Ludmila Müller

Prediction of an HLA-DR-binding peptide derived from Wilms' tumour 1 protein and demonstration of in vitro immunogenicity of WT1(124-138)-pulsed dendritic cells generated according to an optimised protocol.

The Wilms’ tumour 1 (WT1) protein is over-expressed in several types of cancer including leukaemias and might therefore constitute a novel target for immunotherapy. Recently, human leucocyte antigen (HLA) class I-binding WT1 peptides have been identified and shown to stimulate CD8+ T cells in vitro. For maximal CD8 cell efficacy, CD4+ helper T cells responding to major histocompatibility complex (MHC) class II-binding epitopes are required. Here, we report that scanning the WT1 protein sequence using an evidence-based predictive computer algorithm (SYFPEITHI) yielded a peptide WT1(124–138) predicted to bind the HLA-DRB1*0401 molecule with high affinity. Moreover, synthetic WT1(124–138)-peptide-pulsed dendritic cells (DC), generated according to a protocol optimised in the present study, sensitised T cells in vitro to proliferate and secrete interferon-γ (IFN-γ) when rechallenged with specific peptide-pulsed DC, but not with peripheral blood mononuclear cells (PBMC). These results suggest that the WT1 protein may yield epitopes immunogenic to CD4 as well as CD8 T cells, and therefore constitute a novel potential target for specific immunotherapy.

Immunosenescence in vertebrates and invertebrates.

There is an established consensus that it is primarily the adaptive arm of immunity, and the T cell subset in particular, that is most susceptible to the deleterious changes with age known as “immunosenescence”. Can we garner any clues as to why this might be by considering comparative immunology and the evolutionary emergence of adaptive and innate immunity? The immune system is assumed to have evolved to protect the organism against pathogens, but the way in which this is accomplished is different in the innate-vs-adaptive arms, and it is unclear why the latter is necessary. Are there special characteristics of adaptive immunity which might make the system more susceptible to age-associated dysfunction? Given recent accumulating findings that actually there are age-associated changes to innate immunity and that these are broadly similar in vertebrates and invertebrates, we suggest here that it is the special property of memory in the adaptive immune system which results in the accumulation of cells with a restricted receptor repertoire, dependent on the immunological history of the individual’s exposures to pathogens over the lifetime, and which is commonly taken as a hallmark of “immunosenescence”. However, we further hypothesize that this immunological remodelling per se does not necessarily convey a disadvantage to the individual (ie. is not necessarily “senescence” if it is not deleterious). Indeed, under certain circumstances, or potentially even as a rule, this adaptation to the individual host environment may confer an actual survival advantage.

Contribution of neuroinflammation and immunity to brain aging and the mitigating effects of physical and cognitive interventions

It is widely accepted that the brain and the immune system continuously interact during normal as well as pathological functioning. Human aging is commonly accompanied by low-grade inflammation in both the immune and central nervous systems, thought to contribute to many age-related diseases. This review of the current literature focuses first on the normal neuroimmune interactions occurring in the brain, which promote learning, memory and neuroplasticity. Further, we discuss the protective and dynamic role of barriers to neuroimmune interactions, which have become clearer with the recent discovery of the meningeal lymphatic system. Next, we consider age-related changes of the immune system and possible deleterious influences of immunosenescence and low-grade inflammation (inflammaging) on neurodegenerative processes in the normally aging brain. We survey the major immunomodulators and neuroregulators in the aging brain and their highly tuned dynamic and reciprocal interactions. Finally, we consider our current understanding of how physical activity, as well as a combination of physical and cognitive interventions, may mediate anti-inflammatory effects and thus positively impact brain aging.

As we age: Does slippage of quality control in the immune system lead to collateral damage?

The vertebrate adaptive immune system is remarkable for its possession of a very broad range of antigen receptors imbuing the system with exquisite specificity, in addition to the phagocytic and inflammatory cells of the innate system shared with invertebrates. This system requires strict control both at the level of the generation the cells carrying these receptors and at the level of their activation and effector function mediation in order to avoid autoimmunity and mitigate immune pathology. Thus, quality control checkpoints are built into the system at multiple nodes in the response, relying on clonal selection and regulatory networks to maximize pathogen-directed effects and minimize collateral tissue damage. However, these checkpoints are compromised with age, resulting in poorer immune control manifesting as tissue-damaging autoimmune and inflammatory phenomena which can cause widespread systemic disease, paradoxically compounding the problems associated with increased susceptibility to infectious disease and possibly cancer in the elderly. Better understanding the reasons for slippage of immune control will pave the way for developing rational strategies for interventions to maintain appropriate immunity while reducing immunopathology.

