Neuroinflammation creates an immune regulatory niche at the meningeal lymphatic vasculature near the cribriform plate.

Meningeal lymphatics near the cribriform plate undergo lymphangiogenesis during neuroinflammation to drain excess fluid. Here, we hypothesized that lymphangiogenic vessels may acquire an altered phenotype to regulate immunity. Using single-cell RNA sequencing of meningeal lymphatics near the cribriform plate from healthy and experimental autoimmune encephalomyelitis in the C57BL/6 model, we report that neuroinflammation induces the upregulation of genes involved in antigen presentation such as major histocompatibility complex class II, adhesion molecules including vascular cell adhesion protein 1 and immunoregulatory molecules such as programmed cell death 1 ligand 1, where many of these changes are mediated by interferon-γ. The inflamed lymphatics retain CD11c+ cells and CD4 T cells where they capture and present antigen, creating an immunoregulatory niche that represents an underappreciated interface in the regulation of neuroinflammation. We also found discontinuity of the arachnoid membrane near the cribriform plate, which provides unrestricted access to the cerebrospinal fluid. These findings highlight a previously unknown function of local meningeal lymphatics in regulating immunity that has only previously been characterized in draining lymph nodes.

Mycobacterium bovis Bacillus Calmette-Guérin-Infected Dendritic Cells Induce TNF-α-Dependent Cell Cluster Formation That Promotes Bacterial Dissemination through an In Vitro Model of the Blood-Brain Barrier.

CNS tuberculosis (CNSTB) is the most severe manifestation of extrapulmonary tuberculosis infection, but the mechanism of how mycobacteria cross the blood-brain barrier (BBB) is not well understood. In this study, we report a novel murine in vitro BBB model combining primary brain endothelial cells, Mycobacterium bovis bacillus Calmette-Guérin-infected dendritic cells (DCs), PBMCs, and bacterial Ag-specific CD4+ T cells. We show that mycobacterial infection limits DC mobility and also induces cellular cluster formation that has a similar composition to pulmonary mycobacterial granulomas. Within the clusters, infection from DCs disseminates to the recruited monocytes, promoting bacterial expansion. Mycobacterium-induced in vitro granulomas have been described previously, but this report shows that they can form on brain endothelial cell monolayers. Cellular cluster formation leads to cluster-associated damage of the endothelial cell monolayer defined by mitochondrial stress, disorganization of the tight junction proteins ZO-1 and claudin-5, upregulation of the adhesion molecules VCAM-1 and ICAM-1, and increased transmigration of bacteria-infected cells across the BBB. TNF-α inhibition reduces cluster formation on brain endothelial cells and mitigates cluster-associated damage. These data describe a model of bacterial dissemination across the BBB shedding light on a mechanism that might contribute to CNS tuberculosis infection and facilitate treatments.

CXCL13 expressed on inflamed cerebral blood vessels recruit IL-21 producing TFH cells to damage neurons following stroke.

BACKGROUND: Ischemic stroke is a leading cause of mortality worldwide, largely due to the inflammatory response to brain ischemia during post-stroke reperfusion. Despite ongoing intensive research, there have not been any clinically approved drugs targeting the inflammatory component to stroke. Preclinical studies have identified T cells as pro-inflammatory mediators of ischemic brain damage, yet mechanisms that regulate the infiltration and phenotype of these cells are lacking. Further understanding of how T cells migrate to the ischemic brain and facilitate neuronal death during brain ischemia can reveal novel targets for post-stroke intervention. METHODS: To identify the population of T cells that produce IL-21 and contribute to stroke, we performed transient middle cerebral artery occlusion (tMCAO) in mice and performed flow cytometry on brain tissue. We also utilized immunohistochemistry in both mouse and human brain sections to identify cell types and inflammatory mediators related to stroke-induced IL-21 signaling. To mechanistically demonstrate our findings, we employed pharmacological inhibitor anti-CXCL13 and performed histological analyses to evaluate its effects on brain infarct damage. Finally, to evaluate cellular mechanisms of stroke, we exposed mouse primary neurons to oxygen glucose deprivation (OGD) conditions with or without IL-21 and measured cell viability, caspase activity and JAK/STAT signaling. RESULTS: Flow cytometry on brains from mice following tMCAO identified a novel population of cells IL-21 producing CXCR5+ CD4+ ICOS-1+ T follicular helper cells (TFH) in the ischemic brain early after injury. We observed augmented expression of CXCL13 on inflamed brain vascular cells and demonstrated that inhibition of CXCL13 protects mice from tMCAO by restricting the migration and influence of IL-21 producing TFH cells in the ischemic brain. We also illustrate that neurons express IL-21R in the peri-infarct regions of both mice and human stroke tissue in vivo. Lastly, we found that IL-21 acts on mouse primary ischemic neurons to activate the JAK/STAT pathway and induce caspase 3/7-mediated apoptosis in vitro. CONCLUSION: These findings identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and provide a potential new therapeutic target for stroke.

