Stephan Oehen

Antigen localisation regulates immune responses in a dose‐ and time‐dependent fashion: a geographical view of immune reactivity

Summary: This review summarises experimental evidence to illustrate that induction of immune reactivity depends upon antigen reaching and being available in lymphoid organs in a dose‐ and time‐dependent manner. If antigen reaches lymph organs in a localised staggered manner and with a concentration gradient, a response is induced in the draining lymph node. Antigen‐presenting cells are of critical importance to transport antigen from the periphery to local organised lymphoid tissue. If antigen is all over the lymphoid system, then it deletes all specific cells in the thymus or induces them within a few days: because of their limited life‐span they then die off, leaving the repertoire depleted of this specificity. If antigen does not reach lymphoid organs it is ignored by immune cells. Once a response is induced, activated but not resting T cells will reach antigen outside lymphoid organs, whereas activated B cells differentiate into plasma cells in an inducing environment, mostly in lymphoid tissue including bone marrow, but also in chronic lymphoid‐like infiltrations in peripheral organs. In immunopathology (when the infectious agent is known) or in autoimmunity (when the triggering infectious agent is not known or not recognised) lymphoid tissue may become organised close to the antigen (e.g. in organ‐specific autoimmune diseases) and may thereby maintain an autoantigen‐driven disease‐causing immune response for a long time, The notion that naive T cells get induced or silenced in the periphery may be questioned because induction can only occur in lymphoid organs providing anatomical structures where critical cell‐cell interactions are properly guided and where, therefore, cells are likely to meet sufficiently frequently and in a critical milieu. Since overall immune reactivity critically depends upon the localisation of antigens in a dose‐ and time‐dependent manner, it seems more likely ‐ but this remains to be shown ‐ that activated T cells may get exhausted in non‐lymphoid peripheral tissues, whereas they are usually maintained in lymphoid organs. The critical role of antigen in regulating immune responses also has relevance for our understanding of immunological defence against epithelial and mesenchymal tumours, against many infectious diseases and for understanding autoimmunity and immunological memory. Collectively the data indicate that antigen, impendent upon localisation, dose and time, seems to be the simplest regulator of immune responses.

Lifelong protection against hepatitis B: the role of vaccine immunogenicity in immune memory

Long-term protection against hepatitis B (HB) disease is dependent on persistence of a strong immune memory. This paper presents and discusses new knowledge that allows a better understanding of the mechanisms of long-term protection following hepatitis B vaccination. Studies have revealed links between specific lymphoproliferation, the in vivo humoral response and immune memory. The strength of immune memory and of a subsequent secondary immune response can therefore be predicted by the antibody response following primary vaccination. Vaccine antigen dose and structure have been identified as important influences in the primary antibody response and development of immune memory. The data and considerations presented support the use of highly immunogenic HB vaccines in order to provide long-lasting protection against HB disease.

Naïve cytotoxic T lymphocytes spontaneously acquire effector function in lymphocytopenic recipients: A pitfall for T cell memory studies?

Whether memory T cells require persisting antigen for their survival has been a matter of debate. One prominent view that memory T cells do not require persisting antigen is based in part on studies in which T cell populations have been transferred into antigen‐free mice. To generate “space” recipients were often irradiated; the functional properties of the transfused T cells were then evaluated after prolonged periods. In this report we show that transferring cytotoxic T lymphocytes (CTL) into irradiated or T and B cell‐deficient hosts results in their proliferation and a change of their activation state. Moreover, naïve T cell receptor‐ransgenic CTL specific for the lymphocytic choriomeningitis virus glycoprotein spontaneously developed cytotoxic effector function under such conditions. Therefore, some of the conclusions based on transfer of T cell populations into irradiated recipients to investigate T cell memory may have to be reevaluated.

Peptide Antigen Treatment of Naive and Virus-Immune Mice: Antigen-Specific Tolerance Versus Immunopathology

Peptide-specific down-regulation of T cell responses may represent a powerful tool to intervene in autoimmune diseases or graft rejections. It is therefore important to know whether peptide treatment tolerizes both naive and antigen-experienced memory T lymphocytes. Here we show that a major histocompatibility complex class I binding peptide, derived from the glycoprotein (GP33 peptide) of lymphocytic choriomeningitis virus (LCMV), specifically tolerized naive cytotoxic T lymphocytes (CTL) when administered three times intraperitoneally in incomplete Freunds adjuvants. However, in the presence of GP33-specific memory CTL in LCMV-primed mice, the same treatment had a general immunosuppressive effect on unrelated third-party antigen-specific T cell responses and caused severe immunopathological damage to the spleen.

