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Item does not contain fulltext BACKGROUND: Immune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1-2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma. METHODS: In this phase 3, multicenter, open-label trial, we randomly assigned patients with unresectable stage IIIC or IV melanoma in a 1:1 ratio to receive TIL or anti-cytotoxic T-lymphocyte antigen 4 therapy (ipilimumab at 3 mg per kilogram of body weight). Infusion of at least 5×10(9) TILs was preceded by nonmyeloablative, lymphodepleting chemotherapy (cyclophosphamide plus fludarabine) and followed by high-dose interleukin-2. The primary end point was progression-free survival. RESULTS: A total of 168 patients (86% with disease refractory to anti-programmed death 1 treatment) were assigned to receive TILs (84 patients) or ipilimumab (84 patients). In the intention-to-treat population, median progression-free survival was 7.2 months (95% confidence interval [CI], 4.2 to 13.1) in the TIL group and 3.1 months (95% CI, 3.0 to 4.3) in the ipilimumab group (hazard ratio for progression or death, 0.50; 95% CI, 0.35 to 0.72; P

SUMMARY Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1–6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities., In brief Through LPA production, ATX modulates the tumor microenvironment in autocrine-paracrine manners. Matas-Rico et al. show that ATX/LPA is chemorepulsive for T cells with a dominant inhibitory role for Gα12/13-coupled LPAR6. Upon anticancer vaccination, tumor-intrinsic ATX suppresses the infiltration of CD8+ T cells without affecting their cytotoxic quality., Graphical Abstract

For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available., Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro. Here, we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis, M. marinum, or M. leprae. In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo, translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals. IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis.

Mycobacterium tuberculosis infections claim more than a million lives each year and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from the phago-lysosomes to the cytosol upon phagocytosis by macrophages. The translocation from the phago-lysosome into the cytosol is an ESX-1 dependent process as previously shown in vitro. Here we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillo, zebrafish and patient material infected with M. tuberculosis, M. marinum or M. leprae. In contrast, when innate or adaptive immunity was compromised, as in SCID or IL-1R1 deficient mice, a significant number of cytosolic M. tuberculosis bacilli were detected in lungs of infected mice. Taken together, M. tuberculosis infection is controlled by adaptive immune responses as well as IL-1R1-mediated signals that result in clearance of cells containing cytosolic mycobacteria in vivo.ImportanceFor decades, Mycobacterium tuberculosis is one of the deathliest pathogens known. Despite infecting approximately one third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is the subcellular localization, as M. tuberculosis can translocate from the phago-lysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study we establish that high level cytosolic escape of mycobacteria can indeed occur in vivo, but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in clearance of cells containing cytosolic mycobacteria, connects two important fields: cell-biology and immunology which is vital for the understanding of the pathology of M. tuberculosis.

SummaryAutotaxin (ATX) is secreted by diverse cell types to produce lysophosphatidic acid (LPA) that regulates multiple biological functions via G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis mainly via LPAR1; however, its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T-cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T-cell responses but suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from patient samples are consistent with intra-tumor ATX acting as a T-cell repellent. These studies highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T-cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Currently a TCR gene therapy trial to treat stage IV cutaneous and uveal melanoma patients is being conducted at the NKI. The TCR used for T-cell modification is specific for the HLA-A*0201 restricted MART-126-35 epitope, which is expressed on the majority of melanoma cells. Unique to this trial is the use of the combination of anti-CD3/CD28 beads for T-cell activation plus IL-7/ IL-15 for subsequent culture and expansion, instead of the more commonly used strategy that utilizes the combination of anti-CD3 and IL-2. The aim of this alternative production strategy is to generate a “less differentiated“ T-cell product that may have a better engraftment potential and antitumor activity. Thus far, a total of 12 patients have been treated, of whom 5 were uveal melanoma patients. 1 patient treated with 5x109 cells died 9 days after infusion, among others due to a severe cytokine release syndrome. The other 11 patients were treated in 3 different cell doses: 5x107, 25x107and10x107 transduced CD4 and CD8 T-cells. A prolonged engraftment and higher % of gene modified cells in the blood compartment were observed in patients treated with the highest number of cells infused. Gene modified T-cells could be found back in the circulation for up to two months post infusion, even when as few as 5x107 transduced T-cells were infused. In the highest cell dose, transduced T-cells could be detected up to 10 months following infusion. On-target toxicity against MART-1 expressing melanocytes in the skin (rash and vitiligo) was observed in all patients. Most severe skin toxicity, but also uveitis and inner ear toxicity, were observed in patients treated in the highest dose cohort of 2.5x107 transduced T-cells. These patients also developed a cytokine release syndrome requiring tocilizumab (anti-IL6R) treatment. Unexpected toxicity observed at this highest dose level consisted of development of pancytopenia around 6-8 weeks post T-cell infusion. In one of these patients, T-cell clonality of the infused cell product and of the peripheral blood compartment at the time of pancytopenia was analyzed in order to investigate the mechanism behind this toxicity. The highest clonality was found in the endogenous unmodified CD4 population. TCR diversity and evenness of the CD8 modified and unmodified populations were similar, suggesting that the pancytopenia was not associated with specific expansion of a TCR modified T-cell clone. However, this delayed pancytopenia could be related to 1) the conditioning chemotherapy regimen (fludarabine and cyclophosphamide), 2) cytokine release like IL-6, or 3) expansion of an autoreactive clone derived either from the infused T-cell product or newly generated from the endogenous T-cell pool. Of the 12 patients treated thus far, 8 patients had stable disease (SD) and two showed a partial response (PR) (5.2 and 9.5 month). The type of response or the duration of response was not correlated to the amount of cells infused or the peak and duration of engraftment of the infused T-cells. On the basis of these preliminary data, we conclude that TCR-modified T-cells produced by this method have a high engraftment potential and show in vivo activity at low infused cell doses, thereby suggesting the potential value of this protocol for TCR or CAR gene therapy for the treatment of cancer. Citation Format: Raquel Gomez-Eerland, Maartje Rohaan, Joost van den Berg, Maaike van Zon, Renate de Boer, Noor Bakker, Bastiaan Nuijen, Ton N.M. Schumacher, John B.A.G. Haanen. Adoptive transfer of autologous T-cells, modified with a MART-1 specific TCR and cultured in IL-7/IL-15, for the treatment of metastatic melanoma patients [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 B205.

