Dual-Antigen COVID-19 Vaccine Subcutaneous Prime Delivery With Oral Boosts Protects NHP Against SARS-CoV-2 Challenge.

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice.

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.

Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Mycobacterium bovis Bacillus Calmette-Guérin Alters Melanoma Microenvironment Favoring Antitumor T Cell Responses and Improving M2 Macrophage Function.

Intralesional Mycobacterium bovis bacillus Calmette-Guérin (BCG) has long been a relatively inexpensive therapy for inoperable cutaneous metastatic melanoma (CMM), although intralesional BCG skin mechanisms remain understudied. We analyzed intralesional BCG-treated CMM lesions combined with in vitro studies to further investigate BCG-altered pathways. Since macrophages play a pivotal role against both cancer and mycobacterial infections, we hypothesized BCG regulates macrophages to promote antitumor immunity. Tumor-associated macrophages (M2) infiltrate melanomas and impair antitumor immunity. BCG-treated, in vitro-polarized M2 (M2-BCG) showed transcriptional changes involving inflammation, immune cell recruitment, cross talk, and activation pathways. Mechanistic network analysis indicated M2-BCG potential to improve interferon gamma (IFN-γ) responses. Accordingly, frequency of IFN-γ-producing CD4+ T cells responding to M2-BCG vs. mock-treated M2 increased (p < 0.05). Moreover, conditioned media from M2-BCG vs. M2 elevated the frequency of granzyme B-producing CD8+ tumor-infiltrating lymphocytes (TILs) facing autologous melanoma cell lines (p < 0.01). Furthermore, transcriptome analysis of intralesional BCG-injected CMM relative to uninjected lesions showed immune function prevalence, with the most enriched pathways representing T cell activation mechanisms. In vitro-infected MM-derived cell lines stimulated higher frequency of IFN-γ-producing TIL from the same melanoma (p < 0.05). Our data suggest BCG favors antitumor responses in CMM through direct/indirect effects on tumor microenvironment cell types including macrophages, T cells, and tumor itself.

Insights into Local Tumor Microenvironment Immune Factors Associated with Regression of Cutaneous Melanoma Metastases by Mycobacterium bovis Bacille Calmette-Guérin.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is listed as an intralesional (IL) therapeutic option for inoperable stage III in-transit melanoma in the National Comprehensive Cancer Network Guidelines. Although the mechanism is unknown, others have reported up to 50% regression of injected lesions, and 17% regression of uninjected lesions in immunocompetent patients after direct injection of BCG into metastatic melanoma lesions in the skin. BCG and other mycobacteria express ligands capable of stimulating the γ9δ2 T cells. Therefore, we hypothesized that γ9δ2 T cells play a role in promoting BCG-mediated antitumor immunity in patients treated with IL-BCG for in-transit cutaneous melanoma metastases. Indeed, we found γ9δ2 T cell infiltration in melanoma skin lesions during the course of IL-BCG treatment. Gene expression analysis revealed that BCG injection elicits the expression of a vast array of chemokines in tumor lesions, including strong expression of CXCL9, 10, and 11, a set of chemokines that attract T cells expressing the CXCR3 chemokine receptor. In corroboration with our hypothesis, approximately 85% of γδ T cells express high levels of CXCR3 on their surface. Importantly, the injected tumor lesions also express genes whose protein products are the antigenic ligands for γδ T cells (BTN3A1 and MICB), and the cytokines that are the typical products of activated γδ T cells. Interestingly, we also found that γδ T cells infiltrate the regressed lesions that did not receive BCG injections. Our study suggests that γ9δ2 T cells may contribute to melanoma regression induced by IL-BCG treatment.

Characterization of the microbiome of nipple aspirate fluid of breast cancer survivors.

The microbiome impacts human health and disease. Until recently, human breast tissue and milk were presumed to be sterile. Here, we investigated the presence of microbes in the nipple aspirate fluid (NAF) and their potential association with breast cancer. We compared the NAF microbiome between women with a history of breast cancer (BC) and healthy control women (HC) using 16S rRNA gene amplicon sequencing. The NAF microbiome from BC and HC showed significant differences in community composition. Two Operational Taxonomic Units (OTUs) showed differences in relative abundances between NAF collected from BC and HC. In NAF collected from BC, there was relatively higher incidence of the genus Alistipes. By contrast, an unclassified genus from the Sphingomonadaceae family was relatively more abundant in NAF from HC. These findings reflect the ductal source DNA since there were no differences between areolar skin samples collected from BC and HC. Furthermore, the microbes associated with BC share an enzymatic activity, Beta-Glucuronidase, which may promote breast cancer. This is the first report of bacterial DNA in human breast ductal fluid and the differences between NAF from HC and BC. Further investigation of the ductal microbiome and its potential role in breast cancer are warranted.