Molecular Mechanisms of Neuroimmune Crosstalk in the Pathogenesis of Stroke.

Stroke disrupts the homeostatic balance within the brain and is associated with a significant accumulation of necrotic cellular debris, fluid, and peripheral immune cells in the central nervous system (CNS). Additionally, cells, antigens, and other factors exit the brain into the periphery via damaged blood-brain barrier cells, glymphatic transport mechanisms, and lymphatic vessels, which dramatically influence the systemic immune response and lead to complex neuroimmune communication. As a result, the immunological response after stroke is a highly dynamic event that involves communication between multiple organ systems and cell types, with significant consequences on not only the initial stroke tissue injury but long-term recovery in the CNS. In this review, we discuss the complex immunological and physiological interactions that occur after stroke with a focus on how the peripheral immune system and CNS communicate to regulate post-stroke brain homeostasis. First, we discuss the post-stroke immune cascade across different contexts as well as homeostatic regulation within the brain. Then, we focus on the lymphatic vessels surrounding the brain and their ability to coordinate both immune response and fluid homeostasis within the brain after stroke. Finally, we discuss how therapeutic manipulation of peripheral systems may provide new mechanisms to treat stroke injury.

Regional Distribution of CNS Antigens Differentially Determines T-Cell Mediated Neuroinflammation in a CX3CR1-Dependent Manner.

T cells continuously sample CNS-derived antigens in the periphery, yet it is unknown how they sample and respond to CNS antigens derived from distinct brain areas. We expressed ovalbumin (OVA) neoepitopes in regionally distinct CNS areas (Cnp-OVA and Nes-OVA mice) to test peripheral antigen sampling by OVA-specific T cells under homeostatic and neuroinflammatory conditions. We show that antigen sampling in the periphery is independent of regional origin of CNS antigens in both male and female mice. However, experimental autoimmune encephalomyelitis (EAE) is differentially influenced in Cnp-OVA and Nes-OVA female mice. Although there is the same frequency of CD45high CD11b+ CD11c+ CX3CL1+ myeloid cell-T-cell clusters in neoepitope-expressing areas, EAE is inhibited in Nes-OVA female mice and accelerated in CNP-OVA female mice. Accumulation of OVA-specific T cells and their immunomodulatory effects on EAE are CX3C chemokine receptor 1 (CX3CR1) dependent. These data show that despite similar levels of peripheral antigen sampling, CNS antigen-specific T cells differentially influence neuroinflammatory disease depending on the location of cognate antigens and the presence of CX3CL1/CX3CR1 signaling.SIGNIFICANCE STATEMENT Our data show that peripheral T cells similarly recognize neoepitopes independent of their origin within the CNS under homeostatic conditions. Contrastingly, during ongoing autoimmune neuroinflammation, neoepitope-specific T cells differentially influence clinical score and pathology based on the CNS regional location of the neoepitopes in a CX3CR1-dependent manner. Altogether, we propose a novel mechanism for how T cells respond to regionally distinct CNS derived antigens and contribute to CNS autoimmune pathology.

