Julie D. McLeod

'Danger Theory: The Link between AIS and IDS?'

We present ideas about creating a next generation Intrusion Detection System (IDS) based on the latest immunological theories. The central challenge with computer security is determining the difference between normal and potentially harmful activity. For half a century, developers have protected their systems by coding rules that identify and block specific events. However, the nature of current and future threats in conjunction with ever larger IT systems urgently requires the development of automated and adaptive defensive tools. A promising solution is emerging in the form of Artificial Immune Systems (AIS): The Human Immune System (HIS) can detect and defend against harmful and previously unseen invaders, so can we not build a similar Intrusion Detection System (IDS) for our computers? Presumably, those systems would then have the same beneficial properties as HIS like error tolerance, adaptation and self-monitoring. Current AIS have been successful on test systems, but the algorithms rely on self-nonself discrimination, as stipulated in classical immunology. However, immunologist are increasingly finding fault with traditional self-nonself thinking and a new ‘Danger Theory’ (DT) is emerging. This new theory suggests that the immune system reacts to threats based on the correlation of various (danger) signals and it provides a method of ‘grounding’ the immune response, i.e. linking it directly to the attacker. Little is currently understood of the precise nature and correlation of these signals and the theory is a topic of hot debate. It is the aim of this research to investigate this correlation and to translate the DT into the realms of computer security, thereby creating AIS that are no longer limited by self-nonself discrimination. It should be noted that we do not intend to defend this controversial theory per se, although as a deliverable this project will add to the body of knowledge in this area. Rather we are interested in its merits for scaling up AIS applications by overcoming self-nonself discrimination problems.

Apoptotic cells induce dendritic cell‐mediated suppression via interferon‐γ‐induced IDO

Dendritic cells (DC) are sensitive to their local environment and are affected by proximal cell death. This study investigated the modulatory effect of cell death on DC function. Monocyte‐derived DC exposed to apoptotic Jurkat or primary T cells failed to induce phenotypic maturation of the DC and were unable to support CD4+ allogeneic T‐cell proliferation compared with DC exposed to lipopolysaccharide (LPS) or necrotic cells. Apoptotic cells coincubated with LPS‐ or necrotic cell‐induced mature DC significantly suppressed CD80, CD86 and CD83 and attenuated LPS‐induced CD4+ T‐cell proliferation. Reduced levels of interleukin‐12 (IL‐12), IL‐10, IL‐6, tumour necrosis factor‐α and interferon‐γ (IFN‐γ) were found to be concomitant with the suppressive activity of apoptotic cells upon DC. Furthermore, intracellular staining confirmed IFN‐γ expression by DC in association with apoptotic environments. The specific generation of IFN‐γ by DC within apoptotic environments is suggestive of an anti‐inflammatory role by the induction of indoleamine 2,3‐dioxygenase (IDO). Both neutralization of IFN‐γ and IDO blockade demonstrated a role for IFN‐γ and IDO in the suppression of CD4+ T cells. Moreover, we demonstrate that IDO expression within the DC was found to be IFN‐γ‐dependent. Blocking transforming growth factor‐β (TGF‐β) also produced a partial release in T‐cell proliferation. Our study strongly suggests that apoptosis‐induced DC suppression is not an immunological null event and two prime mediators underpinning these functional effects are IFN‐γ‐induced IDO and TGF‐β.

Cooperative automated worm response and detection immune ALgorithm(CARDINAL) inspired by t-cell immunity and tolerance

The role of T-cells within the immune system is to confirm and assess anomalous situations and then either respond to or tolerate the source of the effect. To illustrate how these mechanisms can be harnessed to solve real-world problems, we present the blueprint of a T-cell inspired algorithm for computer security worm detection. We show how the three central T-cell processes, namely T-cell maturation, differentiation and proliferation, naturally map into this domain and further illustrate how such an algorithm fits into a complete immune inspired computer security system and framework.

