Hans Hengartner

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.

Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction

The vertebrate immune system has evolved to protect against infections that threaten survival before reproduction. Clinically manifest tumours mostly arise after the reproductive years and somatic mutations allow even otherwise antigenic tumours to evade the attention of the immune system. Moreover, the lack of immunological co-stimulatory molecules on solid tumours could result in T-cell tolerance; that is, the failure of T cells to respond. However, this may not generally apply. Here we report several important findings regarding the immune response to tumours, on the basis of studies of several tumour types. First, tumour-specific induction of protective cytotoxic T cells (CTLs) depends on sufficient tumour cells reaching secondary lymphatic organs early and for a long enough duration. Second, diffusely invading systemic tumours delete CTLs. Third, tumours that stay strictly outside secondary lymphatic organs, or that are within these organs but separated from T cells by barriers, are ignored by T cells but do not delete them. Fourth, co-stimulatory molecules on tumour cells do not influence CTL priming but enhance primed CTL responses in peripheral solid tumours. Last, cross priming of CTLs by tumour antigens, mediated by major histocompatibility complex (MHC) class I molecules of antigen-presenting host cells, is inefficient and not protective. These rules of T-cell induction and maintenance not only change previous views but also rationales for anti-tumour immunotherapy.

Quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24- or 96-well plates

Titers of lymphocytic choriomeningitis virus (LCMV) were determined on adherent fibroblast cell lines in 24- or 96-well plates. After absorption of virus by cells and 48 h incubation under a methylcellulose overlay, cell monolayers were fixed with 4% formaldehyde in phosphate-buffered saline, permeabilized by incubation in 0.5% Triton X-100 in balanced salt solution and then stained with a monoclonal rat anti-LCMV and a peroxidase-labeled second stage antibody. The sensitivity of the assay is within a factor of 2-4 of conventional plaquing methods. The method also detects poorly or non-plaquing LCMV isolates, and therefore drastically reduces the need for titration of LCMV in mice. The method is quicker (2-3 days), as compared to conventional methods (4-6 days) and less expensive in terms of work and materials.

FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory.

FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride) prolongs survival of solid organ allografts in animal models. Mechanisms of FTY720 immunomodulation were studied in mice infected with lymphocytic choriomeningitis virus (LCMV) to assess T cell responses or with vesicular stomatitis virus to evaluate Ab responses. Oral FTY720 (0.3 mg/kg/day) did not affect LCMV replication and specific CTL and B cells were induced and expanded normally. Moreover, the anti-viral humoral immune responses were normal. However, FTY720 treatment showed first a shift of overall distribution of CTL from the spleen to peripheral lymph nodes and lymphocytopenia was observed. This effect was reversible within 7–21 days. Together with unimpaired T and B cell memory after FTY720 treatment, this finding rendered enhancement of lymphocyte apoptosis by FTY720 in vivo unlikely. Secondly, the delayed-type hypersensitivity reaction to a viral MHC class I-presented peptide was markedly reduced by FTY720. These results were supported by impaired circulation of LCMV specific TCR transgenic effector lymphocytes in the peripheral blood and reduced numbers of tissue infiltrating CTL in response to delayed-type hypersensitivity reaction. Thirdly, in a CD8+ T cell-mediated diabetes model in a transgenic mouse expressing the LCMV glycoprotein in the islets of the pancreas, FTY720 delayed or prevented disease by reducing islet-infiltrating CTL. Thus, FTY720 effectively reduced recirculation of CD8+ effector T cells and their recruitment to peripheral lesions without affecting the induction and expansion of immune responses in secondary lymphoid organs. These properties may offer the potential to treat ongoing organ-specific T cell-mediated immunopathologic disease.

