The prevalence of allergic diseases is rising worldwide. Desensitizing immunotherapy is currently the only available treatment, but it is often ineffective for certain allergies, underscoring the need for new treatments. This study tested in an Ovalbumin (OVA)-mediated allergy mouse model whether direct allergen targeting to DC subsets can induce desensitization. A Standard desensitization protocol was compared with an antibody-OVA fusion protein targeting the conventional cDC1 or cDC2 subsets via their respective receptors CD205 (CD205-Fp) or DCIR2 (DCIR2-Fp). Allergen targeting to cDC2 provided long-lasting protection for up to 56 days after desensitization with only nanogram doses of allergen, whereas the Standard therapy protected for only 20 days, requiring microgram doses and repeated injections with Alum adjuvant. CD205-Fp showed no protective effect. Blocking IL-10 either before administering DCIR2-Fp or before allergen challenge completely abolished protection. Standard therapy had moderate effects on CD4+ T cells, with early expansion of IL-10+ Foxp3- T regulatory type-1 cells (Tr1), followed by an increase in both IL-10+ and IL-10- Foxp3+ regulatory T cells (Foxp3+ Treg). DCIR2-Fp therapy initially expanded IL-10+ and IL-10- Foxp3+ Treg, but these levels returned to baseline. On day 80, we identified Foxp3- CD44high Treg-like CD8+ as the primary source of IL-10. Overall, our data suggest that cDC2 allergen targeting in allergic mice offers a potent, long-lasting IL-10-dependent desensitizing effect.

Tuberculosis (TB) is still one of the deadliest infectious diseases globally. Although i.d. BCG immunization offers limited protection, a vaccine based on Mycobacterium tuberculosis (Mtb) has yet to be approved. Our previous findings demonstrated that s.c. immunization with heat-killed Mtb significantly increased the number of monocytic myeloid-derived suppressor cells (M-MDSC) in mice. Therefore, we hypothesized that the defense against a subsequent BCG infection would be impaired in Mtb-immunized mice. Surprisingly, mice vaccinated with Mtb were protected against a BCG infection and showed elevated frequencies and activation of DC and mycobacteria-specific T cells despite high frequencies and suppressor activity of M-MDSC. Genetic ablation of CCR2+ monocytic cells or pharmacological intervention with all-trans retinoic acid (ATRA) reduced the frequency of Mtb-induced M-MDSC, enhanced frequencies, and activation of dendritic cells (DC) and CD4+ T cells, and resulted in decreased bacterial loads in the lung and spleen. These findings offer fresh perspectives on TB vaccination using heat-killed Mtb despite parallel unwanted vaccine-induced M-MDSC. M-MDSC depletion by ATRA further tips the balance towards immunity and should be considered an adjunct host-directed therapy with TB vaccines in humans. ### Competing Interest Statement The authors have declared no competing interest.

Tumor-draining lymph nodes (TDLNs) are sites of anti-tumor immune priming as well as metastases. Here, we examined how the cellular networks within TDLNs are reorganized in triple-negative breast cancer (TNBC). We found that the frequencies of programmed death ligand 1 high (PD-L1hi) monocytes increased in TDLNs of metastatic TNBC mouse tumors. Fibroblastic reticular cell (FRC) subtypes heightened the expression of the chemokines CCL2 and CCL7, supporting the homing of CCR2+ monocytes. These monocytes suppressed T cells in vitro via PD-L1 and inducible nitric oxide synthase (iNOS). Spatial transcriptomics revealed immunosuppressive FRC-monocyte niches in vascularized and T cell areas. Tumor-associated Toll-like receptor (TLR) 4 ligands induced CCL2 and CCL7 expression by FRCs to promote monocyte recruitment. Localized TLR4 inhibition in combination with anti-programmed cell death protein 1 (αPD-1) therapy reduced monocyte homing and boosted T cell function, ultimately attenuating lung metastases. Monocytes accumulate in human TNBC TDLNs, with evidence of a FRC-monocyte axis, and a TLR4 ligand signature is predictive of poor survival outcomes in TNBC patients. Thus, metastatic TNBC can reprogram lymph nodes (LNs) to facilitate PD-L1-mediated immune evasion and metastasis.

Cellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive- and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus, and SARS-CoV-2, not only in mammalian and insect cells but also in precision-cut lung slices and neuronal organoids. ULK1 activation inhibited enveloped viruses but not EV71. However, receptor presentation at the cytoplasmic membrane remained unaffected, indicating that receptor binding was unchanged, while later stages of endocytosis were targeted via two distinct pathways. Drug-induced activation of the AMPK pathway reduced early endocytic factor TXNIP by suppressing translation. In contrast, the amounts of Rab5 and the late endosomal marker Rab7 decreased due to translation inactivation and ULK1-dependent proteasome activation within minutes. Furthermore, activation of AMPK hindered the late replication steps of SARS-CoV-2 by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction in early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acid and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses.

