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A human islet transplant can cure patients with type 1 diabetes. A drawback of islet transplantation is the life-long immunosuppressive medication, often associated with severe side effects. Moreover, in spite of the immunosuppressive therapy, islets are lost in the majority of transplanted patients over time. An improved small animal model for studies on human islet allograft rejection mechanisms and the development of new measures for its prevention is clearly warranted. Here, we evaluated the potential of Balb/cRag2−/−γc−/− mice carrying a human-like immune system (so-called humanized mice) as a tool for studies on the rejection of transplanted human islets. Human T cells from Balb/cRag2−/−γc−/− mice, which as neonates had been transplanted with CD34+ human cord blood haematopoietic stem cells (HIS mice), proliferated in response to allogeneic human dendritic cells, but failed to reject a human islet allograft transplanted to the renal subcapsular space as assessed by immunohistochemistry and analysis of human serum C-peptide levels. Histological analysis revealed that few if any T cells had migrated to the grafted tissue. These observations question the usefulness of the HIS mouse model for studies on human islet allograft rejection mechanisms and highlight the need for further improvements. [ABSTRACT FROM AUTHOR]

The maintenance of intestinal homeostasis is a fundamental process critical for organismal integrity. Sitting at the interface of the gut microbiome and mucosal immunity, adaptive and innate lymphoid populations regulate the balance between commensal micro-organisms and pathogens. Checkpoint inhibitors, particularly those targeting the CTLA-4 pathway, disrupt this fine balance and can lead to inflammatory bowel disease and immune checkpoint colitis. Here, we show that CTLA-4 is expressed by innate lymphoid cells and that its expression is regulated by ILC subset-specific cytokine cues in a microbiota-dependent manner. Genetic deletion or antibody blockade of CTLA-4 in multiple in vivo models of colitis demonstrates that this pathway plays a key role in intestinal homeostasis. Lastly, we have found that this observation is conserved in human IBD. We propose that this population of CTLA-4-positive ILC may serve as an important target for the treatment of idiopathic and iatrogenic intestinal inflammation., (© 2024. The Author(s).)

Germinal center (GC) dysregulation has been widely reported in the context of autoimmunity. Here, we show that interleukin 21 (IL-21), the archetypal follicular helper T cell (Tfh) cytokine, shapes the scale and polarization of spontaneous chronic autoimmune as well as transient immunization-induced GC. We find that IL-21 receptor deficiency results in smaller GC that are profoundly skewed toward a light zone GC B cell phenotype and that IL-21 plays a key role in selection of light zone GC B cells for entry to the dark zone. Light zone skewing has been previously reported in mice lacking the cell cycle regulator cyclin D3. We demonstrate that IL-21 triggers cyclin D3 upregulation in GC B cells, thereby tuning dark zone inertial cell cycling. Lastly, we identify Foxo1 regulation as a link between IL-21 signaling and GC dark zone formation. These findings reveal new biological roles for IL-21 within GC and have implications for autoimmune settings where IL-21 is overproduced., (© 2023 Petersone et al.)

Efficacy of checkpoint inhibitor therapies in cancer varies greatly, with some patients showing complete responses while others do not respond and experience progressive disease. We aimed to identify correlates of response and progression following PD-1-directed therapy by immunophenotyping peripheral blood samples from 20 patients with advanced malignant melanoma before and after treatment with the PD-1 blocking antibody pembrolizumab. Our data reveal that individuals responding to PD-1 blockade were characterised by increased CD8 T cell proliferation following treatment, while progression was associated with an increase in CTLA-4-expressing Treg. Remarkably, unsupervised clustering analysis of pre-treatment T cell subsets revealed differences in individuals that went on to respond to PD-1 blockade compared to individuals that did not. These differences mapped to expression of the proliferation marker Ki67 and the costimulatory receptor CD28 as well as the inhibitory molecules 2B4 and KLRG1. While these results require validation in larger patient cohorts, they suggest that flow cytometric analysis of a relatively small number of T cell markers in peripheral blood could potentially allow stratification of PD-1 blockade treatment response prior to therapy initiation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.)