Cross-platform comparison of independent datasets identifies an immune signature associated with improved survival in metastatic melanoma.

Platform and study differences in prognostic signatures from metastatic melanoma (MM) gene expression reports often hinder consensus arrival. We performed survival/outcome-based pairwise comparisons of three independent MM gene expression profiles using the threshold-free algorithm rank-rank hypergeometric overlap analysis (RRHO). We found statistically significant overlap for genes overexpressed in favorable outcome (FO) groups, but no overlap for poor outcome (PO) groups. This "favorable outcome signature" (FOS) of 228 genes coinciding on all three overlapping gene lists showed immune function predominated in FO MM. Surprisingly, specific cell signature-enrichment analysis showed B cell-associated genes enriched in FO MM, along with T cell-associated genes. Higher levels of B and T cells (p<0.05) and their relative proximity (p<0.05) were detected in FO-to-PO tumor comparisons from an independent MM patients cohort. Finally, expression of FOS in two independent Stage III MM tumor datasets correctly predicted clinical outcome in 12/14 and 44/70 patients using a weighted gene voting classifier (area under the curve values 0.96 and 0.75, respectively). This RRHO-based, cross-study analysis emphasizes the RRHO approach power, confirms T cells relevance for prolonged MM survival, supports a favorable role for B cells in anti-melanoma immunity, and suggests B cells potential as means of intervention in melanoma treatment.

Clinical Benefit from Ipilimumab Therapy in Melanoma Patients may be Associated with Serum CTLA4 Levels.

Stage IV metastatic melanoma patients historically have a poor prognosis with 5-10% 5-year survival. Ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte antigen 4 (CTLA4), is one of the first treatments to provide beneficial durable responses in advanced melanoma. However, less than 25% of those treated benefit, treatment is expensive, and side effects can be fatal. Since soluble (s) CTLA4 may mediate inhibitory effects previously ascribed to the membrane-bound isoform (mCTLA4), we hypothesized patients benefiting from ipilimumab have higher serum levels of sCTLA4. We found that higher sCTLA4 levels correlated both with response and improved survival in patients treated with ipilimumab in a small patient cohort [patients with (n = 9) and without (n = 5) clinical benefit]. sCTLA4 levels were statistically higher in ipilimumab-treated patients with response to ipilimumab. In contrast, sCTLA4 levels did not correlate with survival in patients who did not receive ipilimumab (n = 11). These preliminary observations provide a previously unrecognized link between serum sCTLA4 levels and response to ipilimumab as well as to improved survival in ipilimumab-treated melanoma patients and a potential mechanism by which ipilimumab functions.

Microbial dysbiosis is associated with human breast cancer.

Breast cancer affects one in eight women in their lifetime. Though diet, age and genetic predisposition are established risk factors, the majority of breast cancers have unknown etiology. The human microbiota refers to the collection of microbes inhabiting the human body. Imbalance in microbial communities, or microbial dysbiosis, has been implicated in various human diseases including obesity, diabetes, and colon cancer. Therefore, we investigated the potential role of microbiota in breast cancer by next-generation sequencing using breast tumor tissue and paired normal adjacent tissue from the same patient. In a qualitative survey of the breast microbiota DNA, we found that the bacterium Methylobacterium radiotolerans is relatively enriched in tumor tissue, while the bacterium Sphingomonas yanoikuyae is relatively enriched in paired normal tissue. The relative abundances of these two bacterial species were inversely correlated in paired normal breast tissue but not in tumor tissue, indicating that dysbiosis is associated with breast cancer. Furthermore, the total bacterial DNA load was reduced in tumor versus paired normal and healthy breast tissue as determined by quantitative PCR. Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer. Lastly, we observed lower basal levels of antibacterial response gene expression in tumor versus healthy breast tissue. Taken together, these data indicate that microbial DNA is present in the breast and that bacteria or their components may influence the local immune microenvironment. Our findings suggest a previously unrecognized link between dysbiosis and breast cancer which has potential diagnostic and therapeutic implications.

Interleukin-1ß triggers the differentiation of macrophages with enhanced capacity to present mycobacterial antigen to T cells.

