Christopher Coleclough

Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35–55 from myelin oligodendrocyte glycoprotein (pMOG35–55) consistently activate a high proportion of CD8+ αβTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG35–55. CNS lesions in pMOG35–55 CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1−/− mice, but not in isogeneic β2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1−/− mice in which disease was induced adoptively with <1 × 106 T cells sensitized to pMOG35–55. It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.

Antibody response to influenza infection of mice: different patterns for glycoprotein and nucleocapsid antigens

Our previous studies of C57BL/6 mice intranasally infected with influenza virus (A/PR8) revealed a spike of virus‐specific immunoglobulin A (IgA)‐secreting antibody‐forming cells (AFC) in the mediastinal lymph node (MLN) 7 days post‐infection. Here we show that these AFC are directed only against viral glycoprotein, and not nucleocapsid antigens. The early IgA spike associates with a decline in glycoprotein‐specific AFC during week 2 post‐infection. In contrast to the glycoprotein‐specific AFC, nucleocapsid‐specific, IgA‐secreting AFC develop gradually in the MLN and persist for more than 3 weeks post‐infection. As peripheral lymph node reactions wane, the nucleocapsid‐specific AFC appear as long‐sustained populations in the bone marrow. Microanatomical examination of the respiratory tract in infected mice shows foci of infection established in the lung 2 days post‐infection, from which virus spreads to infect the entire lining of the trachea by day 3. At this time, viral haemagglutinin can be seen within the MLN, probably on projections from infected dendritic cells. This feature disappears within a day, though viral antigen expression continues to spread throughout the respiratory tract. Total IgA‐ and IgG‐secreting AFC appear histologically in large numbers during the first week post‐infection, significantly preceding the appearance of germinal centres (revealed by peanut agglutinin staining in week 2). To explain these results, we suggest that the initial immunogenic encounter of B cells with viral antigens occurs about 3 days post‐infection in the MLN, with antigens transported by dendritic cells from airway mucosa, the only site of viral replication. Viral glycoproteins expressed as integral membrane components on the surface of infected dendritic cells [probably in the absence of cognate T helper (Th) cells] promote members of expanding relevant B‐cell clones to undergo an IgA switch and terminal local plasmacytoid differentiation. Anti‐glycoprotein specificities are thus selectively depleted from progeny of activated B‐cell clones which are channelled to participate in germinal centre formation (perhaps by cognate T helper cells when they become sufficiently frequent). One product of the germinal centre reaction is the long‐sustained, bone marrow‐resident population, which is accordingly rich in anti‐nucleoprotein, but not anti‐glycoprotein specificities. Of note, we find that AFC responses toward influenza virus and Sendai virus differ, even though viral replication is limited to the airway mucosa in each case. The response towards Sendai virus exhibits neither the early appearance of anti‐glycoprotein AFC expressing IgA in draining lymph nodes, nor the subsequent relative deficit of this specificity from bone marrow AFC populations.

CD4+ T Cell Priming Accelerates the Clearance of Sendai Virus in Mice, but Has a Negative Effect on CD8+ T Cell Memory

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421–436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.

Reciprocal stimulation between TNF-α and nitric oxide may exacerbate CNS inflammation in experimental autoimmune encephalomyelitis

Nitric oxide (NO) and TNF-alpha are both highly active pleotypic modulators of cell function that are abundantly generated during inflammation. Experiments in animal systems have linked the generation of NO and TNF-alpha to autoimmune pathogenesis, and blockade of either NO or TNF-alpha has been shown to impede disease development. In this study, we show that NO and TNF-alpha can act mutually to stimulate each others production. While IFN-gamma alone induces NO release from microglia, astrocytes are provoked into significant NO production only by a combination of IFN-gamma and TNF-alpha. Since both TNF-alpha and NO are abundantly generated during T-glial cell interaction, we asked whether and how NO affects TNF-alpha production. Using an in vitro system in which TNF-alpha secretion is induced in MBP-reactive T cells by co-culture with syngeneic astrocytes, we observed that the efficiency of TNF-alpha secretion was markedly increased, in a dose-dependent fashion, by addition of micromolar concentrations of a chemical generator of NO donor, sodium nitroprusside (SNP). Similarly, low concentrations of SNP significantly enhanced the IL-2 dependent growth of MBP-reactive T cells. These results suggest that autoimmune pathogenesis initiated by inflammatory responses within the CNS may result in part from a vicious cycle in which TNF-alpha and NO mutually provoke each others production.