Blockade of CD28 costimulation with CTLA-4-Ig/Abatacept is used to dampen effector T cell responses in autoimmune and transplantation settings. However, a significant drawback of this approach is impaired regulatory T cell homeostasis that requires CD28 signaling. Therefore, strategies that restrict the effects of costimulation blockade to effector T cells would be advantageous. Here we probe the relative roles of CD28 and IL-2 in maintaining Treg. We find provision of IL-2 counteracts the regulatory T cell loss induced by costimulation blockade while minimally affecting the conventional T cell compartment. These data suggest that combining costimulation blockade with IL-2 treatment may selectively impair effector T cell responses while maintaining regulatory T cells. Using a mouse model of autoimmune diabetes, we show combined therapy supports regulatory T cell homeostasis and protects from disease. These findings are recapitulated in humanised mice using clinically relevant reagents and provide an exemplar for rational use of a second immunotherapy to offset known limitations of the first., (© 2022. The Author(s).)

Background: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies., Methods and Results: A new method for growing P. falciparum in 5% CO 2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 - FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed., Conclusions: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

Follicular helper T (T FH ) cells are implicated in type 1 diabetes (T1D), and their development has been linked to CD28 costimulation. We tested whether T FH cells were decreased by costimulation blockade using the CTLA-4-immunoglobulin (Ig) fusion protein (abatacept) in a mouse model of diabetes and in individuals with new-onset T1D. Unbiased bioinformatics analysis identified that inducible costimulatory molecule (ICOS) + T FH cells and other ICOS + populations, including peripheral helper T cells, were highly sensitive to costimulation blockade. We used pretreatment T FH profiles to derive a model that could predict clinical response to abatacept in individuals with T1D. Using two independent approaches, we demonstrated that higher frequencies of ICOS + T FH cells at baseline were associated with a poor clinical response following abatacept administration. Therefore, T FH analysis may represent a new stratification tool, permitting the identification of individuals most likely to benefit from costimulation blockade.

Background: Umbilical cord blood (UCB) is a source of hematopoietic stem cells for transplantation, offering an alternative for patients unable to find a matched adult donor. UCB is also a versatile source of hematopoietic stem and progenitor cells (hCD34 + HSPCs) for research into hematologic diseases, in vitro expansion, ex vivo gene therapy, and adoptive immunotherapy. For these studies, there is a need to isolate hCD34 + HSPCs from cryopreserved units, and protocols developed for isolation from fresh cord blood are unsuitable., Study Design: This study describes a modified method for isolating hCD34 + HSPCs from cryopreserved UCB. It uses the Plasmatherm system for thawing, followed by CD34 microbead magnetic-activated cell sorting isolation with a cell separation kit (Whole Blood Columns, Miltenyi Biotec). hCD34 + HSPC phenotypes and functionality were assessed in vitro and hematologic reconstitution determined in vivo in immunodeficient mice., Results: Total nucleated cell recovery after thawing and washing was 44.7 ± 11.7%. Recovery of hCD34 + HSPCs after application of thawed cells to Whole Blood Columns was 77.5 ± 22.6%. When assessed in two independent laboratories, the hCD34+ cell purities were 71.7 ± 10.7% and 87.8 ± 2.4%. Transplantation of the enriched hCD34 + HSPCs into NSG mice revealed the presence of repopulating hematopoietic stem cells (estimated frequency of 0.07%) and multilineage engraftment., Conclusion: This provides a simplified protocol for isolating high-purity human CD34 + HSPCs from banked UCB adaptable to current Good Manufacturing Practice. This protocol reduces the number of steps and associated risks and thus total production costs. Importantly, the isolated CD34 + HSPCs possess in vivo repopulating activity in immunodeficient mice, making them a suitable starting population for ex vivo culture and gene editing., (© 2019 AABB.)

