Mara Gerloni

CD4 T cells in tumor immunity

T cell immunity is the key to protective immune responses against tumors. Traditionally, this function has been ascribed to CD8 T lymphocytes with cytotoxic activity, which are restricted by MHC class I molecules. In recent years the realization that CD4 T cells can also play a relevant role in protective anti-tumor responses has received growing attention. Here we will discuss the role of MHC class II-restricted T cells in response to, and in the regulation of, tumor antigens. Emphasis will be placed on four areas: (1) the role of CD4 T cell immunity in tumor protection in animal models and putative mode of action, (2) tumor antigens recognized by human CD4 T cells, (3) the cooperation between two CD4 T cells of different specificity as a new way to jump start the response against sub-immunogenic determinants of tumor antigens in a tolerant environment, and (4) the negative impact of regulatory CD4 T cells on anti-tumor T cell responses. By drawing attention to these four areas, it is our intention to provide the reader with a comprehensive view of issues of contemporary importance for this field, in the expectation that the information will help a better design of therapeutic cancer vaccines.

The Role of relB in Regulating the Adaptive Immune Response

Abstract: Dendritic cells (DCs), which represent a key type of antigen‐presenting cell (APC), are important for the development of innate and adaptive immunity. DCs are involved in T cell activation in at least two main ways: priming via direct processing/presentation of soluble antigen taken up from the microenvironment (conventional priming), and processing/presentation of antigen released from other cells (cross‐priming). relB, a component of the NF‐κB complex of transcription factors, is a critical regulator of the differentiation of DCs. In mice, lack of relB impairs DCs derived from bone marrow both in number and function. Here relB (−/−) bone marrow chimera mice is used to study the APC function of residual DCs in presentation of soluble antigen and cross‐priming. It is found that the DCs in these mice are profoundly deficient in their ability to both prime and cross‐prime T cell responses. It was concluded that the relB gene is involved in regulating the APC function of DCs in vivo.

B lymphocytes as antigen-presenting cell-based genetic vaccines.

Summary:  Inoculation of plasmid DNA is a simple way to immunize, but it is characterized by low immunogenicity, which has hampered the development of effective DNA vaccines for human use. Here, we discuss how poor immunogenicity can be solved and present our proposal: genetically programmed B lymphocytes as antigen‐presenting cell (APC) vaccines. First, we demonstrate that mature B lymphocytes take up plasmid DNA spontaneously, i.e., in the absence of any facilitating molecule or event, spontaneous lymphocyte transgenesis. Second, we demonstrate that transgenic B lymphocytes are easily and reproducibly turned into functional APCs with dual characteristics: upregulation of costimulatory molecules and endogenous synthesis of antigen. Used as immunogens in mice, transgenic B lymphocytes induce robust and long‐lasting T‐cell immunity after single intravenous injection. Surprisingly, immunity and protection against lethal virus challenge can be obtained with a single intravenous injection of 3 × 102 transgenic lymphocytes. The new approach is discussed relative to the advantage of targeting secondary lymphoid organs with genetically programmed B lymphocytes and the advantage offered with respect to low antigen dose. We suggest that these properties reflect on simple characteristics, such as time synchronization and initial localization to secondary lymphoid organs of APCs endowed with protracted synthesis and presentation of antigen to T cells.

Role of T cell help and endoplasmic reticulum targeting in protective CTL response against influenza virus.

We report on the induction of primary and long‐term memory cytotoxic T lymphocyte (CTL) responses against the nucleoprotein of the influenza virus A/PR8/34 in mice immunized with plasmid DNA targeted to B lymphocytes in the spleen. We found that the magnitude of the CTL response and the size of the pool of memory CTL was greater when the CTL response was induced in presence of T cell help. Interestingly, immunization with a signal sequence‐competent transgene was markedly superior to immunization with a transgene lacking the endoplasmic reticulum (ER) targeting sequence, in inducing CTL. We also found a correlation between in vivo protection from lethal virus challenge and (1) the availability of T cell help and (2) ER targeting. Immunization of dendritic cell‐deficient mice suggests that B lymphocytes function as antigen‐presenting cells in this model of immunization. Collectively, the results suggest that somatic transgene immunization is a conceptually new approach to induce effective anti‐viral CTL responses and to assess the parameters critical for long‐lasting and protective CTL responses in vivo.

DNA immunization in relB‐deficient mice discloses a role for dendritic cells in IgM → IgG1 switch in vivo

A single intraspleen inoculation of plasmid DNA coding for an immunoglobulin heavy chain gene initiates immunity and establishes immunologic memory against the antigenic determinants of transgenic immunoglobulins, somatic transgene immunization. During priming mice produce IgM but not IgG1 antibodies. Since IgM → IgG1 class switch occurs spontaneously during the primary immune response to protein antigens we investigated possible mechanisms for failure of spontaneous isotype switch in vivo in this model of immunity. We found that inoculation of plasmid DNA in the form of a chimeric gene coding for granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was able to drive IgG1 class switch readily after priming. Since GM‐CSF activates cells of the dendritic lineage we tested the possibility that dendritic cells (DC) may be involved in regulating IgM → IgG1 switch. To this end we used bone marrow chimeras constructed from mice carrying the null mutation for the relB member of the NF‐κB/Rel family as these mice lack bone marrow‐derived mature DC. RelB (‐/‐) mice and (‐/‐) bone marrow chimeras inoculated with DNA/GM‐CSF did not produce IgG1 antibodies during the primary immune response. Since relB (‐/‐) bone marrow chimeras lack DC of donor origin but possess resident follicular dendritic cells we conclude that Ig class switch in vivo is regulated by the function of interdigitating dendritic cells (IDC). Thus, IDC may contribute to the qualitative aspects of the emerging immune response.