Immune responses in stroke: how the immune system contributes to damage and healing after stroke and how this knowledge could be translated to better cures?

Stroke is one of the leading causes of death and disability worldwide. The long-standing dogma that stroke is exclusively a vascular disease has been questioned by extensive clinical findings of immune factors that are associated mostly with inflammation after stroke. These have been confirmed in preclinical studies using experimental animal models. It is now accepted that inflammation and immune mediators are critical in acute and long-term neuronal tissue damage and healing following thrombotic and ischaemic stroke. Despite mounting information delineating the role of the immune system in stroke, the mechanisms of how inflammatory cells and their mediators are involved in stroke-induced neuroinflammation are still not fully understood. Currently, there is no available treatment for targeting the acute immune response that develops in the brain during cerebral ischaemia. No new treatment has been introduced to stroke therapy since the discovery of tissue plasminogen activator therapy in 1996. Here, we review current knowledge of the immunity of stroke and identify critical gaps that hinder current therapies. We will discuss advances in the understanding of the complex innate and adaptive immune responses in stroke; mechanisms of immune cell-mediated and factor-mediated vascular and tissue injury; immunity-induced tissue repair; and the importance of modulating immunity in stroke.

CCR2-dependent dendritic cell accumulation in the central nervous system during early effector experimental autoimmune encephalomyelitis is essential for effector T cell restimulation in situ and disease progression.

Dendritic cells (DCs)--although absent from the healthy CNS parenchyma--rapidly accumulate within brain and spinal cord tissue during neuroinflammation associated with experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis). Yet, although DCs have been appreciated for their role in initiating adaptive immune responses in peripheral lymphoid organ tissues, how DCs infiltrate the CNS and contribute to ongoing neuroinflammation in situ is poorly understood. In this study, we report the following: 1) CD11c(+) bone marrow-derived DCs and CNS-infiltrating DCs express chemokine receptor CCR2; 2) compared with CCR2(+/+) cells, adoptively transferred CCR2(-/-) bone marrow-derived DCs or DC precursors do not accumulate in the CNS during EAE, despite abundance in blood; 3) CCR2(-/-) DCs show less accumulation in the inflamed CNS in mixed bone marrow chimeras, when compared with CCR2(+/+) DCs; and 4) ablation of CCR2(+/+) DCs during EAE clinical onset delays progression and attenuates cytokine production by infiltrating T cells. Whereas the role of CCR2 in monocyte migration into the CNS has been implicated previously, the role of CCR2 in DC infiltration into the CNS has never been directly addressed. Our data suggest that CCR2-dependent DC recruitment to the CNS during ongoing neuroinflammation plays a crucial role in effector T cell cytokine production and disease progression, and signify that CNS-DCs and circulating DC precursors might be key therapeutic targets for suppressing ongoing neuroinflammation in CNS autoimmune diseases.

Lymphangiogenesis is induced by mycobacterial granulomas via vascular endothelial growth factor receptor-3 and supports systemic T-cell responses against mycobacterial antigen.

Granulomatous inflammation is characteristic of many autoimmune and infectious diseases. The lymphatic drainage of these inflammatory sites remains poorly understood, despite an expanding understanding of lymphatic role in inflammation and disease. Here, we show that the lymph vessel growth factor Vegf-c is up-regulated in Bacillus Calmette-Guerin- and Mycobacterium tuberculosis-induced granulomas, and that infection results in lymph vessel sprouting and increased lymphatic area in granulomatous tissue. The observed lymphangiogenesis during infection was reduced by inhibition of vascular endothelial growth factor receptor 3. By using a model of chronic granulomatous infection, we also show that lymphatic remodeling of tissue persists despite resolution of acute infection and a 10- to 100-fold reduction in the number of bacteria and tissue-infiltrating leukocytes. Inhibition of vascular endothelial growth factor receptor 3 decreased the growth of new vessels, but also reduced the proliferation of antigen-specific T cells. Together, our data show that granuloma-up-regulated factors increase granuloma access to secondary lymph organs by lymphangiogenesis, and that this process facilitates the generation of systemic T-cell responses to granuloma-contained antigens.

