Christina R. Reilly

Mature microglia resemble immature antigen-presenting cells

Owing to the difficulties of isolating adequate numbers of microglia from adult tissue, much of our understanding of their function is based on characterizations of microglia that develop in mixed glial cultures. To learn more about the nature of these cells in vivo, we have compared the phenotypes of murine microglia isolated from adults, neonates, and from mixed glial cultures with spleen cells from fetuses, neonates, and adults. In the adult CNS, the only resident population of cells that express CD45, a protein tyrosine phosphatase, are the F4/80+ and FcR+ cells: the microglia. In contrast to all other differentiated cells of hemopoietic origin, microglial CD45 levels fail to increase from the neonatal period through adulthood. Rather, their levels are indistinguishable from the low levels found on a small population of embryonic day 16 liver cells. Conversely, we find that the F4/80 values of microglia are elevated as compared to splenic macrophages. Strikingly, microglia that develop in mixed glial cultures display a more activated phenotype, with low F4/80 values, weak MHC class II expression, and the appearance of a subset of cells positive for the dendritic cell marker, NLDC145. Additionally, CD45 values are elevated to a level intermediate between that of adult microglia and adult spleen, a level similar to that found on microglia activated in vivo. Consistent with this activated phenotype, indomethacin revealed the ability of mixed glial culture microglia to present a peptide antigen to naive T‐cells expressing a defined T‐cell receptor. Although adult microglia did express costimulatory molecules, B7.2, ICAM‐1, and CD40, and could be induced to express MHC class II, they failed to present antigen in the same assay. Interestingly, these same cells could stimulate T‐cell proliferation in a mixed lymphocyte reaction but not in an allogeneic specific manner. Taken together these data suggest that adult microglia remain in a relatively immature and unactivated state of differentiation as compared to other tissue macrophages. GLIA 22:72–85, 1998.

Cutting Edge: Ectopic Expression of the Chemokine TCA4/SLC Is Sufficient to Trigger Lymphoid Neogenesis

To test whether accumulation of naive lymphocytes is sufficient to trigger lymphoid development, we generated mice with islet expression of the chemokine TCA4/SLC. This chemokine is specific for naive lymphocytes and mature dendritic cells (DC) which express the CCR7 receptor. Islets initially developed accumulations of T cells with DC, with scattered B cells at the perimeter. These infiltrates consolidated into organized lymphoid tissue, with high endothelial venules and stromal reticulum. Infiltrate lymphocytes showed a naive CD44low CD25− CD69− phenotype, though half were CD62L negative. When backcrossed to RAG-1 knockout, DC were not recruited. Interestingly, islet lymphoid tissue developed in backcrosses to Ikaros knockout mice despite the absence of normal peripheral nodes. Our results indicate that TCA4/SLC can induce the development and organization of lymphoid tissue through diffential recruitment of T and B lymphocytes and secondary effects on stromal cell development.

Disproportionate Recruitment of CD8+ T Cells into the Central Nervous System by Professional Antigen-Presenting Cells

Inappropriate immune responses, thought to exacerbate or even to initiate several types of central nervous system (CNS) neuropathology, could arise from failures by either the CNS or the immune system. The extent that the inappropriate appearance of antigen-presenting cell (APC) function contributes to CNS inflammation and pathology is still under debate. Therefore, we characterized the response initiated when professional APCs (dendritic cells) presenting non-CNS antigens were injected into the CNS. These dendritic cells expressed numerous T-cell chemokines, but only in the presence of antigen did leukocytes accumulate in the ventricles, meninges, sub-arachnoid spaces, and injection site. Within the CNS parenchyma, the injected dendritic cells migrated preferentially into the white matter tracts, yet only a small percentage of the recruited leukocytes entered the CNS parenchyma, and then only in the white matter tracts. Although T-cell recruitment was antigen specific and thus mediated by CD4+ T cells in the models used here, CD8+ T cells accumulated in numbers equal to or greater than that of CD4+ T cells. Few of the recruited T cells expressed activation markers (CD25 and VLA-4), and those that did were primarily in the meninges, injection site, ventricles, and perivascular spaces but not in the parenchyma. These results indicate that 1) the CNS modulates the cellular composition and activation states of responding T-cell populations and that 2) myelin-restricted inflammation need not be initiated by a myelin-specific antigen.