The rapid differentiation of monocytes into macrophages (MΦ) and dendritic cells is a pivotal aspect of the innate immune response. Differentiation is triggered following recognition of microbial ligands that activate pattern recognition receptors or directly by pro-inflammatory cytokines. We demonstrate that interleukin-1ß (IL-1ß) induces the rapid differentiation of monocytes into CD209(+) MΦ, similar to activation via Toll-like receptor 2/1, but with distinct phenotypic and functional characteristics. The IL-1ß induced MΦ express higher levels of key markers of phagocytosis, including the Fc-receptors CD16 and CD64, as well as CD36, CD163 and CD206. In addition, IL-1ß-induced MΦ exert potent phagocytic activity towards inert particles, oxidized low-density lipoprotein and mycobacteria. Furthermore, IL-1ß-induced MΦ express higher levels of HLA-DR and effectively present mycobacterial antigens to T cells. Therefore, the ability of IL-1ß to induce monocyte differentiation into MΦ with both phagocytosis and antigen-presenting function is a distinct part of the innate immune response in host defence against microbial infection.

Immune correlates of melanoma survival in adoptive cell therapy.

The advent of immunotherapies for cancer has resulted in robust clinical responses and confirmed that the immune system can significantly inhibit tumor progression. The recent success of adoptive cell therapy against melanoma suggests that endogenous T-cell responses have the potential to control cancer. However, the lack of responses in some patients receiving such therapy indicates a need for a better understanding of the host immune response to solid tumors. In this review, we summarize the current knowledge on the characteristics of adoptively transferred T cells associated with successful anti-melanoma immune responses in humans.

Galectin-3 regulates the innate immune response of human monocytes.

Galectin-3 is a ß-galactoside-binding lectin widely expressed on epithelial and hematopoietic cells, and its expression is frequently associated with a poor prognosis in cancer. Because it has not been well-studied in human infectious disease, we examined galectin-3 expression in mycobacterial infection by studying leprosy, an intracellular infection caused by Mycobacterium leprae. Galectin-3 was highly expressed on macrophages in lesions of patients with the clinically progressive lepromatous form of leprosy; in contrast, galectin-3 was almost undetectable in self-limited tuberculoid lesions. We investigated the potential function of galectin-3 in cell-mediated immunity using peripheral blood monocytes. Galectin-3 enhanced monocyte interleukin 10 production to a TLR2/1 ligand, whereas interleukin 12p40 secretion was unaffected. Furthermore, galectin-3 diminished monocyte to dendritic cell differentiation and T-cell antigen presentation. These data demonstrate an association of galectin-3 with unfavorable host response in leprosy and a potential mechanism for impaired host defense in humans.

Isolation of a distinct Mycobacterium tuberculosis mannose-capped lipoarabinomannan isoform responsible for recognition by CD1b-restricted T cells.

Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.

NOD2 triggers an interleukin-32-dependent human dendritic cell program in leprosy.

It is unclear whether the ability of the innate immune system to recognize distinct ligands from a single microbial pathogen via multiple pattern recognition receptors (PRRs) triggers common pathways or differentially triggers specific host responses. In the human mycobacterial infection leprosy, we found that activation of monocytes via nucleotide-binding oligomerization domain-containing protein 2 (NOD2) by its ligand muramyl dipeptide, as compared to activation via heterodimeric Toll-like receptor 2 and Toll-like receptor 1 (TLR2/1) by triacylated lipopeptide, preferentially induced differentiation into dendritic cells (DCs), which was dependent on a previously unknown interleukin-32 (IL-32)-dependent mechanism. Notably, IL-32 was sufficient to induce monocytes to rapidly differentiate into DCs, which were more efficient than granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived DCs in presenting antigen to major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. Expression of NOD2 and IL-32 and the frequency of CD1b(+) DCs at the site of leprosy infection correlated with the clinical presentation; they were greater in patients with limited as compared to progressive disease. The addition of recombinant IL-32 restored NOD2-induced DC differentiation in patients with the progressive form of leprosy. In conclusion, the NOD2 ligand-induced, IL-32-dependent DC differentiation pathway contributes a key and specific mechanism for host defense against microbial infection in humans.

The role of aggregation in Fusobacterium nucleatum- induced immune cell death.

