Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.

Introduction: It is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2. Methods: To address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease. Results: Although NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies. Discussion: These observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection.

Background: Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection. Methods: We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines. Results: In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells. Conclusion: H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.

Extracellular vesicles in cancer: challenges and opportunities for clinical laboratories.

Extracellular vesicles (EVs) are nano-sized particles secreted by most cells. They transport different types of biomolecules (nucleic acids, proteins, and lipids) characteristic of their tissue or cellular origin that can mediate long-distance intercellular communication. In the case of cancer, EVs participate in tumor progression by modifying the tumor microenvironment, favoring immune tolerance and metastasis development. Consequently, EVs have great potential in liquid biopsy for cancer diagnosis, prognosis and follow-up. In addition, EVs could have a role in cancer treatment as a targeted drug delivery system. The intense research in the EV field has resulted in hundreds of patents and the creation of biomedical companies. However, methodological issues and heterogeneity in EV composition have hampered the advancement of EV validation trials and the development of EV-based diagnostic and therapeutic products. Consequently, only a few EV biomarkers have moved from research to clinical laboratories, such as the ExoDx Prostate IntelliScore (EPI) test, a CLIA/FDA-approved EV prostate cancer diagnostic test. In addition, the number of large-scale multicenter studies that would clearly define biomarker performance is limited. In this review, we will critically describe the different types of EVs, the methods for their enrichment and characterization, and their biological role in cancer. Then, we will specially focus on the parameters to be considered for the translation of EV biology to the clinic laboratory, the advances already made in the field of EVs related to cancer diagnosis and treatment, and the issues still pending to be solved before EVs could be used as a routine tool in oncology.

SARS-CoV-2 membrane protein-specific antibodies from critically ill SARS-CoV-2-infected individuals interact with Fc receptor-expressing cells but do not neutralize the virus.

The membrane (M) glycoprotein of SARS-CoV-2 is one of the key viral proteins regulating virion assembly and morphogenesis. Immunologically, the M protein is a major source of peptide antigens driving T cell responses, and most individuals who have been infected with SARS-CoV-2 make antibodies to the N-terminal, surface-exposed peptide of the M protein. We now report that although the M protein is abundant in the viral particle, antibodies to the surface-exposed N-terminal epitope of M do not appear to neutralize the virus. M protein-specific antibodies do, however, activate antibody-dependent cell-mediated cytotoxicity and cytokine secretion by primary human natural killer cells. Interestingly, while patients with severe or mild disease make comparable levels of M antigen-binding antibodies, M-specific antibodies from the serum of critically ill patients are significantly more potent activators of antibody-dependent cell-mediated cytotoxicity than antibodies found in individuals with mild or asymptomatic infection.

NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and in vivo but fail to eliminate leukemia initiating cells.

Introduction: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation. Methods: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells' cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality. Results: In vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells. Discussion: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.

Intravenous administration of BCG in mice promotes natural killer and T cell-mediated antitumor immunity in the lung.

Intravesical administration of Bacillus Calmette-Guérin (BCG) was one of the first FDA-approved immunotherapies and remains a standard treatment for bladder cancer. Previous studies have demonstrated that intravenous (IV) administration of BCG is well-tolerated and effective in preventing tuberculosis infection in animals. Here, we examine IV BCG in several preclinical lung tumor models. Our findings demonstrate that BCG inoculation reduced tumor growth and prolonged mouse survival in models of lung melanoma metastasis and orthotopic lung adenocarcinoma. Moreover, IV BCG treatment was well-tolerated with no apparent signs of acute toxicity. Mechanistically, IV BCG induced tumor-specific CD8+ T cell responses, which were dependent on type 1 conventional dendritic cells, as well as NK cell-mediated immunity. Lastly, we also show that IV BCG has an additive effect on anti-PD-L1 checkpoint inhibitor treatment in mouse lung tumors that are otherwise resistant to anti-PD-L1 as monotherapy. Overall, our study demonstrates the potential of systemic IV BCG administration in the treatment of lung tumors, highlighting its ability to enhance immune responses and augment immune checkpoint blockade efficacy.

BCG-activation of leukocytes is sufficient for the generation of donor-independent innate anti-tumor NK and γδ T-cells that can be further expanded in vitro.