Nonactivated astrocytes downregulate T cell receptor expression and reduce antigen-specific proliferation and cytokine production of myelin basic protein (MBP)-reactive T cells

Astrocytes express variable levels of MHC class II antigens depending on their activation status or exposure to certain cytokines, notably IFN-gamma. When they are induced to express higher surface densities of MHC class II molecules, astrocytes are capable of stimulating syngeneic myelin basic protein (MBP)-reactive T cells to proliferate at a modest rate and to secrete proinflammatory cytokines, such as TNF-alpha, in response to antigen. In the present investigation evidence is presented that uninduced astrocytes, whether fresh or established as clones, on which surface MHC class II molecules are expressed at a very low density, promote an antigen-dependent reduction of TCR on the surface of syngeneic T cells. Accompanying this effect on the TCR is an induction of T cell hyporeactivity and little or no production of proinflammatory cytokines. These observations suggest that the ability of the astrocyte, through varying their surface MHC class II molecules, can control the effect of antigen-induced T cell responses. In their normal state of low MHC II expression astrocytes are expected to induce no or partial, rather than full, activation of autoreactive T cells that enter the CNS, resulting in T cell hyporeactivity. Since astrocytes usually diminish the production of proinflammatory cytokines by T cells that enter the CNS, the status and control of MHC class II expression on astrocytes should be important determinants of the suppression or enhancement of in situ immune responses in the CNS.

Production of tumor necrosis factor-? as a result of glia-T-Cell interaction correlates with the pathogenic activity of myelin basic protein-reactive T cells in experimental autoimmune encephalomyelitis

Tumor necrosis factor‐α (TNF‐α) has attracted the greatest attention as a major factor in experimental autoimmune encephalomyelitis (EAE) pathogenesis. We compared rats undergoing EAE with manipulated but healthy animals by examining TNF‐α gene expression in cells recovered from the brain. We used reverse transcriptase‐polymerase chain reaction (RT‐PCR) as a sensitive assay for detection and Northern blot hybridization as a reliable quantitative assay of TNF‐α mRNA. TNF‐α gene expression was consistently detected in rats immunized with myelin basic protein (MBP) emulsified in complete Freund adjuvant (CFA), but not in rats immunized with MBP emulsified in incomplete Freund adjuvant (IFA), which does not induce EAE. Similarly, brain‐derived cells from rats injected with cloned encephalitogenic T cells contained increased amounts of TNF‐α mRNA compared with rats injected with nonencephalitogenic T cell clones similar in antigen specificity and in vitro lymphokine‐producing capacity. Considering that the differing pathogenic capacity of MBP‐reactive T cells might result from differing patterns of interaction with glia, we examined the impact of T‐cell‐glia interaction in vitro on cytokine gene expression in both cell types. Glial components were efficient in inducing TNF‐α expression in T cells; T cells and T‐cell‐derived cytokines could elicit expression of several lymphokine genes in glial cells. Comparison of RT‐PCR and blot hybridization assays, however, suggested that cytokine expression was much more efficient, on a per cell basis, in T cells than in glia. TNF‐α was shown to have direct cytotoxic effect on glial cells, which was greatly enhanced by small amounts of interferon‐γ (IFN‐γ).

Limited Breadth of a T-Helper Cell Response to a Human Immunodeficiency Virus Envelope Protein

ABSTRACT Single-envelope human immunodeficiency virus (HIV) vaccines have been studied for more than a decade, with some successes in homologous challenge experiments in nonhuman primates but with no clear successes in clinical trials. To gain insight into the breadth of the immunity elicited by such vaccines, we have dissected the T-helper cell response of C57BL/6 mice to an individual, molecularly cloned envelope protein. Here, we report that T-helper cells responsive to HIV type 1 1035 envelope are very highly restricted in C57BL/6 animals: seven different hybridomas recovered from five separate mice recognized the same peptide, PKVSFEPIPIHYCAP, located in the C2 region of gp120. Three of these hybridomas were tested on a natural variant of the peptide but failed to respond. A more extensive analysis of whole splenic populations from other C57BL/6 mice immunized with the 1035 envelope reproducibly confirmed that the gp120-specific T-helper response was almost exclusively focused on a single epitope. We conclude that single-envelope vaccines may frequently fail to provoke an immune response sufficiently diverse to recognize variant sequences among circulating HIV. The results encourage the inclusion of more than one envelope in future vaccines to enhance the potential diversity and respective surveillance capacities of responding T-helper cell populations.