Tuberculosis (TB) remains one of the deadliest infectious diseases globally. Although the approved human Bacille-Calmette-Guérin (BCG) vaccines provide limited protection, a vaccine based on Mycobacterium tuberculosis (Mtb) has yet to be approved. Our previous findings demonstrated that s.c. immunization with heat-killed Mtb significantly increased the number of monocytic myeloid-derived suppressor cells (M-MDSC) in mice. Thus, we hypothesized that the defense against a subsequent BCG infection would be compromised in Mtb-immunized mice. Surprisingly, mice vaccinated with Mtb were protected against BCG infection and exhibited elevated frequencies and activation of dendritic cells (DC) and mycobacteria-specific T cells, despite high frequencies and suppressor activity of M-MDSC. Genetic ablation of CCR2+ monocytic cells or pharmacological intervention with all-trans retinoic acid (ATRA) reduced the frequency of Mtb-induced M-MDSC, enhanced the frequencies and activation of DC and CD4+ T cells, and resulted in decreased bacterial loads in the lungs and spleen. These findings provide new insights into TB vaccination using heat-killed Mtb despite the concurrent unwanted effects of vaccine-induced M-MDSC. M-MDSC depletion via ATRA further shifts the balance toward immunity and should be considered an adjunct host-directed therapy alongside TB vaccines in humans.

AbstractCellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran-uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus and SARS-CoV-2 not only in mammalian and insect cells but in precision-cut lung slices and neuronal organoids. However, receptor presentation at the cytoplasmic membrane was unaffected, indicating that receptor-binding remained unaltered and later steps of endocytosis were targeted. Indeed, AMPK pathway activation reduced early endocytic factors TXNIP, Rab5 and the late endosomal marker Rab7 amounts. Furthermore, AMPK activation impaired SARS-CoV-2 late-replication steps by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction of early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acids and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses.

Myeloid-derived suppressor cells (MDSC) represent major regulators of immune responses, which can control T cells via their inducible nitric oxide synthase (iNOS)- and arginase 1 (Arg1)-mediated effector functions. While GM-CSF is well documented to promote MDSC development, little is known about this potential of IL-3, an established growth factor for mast cells. Here, we show that IL-3, similar to GM-CSF, generates monocytic MDSC (M-MDSC) from murine bone marrow (BM) cells after 3 days of in vitro culture. At this time point, predominantly CD11b+ CD49a+ monocytic and CD11b+ CD49a- FcεR I- neutrophilic cells were detectable, while CD11blow/neg FcεR I+ mast cells accumulated only after extended culture periods. Both growth factors were equivalent in generating M-MDSC with respect to phenotype, cell yield and typical surface markers. However, IL-3 generated M-MDSC produced less TNF, IL-1β and IL-10 after activation with LPS + IFN-γ but showed higher Arg1 expression compared to GM-CSF generated M-MDSC. Arg1 was further induced together with iNOS after MDSC activation. Accordingly, an increased Arg1-dependent suppressor activity by the IL-3 generated M-MDSC was observed using respective iNOS and Arg1 inhibitors. Together, these data indicate that M-MDSC can be generated in vitro by IL-3, similar to GM-CSF, but with increased Arg1 expression and Arg1-mediated suppression capacity. This protocol now allows further in vitro studies on the role of IL-3 for MDSC biology.