Direct quantitation of rapid elimination of viral antigen-positive lymphocytes by antiviral CD8 + T cells in vivo

Lysis of infected cells by CD8+ T cells is an important mechanism for the control of virus infections, but remains difficult to quantify in vivo. Here, we study the elimination kinetics of viral antigen‐positive lymphocytes by antiviral CD8+ T cells using flow cytometry and mathematical analysis. In mice acutely infected with lymphocytic choriomeningitis virus, more than 99.99 % of target cells were eliminated each day, corresponding to a half‐life of 1.4 h. Even in mice exposed to virus 300 days previously, and with no ex vivo killing activity, 84 % of the target cells were eliminated per day. Unexpectedly, the elimination kinetics of antigen‐positive lymphocytes was not significantly impaired in mice deficient in either perforin‐, CD95 ligand‐ or TNF‐mediated cytotoxicity. For viruses with a particular tropism for lymphocytes, such as Epstein‐Barr virus or HIV, our results illustrate how effectively CD8+ T cell‐mediated elimination of target cells can potentially contribute to virus control and immunosuppression.

A simple method for evaluating the rejection of grafted spleen cells by flow cytometry and tracing adoptively transferred cells by light microscopy

In this report we describe a simple method using the vital dye 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) to follow splenic graft rejection by flow cytometry. CFSE-labelled spleen cell suspensions were injected intravenously into various recipients and blood samples were taken at different time points to follow the transferred cells. We found that the labelled cells could be readily detected by flow cytometry for up to eleven weeks. The loss of these labelled cells in various transfusion experiments with different major and minor histocompatibility differences followed the rejection kinetics previously described for skin transplants. Thus, this method offers a simple tool to test the histocompatibility of donor cells/grafts with the host in adoptive/transplantation experiments in which donor and host are not completely syngeneic. Furthermore, we developed a method to trace adoptively transferred fluorescent CFSE-labelled cells by light microscopy by converting in situ immunofluorescence staining into immunoenzyme staining.

Marginal zone macrophages and immune responses against viruses

The effective establishment of antiviral protection requires a coordinated interplay between the innate and adaptive immune system. Using osteopetrotic (op−/−) mice, this study investigated the influence of marginal zone macrophages in controlling and initiating a protective immune response against a cytopathic vs a non- or low-cytopathic virus. Despite the generation of potent adaptive immune responses, antiviral protection against cytopathic vesicular stomatitis virus critically depended on the presence of marginal zone macrophages. Infection with low doses (100 PFU) of non- or low-cytopathic lymphocytic choriomeningitis virus was rarely cleared and usually resulted in a carrier state in the majority of mice. This shows that the early innate immune system provides an important preparatory phase to the adaptive immune system and is particularly important for antiviral protection.

Bone Marrow Transfer from Wild-Type Mice Reverts the Beneficial Effect of Genetically Mediated Immune Deficiency in Myelin Mutants

Inherited demyelinating neuropathies are chronically disabling human disorders caused by various genetic defects, including deletions, single site mutations, and duplications in the respective myelin genes. We have shown in a mouse model of one distinct hereditary demyelinating neuropathy (heterozygous P0-deficiency, P0+-) that an additional null mutation in the recombination activating gene-1 (RAG-1--) leads to a substantially milder disorder, indicating a disease modifying role of T-lymphocytes. In the present study, we addressed the role of lymphocytes in the mouse model by reconstituting bone marrow of P0+-/RAG-1-- mice with bone marrow from immunocompetent wild-type mice. We compared the pathology and nerve conduction in double mutant mice (P0+-/RAG-1-- on a C57BL/6 background) with that in double mutants after receiving a bone marrow transplant. We found that the milder demyelination seen in the lymphocyte-deficient P0+-/RAG-1-- mutants was reverted to the more severe pathology by reestablishing a competent immune system by bone marrow transfer. These data corroborate the concept that the immune system contributes substantially to the pathologic process in this mouse model and may open new avenues to ameliorate human hereditary neuropathies by exploiting immunosuppressive treatments.

Donor cell persistence and activation-induced unresponsiveness of peripheral CD8 + T cells

We studied the impact of the duration of donor cell persistence on CD8+ T cell responsiveness after adoptive transfer of antigen‐expressing lymphoid cells. Naive or immunized female mice were treated by adoptive transfer of spleen cells from mice ubiquitously expressing a lymphocytic choriomeningitis virus‐derived cytotoxic T lymphocyte (CTL) epitope (gp33 – 41) either alone or in combination with the male H‐Y antigen providing additional antigenic CTL and T helper cell determinants. Low doses of male spleen cells (or sorted B cells) primed CTL, while high doses of the same cells rendered them unresponsive. CTL unresponsiveness induced by high numbers of male spleen cells was dependent upon prolonged persistence of antigen‐expressing donor cells. Unresponsive CTL reverted to a state of activation when the duration of donor cell chimerism was limited. Memory CTL could be rendered unresponsive if antigen‐expressing donor cells were allowed to persist. These results suggest that, irrespective of the type of antigen‐presenting cell and the functional state of the responding T cell, activation and unresponsiveness can represent two different outcomes critically determined by quantitative and kinetic differences of antigen persistence.