Evan R. Jellison

CD4+ T cell regulation of CD25 expression controls development of short-lived effector CD8+ T cells in primary and secondary responses

Both CD4+ T cell help and IL-2 have been postulated to “program” activated CD8+ T cells for memory cell development. However, the linkage between these two signals has not been well elucidated. Here we have studied effector and memory CD8+ T cell differentiation following infection with three pathogens (Listeria monocytogenes, vesicular stomatitis virus, and vaccinia virus) in the absence of both CD4+ T cells and IL-2 signaling. We found that expression of CD25 on antigen-specific CD8+ T cells peaked 3–4 days after initial priming and was dependent on CD4+ T cell help, likely through a CD28:CD80/86 mediated pathway. CD4+ T cell or CD25-deficiency led to normal early effector CD8+ T cell differentiation, but a subsequent lack of accumulation of CD8+ T cells resulting in overall decreased memory cell generation. Interestingly, in both primary and recall responses KLRG1high CD127low short-lived effector cells were drastically diminished in the absence of IL-2 signaling, although memory precursors remained intact. In contrast to previous reports, upon secondary antigen encounter CD25-deficient CD8+ T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. Thus, these results demonstrated that CD4+ T cell help and IL-2 signaling were linked via CD25 up-regulation, which controls the expansion and differentiation of antigen-specific effector CD8+ T cells, rather than “programming” memory cell traits.

Cutting Edge: MHC Class II-Restricted Killing In Vivo during Viral Infection

Class II-restricted CD4 T cell-mediated killing of target cells has previously been documented in vitro but not in vivo. In this study, we demonstrate CD4-dependent MHC class II-restricted killing in lymphocytic choriomeningitis virus-infected mice in vivo using an in vivo cytotoxicity assay that features class II-expressing B cells as targets.

Pathogen induced inflammatory environment controls effector and memory CD8+ T cell differentiation

In response to infection, CD8+ T cells integrate multiple signals and undergo an exponential increase in cell numbers. Simultaneously, a dynamic differentiation process occurs, resulting in the formation of short-lived effector cells (SLECs; CD127lowKLRG1high) and memory precursor effector cells (CD127highKLRG1low) from an early effector cell that is CD127lowKLRG1low in phenotype. CD8+ T cell differentiation during vesicular stomatitis virus infection differed significantly than during Listeria monocytogenes infection with a substantial reduction in early effector cell differentiation into SLECs. SLEC generation was dependent on Ebi3 expression. Furthermore, SLEC differentiation during vesicular stomatitis virus infection was enhanced by administration of CpG-DNA, through an IL-12–dependent mechanism. Moreover, CpG-DNA treatment enhanced effector CD8+ T cell functionality and memory subset distribution, but in an IL-12–independent manner. Population dynamics were dramatically different during secondary CD8+ T cell responses, with a much greater accumulation of SLECs and the appearance of a significant number of CD127highKLRG1high memory cells, both of which were intrinsic to the memory CD8+ T cell. These subsets persisted for several months but were less effective in recall than memory precursor effector cells. Thus, our data shed light on how varying the context of T cell priming alters downstream effector and memory CD8+ T cell differentiation.

Age- and location-related changes in microglial function

Inflammation in the central nervous system (CNS) is primarily regulated by microglia. No longer considered a homogenous population, microglia display a high degree of heterogeneity, immunological diversity and regional variability in function. Given their low rate of self-renewal, the microenvironment in which microglia reside may play an important role in microglial senescence. This study examines age-related changes in microglia in the brain and spinal cord. Using ex-vivo flow cytometry analyses, functional assays were performed to assess changes in microglial morphology, oxidative stress, cytokine production, and phagocytic activity with age in both the brain and spinal cord. The regional CNS environment had a significant effect on microglial activity with age. Blood-CNS barrier permeability was greater in the aging spinal cord compared with aging brain; this was associated with increased tissue cytokine levels. Aged microglia had deficits in phagocytosis at baseline and after stimulus-induced activation. The identification of age-specific, high scatter microglia together with the use of ex-vivo functional analyses provides the first functional characterization of senescent microglia. Age and regional-specificity of CNS disease should be taken into consideration when developing immune-modulatory treatments.

Cell-selective knockout and 3D confocal image analysis reveals separate roles for astrocyte-and endothelial-derived CCL2 in neuroinflammation

BackgroundExpression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood–brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s).MethodsMice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature.ResultsCell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen.ConclusionsThese results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease.