The Cooperation between Two CD4 T Cells Induces Tumor Protective Immunity in MUC.1 Transgenic Mice

Immunity and tumor protection in mice transgenic for human MUC.1, a glycoprotein expressed in the majority of cancers of epithelial origin in humans, were induced by vaccination with B lymphocytes genetically programmed to activate MUC.1-specific CD4 T cells. Their activation required a functional cooperation between two Th cells, one specific for a self (MUC.1) and the other for a nonself T cell determinant. The immunological switch provided by Th-Th cooperation was sufficient to induce MUC.1-specific CD4 and CD8 T cell responses in MUC.1-transgenic mice, and protect them permanently from tumor growth. CD4 T cells specific for MUC.1 lacked cytolytic function, but produced IFN-γ upon restimulation with Ag. We conclude that immunity against tumor self-Ags and tumor protection can be regulated exploiting an inherent property of the immune system.

Immunological memory after somatic transgene immunization is positively affected by priming with GM‐CSF and does not require bone marrow‐ derived dendritic cells

Inoculation of plasmid DNA is a promising vaccination approach but optimal regimes and ways to enhance immunogenicity remain to be established. Among natural immunological adjuvants, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was shown to increase the potency of immunization against tumor cells and protein antigens. Here we studied the effect of GM‐CSF on memory responses against a 12‐mer B cell epitope in mice primed with a single DNA inoculation. The results show that GM‐CSF given at priming as a DNA/GM‐CSF chimeric vaccine enhances the magnitude of the anamnestic response irrespective of the form of antigen used subsequently in the booster immunization. Using mice lacking bone marrow‐derived dendritic cells we also determined that the enhancing effect is not strictly dependent on these cells. These results expand our understanding of the activity of GM‐CSF in vivo as a modulator of the immune response including immunological memory.

CD8 T cell priming by B lymphocytes is CD4 help dependent.

While it is generally accepted that B lymphocytes can present antigen and activate CD4 T cells, priming of CD8 T cells by B lymphocytes remains controversial. Recently, we showed that mice injected with genetically programmed B lymphocytes generate antigen specific CD4 and CD8 T cell responses in vivo that could also be induced in mice lacking functional dendritic cells. To gain further insights into the requirements for T cell priming by antigen‐presenting B lymphocytes, in vitro experiments were performed using ovalbumin (OVA) and OVA‐specific TCR‐transgenic CD4 and CD8 T cells. We found that while B lymphocytes can directly prime CD4 T cells, the activation of CD8 T cells requires T cell help. Transfer experiments show that help can either be contact dependent or be mediated by soluble factors in the supernatants of activated OVA‐specific CD4 T cells. Furthermore, the effect of activated CD4 T cells can be replaced by soluble recombinant IL‐4. Collectively, the data show the existence of different requirements for priming of CD4 and CD8 T cells and point to the previously unappreciated fact that the induction of CD8 T cell responses by B lymphocytes requires T cell help.

Durable immunity and immunologic memory to a parasite antigen induced by somatic transgene immunization

Somatic transgene immunization (STI) is an alternative approach to immunization mediated by inoculation of plasmid DNA. In the experiments presented here we show that inoculation of plasmid DNA carrying an immunoglobulin heavy chain gene under the control of tissue-specific regulatory elements, leads to immunity and persistent immunologic memory against a peptide epitope encoded in the third complementarity-determining region. The epitope consists in three repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP) and is the immunodominant B cell epitope expressed at the surface of Plasmodium falciparum malaria parasite. When inoculated directly in the spleen the plasmid DNA initiated a specific anti-NANP response which lasted for 2 years. During the initial phase of priming the anti-NANP response was higher than that induced by immunization with recombinant protein in immunologic adjuvants. The establishment of immunologic memory was probed by single booster injection at various times after priming. We found that STI induces persistent immunologic memory up to 2 years. The immunologic characteristics of this new model are examined with respect to the requirement for the induction of B cell memory.

Circumvention of MHC class II restriction by genetic immunization.

The fate of T cell responses to peptide-based vaccination is subject to constraints by the major histocompatibility complex (MHC), MHC restriction. Using as a model system of T and B cell epitopes from the circumsporozoite protein of Plasmodium falciparum malaria parasite, we show that vaccination by somatic transgene immunization readily primes Balb/c mice (H-2(d)) a strain previously reported to be non-responder to immunization with a synthetic peptide vaccine encompassing these epitopes. Following genetic vaccination Balb/c mice developed a primary T cell response comparable to that of the responder strain C57Bl/6 (H-2(b)). Following booster immunization on day 45 Balb/c mice responded with a typical T cell memory response. Priming induced the formation of specific antibodies, which rose sharply after booster immunization. These findings suggests that genetic immunization can circumvent MHC class II restriction.