD8(+) and CD11c(+)CD8(+) T cells were present in the periphery than in primary lymphoid organs; in contrast, the F4/80(+)CD8(+) T cell population was more prevalent in the spleen. All three myeloid markers were detected on virus-specific CTL. The expression of CD11b and CD11c on CD8(+) T cells correlated with their level of CTL activity, whereas the F4/80(+)CD8(+) T cell population increased after the peak of the CTL response but did not have higher CTL activity. These data suggest that there is a differential induction of CD11b, CD11c, and F4/80 on virus-specific CD8(+) T cells following an acute virus infection.

CD1d molecules are lipid antigen-presenting molecules. They are involved in presenting these antigens to a unique subpopulation of T cells called natural killer T (NKT) cells, which have the capacity to produce both T helper (Th) 1 and Th2 cytokines. Thus, it is possible that the antigens presented by CD1d and/or the level at which they are presented could have profound effects on the immunoregulation of autoimmune and infectious diseases, as well as cancer. Because of the ability of CD1d-binding ligands to modulate NKT cell responses, targeting CD1d-mediated antigen presentation as a novel approach for new therapies in these and other diseases holds great promise.

The CD28− subset of CD8+ T cells is associated with cytotoxic T lymphocyte (CTL) effector function. We investigated a potential role for 4-1BB, a costimulatory molecule structurally related to members of the tumor necrosis factor (TNF) receptor family, in the generation and functional activation of CD28− CTLs by using human cord blood (CB) cells composed exclusively of naive CD8+ T cells with few or no CD28− CTLs. The 4-1BB was induced preferentially on the CB CD28−CD8+ T cells when CD28 down-regulation was induced by interleukin 15 (IL-15) and IL-12 stimulation. Anti–4-1BB costimulation induced dramatic phenotypic changes in the CD28− CTLs, including restoration of CD28 expression as well as that of memory markers such as CD45RO and CC chemokine receptor 6 (CCR6). Anti–4-1BB costimulation also promoted long-term survival of CD28− CTLs, which were sensitive to activation-induced cell death upon anti-CD3 stimulation. The memory-type CD28+CTLs induced by anti–4-1BB costimulation acquired a greatly enhanced content of granzyme B, a cytolytic mediator, and enhanced cytotoxic activity as compared with CD28− CTLs. Strong cytotoxicity of memory-type CTLs to a 4-1BB ligand–expressing Epstein-Barr virus (EBV)–transformed B-cell line was almost completely abrogated by 4-1BB–Fc, a soluble form of 4-1BB, suggesting involvement of 4-1BB in cytolytic processes. Taken all together, our results suggest that 4-1BB plays a role in the differentiation of effector memory CTLs.

We have reported that lung allograft rejection involves an immune response to a native protein in the lung, type V collagen (col(V)), and that col(V)-induced oral tolerance prevented acute and chronic rejection. In support of these findings col(V) fragments were detected in allografts during rejection, but not in normal lungs. The purpose of the current study was to isolate and characterize col(V)-specific allograft-infiltrating T cells and to determine their contribution to the rejection response in vivo. Two col(V)-specific T cell lines, LT1 and LT3, were isolated from F344 (RT1lv1) rat lung allografts during rejection that occurred after transplantation into WKY (RT1l) recipients. Both cell lines, but not normal lung lymphocytes, proliferated in response to col(V). Neither LT1 nor LT3 proliferated in response to alloantigens. LT1 and LT3 were CD4+CD25− and produced IFN-γ in response to col(V). Compared with normal CD4+ T cells, both cell lines expressed a limited V-β TCR repertoire. Each cell strongly expressed V-β 9 and 16, but differed in expression of other V-βs. Adoptive transfer of each cell line did not induce pathology in lungs of normal WKY rats. In contrast, adoptive transfer of LT1, but not LT3, caused marked peribronchiolar and perivascular inflammation in isograft (WKY) lungs and abrogated col(V)-induced oral tolerance to allograft (F344) lungs. Collectively, these data show that lung allograft rejection involves both allo- and autoimmune responses, and graft destruction that occurs during the rejection response may expose allograft-infiltrating T cells to potentially antigenic epitopes in col(V).

