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Although laboratory indices, such as drug levels, are in routine use for monitoring immunosuppression, these do not always correlate well with an individual's risk of toxicity. In practice individual organs are monitored for toxicity with a combination of laboratory and clinical methods. These methods are limited by the fact that one has to await compromise before making therapeutic changes and there are often idiosyncrasies in the way individuals respond. Ocular complications can be sight threatening and many have proposed routine ocular monitoring in immunosuppressed patients. This study prospectively monitored a cohort of patients receiving high levels of immunosuppression for the prevention of rejection of heart, lung and heart-lung transplants for the development of ocular complications and to assess ocular morbidity. Specific surrogate markers that gave more information about the level of immunosuppression in a particular patient would improve patient care. Cytokines have the advantage of being directly generated by the immune response and offer promise as surrogate markers. Various, and not always consistent, cytokine profiles have been described for a number of conditions. Flow cytometry with intracellular cytokine staining allows quantification of the amount of cytokine present. These studies used this technique to describe the cytokine profile and changes in cytokine profile seen in patients during treatment for autoimmune uveitis and in patients with human immunodeficiency virus (HIV) receiving combination anti-retroviral therapy (ART). These studies emphasized the importance of prompt and careful clinical examination in the presence of clinical symptoms but did not support routine screening of patients on high levels of immunosuppression. Accurate measurements of interleukin-2 (IL-2) and interferon-gamma (IFNgamma) in patients with uveitis did not correlate well with disease activity. In contrast patients with different HIV profiles did show measurably different Thl cytokine expression providing information on the T cell deficits that persist despite treatment with ART.

T lymphocyte numbers in the human body are kept constant by homeostatic mechanisms balancing cell gain and loss. These mechanisms eventually fail in HIV infection, which is characterized by progressive immune deficiency attributable to a slow but relentless depletion of CD4+ T cells, the main viral targets. HIV infection is also associated with increased T cell turnover and a state of generalized immune activation. One of the fundamental questions in the field of HIV research is the relation between CD4+ T cell depletion and immune activation. It has been suggested that the virus has a direct effect by killing CD4+ T cells and increased T cell turnover reflects a homeostatic response to CD4+ T cell depletion. Alternatively, chronic immune activation may lead to enhanced turnover of T cells by ongoing proliferation-differentiation and cell death. In both cases, AIDS is the result of an exhaustion of the regenerative capacity of the immune system. To address these questions we examined the consequences of activated CD4+ T cell killing in a virus-free mouse model. Immunodeficiency viruses are highly selective for activated/memory and regulatory CD4+ T cells due to restricted expression of CCR5, the co-receptor for HIV and SIV, or CD134 (OX40, TNFRSF4), the cellular receptor for FIV. Activated CD4+ T cells were depleted by conditional reactivation of diphtheria toxin gene mediated by Tnfrsf4-driven Cre recombinase expression. Conditional ablation of activated CD4+ T cells resulted in accelerated turnover, with only a minimal apparent effect on their numbers, and was associated with a reduction in CD4:CD8 ratio and development of CD4+ T cell immune deficiency, resembling HIV infection. Importantly, activated CD4+ T cell killing also resulted in generalized immune activation, including lymph node enlargement, B cell expansion, elevated serum levels of proinflammatory cytokines and increased turnover and activation of CD8+ T cells, characteristic of HIV infection. CD8+ T cell activation correlated with lack of regulatory CD4+ T cell function and was prevented upon regulatory CD4+ T cell reconstitution. We therefore propose a causal link between memory and regulatory T cell depletion and immune deficiency and immune activation, respectively.

Immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical SCT, but infection and leukaemic relapse remain problematic. To develop a rational approach to refining allodepletion, we characterized the expression of surface markers and cytokines on proliferating alloreactive T-cells flow cytometrically. CD25 was expressed on 83 % of CFSE-dim alloreactive T-cells, confirming this as an excellent target for allodepletion. 70 % of the alloreactive CD25-ve population expressed CD71, identifying this as a novel marker to target alloreactive T-cells that persist after CD25 depletion. We compared residual alloreactivity to host or 3rd party after CD25 vs combined CD25/71 immunomagnetic depletion in 8 HLA-mismatched donor-recipient pairs. In 1o MLRs, residual responses to host were undetectable after CD25/71 depletion. In 2o MLRs, CD25/71 depletion resulted in significantly lower residual proliferative response to host than CD25 depletion (median 4.8% of the response of unmanipulated PBMC vs 9.9%, p < 0.01). Likewise, the median residual reactivity to host in IFN-γ ELISPOT assays was significantly lower after combined CD25/71 than CD25 allodepletion (14.1 % vs 54.6%, p < 0.05). Third party responses after CD25/71 allodepletion were equivalent to unmanipulated PBMCs in both assays. In pentamer and IFN-γ ELISPOT assays, anti-viral responses to CMV, EBV and adenovirus were preserved after combined CD25/71 allodepletion. Finally, we showed that CD25/71 allodepleted T-cells can be redirected to recognize and secrete IFN-γ and granzyme B in response to CD19 cell lines and primary ALL blasts through lentiviral transfer of a chimeric αCD19ζ TCR. This strategy may facilitate immunotherapy with larger doses of allodepleted T-cells after haplo-SCT, enhancing graft versus leukaemia and anti-viral effects.

This work investigated the effects of ageing on the cutaneous response to the injection of bacterial, fungal and viral antigens in humans. Previous studies have shown reduced cutaneous delayed type hypersensitivity (DTH) responses in the old and it has been assumed that this is due to an overall decline in cell-mediated immunity that occurs with ageing. However, it has remained unclear as to whether DTH responses in old skin are truly reflective of immune responses in all compartments of the immune system. Reduced clinical responsiveness in the skin to the intradermal injection of antigen was identified despite good peripheral T cell responses in the old and it was hypothesised that this may be reflective of a skin-specific defect that occurs during ageing. Possible mechanisms for this were investigated by performing both skin suction blisters and skin biopsies following injection of antigen into the skin. In the old there was a significant reduction in the infiltration of CD4+ lymphocytes in the skin that correlated with a reduced clinical response. A reduction in CXCR3 expression was found on CLA+CD4+ T cells isolated from the peripheral blood in old subjects. In addition, a reduction in the levels of cutaneous pro-inflammatory mediators was identified. This was associated with reduced CD4+ T cell activation and proliferation in situ as determined by CD69 and Ki67 expression on lymphocytes isolated directly ex vivo from the skin. A defect in initiation of the DTH response was considered. Although a possible decrease in the numbers of CD14+ monocytes was identified, no difference in monocyte activation or the numbers of CD1a or DC-SIGN positive dendritic cells was found in the old. The data suggests that both decreased cell migration in to the skin in addition to decreased expansion of infiltrating cells contributes to the defective cutaneous response to antigenic challenge during ageing.