CTLA-4 is a critical negative regulator of the immune system and a major target for immunotherapy. However, precisely how it functions in vivo to maintain immune homeostasis is not clear. As a highly endocytic molecule, CTLA-4 can capture costimulatory ligands from opposing cells by a process of transendocytosis (TE). By restricting costimulatory ligand expression in this manner, CTLA-4 controls the CD28-dependent activation of T cells. Regulatory T cells (T regs ) constitutively express CTLA-4 at high levels and, in its absence, show defects in TE and suppressive function. Activated conventional T cells (T conv ) are also capable of CTLA-4-dependent TE; however, the relative use of this mechanism by T regs and T conv in vivo remains unclear. Here, we set out to characterize both the perpetrators and cellular targets of CTLA-4 TE in vivo. We found that T regs showed constitutive cell surface recruitment of CTLA-4 ex vivo and performed TE rapidly after TCR stimulation. T regs outperformed activated T conv at TE in vivo, and expression of ICOS marked T regs with this capability. Using TCR transgenic T regs that recognize a protein expressed in the pancreas, we showed that the presentation of tissue-derived self-antigen could trigger T regs to capture costimulatory ligands in vivo. Last, we identified migratory dendritic cells (DCs) as the major target for T reg -based CTLA-4-dependent regulation in the steady state. These data support a model in which CTLA-4 expressed on T regs dynamically regulates the phenotype of DCs trafficking to lymph nodes from peripheral tissues in an antigen-dependent manner., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Co-ordinated interaction between distinct cell types is a hallmark of successful immune function. A striking example of this is the carefully orchestrated cooperation between helper T cells and B cells that occurs during the initiation and fine-tuning of T-cell dependent antibody responses. While these processes have evolved to permit rapid immune defense against infection, it is becoming increasingly clear that such interactions can also underpin the development of autoimmunity. Here we discuss a selection of cellular and molecular pathways that mediate T cell/B cell collaboration and highlight how in vivo models and genome wide association studies link them with autoimmune disease. In particular, we emphasize how CTLA-4-mediated regulation of CD28 signaling controls the engagement of secondary costimulatory pathways such as ICOS and OX40, and profoundly influences the capacity of T cells to provide B cell help. While our molecular understanding of the co-operation between T cells and B cells derives from analysis of secondary lymphoid tissues, emerging evidence suggests that subtly different rules may govern the interaction of T and B cells at ectopic sites during autoimmune inflammation. Accordingly, the phenotype of the T cells providing help at these sites includes notable distinctions, despite sharing core features with T cells imparting help in secondary lymphoid tissues. Finally, we highlight the interdependence of T cell and B cell responses and suggest that a significant beneficial impact of B cell depletion in autoimmune settings may be its detrimental effect on T cells engaged in molecular conversation with B cells.

Recent developments in mouse models that harbor part of a human immune system have proved extremely valuable to study the in vivo immune response to human specific pathogens such as Epstein-Barr virus. Over the last decades, advances in immunodeficient mouse strains that can be used as recipients for human immune cells have greatly enhanced the use of these models. Here, we describe the generation of mice with reconstituted human immune system (HIS mice) using immunocompromised mice transplanted with human CD34 + hematopoietic stem cells. We will also describe how such mice, in which human immune cells are generated de novo, can be used to study EBV infection.

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T-cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory T cells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is an essential regulator of T-cell responses, and its absence precipitates lethal T-cell hyperactivity. However, whether CTLA-4 acts simply to veto the activation of certain clones or plays a more nuanced role in shaping the quality of T-cell responses is not clear. Here we report that T cells in CTLA-4-deficient mice show spontaneous T-follicular helper (T(FH)) differentiation in vivo, and this is accompanied by the appearance of large germinal centers (GCs). Remarkably, short-term blockade with anti-CTLA-4 antibody in wild-type mice is sufficient to elicit T(FH) generation and GC development. The latter occurs in a CD28-dependent manner, consistent with the known role of CTLA-4 in regulating the CD28 pathway. CTLA-4 can act by down-regulating CD80 and CD86 on antigen presenting cells (APCs), thereby altering the level of CD28 engagement. To mimic reduced CD28 ligation, we used mice heterozygous for CD28, revealing that the magnitude of CD28 engagement is tightly linked to the propensity for T(FH) differentiation. In contrast, other parameters of T-cell activation, including CD62L down-regulation and Ki67 expression, were relatively insensitive to altered CD28 level. Altered T(FH) generation as a result of graded reduction in CD28 was associated with decreased numbers of GC B cells and a reduction in overall GC size. These data support a model in which CTLA-4 control of immunity goes beyond vetoing T-cell priming and encompasses the regulation of T(FH) differentiation by graded control of CD28 engagement.