INTRODUCTION: Fusobacterium nucleatum, an anaerobic oral bacterium, has been shown to be highly abundant in endodontic infections. Its role in these infections remains unclear. Previous studies have shown that F. nucleatum could aggregate immune cells. We have demonstrated that F. nucleatum can induce significant apoptosis in peripheral blood mononuclear cells (PBMCs). In this in vitro study, we sought to determine what role this aggregation phenomenon has on the induction of apoptosis in PBMCs. METHODS: F. nucleatum bacteria were treated as follows: formaldehyde-fixed, heat-treated, or sonicated before co-culturing with PBMCs. Cell aggregation and apoptosis of the PBMCs were assessed under light microscopy and analyzed by flow cytometry, respectively. PBMCs were then immobilized with a Matrigel matrix before treatment with F. nucleatum. Aggregation and apoptosis were assessed as before. Surface staining of activation marker CD69 was assessed by flow cytometry. The apoptosis and CD69 data underwent one-way analysis of variance, followed by post hoc Bonferroni test and χ(2) test, respectively, to determine statistical significance. RESULTS: Viable and formaldehyde-treated but not sonicated or heat-treated F. nucleatum bacteria were able to cause severe aggregation and apoptosis of the immune cells. Disruption of F. nucleatum mediated aggregation by immobilization of the cells with a Matrigel matrix resulted in a significant diminution of cell death but not cell activation when assessed by using surface expression of CD69 early activation antigen. CONCLUSIONS: F. nucleatum's ability to induce cell death in immune cells appears to be mediated through the immune cells being aggregated, which might have important implications for its pathogenesis.

Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages.

Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D-dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D-sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D-deficient serum with 25-hydroxyvitamin D3 restored IFN-γ-induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection.

Structural differences in lipomannans from pathogenic and nonpathogenic mycobacteria that impact CD1b-restricted T cell responses.

Mannosylated molecules on the Mycobacterium tuberculosis surface are important determinants in the immunopathogenesis of tuberculosis. To date, much attention has been paid to mannose-capped lipoarabinomannan, which mediates phagocytosis and intracellular trafficking of M. tuberculosis by engaging the macrophage mannose receptor and subsequently binds to intracellular CD1b molecules for presentation to T cells. Another important mannosylated lipoglycan on the M. tuberculosis surface is lipomannan (LM). Comparative structural detail of the LMs from virulent and avirulent strains is limited as is knowledge regarding their differential capacity to be recognized by the adaptive immune response. Here, we purified LM from the avirulent M. smegmatis and the virulent M. tuberculosis H(37)R(v), performed a comparative structural biochemical analysis, and addressed their ability to stimulate CD1b-restricted T cell clones. We found that M. tuberculosis H(37)R(v) produces a large neutral LM (TB-LM); in contrast, M. smegmatis produces a smaller linear acidic LM (SmegLM) with a high succinate content. Correspondingly, TB-LM was not as efficiently presented to CD1b-restricted T cells as SmegLM. Thus, here we correlate the structure-function relationships for LMs with CD1b-restricted T cell responses and provide evidence that the structural features of TB-LM contribute to its diminished T cell responsiveness.

Activation of Fc gamma RI on monocytes triggers differentiation into immature dendritic cells that induce autoreactive T cell responses.

The formation of immune complexes results in activation of the innate immune system and subsequent induction of host inflammatory responses. In particular, the binding of IgG immune complexes to FcgammaR on monocytes triggers potent inflammatory responses leading to tissue injury in disease. We investigated whether activation of monocytes via FcgammaR induced cell differentiation, imparting specific inflammatory functions of the innate immune response. Human IgG alone induced monocytes to differentiate into cells with an immature dendritic cell (iDC) phenotype, including up-regulation of CD1b, CD80, CD86, and CD206. Differentiation into CD1b(+) iDC was dependent on activation via CD64 (FcgammaRI) and induction of GM-CSF. The human IgG-differentiated iDC were phenotypically different from GM-CSF-derived iDC at the same level of CD1b expression, with higher cell surface CD86, but lower MHC class II, CD32, CD206, and CD14. Finally, in comparison to GM-CSF-derived iDC, IgG-differentiated iDC were more efficient in activating T cells in both autologous and allogeneic mixed lymphocyte reactions but less efficient at presenting microbial Ag to T cells. Therefore, activation of FcgammaRI on monocytes triggers differentiation into specialized iDC with the capacity to expand autoreactive T cells that may contribute to the pathogenesis of immune complex-mediated tissue injury.

Host-derived oxidized phospholipids and HDL regulate innate immunity in human leprosy.

Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.

Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation.

CD4(+) T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4(+) T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by alpha-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.