Cytokines and Antitumor Immunity

Currently, the notion of immunosurveillance against tumors is enjoying something of a renaissance. Even if we still refuse to accept that tumors arising in the normal host are unable to trigger an immune response because of the lack of initiation (“danger”) signals, there is no doubt that the immune system can be manipulated experimentally and by implication therapeutically to exert anti-tumor effects. For this activity to be successful, the appropriate cytokine milieu has to be provided, making cytokine manipulation central to immunotherapy. On the other hand, the major hurdle currently preventing successful immunotherapy is the ability of tumors to evolve resistant variants under the pressure of immune selection. Here, too, the cytokine milieu plays an essential role. The purpose of this brief review is to consider the current status of the application of cytokines in facilitating antitumor immunity, as well their role in inhibiting responses to tumors. Clearly, encouraging the former but preventing the latter will be the key to the effective clinical application of cancer immunotherapy.

Chronic phase CML patients possess T cells capable of recognising autologous tumour cells.

Much circumstantial evidence points to the immunogenicity of chronic myloid leukemia (CML) cells, most impressively the well-established T cell-dependent GvL effect seen in bone marrow transplantation. However, only a small number of shared antigens expressed by CML cells have been identified as potential targets for T cell-mediated immune responses which might be exploited for immunotherapy. It may be that unique antigens expressed by individual tumours are more potent rejection antigens if the patients own T cells could be encouraged to react against them. Work is reviewed here which documents that in vitro mixed cultures between autologous T cells and dendritic cells of chronic-phase CML patients can give rise to sensitised T cells capable of recognising the patients tumour cells. Additionally, mixed autologous tumour cell/lymphocyte cultures, modified by the addition of cytokine cocktails, may also result in the generation of similarly sensitised T cells. These results could be exploited for adoptive immunotherapy, and possibly, after identification of the antigens recognised, also for active immunotherapy, i.e. including therapeutic vaccination.

Generation of chronic myelogenous leukemia-specific T cells in cytokine-modified autologous mixed lymphocyte/tumor cell cultures.

Chronic myelogenous leukemia (CML) may be amenable to cell-based adoptive immunotherapy, as suggested by the graft-versus-leukemia effect of bone marrow transplantation and the therapeutic benefit of donor leukocyte infusions. Specific adoptive immunotherapy without bone marrow transplantation might be more effective and less cost-intensive. Professional antigen-presenting cells, the dendritic cells, from patients with CML are derived from the malignant clone and may stimulate antileukemia T-cell responses. Autologous T cells may also be able to recognize tumor antigens on CML cells directly. Here, the authors show that CD4 and CD8 T-cell responses to autologous CML cells can be generated in vitro rapidly and effectively by performing modified autologous mixed lymphocyte/tumor cell cultures (MLTC) in serum-free medium in the presence of cytokines known to support dendritic cell differentiation. MLTC-sensitized T cells secreted large amounts of the type 1 cytokine interferon-&ggr;, as well as interleukin (IL)-2. However, they also secreted a variety of other cytokines, including the type 2-subtype cytokine IL-13 but not the classic type 2 cytokines IL-4, IL-5, and IL-10. Monoclonal populations of CML-specific CD4 cells could be derived from these lines in limited numbers but showed markedly enhanced reactivity. This suggests that CML-specific T cells are relatively rare in these autologous MTLC-derived sensitized populations, but that their isolation and propagation would yield much more potent antitumor effector cells for use in adoptive immunotherapy without the need for bone marrow transplantation.

Progress in vaccination against cancer (PIVAC) 2001, Robinson College, University of Cambridge, UK