Virus-specific major MHC class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection

A transgenic mouse expressing MHC class II‐restricted TCR with specificity for a lymphocytic choriomeningitis virus (LCMV) glycoprotein‐derived T helper cell epitope was developed to study the role of LCMV‐specific CD4+ T cells in virus infection in vivo. The majority of CD4+ T cells in TCR transgenic mice expressed the transgenic receptor, and LCMV glycoprotein‐specific TCR transgenic CD4+ T cells efficiently mediated help for the production of LCMV glycoprotein‐specific isotype‐switched antibodies. In contrast, LCMV glycoprotein‐specific TCR transgenic mice exhibited a drastically reduced ability to provide help for the generation of antibody responses specific for the virus‐internal nucleoprotein, indicating that intramolecular/intrastructural help is limited to antigens that are accessible to B cells on the viral surface. Antiviral cellular immunity was studied with noncytopathic LCMV and recombinant cytopathic vaccinia virus expressing the LCMV glycoprotein. TCR transgenic mice failed to efficiently control LCMV infection, demonstrating that functional LCMV‐specific CD4+ T cells – even if activated and present at extremely high frequencies – cannot directly mediate protective immunity against LCMV. Despite the fact that LCMV‐primed CD4+ T cells from TCR transgenic mice as well as from control mice showed low MHC class II‐restricted cytotoxic activity in vivo, this did not correlate with protection against LCMV replication in vivo. In contrast, CD4+ T cells from TCR‐transgenic mice mediated efficient protection against infection with recombinant vaccinia virus. These results further support the need for different immune effector functions for protective immunity against different viral infections.

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.

Protective Immunity Does Not Correlate with the Hierarchy of Virus-specific Cytotoxic T Cell Responses to Naturally Processed Peptides

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) stimulates major histocompatibility complex class I–restricted cytotoxic T cells (CTLs), which normally resolve the infection. Three peptide epitopes derived from LCMV have been shown to bind the mouse class I molecule H-2 Db and to stimulate CTL responses in LCMV-infected mice. This report describes the identity and abundance of each CTL epitope after their elution from LCMV-infected cells. Based on this information, peptide abundance was found to correlate with the magnitude of each CTL response generated after infection with LCMV. Subsequent experiments, performed to determine the antiviral capacity of each CTL specificity, indicate that the quantitative hierarchy of CTL activity does not correlate with the ability to protect against LCMV infection. This report, therefore, indicates that immunodominant epitopes should be defined, not only by the strength of the CTL response that they stimulate, but also by the ability of the CTLs to protect against infection.

T-cell-mediated immunopathology versus direct cytolysis by virus: implications for HIV and AIDS

It has been much debated as to whether CD4+ T-cell depletion and the pathogenesis of AIDS is the result of direct cytolytic effects of human immunodeficiency virus (HIV), T-cell apoptosis by nonspecific activation, dysregulation of cytokine production, or autoimmunity. In this context, Rolf Zinkernagel and Hans Hengartner discuss data from model infections with non-cytopathic viruses. They suggest that HIV may cause immunosuppression, not by direct cytolytic effects, but rather by a conventional virus-specific cytotoxic T-cell-mediated immunopathology.

Antiviral antibody responses: the two extremes of a wide spectrum.

Viruses elicit a diverse spectrum of antiviral antibody responses. In this review, we discuss two widely used experimental model systems for viral infections — non-cytopathic lymphocytic choriomeningitis virus (LCMV) and acutely cytopathic vesicular stomatitis virus (VSV) — to analyse two fundamentally different types of antiviral antibody response. The basic principles found in these model infections are discussed in the context of other viral infections, and with regard to protective neutralizing versus non-protective enzyme-linked immunosorbent assay (ELISA)-detected antibody responses. Issues of antibody specificity, affinity and avidity, maturation and escape are discussed in the context of co-evolution of the host and viruses.

IgA production without |[mu]| or |[delta]| chain expression in developing B cells

Surface, membrane-bound, immunoglobulin M (IgM) or IgD expression early in B cell ontogeny is considered essential for the differentiation of antibody-producing cells in mammals; only in IgM+ B cells is the heavy chain locus rearranged to express antibodies of other classes. We show here that IgA is selectively expressed in μMT mice, which lack IgM or IgD expression and have a pro-B cell developmental block. μMT IgA binds proteins of commensal intestinal bacteria and is weakly induced by Salmonella infection, although not through conventional immunization. This μMT IgA pathway requires extrasplenic peripheral lymphoid tissues and may be an evolutionarily primitive system in which immature B cells switch to IgA production at peripheral sites.