Neoplastic transformation of cells is accompanied by an aberration of cell surface glycolipid composition. These tumor-associated, altered glycosphingolipids are often shed into the tumor cell microenvironment and mediate immunosuppressive activity. The nature and form of glycolipids shed by a variety of tumor cell lines and the mechanism(s) of shedding have been well characterized. The murine T cell lymphoma line, L5178Y-R, is known to shed a tumor-associated glycolipid, gangliotriaosylceramide, into the culture medium. We analyzed the effect of glycolipids from L5178Y-R on antigen presentation by murine CD1d1 molecules. CD1d1 molecules present glycolipid antigens to a specialized class of T cells called natural killer T (NKT) cells that mainly express a T cell receptor α chain (Vα14Jα281) associated with Vβ chains of limited diversity. In the current report, we found that L5178Y-R cells express CD1 on their cell surface yet are unable to stimulate CD1d1-specific NKT cells. We hypothesized that the glycolipid(s) shed by L5178Y-R inhibited antigen presentation by CD1d1. Pretreatment of CD1d1+cells with conditioned medium from L5178Y-R inhibited CD1-specific stimulation of canonical (Vα14+) but not noncanonical (Vα5+) NKT cells. Exogenous addition of lipids extracted from L5178Y-R cells as well as purified gangliotriaosylceramide mimicked this effect. Inhibition of glycolipid shedding in L5178Y-R cells withd-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol resulted in the rescue of CD1d1 recognition by canonical (but not noncanonical) NKT cells. These results suggest that one means by which certain tumor cells can evade the host's innate antitumor immune response is by shedding glycolipids that inhibit CD1-mediated antigen presentation to NKT cells.

Obesity is a major risk factor for certain types of cancer and the mechanisms by which obesity contributes to tumor metastasis are not well understood. Whereas the immune system is essential for fighting against tumors, in the context of obesity, systemic chronic inflammation with altered immune responses are found. Recent studies have revealed that the gut microbiota also contributes to obesity and the pathology associated with that disease. Mucosal-associated innate T (MAIT) cells are a unique subpopulation of T cells that are characterized by the expression of a semi-invariant TCR a chain (Vα19 in mice; Vα7.2 in humans). Although the development and maturation of MAIT cells require the gut microbiota and antigen presenting molecule MR1, the actual function of MAIT cells has not been well studied. We have found reduced circulating MAIT cells in obese patients as compared to age- and gender-matched healthy donors. Using real-time PCR to determine the expression levels of the invariant TCR of MAIT cells and classical T cell transcription factors, we found that, although no difference was observed in any gene expression analyzed between samples from obese patients or healthy donors, mRNA expression of the Vα7.2 TCR positively correlated with the transcription factor TBX21 (found in T helper 1 cells) and BCL6 (found in follicular helper T cells). Our work suggests that obesity alters the number of MAIT cells. The correlation of MAIT cell TCR gene expression with transcription factors of classical effector T cells, suggests there may be transcriptional pathways linking MAIT cells to the function of conventional T cells. Future work will focus on determining how obesity impacts the function of MAIT cells.

We previously reported that posttransplant alloantibody production in CD8-deficient hosts is IL-4+ CD4+ T cell-dependent and IgG1 isotype-dominant. The current studies investigated the hypothesis that IL-4-producing natural killer T cells (NKT cells) contribute to maximal alloantibody production. To investigate this, alloantibody levels were examined in CD8-deficient WT, CD1d KO and Jα18 KO transplant recipients. We found that the magnitude of IgG1 alloantibody production was critically dependent on the presence of type I NKT cells, which are activated by day 1 posttransplant. Unexpectedly, type I NKT cell contribution to enhanced IgG1 alloantibody levels was interferon-γ-dependent and IL-4-independent. Cognate interactions between type I NKT and B cells alone do not stimulate alloantibody production. Instead, NKT cells appear to enhance maturation of IL-4+ CD4+ T cells. To our knowledge, this is the first report to substantiate a critical role for type I NKT cells in enhancing in vivo antibody production in response to endogenous antigenic stimuli.

Current knowledge of the processing of viral Ags into MHC class I-associated ligands is based almost completely on in vitro studies using nonprofessional APCs (pAPCs). This is two steps removed from real immune responses to pathogens and vaccines, in which pAPCs activate naive CD8+ T cells in vivo. Rational vaccine design requires answers to numerous questions surrounding the function of pAPCs in vivo, including their abilities to process and present peptides derived from endogenous and exogenous viral Ags. In the present study, we characterize the in vivo dependence of Ag presentation on the expression of TAP by testing the immunogenicity of model Ags synthesized by recombinant vaccinia viruses in TAP1−/− mice. We show that the efficiency of TAP-independent presentation in vitro correlates with TAP-independent activation of naive T cells in vivo and provide the first in vivo evidence for proteolytic processing of antigenic peptides in the secretory pathway. There was, however, a clear exception to this correlation; although the presentation of the minimal SIINFEKL determinant from chicken egg OVA in vitro was strictly TAP dependent, it was presented in a TAP-independent manner in vivo. In vivo presentation of the same peptide from a fusion protein retained its TAP dependence. These results show that determinant-specific processing pathways exist in vivo for the generation of antiviral T cell responses. We present additional findings that point to cross-priming as the likely mechanism for these protein-specific differences.