Integrating innate and adaptive immunity in the whole animal

Summary: The mammalian defense system can respond to a variety of threats, but this capability is not just a simple alarm system for triggering antigen‐presenting cells and initiating cellular immunity. Instead, the body is at integrated system in which nearly every ceil type can relay the alarm through the production of chemokines, which recruit specific inflammatory cells to the target tissues. This chemokine production is carefully regulated at several levels so that the kinetics and character of local tissue inflammation is tailored to the specific threat. First, the production of nuclear factor‐KB‐regulated chemokines can be modulated in non‐bone marrow‐derived cells through transcriptional repression mediated by RelB. RelB is also implicated in the differentiation of lymphoid dendritic cells, suggesting that this gene regulates the transition from acute inflammation to adaptive immunity. Second, tissue parenchymal cells, in their capacity as sentinel cells, are able to produce different patterns of chemokines in response to different alarm stimuli. Third, cells from different tissues also show distinct potentials for chemokine responses so that the non‐specific damage from inflammation might he avoided in some cases. Finally, the differentiation of T‐cell effectors allows for further regulation of local inflammation as their cytokines can also affect chemokine production. This integration of innate and adaptive immunity allows for both rapid responses and dynamic regulation of inflammation in vivo.

Thymic stromal cell specialization and the T-cell receptor repertoire.

Ten years ago, we proposed a model for thymus function in which thymic epithelial cells are primarily responsible for imprinting major histocompatibility complex (MHC)-restricted specificity, and bone marrow-derived macrophages or dendritic cells are responsible for the induction of self-tolerance. Since then, transgenic and knockout models have allowed for a dissection of thymic stromal components in vivo, leading to a new understanding of their specialized functions. We have determined that with regard to class II-restricted CD4 T-cell development, two distinct subsets of thymic epithelium help shape the repertoire: Cortical epithelium appears solely responsible for positive selection, whereas a fucose-bearing subset of medullary epithelium is specialized for negative selection. This absolute separation of positive and negative selection into two distinct spatial and temporal compartments leads to a much simpler view of the process of repertoire selection. Finally, a novel view of the function of the thymic medulla is discussed.

Potent effects of low levels of MHC class II-associated invariant chain on CD4+ T cell development.

Invariant chain (Ii)-negative mice exhibit defects in MHC class II assembly and transport that results in reduced levels of surface class II, altered antigen presentation, and inefficient positive selection of CD4+ T cells. Many CD4+ T cells that do mature in Ii-negative mice express a cell surface phenotype consistent with aberrant positive selection or peripheral activation. Reconstitution of these mice with low levels of either the p31 or p41 form of Ii does not restore transport of the bulk of class II or class II surface expression, but surprisingly does restore positive selection as measured by numbers and surface phenotype of CD4+ T cells. Thus, an Ii-dependent process, independent of effects on class II surface density, appears to be required for normal positive selection of CD4+ T cells.