Avidity maturation of memory CD8 T cells is limited by self-antigen expression

Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. We developed a system that revealed a population of self-specific CD8 T cells within the endogenous T cell repertoire. Immunization of ovalbumin (OVA)-expressing transgenic mice with recombinant viruses expressing OVA-peptide variants induced self-reactive T cells in vivo that matured into memory T cells able to respond to secondary infection. However, whereas the avidity of memory cells in normal mice increased dramatically with repeated immunizations, avidity maturation was limited for self-specific CD8 T cells. Despite decreased avidity, such memory cells afforded protection against infection, but did not induce overt autoimmunity. Further, up-regulation of self-antigen expression in dendritic cells using an inducible system promoted programmed death-1 expression, but not clonal expansion of preexisting memory cells. Thus, the self-reactive T cell repertoire is controlled by overlapping mechanisms influenced by antigen dose.

High Frequencies of Virus-Specific CD8+ T-Cell Precursors

ABSTRACT A productive CD8+ T-cell response to a viral infection requires rapid division and proliferation of virus-specific CD8+ T cells. Tetramer-based enrichment assays have recently given estimates of the numbers of peptide-major histocompatibility complex-specific CD8+ T cells in naïve mice, but precursor frequencies for entire viruses have been examined only by using in vitro limiting-dilution assays (LDAs). To examine CD8+ T-cell precursor frequencies for whole viruses, we developed an in vivo LDA and found frequencies of naïve CD8+ T-cell precursors of 1 in 1,444 for vaccinia virus (VV) (∼13,850 VV-specific CD8+ T cells per mouse) and 1 in 2,958 for lymphocytic choriomeningitis virus (LCMV) (∼6,761 LCMV-specific CD8+ T cells per mouse) in C57BL/6J mice. In mice immune to VV, the number of VV-specific precursors, not surprisingly, dramatically increased to 1 in 13 (∼1,538,462 VV-specific CD8+ T cells per mouse), consistent with estimates of VV-specific memory T cells. In contrast, precursor numbers for LCMV did not increase in VV-immune mice (1 in 4,562, with ∼4,384 LCMV-specific CD8+ T cells per VV-immune mouse). Using H-2Db-restricted LCMV GP33-specific P14-transgenic T cells, we found that, after donor T-cell take was accounted for, approximately every T cell transferred underwent a full proliferative expansion in response to LCMV infection. This high efficiency was also seen with memory populations, suggesting that most antigen-specific T cells will proliferate extensively at a limiting dilution in response to infections. These results show that frequencies of naïve and memory CD8+ T cell precursors for whole viruses can be remarkably high.

Age-Associated Resident Memory CD8 T Cells in the Central Nervous System Are Primed To Potentiate Inflammation after Ischemic Brain Injury

Aging is associated with an increase in basal inflammation in the CNS and an overall decline in cognitive function and poorer recovery following injury. Growing evidence suggests that leukocyte recruitment to the CNS is also increased with normal aging, but, to date, no systematic evaluation of these age-associated leukocytes has been performed. In this work, the effect of aging on CNS leukocyte recruitment was examined. Aging was associated with more CD45high leukocytes, primarily composed of conventional CD8+ T cells. These results were strain independent and seen in both sexes. Intravascular labeling and immunohistology revealed the presence of parenchymal CD8+ T cells in several regions of the brain, including the choroid plexus and meninges. These cells had effector memory (CD44+CD62L−) and tissue-resident phenotypes and expressed markers associated with TCR activation. Analysis of TCRvβ repertoire usage suggested that entry into the CNS is most likely stochastic rather than Ag driven. Correlational analyses revealed a positive association between CD8 T cell numbers and decreased proinflammatory function of microglia. However, the effects of cerebral ischemia and ex vivo stimulation of these cells dramatically increased production of TNF, IFN-γ, and MCP-1/CCL2. Taken together, we identified a novel population of resident memory, immunosurveillant CD8 T cells that represent a hallmark of CNS aging and appear to modify microglia homeostasis under normal conditions, but are primed to potentiate inflammation and leukocyte recruitment following ischemic injury.

Splenic Morphological Changes Are Accompanied by Altered Baseline Immunity in a Mouse Model of Sickle-Cell Disease

Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.

Early Effector CD8 T Cells Display Plasticity in Populating the Short- Lived Effector and Memory- Precursor Pools Following Bacterial or Viral Infection

Naïve antigen-specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations, which include memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). In the days before the peak of the T cell response, a third population called early effector cells (EECs) predominate the antigen-specific response. However, the contribution of the EEC population to the CD8 T cell differentiation program during an antimicrobial immune response is not well understood. To test if EEC populations were pre-committed to either an MPEC or SLEC fate, we purified EECs from mice infected with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV), where the relative frequency of each population is known to be different at the peak of the response. Sorted EECs transferred into uninfected hosts revealed that EECs were pre-programmed to differentiate based on early signals received from the distinct infectious environments. Surprisingly, when these same EECs were transferred early into mismatched infected hosts, the transferred EECs could be diverted from their original fate. These results delineate a model of differentiation where EECs are programmed to form MPECs or SLECs, but remain susceptible to additional inflammatory stimuli that can alter their fate.