Citations

Abstract CD8 T cell responses are essential for clearance of infections and tumors in the central nervous system (CNS). Under homeostatic conditions, T cell access to the CNS parenchyma is restricted. An intricate process of T cell reactivation by antigen presenting cells (APCs) at CNS barriers is thought to allow activated antigen-specific T cells to infiltrate the parenchyma during neurological disease. The role of CNS-resident APCs during CD8 T cell infiltration of the CNS parenchyma is unclear. We hypothesized that microglia and brain-associated macrophages (BAMs) are APCs critical for antigen specific CD8 T cell reactivation and CNS infiltration. To test this hypothesis, we generated mice with tamoxifen-inducible deletion of MHC class I on microglia and BAMs by crossing CX3CR1creER mice with H-2Db fl/fl mice (Db cKO). We challenged these mice with intracranial infection of Theiler’s murine encephalomyelitis virus (TMEV), which generates a robust CD8 T cell response against a Db restricted antigen (VP2121–130) in the brain. At 7 days post infection (dpi), brain infiltrating Db:VP2-specific CD8 T cells were reduced in Db cKO mice compared to control, while peripheral priming of CD8 T cells was unaffected. Unexpectedly, inflammatory monocytes infiltrating the brain at 7 dpi were also reduced in Db cKO mice, indicating a role for microglia/BAMs and CD8 T cells in recruitment of inflammatory monocytes. Db cKO mice were able to clear virus, indicating functional antiviral responses against TMEV occur despite reduced immune infiltration. These results demonstrate that local antigen presentation by microglia and BAMs facilitates inflammatory monocyte recruitment and antigen-specific CD8 T cell responses in the brain after TMEV infection.

Abstract CD8 T cell priming after viral infection of the central nervous system (CNS) is incompletely understood. In peripheral infections, T cell priming generally relies upon stimulation by antigen-bearing dendritic cells (DCs) migrating into draining lymph nodes. The parenchyma of the brain, however, is largely devoid of dendritic cells and evidence demonstrating that activated DCs leave the brain parenchyma is lacking. We therefore sought to understand the role of DCs and specific MHC class I molecules in priming CD8 T cells following CNS infection. We employed H-2Db−/− H-2Kb−/− (MHC-I deficient) mice that express a floxed H-2Db or floxed Kb molecule, a unique transgenic mouse system developed by our research program. Crossed with an MHC class I deficient CD11c-Cre expressing animal results in DC-specific ablation of Db or Kb Class I restricted antigen presentation ability. We found that CD11c+ APCs are critical for the early priming of CD8 T cells against the Db restricted VP2121–130 epitope of the model pathogen, TMEV. However, DCs are ultimately dispensable for long-term development of these antigen specific T cells. Discordantly, the loss of MHC class I restricted antigen presentation ability on CD11c+ APCs does not impact the priming of T cells against the H-2Kb restricted OVA257–264 epitope following intracranial TMEV-OVA infection, but does impact the ability of these T cells to successfully occupy the CNS. This work suggests that differing patterns of MHC class I restriction for CNS pathogen-derived epitopes might have functional consequences on the APC requirements for a successful CD8 T cell response against neurotropic viruses.

Abstract Immunosuppression following damage to the CNS is a common, yet poorly understood, feature of neurological diseases as diverse as stroke, traumatic brain injury, and glioblastoma. This immunosuppression is a barrier to successful patient outcomes. We sought to define the effect of various brain insults on the thymus and T-cell responses. We tested thymic function following various neurological insults, including viral infection, brain tumor, sterile inflammation, physical injury, and seizures. All brain insults resulted in significant thymic involution that was reversible if the insult was cleared. Thymic involution did not occur following similar peripheral insults. Using parabiosis, we demonstrated that thymic involution was transferable via circulatory routes from glioma-bearing to non-tumor-bearing parabionts. Similarly, serum obtained from mice with ongoing neurological insults potently inhibited T-cell proliferation in vitro. We next fractionated the serum based on molecular weight and tested the resulting fractions’ immunosuppressive potential. Interestingly, we found that serum fractions with large molecular weights of greater than 100 kiloDaltons were responsible for the immunosuppressive properties of serum obtained from glioma-bearing mice. In short, CNS-specific insults, regardless of nature, induce immunosuppression by prompting thymic involution and systemic immunosuppression mediated through circulating factors with large molecular weight. These studies provide evidence of the mechanisms leading to immune deficiencies observed in patients following neurological injuries.