Infusion of tumor-infiltrating T-lymphocytes was developed as treatment for metastatic melanoma by Dr. S. Rosenberg (Surgery Branch, NIH, Bethesda, MD, USA) in the late 90's of the past century. This therapy was based on the finding that melanomas oftentimes have infiltrates of CD8 T cells that show recognition of melanoma cells. Many tumor antigens have been characterized for which specific T cells can be found within the tumor or in peripheral blood. As tumors may use mechanisms to induce local T cell unresponsiveness, isolation of TILs and in vitro activation and expansion was shown to overcome this anergic state both in preclinical models and in clinical trials. In order for the TILs to survive, persist, and be cytolytic after infusion, culture time should be as short as possible (to obtain cells with a less terminally differentiated phenotype), patients should receive lymphodepleting treatment (chemotherapy +/- TBI) prior to infusion and interleukin-2 as a survival factor post infusion. Up to date several hundreds of metastatic melanoma patients have been treated with TIL therapy in centers within the US, Europe and Israel. Objective response rates vary between 38 and 72% depending on the phase I/II trial with 10-20% of patients acquiring a CR, which were most often very durable. Many of these oftentimes heavily pretreated patients have benefitted from TIL therapy. As melanomas are heavily mutated cancers, the idea that T cells may react to epitopes derived from mutated proteins could be investigated with new technologies developed at NKI. Using whole exome sequencing, the total mutational burden could be measured for each melanoma. Only those mutations present in expressed genes were used for epitope prediction for every MHC class I molecule of the patient using validated prediction algorithms. Filters were used to select those epitopes with high enough binding affinity for MHC that were subsequently synthesized for production of peptide-MHC multimers that could be used to screen for neo-antigen specific T cells among peripheral blood T- cells or TIL using a multiplex flow cytometric technology. Together with the NCI, using a somewhat different approach, we were able to show that in the vast majority of melanoma TIL, CD8 T cells specific for epitopes resulting from UV light induced DNA damage can be found, sometimes in high frequencies (up to 25-30%), strongly suggesting that neo-antigen-specific T cells play an important role in the success of TIL therapy and possibly other immunotherapies, including checkpoint inhibitors. So far TIL therapy has been mostly used in metastatic melanoma patients pretreated with BRAF inhibitors, anti-CTLA4, high-dose IL-2, and demonstrated activity irrespective of these treatments, although isolation of TIL appeared less successful in patients progressing upon BRAFi. Early results in patients pretreated with PD1 blocking antibodies are promising as well, although the objective response rate is still unknown. Next to metastatic melanoma, TIL can be grown from other cancers as well and promising results have been obtained in patients with cervical cancer and in a patient with cholangiocarcinoma. Trials are planned for patients with epithelial ovarian cancer as well. Citation Format: John B.A.G. Haanen, Joost Van den Berg, Maaike Van Zon, Noor Bakker, Marnix Geukes, Hans Van Thienen, Christian Blank, Pia Kvistborg, Ton Schumacher. TIL therapy in the era of immune checkpoint blockade. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL01-03.