The prominence of dendritic cell (DC)-based approaches to vaccination was clearly apparent from the results of both experimental murine and human studies. The maturation of DCs, several sub-types of which are now known, determines whether they interact efficiently with effector and regulatory T lymphocytes (G. Adema, Nijmegen, the Netherlands; S. McArdle, Nottingham, UK; L. Müller, Tübingen, Germany; D. Schadendorf, Heidelberg, Germany). Immature DCs take up antigen efficiently and in this state may induce regulatory T cells, in contrast to fully matured DCs which preferentially activate CD4 helper and CD8 cytolytic (CTL) T cells. DCs present in the germinal centres of lymph nodes attract naive (CD45RA, RO) T cells and possibly B lymphocytes with a preference for naive B cells through the production of large amounts (30–50 fg/d per DC) of a chemokine, DC-CK1 (G. Adema, Nijmegen, the Netherlands); however, the precise role of this sub-population in cancer therapy and/or prophylaxis has not yet been established. DC-CK1 is expressed both in T cell regions as well as in B cell follicles of the secondary lymphoid organs. Investigation of the number of DC-CK1-expressing cells in the germinal centres in tonsils has shown that DC-CK1 expression is dependent on the activation status of the germinal centres. DC-based vaccination is largely undertaken with mature DCs pulsed with tumour lysate or peptides, and in a European study of 32 melanoma patients evidence of clinical tumour regression was clearly apparent (three complete remissions [CR] and five partial remissions [PR]), with almost 50% of patients showing stable disease status or regression (D. Schadendorf, Heidelberg, Germany). This programme is currently being extended into a prospective randomised trial using a peptide cocktail for DC pulsing which includes both MHC class I-restricted CTL epitopes and several MHC class II-binding T helper cell epitopes. DCs can also take up apoptotic bodies and present tumour antigens to the immune system; some previously controversial results in this area might be reconciled by the findings of N. Labarrière (Nantes, France) who showed that the method chosen for inducing apoptosis (UV, butyrate, etc.) affects DCmaturation and function. Experimentally it can also be shown that increasing the number of mature DCs at the tumour site can result in enhanced tumour regression induced by immunotherapy (S. Ali, Nottingham, UK). Other vaccination approaches combining specific stimuli with inflammatory ‘danger’ signalling include the induction of lytic cell death induced by viral infection, or the administration of CpG oligonucleotides. Certain Cancer Immunol Immunother (2002) 51: 58–61 DOI 10.1007/s00262-001-0252-4

The Role of CMV in Immunosenescence

The term “immunosenescence” is commonly taken to mean age-associated changes in immune parameters hypothesized to contribute to increased susceptibility and severity of the older adult to infectious disease, autoimmunity and cancer. In humans, it is characterized by lower numbers and frequencies of naive T and B cells and higher numbers and frequencies of late-differentiated T cells, especially CD8+ T cells, in the peripheral blood. The latter may be very noticeable, but intriguingly, only in people infected by human herpesvirus 5 (Cytomegalovirus, CMV). Almost all human studies have been cross-sectional, thus documenting differences between old and young populations, but not necessarily changes over time. Nonetheless, limited longitudinal studies have provided data consistent with gradually decreasing naive T and B cells, and increasing late-differentiated T cells over time, and in rare instances associating these changes with increasing frailty and incipient mortality in the elderly. Low numbers of naive cells render the aged highly susceptible to pathogens to which they have not been previously exposed, but are not otherwise associated with an “immune risk profile” predicting earlier mortality. Whether the accumulations of late-differentiated T cells driven primarily by CMV contribute to frailty and mortality or are only adaptive responses to the persistent virus remains controversial. Either way, there is currently little direct evidence that “immunosenescence” contributes to either autoimmunity or cancer in the aged. This chapter reviews some of the studies implicating CMV infection in immunosenescence and its consequences for ageing trajectories in humans.

Introduction to ageing of the adaptive immune system

Like other somatic tissues and organs, the vertebrate immune system manifests age-associated alterations to its components and their functions. Unlike in invertebrates, in addition to the innate arm, vertebrates also possess adaptive immunity mediated by both cellular and humoral components. This chapter reviews data on age-associated alterations to adaptive immunity specifically in humans, mostly originating from cross-sectional studies (i.e., comparing young with old people). We summarise what is known about the effects of age on the different components of the adaptive immune system, particularly T cells, which appear most obviously different in the elderly. We consider the serious limitations inherent in cross-sectional studies, and discuss the crucial requirement to perform longitudinal studies (i.e., following the same individuals over time). Despite the logistical and financial constraints, longitudinal follow-up has provided the most biologically meaningful information about which of the many biomarkers apparently changing with age are actually relevant to medical parameters and for late-life health and longevity, and which, in contrast, may change with age but without clinical relevance. Given the lack of consistent data currently available, as a result of performing studies on heterogeneous populations using different analytical techniques, we emphasize the necessity for more numerous, more extensive and more detailed studies including assessments of the impact of psychosocial, nutritional and other thus-far rarely considered parameters on immunological and other biomarkers in longitudinal studies.