Thymic skewing of the CD4/CD8 ratio maps with the T-cell receptor α-chain locus

Abstract The thymic preference for CD4 + T cells over CD8 + T cells is often attributed to a default pathway favouring CD4 + T cells [1] or to homeostatic mechanisms [2,3]. It is also clear, however, that T-cell receptor (TCR) preferences for major histocompatibility complex (MHC) class I versus class II binding will strongly influence an individual clones skewing to the CD4 or CD8 subset [4]. The variable region of each TCR α chain (V α ) studied to date is found to be overrepresented in either CD4 + [5–7] or CD8 + cells [7–9], suggesting that each V α element can interact more favourably with either MHC class I or class II molecules [10]. Indeed, TCRs appear to have an intrinsic ability to interact with MHC molecules [11,12], and single amino acid residues present in germline-encoded complementarity determining region 1 (CDR1) and CDR2 of the V α element can be responsible for determining MHC specificity [13]. Interestingly, the degree of CD4/CD8 skewing is variable among different mouse strains [14,15] and in human populations [16]. Here, we have shown that polymorphism in CD4/CD8 skewing between B6 and BALB/c mice is determined by the stem cell genotype and not by environmental effects, and that it maps in or near the TCR α -chain complex, Tcra . This was confirmed by comparing Tcra b with Tcra a or Tcra c haplotypes in congenic mice. We propose that the array of V α genes in various Tcra haplotypes exerts influence over the proportion of CD4 and CD8 subsets generated and may account in part for the observed thymic skewing. Thus, while it has been suggested that the TCR genes have been selected by evolution for MHC binding, our results further indicate selection for class II MHC preference.

Transgenic Expression of Ly-49A in Thymocytes Alters Repertoire Selection

A T cell-specific Ly-49A transgene inhibits TCR-mediated activation in the presence of H-2Dd. Expression of this transgene by developing thymocytes impairs negative selection evidenced by a failure to delete potentially autoreactive T cells and development of a graft-vs-host-disease-like syndrome. In mice carrying both the Ly-49A and a class II-restricted TCR transgene, positive selection was lost, but only when H-2Dd was present on thymic epithelium. These results are consistent with models suggesting that thymic selection is dependent on the perceived intensity of TCR signaling. More interestingly, these results show that Ly-49A does not simply provide a strict on/off switch for T cell responses. Since Ly-49A may shift the signaling threshold of TCR-induced triggering, inducible expression of Ly-49A may regulate peripheral memory/activated T cells by raising the threshold for T cell reactivation.

Discrimination between thymic epithelial cells and peripheral antigen-presenting cells in the induction of immature T cell differentiation.

During their intrathymic migration, immature CD4+ CD8+ thymocytes that express a TCR able to recognize the expressed MHC molecules are positively selected, i.e., complete their differentiation program and become mature T cells. Using the immature CD4+CD8+ T cell line DPK, which can be induced to differentiate in culture, we show here that a subset of isolated thymic epithelial cells, but not peripheral antigen-presenting cells, can induce differentiation, suggesting a unique function of these cells in T cell development. In addition, analysis of activation markers induced by thymic epithelial cells versus specific antigen gives the first direct evidence that positive selection is associated with low level cell activation. In contrast with strict affinity-avidity models of thymic selection, we propose that a specialized antigen-presenting cell environment is an essential contributor to TCR-mediated differentiation in the thymus.

Dendritic-Like Cells from relB Mutant Mice

Mice deficient in the NF-kappa B transcription factor relB appear to have defects in the production of mature dendritic cells, as secondary lymphoid tissues are absent, and spleen cells show a significant loss of antigen presenting function. Moreover, the thymus appears to be impaired in negative selection, and immune responses in vivo are poor. Since dendritic cell precursors such as skin Langerhans cells appear to be normal, we sought information on the nature of the dendritic cell defect in these mice. Cultures of mutant bone marrow in the presence of GM-CSF revealed a delay in the accumulation of cells with dendritic cell features relative to controls; however, these cells were nearly as potent on a per cell basis as wild type cells in the stimulation of allogeneic mixed lymphocyte cultures. Similarly, skin Langerhans cells from mutant mice also showed significant ability to stimulate allogeneic T cells in culture. Since these findings cannot explain the defect in immune responses and the absence of secondary lymphoid tissues, we also looked at the ability of the relB mutant dendritic-like cells to form aggregates in vitro with naive syngeneic T cells. In this case, while wild type dendritic cells generated compact aggregates with T cells, relB mutant cells only formed irregular small aggregates. Thus, while relB mutant dendritic-like cells have some functions of mature dendritic cells, other functions are deficient. Understanding the role of relB in regulation of these functions should lead to a greater understanding of the molecular basis of dendritic cell development and function.