Myeloid-derived suppressor cells (MDSC) play a crucial role in controlling T-cell responses, but their development and suppressor mechanisms are not fully understood. To study the molecular functions of MDSC, a large number of standardized cells are required. Traditionally, bone marrow (BM) has been used to generate myeloid cell types, including MDSC. In this study, we demonstrate that a previously described protocol for generating monocytic MDSC (M-MDSC) from murine BM with GM-CSF can be fully transferred to BM cells that are conditionally transformed with HoxB8 gene (HoxB8 cells). HoxB8 cells have an extended lifespan and efficiently differentiate into MDSC that are quantitatively and qualitatively comparable to M-MDSC from BM cells. Flow cytometric analyses of LPS/IFN-γ activated cultures revealed the same iNOS+ and/or Arg1+ PD-L1high M-MDSC subsets in similar frequencies from BM or HoxB8 cells. In vitro suppression of CD4+ and CD8+ T-cell proliferations was also largely comparable in their efficacy and its iNOS- or Arg1-dependent suppressor mechanisms, which was confirmed by the similar amounts of nitric oxide (NO) secretion measured from the suppressor assay. Therefore, our data suggest that murine M-MDSC generation from HoxB8 cells with GM-CSF can be used to substitute BM cultures.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Regulatory CD4+CD25+FoxP3+ T cells (Treg) are a subgroup of T lymphocytes involved in maintaining immune balance. Disturbance of Treg number and impaired suppressive function of Treg correlate with Parkinson’s disease severity. Superagonistic anti-CD28 monoclonal antibodies (CD28SA) activate Treg and cause their expansion to create an anti-inflammatory environment. Using the AAV1/2-A53T-α-synuclein Parkinson’s disease mouse model that overexpresses the pathogenic human A53T-α-synuclein (hαSyn) variant in dopaminergic neurons of the substantia nigra, we assessed the neuroprotective and disease-modifying efficacy of a single intraperitoneal dose of CD28SA given at an early disease stage. CD28SA led to Treg expansion 3 days after delivery in hαSyn Parkinson’s disease mice. At this timepoint, an early pro-inflammation was observed in vehicle-treated hαSyn Parkinson’s disease mice with elevated percentages of CD8+CD69+ T cells in brain and increased levels of interleukin-2 (IL-2) in the cervical lymph nodes and spleen. These immune responses were suppressed in CD28SA-treated hαSyn Parkinson’s disease mice. Early treatment with CD28SA attenuated dopaminergic neurodegeneration in the SN of hαSyn Parkinson’s disease mice accompanied with reduced brain numbers of activated CD4+, CD8+ T cells and CD11b+ microglia observed at the late disease-stage 10 weeks after AAV injection. In contrast, a later treatment 4 weeks after AAV delivery failed to reduce dopaminergic neurodegeneration. Our data indicate that immune modulation by Treg expansion at a timepoint of overt inflammation is effective for treatment of hαSyn Parkinson’s disease mice and suggest that the concept of early immune therapy could pose a disease-modifying option for Parkinson’s disease patients.

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAM(low) CD103(-) XCR1(-) CD172a(+) CD11b(+) cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4(-/-)mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-. production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BMcells selectively promotes CDP-derived ESAM(low) cDC2 (cDC2B) development and survival in vitro.

Acute graft versus host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, Treg transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre–expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cell activation in the effector phase, resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant NK T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated costimulatory surface receptors, and although they degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in 2 mouse models of aGvHD. In summary, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlight a framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.

Background: Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson's disease (PD) pathogenesis, alpha-synuclein is a known culprit. Evidence for alpha-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these alpha-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of alpha-synuclein-specific immune responses in PD in a mouse model. Methods: We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-alpha-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)(-/-) mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4(+)/CD8(-), CD4(-)/CD8(+), or CD4(+)/CD8(+) (JHD(-/-)) mice into the RAG-(1-/-) mice was also employed. In addition to the in vivo studies, a newly developed A53T-alpha-synuclein-expressing neuronal cell culture/immune cell assay was utilized. Results: AAV-based overexpression of pathogenic human A53T-alpha-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a proinflammatory gene profile. T cell responses were directed against A53T-alpha-synuclein-peptides in the vicinity of position 53 (68-78) and surrounding the pathogenically relevant S129 (120-134). T cells were required for alpha-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration. Conclusions: Using T cell and/or B cell deficient mice and a newly developed A53T-alpha-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic alpha-synuclein peptidespecific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology.

The original concept stated that immature dendritic cells (DC) act tolerogenically whereas mature DC behave strictly immunogenically. Meanwhile, it is also accepted that phenotypically mature stages of all conventional DC subsets can promote tolerance as steady-state migratory DC by transporting self-antigens to lymph nodes to exert unique functions on regulatory T cells. We propose that in vivo 1) there is little evidence for a tolerogenic function of immature DC during steady state such as CD4 T cell anergy induction, 2) all tolerance as steady-state migratory DC undergo common as well as subset-specific molecular changes, and 3) these changes differ by quantitative and qualitative markers from immunogenic DC, which allows one to clearly distinguish tolerogenic from immunogenic migratory DC.

Multiple myeloma remains a largely incurable disease of clonally expanding malignant plasma cells. The bone marrow microenvironment harbors treatment-resistant myeloma cells, which eventually lead to disease relapse in patients. In the bone marrow, CD4 + FoxP3 + regulatory T cells (Tregs) are highly abundant amongst CD4 + T cells providing an immune protective niche for different long-living cell populations, e.g., hematopoietic stem cells. Here, we addressed the functional role of Tregs in multiple myeloma dissemination to bone marrow compartments and disease progression. To investigate the immune regulation of multiple myeloma, we utilized syngeneic immunocompetent murine multiple myeloma models in two different genetic backgrounds. Analyzing the spatial immune architecture of multiple myeloma revealed that the bone marrow Tregs accumulated in the vicinity of malignant plasma cells and displayed an activated phenotype. In vivo Treg depletion prevented multiple myeloma dissemination in both models. Importantly, short-term in vivo depletion of Tregs in mice with established multiple myeloma evoked a potent CD8 T cell- and NK cell-mediated immune response resulting in complete and stable remission. Conclusively, this preclinical in-vivo study suggests that Tregs are an attractive target for the treatment of multiple myeloma.