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC-MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.

In the past few years, we have shown in cell cultures that the localization of Mycobacterium tuberculosis is crucial for its pathogenicity [1]. While vaccine strain M. bovis BCG and other non-patho...

Item does not contain fulltext Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. 01 augustus 2012

TPS9602Background: Since the introduction of targeted therapy and checkpoint inhibitors, the historically poor prognosis of patients with unresectable stage IIIc/IV melanoma has greatly improved, w...

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC-MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5 - two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators - during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. Copyright © 2011 by The American Association of Immunologists, Inc.

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins., Author Summary The genus Mycobacterium contains a number of important pathogens, such as Mycobacterium tuberculosis. The highly characteristic cell envelope of these bacteria plays a crucial role in the infection process. The most apparent difference with other bacteria is the recently described outer membrane composed of unique (glyco)lipids. However, on top of this membrane mycobacteria also have an ill-defined capsular layer. In this paper, we studied this capsular layer using different electron microscopy techniques and mass spectrometry. Using close to native state preparation method, we show that both pathogenic and non-pathogenic mycobacteria have a labile capsular layer that covers the outer membrane. This capsular layer, in addition to containing arabinogalactan, glycan and mannose-containing glyco-lipids, also surprisingly contains a large amount of ESX-1-secreted proteins in Mycobacterium marinum. Furthermore, we also show that the capsule plays a role in the binding of macrophages and the induction of cytokines. Collectively, these results show for the first time that the capsule can be visualized on both pathogenic and non-pathogenic mycobacteria. In addition, growing mycobacteria under standard laboratory conditions in the presence of detergent with agitation promotes capsular shedding and influences the biological characteristics of the bacteria.

Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find that in Mycobacterium tuberculosis (Mtb)-infected DCs, CD1 presents antigens quickly. Surprisingly, rapid DC maturation results in early failure and delay in MHCII presentation. Whereas both CD1b and MHCII localize to bacterial phagosomes early after phagocytosis, MHCII traffics from the phagosome to the plasma membrane with a rapid kinetic that can precede antigen availability and loading. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow organisms to use DC maturation as a mechanism of immune evasion. In contrast, CD1 antigen presentation occurs in the face of Mtb infection and rapid DC maturation because a pool of CD1 molecules remains available on the phagolysosome membrane that is able to acquire lipid antigens and deliver them to the plasma membrane.

One of the focal points of our group is to reveal the macromolecular organisation of cells by means of cryo-electron tomography. This is the only method that can be used to obtain molecular resolution information of intact cells constituencies in a near-native situation. The tomogram contains a 3D map of the cellular proteome and we are just beginning to explore its potential after we worked for a few years on some technical hurdles of specimen preparation. The other central point of our group is to localize gene product in cellular structures by EM at the highest resolution with gold probes on cryosections. Our main focus is on understanding the molecular machinery and organization within the endomembrane system. We concentrate our work on the transport mechanism within the endocytic pathway and the link to pathogenesis. Cryo-electron tomography of unfixed frozen hydrated cryo-sections and cryo immunogold-EM of aldehyde fixed cells are our main techniques, which allow the subcellular detection and visualization of molecular machines at the highest resolution.

The ESX-5 secretion system of pathogenic mycobacteria is responsible for the secretion of various PPE and PE-PGRS proteins. To better understand the role of ESX-5 effector proteins in virulence, we analyzed the interactions of Mycobacterium marinum ESX-5 mutant with human macrophages (Mφ). Both wild-type bacteria and the ESX-5 mutant were internalized and the ESX-5 mutation did not affect the escape of mycobacteria from phagolysosomes into the cytosol, as was shown by electron microscopy. However, the ESX-5 mutation strongly effected expression of surface Ags and cytokine secretion. Whereas wild-type M. marinum actively suppressed the induction of appreciable levels of IL-12p40, TNF-α, and IL-6, infection with the ESX-5 mutant resulted in strongly induced production of these proinflammatory cytokines. By contrast, infection with M. marinum wild-type strain resulted in a significant induction of IL-1β production as compared with the ESX-5 mutant. These results show that ESX-5 plays an essential role in the modulation of immune cytokine secretion by human Mφ. Subsequently, we show that an intact ESX-5 secretion system actively suppresses TLR signaling-dependent innate immune cytokine secretion. Together, our results show that ESX-5 substrates, directly or indirectly, strongly modulate the human Mφ response at various critical steps.

SummaryM. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.