Abstract Brain resident memory T cells (TRM) have recently been phenotypically characterized. However, the function of these cells in the brain, and their changes during neurological insults, is not well understood. Using the described phenotype of brain TRMs (TCRB+, CD69+, CD4 or CD8+, CD103−, CD44+), we evaluated cellular responses of this population in the naïve brain and upon various neurological insults. We found that numbers of brain TRMs increased as a function of age. Additionally, TRM cells in the naïve brain produce TNFα constitutively, setting them apart from TRMs in other organs. Parabiosis studies revealed that circulating pools of memory T cells contributes to maintenance of TRM cells in the naïve brain. During neurological injury, we observed an increase in numbers of TRMs as early as 24 hours following physical insult induced by intracranial injection of PBS. During brain infection with Theiler’s Murine Encephalomyelitis virus, brain TRMs increased in number prior to detection of any virus-specific CD8 T cells. This implies TRMs respond to CNS viral infections prior to generation of virus antigen specific responses. This response is due to proliferation of TRM cells in the brain, as treatment with FTY720 did not inhibit TRM proliferation 24 hours post infection. In short, naïve brains harbor populations of TNFα producing TRM cells. And TRMs rapidly respond to neurological injuries through proliferation in an antigen independent manner. Understanding brain TRMs is crucial in investigating their role in neurodegenerative disorders or targeting this potent population of brain resident T cells in cancers of the CNS.

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12 months post diagnosis despite aggressive treatment. Immunotherapy approaches to treat GBM are being developed, but the unique immune environment of the brain and the immunosuppressive features of GBM have complicated early efforts. Even αPD-1 checkpoint blockade, highly useful in the context of melanoma, has proved ineffective in the treatment of GBM. In order to promote the efficacy of αPD-1 against the GL261 murine model of GBM, we supplemented this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated dangerously high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. While neither αPD-1 nor MSA-IL-2 are effective monotherapies against the GL261 model tumor, we have found this combination immunotherapy to provide durable tumor clearance and to create immunologic memory capable of resisting GL261 rechallenge. Most strikingly, this combination therapy is independent of the CD8 T cell function, clearing established GL261 tumors in mice incapable of MHC class I restricted antigen presentation. Independence of MHC class I restricted antigen presentation makes this combination immunotherapy using αPD-1 checkpoint blockade both highly novel and translatable to clinical use.

Abstract Blood brain barrier (BBB) disruption is a feature of multiple neurological disorders for which there is an increasingly recognized role for CD8+ T cells. Theiler’s murine encephalomyelitis virus (TMEV) infection serves as an excellent model to study CD8+ T cell mediated BBB disruption because of the well-characterized immune response to the immunodominant VP2121–130 peptide presented in the context of the H-2Db class I molecule. Intravenous injection of VP2121–130 peptide seven days post TMEV infection results in severe BBB disruption. BBB disruption is dependent on CD8+ T cell interaction with the Db: VP2121–130 epitope, but the antigen presenting cell required for BBB disruption is undefined. We hypothesized that dendritic cells are responsible for presentation of the immunodominant VP2121–130 peptide. We tested this hypothesis using a novel H-2Db Cre-LoxP conditional knockout mouse bred to MHC class I deficient CD11c-Cre expressing mice. We then evaluated these animals at 7 days post infection for induction of BBB disruption, following VP2121–130 peptide administration, using gadolinium enhanced MRI, fluorescent albumin leakage into the brain, and confocal microscopy for the tight junction protein occludin. Control CD11c-Cre(−) mice generated robust Db: VP2121–130 epitope specific CD8+ T cell responses and displayed extensive BBB disruption. In contrast, CD11c-Cre(+) littermates generated no brain infiltrating antiviral CD8+ T cell responses and were protected from BBB disruption. We conclude that CD11c+ dendritic cells are required for priming of CD8+ T cell responses and are necessary for BBB disruption during neuroinflammation in this model.