The strong genetic association between particular HLA alleles and type 1 diabetes (T1D) indicates a key role for CD4+ T cells in disease; however, the differentiation state of the responsible T cells is unclear. T cell differentiation originally was considered a dichotomy between Th1 and Th2 cells, with Th1 cells deemed culpable for autoimmune islet destruction. Now, multiple additional T cell differentiation fates are recognized with distinct roles. Here, we used a transgenic mouse model of diabetes to probe the gene expression profile of islet-specific T cells by microarray and identified a clear follicular helper T (Tfh) cell differentiation signature. Introduction of T cells with a Tfh cell phenotype from diabetic animals efficiently transferred diabetes to recipient animals. Furthermore, memory T cells from patients with T1D expressed elevated levels of Tfh cell markers, including CXCR5, ICOS, PDCD1, BCL6, and IL21. Defects in the IL-2 pathway are associated with T1D, and IL-2 inhibits Tfh cell differentiation in mice. Consistent with these previous observations, we found that IL-2 inhibited human Tfh cell differentiation and identified a relationship between IL-2 sensitivity in T cells from patients with T1D and acquisition of a Tfh cell phenotype. Together, these findings identify a Tfh cell signature in autoimmune diabetes and suggest that this population could be used as a biomarker and potentially targeted for T1D interventions.

Unlabelled: Human B cells, the main target of Epstein-Barr virus (EBV), can display several types of latent viral protein expression, denoted 0, I, IIa, IIb, or III. Of these, only type III expression induces proliferation of cells in vitro. These latency types are present at specific stages of infection and are also characteristic of different tumor types, but their generation is not fully understood. In this study, we analyzed the role of T cells in the regulation of EBV viral latency by using humanized NOD/SCID/IL2Rγ(-/-) mice. Several spleens presented macroscopic tumors 4 weeks after infection. Explanted spleen B cells from some of the EBV-infected mice proliferated in vitro, but this was usually lowered when cyclosporine was added to the cultures. This suggested that the in vitro growth of EBV-infected B cells required T cell help; thus, cells other than type III cells were also present in the spleens. Quantitative PCR analysis of promoter activities specific for the different EBV latency types confirmed that in addition to type III cells, type IIa and type I cells were present in the spleen. The relative usage of the viral promoter specific for I and IIa latency types (Q promoter) was higher in CD8(+) cell-depleted mice, and it was absent from CD4(+) cell-depleted mice. These results indicate that CD4(+) T cells are necessary for the generation/maintenance of cells with latency I/IIa in the humanized mice. CD4(+) T cells contributed to this process through their CD40L expression., Importance: At primary infection with EBV, the infected B cells are proliferating and express viral proteins that have transforming potential. However, when the acute infection is resolved, in healthy individuals EBV is carried by a small fraction of B cells that express a restricted number of viral proteins unable to induce proliferation. Understanding the details of this transition is of fundamental importance. We studied this question in humanized mice by manipulating their different T cell compartments before and during infection with EBV. Our results indicate that CD4(+) T cells are responsible for the switch to a nonproliferating EBV program during primary infection with EBV.