Myeloid-derived suppressor cells (MDSC) are important immune-regulatory cells but their identification remains difficult. Here, we provide a critical view on selected surface markers, transcriptional and translational pathways commonly used to identify MDSC by specific, their developmental origin and new possibilities by transcriptional or proteomic profiling. Discrimination of MDSC from their non-suppressive counterparts is a prerequisite for the development of successful therapies. Understanding the switch mechanisms that direct granulocytic and monocytic development into a pro-inflammatory or anti-inflammatory direction will be crucial for therapeutic strategies. Manipulation of these myeloid checkpoints are exploited by tumors and pathogens, such as M. tuberculosis (Mtb), HIV or SARS-CoV-2, that induce MDSC for immune evasion. Thus, specific markers for MDSC identification may reveal also novel molecular candidates for therapeutic intervention at the level of MDSC.

T cell anergy is a common mechanism of T cell tolerance. However, although anergic T cells are retained for longer time periods in their hosts, they remain functionally passive. Here, we describe the induction of anergic CD4+ T cells in vivo by intravenous application of high doses of antigen and their subsequent conversion into suppressive Foxp3- IL-10+ Tr1 cells but not Foxp3+ Tregs. We describe the kinetics of up-regulation of several memory-, anergy- and suppression-related markers such as CD44, CD73, FR4, CD25, CD28, PD-1, Egr-2, Foxp3 and CTLA-4 in this process. The conversion into suppressive Tr1 cells correlates with the transient intracellular CTLA-4 expression and required the restimulation of anergic cells in a short-term time window. Restimulation after longer time periods, when CTLA-4 is down-regulated again retains the anergic state but does not lead to the induction of suppressor function. Our data require further functional investigations but at this stage may suggest a role for anergic T cells as a circulating pool of passive cells that may be re-activated into Tr1 cells upon short-term restimulation with high and systemic doses of antigen. It is tentative to speculate that such a scenario may represent cases of allergen responses in non-allergic individuals.

Myeloid-derived suppressor cells (MDSCs) represent a major population controlling T cell immune responses. However, little is known about their molecular requirements for homing and T cell interaction to mediate suppression. Here, we investigated the functional role of the homing and collagen IV receptor VLA-1 (α1β1-integrin) on in vitro GM-CSF generated murine MDSCs from wild-type (WT) and CD49a/α1-integrin (Itga1−/−) gene-deficient mice. Here, we found that effector (Teff) but not naive (Tn) CD4+ T cells express VLA-1 and monocytes further up-regulated their expression after culture in GM-CSF when they differentiated into the monocytic subset of resting MDSCs (R-MDSCs). Subsequent activation of R-MDSCs by LPS+IFN-γ (A-MDSCs) showed increased in vitro suppressor potential, which was independent of VLA-1. Surprisingly, VLA-1 deficiency did not influence A-MDSC motility or migration on collagen IV in vitro. However, interaction times of Itga1−/− A-MDSCs with Teff were shorter than with WT A-MDSCs on collagen IV but not on fibronectin substrate in vitro. After injection, A-MDSCs homed to the splenic red pulp where they co-localized with Teff and showed immediate suppression already after 6 h as shown by inhibition of T cell proliferation and induction of apoptosis. Injection of A-MDSCs from Itga1−/− mice showed equivalent homing into the spleen but a reduced suppressive effect. Interaction studies of A-MDSCs with Teff in the subcapsular red pulp with intravital two-photon microscopy revealed also here that MDSC motility and migration parameters were not altered by VLA-1 deficiency, but the interaction times with Teff were reduced. Together, our data point to a new role of VLA-1 adhesion to collagen IV as a prerequisite for extended contact times with Teff required for suppression.

Myeloid-derived suppressor cells (MDSC) are induced during active TB disease to restore immune homeostasis but instead exacerbate disease outcome due to chronic inflammation. Autophagy, in conventional phagocytes, ensures successful clearance of M.tb. However, autophagy has been demonstrated to induce prolonged MDSC survival. Here we investigate the relationship between autophagy mediators and MDSC in the context of active TB disease and during anti-TB therapy. We demonstrate a significant increase in MDSC frequencies in untreated active TB cases with these MDSC expressing TLR4 and significantly more mTOR and IL-6 than healthy controls, with mTOR levels decreasing during anti-TB therapy. Finally, we show that HMGB1 serum concentrations decrease in parallel with mTOR. These findings suggest a complex interplay between MDSC and autophagic mediators, potentially dependent on cellular localisation and M.tb infection state.