Bacillus Calmette-Guérin (BCG), the nonpathogenic Mycobacterium bovis strain used as tuberculosis vaccine, has been successfully used as treatment for non-muscle invasive bladder cancer for decades, and suggested to potentiate cellular and humoral immune responses. However, the exact mechanism of action is not fully understood. We previously described that BCG mainly activated anti-tumor cytotoxic NK cells with upregulation of CD56 and a CD16+ phenotype. Now, we show that stimulation of human peripheral blood mononuclear cells with iBCG, a preparation based on BCG-Moreau, expands oligoclonal γδ T-cells, with a cytotoxic phenotype, together with anti-tumor CD56high CD16+ NK cells. We have used scRNA-seq, flow cytometry, and functional assays to characterize these BCG-activated γδ T-cells in detail. They had a high IFNγ secretion signature with expression of CD27+ and formed conjugates with bladder cancer cells. BCG-activated γδ T-cells proliferated strongly in response to minimal doses of cytokines and had anti-tumor functions, although not fully based on degranulation. BCG was sufficient to stimulate proliferation of γδ T-cells when cultured with other PBMC; however, BCG alone did not stimulate expansion of purified γδ T-cells. The characterization of these non-donor restricted lymphocyte populations, which can be expanded in vitro, could provide a new approach to prepare cell-based immunotherapy tools.

NKG2D Ligands in Liquid Biopsy: The Importance of Soluble and Vesicle-Bound Proteins for Immune Modulation.

The identification of biomarkers allowing diagnostics, prognostics and patient classification is still a challenge in oncological research for patient management. Improvements in patient survival achieved with immunotherapies substantiate that biomarker studies rely not only on cellular pathways contributing to the pathology, but also on the immune competence of the patient. If these immune molecules can be studied in a non-invasive manner, the benefit for patients and clinicians is obvious. The immune receptor Natural Killer Group 2 Member D (NKG2D) represents one of the main systems involved in direct recognition of tumor cells by effector lymphocytes (T and Natural Killer cells), and in immune evasion. The biology of NKG2D and its ligands comprises a complex network of cellular pathways leading to the expression of these tumor-associated ligands on the cell surface or to their release either as soluble proteins, or in extracellular vesicles that potently inhibit NKG2D-mediated responses. Increased levels of NKG2D-ligands in patient serum correlate with tumor progression and poor prognosis; however, most studies did not test the biochemical form of these molecules. Here we review the biology of the NKG2D receptor and ligands, their role in cancer and in patient response to immunotherapies, as well as the changes provoked in this system by non-immune cancer therapies. Further, we discuss the use of NKG2D-L in liquid biopsy, including methods to analyse vesicle-associated proteins. We propose that the evaluation in cancer patients of the whole NKG2D system can provide crucial information about patient immune competence and risk of tumor progression.

CyTOF analysis identifies unusual immune cells in urine of BCG-treated bladder cancer patients.

High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical parameter predictive of treatment response, and when treatment fails, the prognosis is very poor. Further, a high percentage of NMIBC patients treated with BCG suffer unwanted effects that force them to stop treatment. Thus, early identification of patients in which BCG treatment will fail is really important. Here, to identify early stage non-invasive biomarkers of non-responder patients and patients at risk of abandoning the treatment, we longitudinally analysed the phenotype of cells released into the urine of bladder cancer patients 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed a large proportion of granulocytes and monocytes, mostly expressing activation markers. A novel population of CD15+CD66b+CD14+CD16+ cells was highly abundant in several samples; expression of these markers was confirmed using flow cytometry and qPCR. A stronger inflammatory response was associated with increased cell numbers in the urine; this was not due to hematuria because the cell proportions were distinct from those in the blood. This pilot study represents the first CyTOF analysis of cells recruited to urine during BCG treatment, allowing identification of informative markers associated with treatment response for sub-selection of markers to confirm using conventional techniques. Further studies should jointly evaluate cells and soluble factors in urine in larger cohorts of patients to characterise the arms of the immune response activated in responders and to identify patients at risk of complications from BCG treatment.

Novel intravesical bacterial immunotherapy induces rejection of BCG-unresponsive established bladder tumors.

BACKGROUND: Intravesical BCG is the gold-standard therapy for non-muscle invasive bladder cancer (NMIBC); however, it still fails in a significant proportion of patients, so improved treatment options are urgently needed. METHODS: Here, we compared BCG antitumoral efficacy with another live attenuated mycobacteria, MTBVAC, in an orthotopic mouse model of bladder cancer (BC). We aimed to identify both bacterial and host immunological factors to understand the antitumoral mechanisms behind effective bacterial immunotherapy for BC. RESULTS: We found that the expression of the BCG-absent proteins ESAT6/CFP10 by MTBVAC was determinant in mediating bladder colonization by the bacteria, which correlated with augmented antitumoral efficacy. We further analyzed the mechanism of action of bacterial immunotherapy and found that it critically relied on the adaptive cytotoxic response. MTBVAC enhanced both tumor antigen-specific CD4+ and CD8+ T-cell responses, in a process dependent on stimulation of type 1 conventional dendritic cells. Importantly, improved intravesical bacterial immunotherapy using MBTVAC induced eradication of fully established bladder tumors, both as a monotherapy and specially in combination with the immune checkpoint inhibitor antiprogrammed cell death ligand 1 (anti PD-L1). CONCLUSION: These results contribute to the understanding of the mechanisms behind successful bacterial immunotherapy against BC and characterize a novel therapeutic approach for BCG-unresponsive NMIBC cases.

Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.

Cross-reactive cellular, but not humoral, immunity is detected between OC43 and SARS-CoV-2 NPs in people not infected with SARS-CoV-2: Possible role of cTFH cells.

Multiple questions about SARS-CoV-2 humoral and cellular immunity remain unanswered. One key question is whether preexisting memory T or B cells, specific for related coronaviruses in SARS-CoV-2-unexposed individuals, can recognize and suppress COVID-19, but this issue remains unclear. Here, we demonstrate that antibody responses to SARS-CoV-2 antigens are restricted to serum samples from COVID-19 convalescent individuals. In contrast, cross-reactive T cell proliferation and IFN-γ production responses were detected in PBMCs of around 30% of donor samples collected prepandemic, although we found that these prepandemic T cell responses only elicited weak cTFH activation upon stimulation with either HCoV-OC43 or SARS-CoV-2 NP protein. Overall, these observations confirm that T cell cross-reactive with SARS-CoV-2 antigens are present in unexposed people, but suggest that the T cell response to HCoV-OC43 could be deficient in some important aspects, like TFH expansion, that might compromise the generation of cross-reactive TFH cells and antibodies. Understanding these differences in cellular responses may be of critical importance to advance in our knowledge of immunity against SARS-CoV-2.

ALK-Fusion Transcripts Can Be Detected in Extracellular Vesicles (EVs) from Nonsmall Cell Lung Cancer Cell Lines and Patient Plasma: Toward EV-Based Noninvasive Testing.

BACKGROUND: ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection. METHODS: EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation. RESULTS: Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions. CONCLUSIONS: ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.

A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma.

BACKGROUND: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. RESULTS: Here we describe a method that, using just a few microliters of patient's plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. CONCLUSIONS: This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.

MAPK inhibitors dynamically affect melanoma release of immune NKG2D-ligands, as soluble protein and extracellular vesicle-associated.

Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients.

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry.

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.

An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles.

Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, isolating and characterizing tumor-derived EVs has attracted great interest as an invaluable source of biomarkers, both for diagnosis and stratification of cancer. In this chapter, we describe a method for flow cytometry assessment of melanoma-derived EVs which are firstly captured onto antibody-coated beads recognizing either a common EV marker, namely, a tetraspanin, or a tumor antigen like the stress-related molecules MICA or PDL1. Then, after staining with a fluorophore-conjugated antibody directed against a different protein present on the EV surface, the EV-bead complex can be visualized in a conventional flow cytometer. The technique allows detection of proteins present on EVs isolated from tissue culture supernatants of melanoma cell lines and, more importantly, directly from plasma.

Natural Killer Anti-Tumor Activity Can Be Achieved by In Vitro Incubation With Heat-Killed BCG.

Natural Killer cell receptors allow this heterogeneous immune population to efficiently fight both tumors and infection, so their use as immunotherapy agents is an active field of research. Cytokine activation, particularly by myeloid cell-derived IL15, can induce potent NK anti-tumor responses. While studying the mechanism of action of intravesical instillations of Bacille Calmette-Guérin (BCG) as therapy for patients with high risk non-muscle invasive bladder cancer, we showed that BCG can activate a cytotoxic CD56bright NK cell population which efficiently recognized bladder cancer cells. This pioneer immunotherapy provides an invaluable model to understand the role of different immune populations in tumor elimination. However, during the propagation of BCG worldwide a large number of genetically diverse BCG substrains developed. Here, we investigated the capacity of different BCG substrains to promote NK cell activation and confirmed that they were able to activate lymphocytes. Tice, Connaught and Moreau were the substrains with a stronger NK activation effect as measured by CD56 upregulation. Surprisingly, dead mycobacteria also stimulated PBMC cultures and we further demonstrate here that subcellular fractions of BCG-Tice, in the absence of live mycobacteria, could also induce an NK cell response. Lipids from BCG-Tice, but not from Mycobacterium bovis, stimulated NK cell activation and degranulation, whereas the aqueous fraction of either bacteria did not activate lymphocytes. However, delipidated BCG-Tice bacteria were able to activate effector cells (CD3+CD56+ and NK, CD3-CD56+). These data demonstrate that different components of mycobacteria can stimulate different immune subpopulations resulting in phenotypes suitable for cancer elimination.