The efficacy of DNA vaccines is highly dependent on the methods used for their delivery and the choice of delivery sites/targets for gene injection, pointing at the necessity of a strict control over the gene delivery process. Here, we have investigated the effect of the injection site on gene expression and immunogenicity in BALB/c mice, using as a model a weak gene immunogen, DNA encoding firefly luciferase (Luc) delivered by superficial or deep injection with subsequent electroporation (EP). Immunization was assessed by monitoring the in vivo expression of luciferase by 2D- and 3D-bioluminescence imaging (BLI) and by the end-point immunoassays. Anti-Luc antibodies were assessed by ELISA, and T-cell response by IFN-γ and IL-2 FluoroSpot in which mouse splenocytes were stimulated with Luc or a peptide representing its immunodominant CD8+ T-cell epitope GFQSMYTFV. Monitoring of immunization by BLI identified EP parameters supporting the highest Luc gene uptake and expression. Superficial injection of Luc DNA followed by optimal EP led to a low level Luc expression in the mouse skin, and triggered a CD8+ T-cell response characterized by the peptide-specific secretion of IFN-γ and IL-2, but no specific antibodies. Intramuscular gene delivery resulted in a several-fold higher Luc expression and anti-Luc antibody, but induced low IL-2 and virtually no specific IFN-γ. Photon flux from the sites of Luc gene injection was inversely proportional to the immune response against GFQSMYTFV (p<0.05). Thus, BLI permitted to control the accuracy of gene delivery and transfection with respect to the injection site as well as the parameters of electroporation. Further, it confirmed the critical role of the site of DNA administration for the type and magnitude of the vaccine-specific immune response. This argues for the use of luminescent reporters in the preclinical gene vaccine tests to monitor both gene delivery and the immune response development in live animals.

We have used humanized mice, in which human immune cells differentiate de novo from transplanted cord blood progenitor cells, to study the human immune responses to infection with Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis. Granulomas with a core containing giant cells, human CD68(+) macrophages, and high bacilli numbers surrounded by a layer of CD3(+) T cells and a fibrotic response encapsulating the lesions were observed in livers and lungs from bacillus Calmette-Guérin-infected humanized mice but not in nonhumanized infected controls. Paradoxically, humanized mice contained higher mycobacterial numbers in organs than nonhumanized controls. The enhancement of bacterial load was mediated by human CD4(+) cells and associated to an increased expression of Programmed Death-1 protein and CD57 on T cells, molecules associated with inhibition and senescence. The lesions from mice depleted of CD4(+) cells were scarcer, minimal, and irregular compared with those from mice depleted of CD8(+) cells or nondepleted controls. Granulomas of bacillus Calmette-Guérin-infected humanized mice administered with a TNF-neutralizing TNF receptor fusion molecule preserved their structure, but contained higher levels of intracellular bacilli. Extended necrosis was observed in granulomas from M. tuberculosis- but not bacillus Calmette-Guérin-infected humanized mice. Our data indicate that humanized mice can be used as a model to study the formation and maintenance of human granuloma in tuberculosis and other infectious or noninfectious diseases.

Following infection with Epstein-Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4(+) T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4(+) T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4(+) T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4(+) T cells can control the EBV-induced B-cell proliferation.

Natural killer (NK) cells are lymphocytes of the innate immune system that, following differentiation from CD56(bright) to CD56(dim) cells, have been thought to retain fixed functional and phenotypic properties throughout their lifespan. In contrast to this notion, we here show that CD56(dim) NK cells continue to differentiate. During this process, they lose expression of NKG2A, sequentially acquire inhibitory killer cell inhibitory immunoglobulin-like receptors and CD57, change their expression patterns of homing molecules, and display a gradual decline in proliferative capacity. All cellular intermediates of this process are represented in varying proportions at steady state and appear, over time, during the reconstitution of the immune system, as demonstrated in humanized mice and in patients undergoing hematopoietic stem cell transplantation. CD56(dim) NK-cell differentiation, and the associated functional imprint, occurs independently of NK-cell education by interactions with self-human leukocyte antigen class I ligands and is an essential part of the formation of human NK-cell repertoires.

Monitoring the spread of mycobacterium in vivo using biophotonic imaging provides a fast, reliable and sensitive method to evaluate the distribution of the infection. Moreover, this technique allows for a significant reduction in the number of animals required in comparison to conventional anatomopathological studies. Here, we describe for the first time and validate the use of a luciferase-tagged recombinant Mycobacterium bovis BCG for non-invasive bioluminescent imaging of 1) bacterial dissemination in tissues, 2) the efficacy of treatment with anti-mycobacterial drugs and 3) the role of adaptive immune responses in controlling mycobacterial infection in vivo.

We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1(-/-) mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480(+) Gr1(+) inflammatory monocytes and lower numbers of F480(-) Gr1(+) neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1(-/-) mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1(-/-) BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1(-/-) BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.