Age associated decline in CD25 and CD28 expression correlate with an increased susceptibility to CD95 mediated apoptosis in T cells

Immunosenescence is believed to contribute to increase susceptibility to infectious diseases and cancer in the elderly, and is caused mainly by changes in the T cell compartment. Longitudinal studies were undertaken to examine T cell surface receptor expression and apoptotic susceptibility using Staphylococcal enterotoxin B (SEB) activated human T cells as an in vitro model of an ageing T cell culture. An intracellular stain Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to assess the number of population divisions (PD) occurring in the ageing T cell culture. One major biomarker of aged T cells is a decrease in expression of CD28 and since this is an essential co-stimulatory molecule, its decreasing expression with age could compromise their activation and apoptotic capacity. Activation of T cells resulted in initial up-regulation of CD25, CD95 and CD28, although expression of CD25 and CD28 subsequently decreased with increasing PD. CD4 and CD8 T cells expressed similar CD25 profiles although CD28 expression was unique in each subset. CD4+ cells expressed the highest CD28 levels, and showed a gradual decline in expression with increasing PD, whereas CD8+ cells were low CD28 expressers, but did not appear to lose their expression as they aged. To determine T cell susceptibility to apoptosis via CD95/CD95-L interactions with increasing age, cells were challenged with CD95-L transfected CHO cells at various PD. Increased death was observed as they aged, which correlated with the decreased expression of activation markers CD25 and CD28.

Nociceptin/orphanin FQ modulates human T cell function in vitro

Although nociceptin/orphanin FQ (N/OFQ) and its receptor (ORL-1) are widely distributed throughout the immune system, its role has yet to be elucidated. This study shows that N/OFQ (10(-14)-10(-12) M) modulates T cell activation by up-regulating activation marker expression, e.g. CD28, leading to enhanced proliferation and modulation of TNFalpha secretion. However, on re-stimulated T cells N/OFQ causes inhibition of proliferation, which could be linked with N/OFQ up-regulating CTLA-4 expression. We have also shown that some of these effects are partly prostaglandin-dependent and that N/OFQ induces prostaglandin synthesis. This report suggests that N/OFQ could exert a key modulatory role in human T cell functions.

Apoptotic capability in ageing T cells

The ageing immune system shows a gradual decline in responsiveness to antigens and tumours due to the emergence of immunosenescence. The main functions of T cells are activation, anergy and apoptosis and these are all affected during ageing. Apoptosis is vital in controlling cell numbers, deleting self-reactive T cells and maintaining immune surveillance. One of the principle instigators of death involves the CD95:CD95-ligand interaction and as T cells age both receptor and ligand levels increase. This view will describe the current knowledge of the apoptotic susceptibility of ageing T cells and evaluate the factors that may affect the apoptotic capability of immunosenescent T cells.

Dendritic cell migration assay: A potential prediction model for identification of contact allergens

This manuscript describes methodology and a prediction model for the MUTZ-LC migration assay. The assay represents the physiological change in Langerhans cell (LC) behavior after exposure to a sensitizing chemical, resulting in LC migration from the epidermis to the dermis. MUTZ-LC are derived from the commercially available MUTZ-3 cell line. Upon exposure to a sensitizer MUTZ-LC migrate preferentially towards CXCL12 whereas upon exposure to a non-sensitizer MUTZ-LC migrate towards CCL5. A CXCL12/CCL5 ratio >1.10 in 2/3 independent experiments is indicative of a sensitizer, whereas a CXCL12/CCL5 ratio ≤1.10 is indicative of a non-sensitizer. At non cytotoxic chemical concentrations 9 sensitizers (2,4-dinitrochlorobenzene, paraphenylendiamine, cinnamaldehyde, isoeugenol, nickel-sulfate, tetramethylthiuram disulfide, eugenol, cinnamic-alcohol, ammonium-hexachloroplatinate) were distinguished from 4 non sensitizers (sodium lauryl sulfate, salicylic acid, phenol, octanoic acid). Critical points in assay performance are (i) MUTZ-3 passage number after thawing (p6-p40); (ii) cell viability (>80%); (iii) standard curve to optimize correlation of fluorescence with cell number; and (iv) optimization of the concentration of rhCXCL12 and rhCCL5 in transwell. The protocol has been tested in three European laboratories and results suggest that it may provide working conditions for performing the DC migration assay which is aimed at distinguishing sensitizers from non sensitizers.