Deficient import of acetyl-CoA into the ER lumen causes neurodegeneration and propensity to infections, inflammation, and cancer.

The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the N(ε)-lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1S113R is unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into the ER lumen results in reduced acetylation of ER proteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for malignancies.

Immune cells as messengers from the CNS to the periphery: the role of the meningeal lymphatic system in immune cell migration from the CNS.

In recent decades there has been a large focus on understanding the mechanisms of peripheral immune cell infiltration into the central nervous system (CNS) in neuroinflammatory diseases. This intense research led to several immunomodulatory therapies to attempt to regulate immune cell infiltration at the blood brain barrier (BBB), the choroid plexus (ChP) epithelium, and the glial barrier. The fate of these infiltrating immune cells depends on both the neuroinflammatory environment and their type-specific interactions with innate cells of the CNS. Although the fate of the majority of tissue infiltrating immune cells is death, a percentage of these cells could become tissue resident immune cells. Additionally, key populations of immune cells can possess the ability to "drain" out of the CNS and act as messengers reporting signals from the CNS toward peripheral lymphatics. Recent data supports that the meningeal lymphatic system is involved not just in fluid homeostatic functions in the CNS but also in facilitating immune cell migration, most notably dendritic cell migration from the CNS to the meningeal borders and to the draining cervical lymph nodes. Similar to the peripheral sites, draining immune cells from the CNS during neuroinflammation have the potential to coordinate immunity in the lymph nodes and thus influence disease. Here in this review, we will evaluate evidence of immune cell drainage from the brain via the meningeal lymphatics and establish the importance of this in animal models and humans. We will discuss how targeting immune cells at sites like the meningeal lymphatics could provide a new mechanism to better provide treatment for a variety of neurological conditions.

Dual role of Vascular Endothelial Growth Factor-C (VEGF-C) in post-stroke recovery.

Using a mouse model of ischemic stroke, this study characterizes stroke-induced lymphangiogenesis at the cribriform plate (CP). While blocking CP lymphangiogenesis with a VEGFR-3 inhibitor improves stroke outcome, administration of VEGF-C induced larger brain infarcts. Abstract: Cerebrospinal fluid (CSF), antigens, and antigen-presenting cells drain from the central nervous system (CNS) into lymphatic vessels near the cribriform plate and dural meningeal lymphatics. However, the pathological roles of these lymphatic vessels surrounding the CNS during stroke are not well understood. Using a mouse model of ischemic stroke, transient middle cerebral artery occlusion (tMCAO), we show that stroke induces lymphangiogenesis near the cribriform plate. Interestingly, lymphangiogenesis is restricted to lymphatic vessels at the cribriform plate and downstream cervical lymph nodes, without affecting the conserved network of lymphatic vessels in the dura. Cribriform plate lymphangiogenesis peaks at day 7 and regresses by day 14 following tMCAO and is regulated by VEGF-C/VEGFR-3. These newly developed lymphangiogenic vessels transport CSF and immune cells to the cervical lymph nodes. Inhibition of VEGF-C/VEGFR-3 signaling using a blocker of VEGFR-3 prevented lymphangiogenesis and led to improved stroke outcomes at earlier time points but had no effects at later time points following stroke. Administration of VEGF-C after tMCAO did not further increase post-stroke lymphangiogenesis, but instead induced larger brain infarcts. The differential roles for VEGFR-3 inhibition and VEGF-C in regulating stroke pathology call into question recent suggestions to use VEGF-C therapeutically for stroke.

TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4.