Abstract Microglia are central nervous system (CNS) resident immune cells for which the impact of MHC class I antigen presentation is still being defined. To directly address if microglia contribute to CD8 T cell-mediated immunity of the CNS, we generated a novel tamoxifen inducible CX3CR1-creER H-2Kb MHC class I conditional knockout mouse. In this transgenic mouse, the H-2Kb MHC class I molecule is deleted on all CX3CR1-expressing cells following tamoxifen treatment. By two weeks post tamoxifen treatment, peripheral CX3CR1-expressing cells repopulate, restoring normal expressing of H-2Kb. This leaves only microglia as the sole MHC class I-deficient cell type. We hypothesized that deletion of H-2Kb on microglia would reduce CD8 T cell responses in the CNS. We challenged these animals and cre-negative littermates with intracranial picornavirus infection with Theiler’s murine encephalomyelitis virus expressing ovalbumin (TMEV-OVA). We also challenged mice with experimental GL261 gliomas. We found that microglia-specific H-2Kb deletion reduces the CD8 T cell response in the CNS and attenuates the activation status of brain-infiltrating CD8 T cells in both models of neuroinflammation. We conclude that microglia are functional antigen presenting cells, and are required for a complete antigen-specific CD8 T cell response in the brain. These findings are the first to definitively address the role of microglia-expressed MHC class I in CNS immunological challenges.

Abstract TCR interactions with agonist pMHCs induce a conformational change in the tails of the associated CD3 complex (CD3Δc) that contributes to efficient TCR/CD3 triggering and productive T cell responses. In contrast, TCR engagement by antagonistic pMHCs does not induce either CD3Δc or full TCR/CD3 signaling in mature T cells. Here, we have used a monovalent Fab fragment (Mono-Fab) specific for the murine CD3 complex to provide induction of CD3Δc to mature T cells engaging pMHC ligands. This Mono-Fab does not interfere with TCR capacity to bind pMHC and fails to elicit signaling in the absence of TCR/MHC interactions. Using OT-I Tg T cells and a collection of altered peptide ligands derived of the agonist peptide OVA, we show that addition of CD3Δc to OT-I T cells using Mono-Fab enables T cell responses to antagonist ligands that fail to induce CD3Δc on their own. Moreover, using a mouse model of metastatic melanoma, we show that Mono-Fab promotes in vivo T cell dependent anti-melanoma responses that are antigen-specific. This Mono-Fab can also be combined with established therapies targeting signals 2 and 3 involved in T cell activation for a synergistic effect. Our data suggests that in-trans CD3Δc provision decreases the activation threshold of T cells. We propose that targeting the CD3 complex for the induction of CD3Δc could be exploited to achieve efficient TCR/CD3 triggering by poorly immunogenic antigens in the context of vaccination against cancer and infectious diseases.

Abstract Self-tolerant T cells can cross-react weakly on self, but are usually incapable of activation. However, when activated these T cells can cross-react effectively on cells expressing self antigens. We have observed that mutants of the MHC class I molecule H2-Kb have structural differences resulting in altered antigen presentation. We hypothesize that structural manipulation of MHC class I can increase stabile amino acid contacts with the TCR. Increasing stabile contacts may decrease the energy barrier required to overcome the threshold for activation, breaking tolerance for self antigens. Using computer modeling techniques, amino acid substitutions at specific sites of MHC class I were predicted to result in stabilization of the TCR:pMHC complex. These altered MHC molecules were made and expressed in cell lines. We have shown that altered MHC enhanced TCR binding. Altered MHC enhanced activation and proliferation of naïve T cells in response to a non-stimulatory, weak antigen. Altered MHC also induced CTL killing against self-antigens. In an in vivo transplant model, protective immunity was induced against a native tumor after an altered MHC expressing tumor challenge. By altering the structure of MHC class I we have activated normally tolerant T cells that are capable of cross-reacting on self.

Abstract One limitation to the development of targeted tumor immunity is the availability of safe vectors that can drive strong T-cell responses capable of eradicating tumors. We have designed viral vectors using Theiler’s murine encephalomyelitis virus (TMEV) that effectively inhibit melanoma outgrowth. By cloning the TMEV genome into a eukaryotic expression vector that can easily be manipulated we were able to generate several novel tumor specific virus vaccines. We find that incorporation of defined MHC class I epitopes into sites within the leader protein of the virus can drive strong CD8+ T-cell responses against these target peptides. Further, we find that the site of incorporation can change the elicited cytokine profile and the virulence of the virus. Incorporation of the ovalbumen peptide after the third amino acid of the L protein (p3) generates a strong IFNβ response, whereas insertion within the downstream XhoI restriction site elicits a strong IL6 response similar to wild-type TMEV. Further, the p3 virus is not lethal to RAG-deficient mice, where the XhoI and wild-type TMEV virus are. However, p3 virus persists and kills IFNα/β receptor-deficient mice, suggesting that innate immune responses are responsible for viral clearance. In spite of these differences the p3 and XhoI viruses can stimulate antigen specific T-cell responses and inhibit tumor outgrowth equivalently, demonstrating the efficacy of TMEV and its potential as a therapeutic intervention.