Respiratory vaccination of mice against influenza virus: dissection of T- and B-cell priming functions.

We find that a single respiratory administration of replicationally inactivated influenza A viral particles most often elicits a waning serum antibody response, as the long‐sustained bone marrow antiviral plasma cell populations characteristically induced by viral infection are lacking, though antiviral plasma cells at other sites may occasionally persist for a long time. To determine whether this alteration in the pattern of the B‐cell response is a reflection of the nature of T‐helper (Th) priming, we simultaneously primed B cells with inactivated influenza A/PR8(H1N1) and Th cells with infectious A/x31(H3N2). We show that Th cells cross‐react extensively between these two viruses, although the antibody response to viral envelope glycoproteins is completely non‐cross‐reactive. Th cells primed by infectious A/x31 have little impact on the antibody response specifically elicted from naïve B cells by inactivated A/PR8 viruses, suggesting that the characteristic vigour of the antibody response to influenza viral infection depends on the direct interaction of antiviral B cells with virally infected dendritic cells. Memory B cells primed by inactivated influenza viral particles however, respond rapidly to secondary challenge with live or inactivated viruses, promptly populating bone marrow with antiviral plasma cells. Moreover, Th cells primed by previous live A/x31 viral challenge alter the pattern of the response of naïve B cells to live A/PR8 challenge by accelerating the appearance of anti‐H1/N1 plasma cells in bone marrow, eliminating the early spike of anti‐H1/N1 plasma cells in the mediastinal node, and generally diminishing the magnitude of the lymph node response. Inactivated A/PR8 and infectious A/x31 are both effective vaccines against A/PR8 infection, as mice preimmunized with either vaccine exhibit much more rapid viral clearance from the lung after infectious A/PR8 challenge. In fact, even when given during a course of anti‐CD8 treatment to preempt cross‐reactive cytotoxic T cells, live A/x31 is a more effective vaccine against A/PR8 infection than is inactivated A/PR8 itself.

Global analysis of gene expression in cells of the immune system II. Cell-free translation products and high-density filter hybridization data.

We have developed an experimental system for linking information on cell‐free transcription and translation products from cDNA clones with data obtained from hybridization signals from complex probes. The work described in this paper consists of two distinct processes, one being the construction of a system of clonal addresses and the other the identification of expressed genes involved in the studied processes. We describe the use of this system to identify genes involved in thymus development. Complex probes from fetal thymuses (GD15, 17 and newborn) of Balb/c mice were used to identify genes, which are up‐ or downregulated during the process of differentiation. The full set of information is available in the Clone‐base of the Basel Institute for Immunology and will be retrievable from the website of the collaborating laboratories.

Analysis of a randomly assorted cDNA library from BW 5147 lymphoid cells. Frequency estimates based on computer aided concatenation matching of 2D gel polypeptide products

A cDNA library was divided into 291 sectors of low complexity, 800-1000 recombinant phage plaques per sector. Aliquots of DNA from sectors prepared from high titer phage were subjected to in vitro transcription using T7 polymerase and thereafter RNA was translated in a cell-free rabbit reticulocyte system in the presence of [35S] methionine. The resulting polypeptides were separated by 2D gel electrophoresis. Radiofluorographs of the gels prepared from 10 sectors were subjected to scanning and image analysis. A multilevel spot matching procedure was employed allowing us to recognise spot occurrence in the 10 sectors. The number of detected polypeptide spots per sector varied between 147 and 325. Overall, 1082 different spots were counted totalling 1983 spots considering multiple entries. The distribution of the spots and the frequency distribution of the RNA population among the 10 sectors are shown. The highest detected frequency is 10(-2), while one half of the RNA molecular species are present at a frequency of 10(-3) or lower.