TLR9 activation is important for the maintenance of mycobacteria-elicited pulmonary granulomatous responses, hallmarks of protective immune responses following mycobacterial infection. However, the mechanism or mechanisms underlying this effect of TLR9 are not clear. Here, we show that Tlr9-deficient mice challenged with a Mycobacterium antigen display an altered Th17 cytokine profile, decreased accumulation of granuloma-associated myeloid DCs, and profoundly impaired delta-like 4 (dll4) Notch ligand expression. Mechanistic analysis revealed that WT bone marrow-derived DCs but not macrophages promoted the differentiation of Th17 cells from bacillus Calmette-Guérin-challenged (BCG-challenged) lung CD4+ T cells. Both lung and bone marrow DCs isolated from Tlr9-deficient mice inoculated with Mycobacterium antigen expressed lower levels of dll4 Notch ligand than the same cells isolated from WT mice. Passively immunizing WT mice with neutralizing antibodies specific for dll4 during granuloma formation resulted in larger granulomas and lower levels of Th17-related cytokines. In addition, dll4 specifically regulated Th17 activation in vitro. Together, these results suggest dll4 plays an important role in promoting Th17 effector activity during a mycobacterial challenge. Furthermore, TLR9 seems to be required for optimal dll4 expression and the regulation of Mycobacterium antigen-elicited granuloma formation in mice.

CC chemokine receptor 4 contributes to innate NK and chronic stage T helper cell recall responses during Mycobacterium bovis infection.

Cysteine-cysteinyl chemokine receptor 4 (CCR4) is expressed by a variety of T-cell subsets and leukocytes. This study examined the participation of CCR4 in response to pulmonary infection with Mycobacterium bovis Bacille-Calmette-Guerin (BCG). Constitutive and induced CCR4 agonist expression was detected among large mononuclear cells. The course of infection and mobilization of effector cell populations were then analyzed in CCR4 knockout (CCR4(-/-)) mice. Compared with controls, CCR4(-/-) mice displayed delayed innate stage (<2 weeks) bacterial clearance and reduced late stage inflammation. Innate impairment was associated with reduced natural killer cell activation. In the adaptive phase, CCR4(-/-) mice generated effector T cells in draining lymph nodes and accumulated effector T cells in lungs, which resulted in normal adaptive stage bacterial elimination at 2 to 4 weeks. However, during the late stage, CCR4(-/-) mice had reduced interferonγ+CD4(+)α/ß+ (Th1) and interleukin (IL)-17+CD4(+)α/ß+ (Th17) T helper cells in lungs. In contrast, IL-17+ γ/δ T cells in lungs were unaffected. When challenged with mycobacterial antigen- (Ag-) Ag-coated beads to elicit a recall granulomatous response, CCR4(-/-) mice displayed abrogated recall granuloma formation and reduced interferon γ+ Th1 cells. These findings indicate that CCR4 supports innate natural killer cell activation and sustains later CD4(+) Th effector/memory antimycobacterial responses in the lung but is redundant in the early adaptive elimination phase.

Monocyte-derived inflammatory dendritic cells in the granuloma during mycobacterial infection.

The monocyte-derived, inflammatory dendritic cell subset plays an important role during immune responses against infections. This review will focus on the complex, changing role of this subset during mycobacterial infection. Studies demonstrate that in addition to sustaining a systemic anti-mycobacterial response, the inflammatory dendritic cell subset present in Mycobacterium-induced granulomas also influences local immune regulation within the granuloma over the course of infection. This review will also survey the literature on how similar and different inflammatory dendritic cell subsets during other infections.

Innate-adaptive crosstalk: how dendritic cells shape immune responses in the CNS.

Dendritic cells (DCs) are a heterogeneous group of professional antigen presenting cells that lie in a nexus between innate and adaptive immunity because they recognize and respond to danger signals and subsequently initiate and regulate effector T-cell responses. Initially thought to be absent from the CNS, both plasmacytoid and conventional DCs as well as DC precursors have recently been detected in several CNS compartments where they are seemingly poised for responding to injury and pathogens. Additionally, monocyte-derived DCs rapidly accumulate in the inflamed CNS where they, along with other DC subsets, may function to locally regulate effector T-cells and/or carry antigens to CNS-draining cervical lymph nodes. In this review we highlight recent research showing that (a) distinct inflammatory stimuli differentially recruit DC subsets to the CNS; (b) DC recruitment across the blood-brain barrier (BBB) is regulated by adhesion molecules, growth factors, and chemokines; and (c) DCs positively or negatively regulate immune responses in the CNS.