Inter-laboratory study of the in vitro dendritic cell migration assay for identification of contact allergens

The aim of this study was to investigate the transferability of technology and reproducibility of MUTZ-3 derived Langerhans Cell (MUTZ-LC) migration assay. The protocol was transferred from the NL-lab to two Sens-it-iv project partners (UK-lab, Italy-lab). Intra- and inter-laboratory variation with regards to MUTZ-3 progenitor culture, differentiation to MUTZ-LC, maturation and migration assay were investigated. In the transwell-migration-assay, preferential migration of sensitizer-exposed MUTZ-LC towards CXCL12 was observed (three sensitizers), whereas non-sensitizer-exposed MUTZ-LC only migrated towards CCL5 (two non-sensitizers). Four pre-pro-haptens were also identified by UK-lab. When taking the arbitrary criteria of at least two of three independent repetitions per laboratory having to have a CXCL12/CCL5 ratio>1.1 for classification as a sensitizer, all sensitizers tested in all labs were easily distinguished from all non-sensitizers. The number of repetitions giving false negative or false positive was very low (only 7 out of a total of 54 repetitions), indicating that both intra- and inter-laboratory variation was extremely low. Even though only a few chemicals were tested in this study, we show clearly that the in vitro DC migration assay is transferable between laboratories. The results were consistent between the laboratories, and the dose response data were reproduced in the three laboratories.

Nociceptin-induced modulation of human T cell function.

There is an accumulating evidence for the immunoregulatory role of the neuropeptide, nociceptin/orphanin FQ (N/OFQ) however its role on T cell function requires elucidation. This study has demonstrated an inhibitory role for N/OFQ on SEB-activated T cell function. N/OFQ decreases T cell proliferation, which is abrogated when the costimulatory receptors CD80 and CD86 are blocked. In addition, evidence suggests that the immunoregulatory cytokines TGF-beta, IFN-gamma and nitric oxide (NO) are involved in the N/OFQ effect. N/OFQ also, through involvement of IFN and NO, induces the expression of the immunosuppressive modulator indoleamine 2,3-dioxygenase (IDO), suggesting a central role for IDO in the N/OFQ effect on T cell proliferation. The data presented in this report indicate a multi-faceted mechanism of action used by N/OFQ to modulate T cell function.

Engineering Anticancer T Cells for Extended Functional Longevity

Abstract: Like other somatic cells, human T lymphocytes have a finite replicative capacity in vitro, and, by implication and consistent with the limited data available, in vivo as well. An accumulation of dysfunctional T cells may be detrimental under conditions of chronic antigenic stress (chronic infection, cancer, autoimmunity). Using T cells from young donors to model the process of T cell clonal expansion in vitro under these conditions reveals age‐associated increasing levels of oxidative DNA damage and microsatellite instability (MSI), coupled with decreasing DNA repair capacity, telomerase induction and telomere length, decreased levels of expression of the T cell costimulator CD28 and consequently reduced secretion of the T cell growth factor interleukin‐2 (IL‐2). However, data from similar experiments using T cell clones (TCCs) derived from extremely healthy very elderly donors (“successfully aged”) indicate that DNA repair is better maintained, MSI less prevalent, and (already short) telomere lengths are maintained. Nonetheless, oxidative DNA damage is seen to the same extent, and clonal longevity is also similar in these clones. DNA damage levels are reduced by culture in 5% oxygen, but longevity is not improved. This may be because of the requirement for intermittent reactivation via receptor pathways dependent on free radical production in T cells. These recent findings from our international immunosenescence research consortium suggest that strategies other than telomere maintenance, better protection against free radicals, or improved DNA repair will be required for functional longevity extension of human TCCs. To obtain sufficient cells for adoptive immunotherapy of cancer, alternative avenues need exploration; currently, these include enforced expression of certain heat shock proteins and proteasome components, and interference with the expression of negative regulatory receptors expressed by T cells.