Modeling infectious diseases of the central nervous system with human brain organoids.

Bacteria, fungi, viruses, and protozoa are known to infect and induce diseases in the human central nervous system (CNS). Modeling the mechanisms of interaction between pathogens and the CNS microenvironment is essential to understand their pathophysiology and develop new treatments. Recent advancements in stem cell technologies have allowed for the creation of human brain organoids, which more closely resembles the human CNS microenvironment when compared to classical 2-dimensional (2D) cultures. Now researchers can utilize these systems to investigate and reinvestigate questions related to CNS infection in a human-derived brain organoid system. Here in this review, we highlight several infectious diseases which have been tested in human brain organoids and compare similarities in response to these pathogens across different investigations. We also provide a brief overview of some recent advancements which can further enrich this model to develop new and better therapies to treat brain infections.

Pin1 regulates TGF-beta1 production by activated human and murine eosinophils and contributes to allergic lung fibrosis.

Eosinophilic inflammation is a cornerstone of chronic asthma that often culminates in subepithelial fibrosis with variable airway obstruction. Pulmonary eosinophils (Eos) are a predominant source of TGF-beta1, which drives fibroblast proliferation and extracellular matrix deposition. We investigated the regulation of TGF-beta1 and show here that the peptidyl-prolyl isomerase (PPIase) Pin1 promoted the stability of TGF-beta1 mRNA in human Eos. In addition, Pin1 regulated cytokine production by both in vitro and in vivo activated human Eos. We found that Pin1 interacted with both PKC-alpha and protein phosphatase 2A, which together control Pin1 isomerase activity. Pharmacologic blockade of Pin1 in a rat asthma model selectively reduced eosinophilic pulmonary inflammation, TGF-beta1 and collagen expression, and airway remodeling. Furthermore, chronically challenged Pin1(-/-) mice showed reduced peribronchiolar collagen deposition compared with wild-type controls. These data suggest that pharmacologic suppression of Pin1 may be a novel therapeutic option to prevent airway fibrosis in individuals with chronic asthma.

CD4+ T cells sensitized by vascular smooth muscle induce vasculitis, and interferon gamma is critical for the initiation of vascular pathology.

Primary vasculitis is the result of idiopathic inflammation in blood vessel walls. T cells are believed to play a critical role, but the nature of the pathological T-cell response remains obscure. In this study, we provide evidence that CD4(+) T lymphocytes, activated in the presence of syngeneic vascular smooth muscle cells, were sufficient to induce vasculitic lesions after adoptive transfer to recipient mice. Additionally, the disease is triggered in the absence of antibodies in experiments in which both the donors of stimulated lymphocytes and the transfer recipients were mice that were deficient in B cells. Tracking and proliferation of the transferred cells and their cytokine profiles were assessed by fluorescence tagging and flow cytometry. Proliferating CD4(+) T cells were evident 3 days after transfer, corresponding to the occurrence of vasculitic lesions in mouse lungs. The transferred T lymphocytes exhibited Th1 and Th17 cytokine profiles and minimal Th2. However, 1 week after vasculitis induction, effector functions could be successfully recalled in Th1 cells, but not in Th17 cells. Additionally, in the absence of constitutive interferon-γ expression, T cells sensitized by vascular smooth muscle cells failed to induce vasculitis. In conclusion, our results show that Th1 cells play a key role in eliciting vasculitis in this murine model and that induction of the disease is possible in the absence of pathogenic antibodies.

C3 promotes expansion of CD8+ and CD4+ T cells in a Listeria monocytogenes infection.

It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of Ag-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of dendritic cells or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild-type mice showed that these T cells do not have intrinsic proliferative defects, and a paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on APC. On the basis of these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens.