New assay methods have enabled the design of experiments addressing different stages involved in immune-cell activation and the study of effects on signalling pathways, which may then lead to the characterization of causal relationships. Table 2.2 outlines some of the types of methods currently in use. Most investigations begin with a general assessment of how a nutrient or altered nutritional state affects the general parameters of the immune system, immune-cell subsets and function. Measurement of changes in frequency and number of circulating lymphocyte subpopulations in the course of observation or dietary intervention is now accepted as a useful and widely comparable procedure, but attention must be given to the issue of controls This analysis should include standardized performance of immunophenotyping, using correction for purity of the gating region, quantitative recovery of the cell type and positive identification of cellular subsets. For human studies, a complete blood count and differential are needed to quantify effects on absolute numbers of cells. Although there is frequently a limitation on blood to be drawn for nutritional studies, it is essential that the baseline evaluation includes parallel studies providing a complete blood count, haematological analysis of haemoglobin, haematocrit, etc., on an aliquot of the same specimen of blood.
Table 2.2. Assessment of functional immune response.
Determinant of specificity Principle
Early activation event
Proliferation: magnitude of cellular response to signal
Cytokine response Cytokine pattern
Immune-cell subsets Antigen-specific cells
Antibody secretion Cytotoxicity
Response to signal
Specific cytokine Th1/Th2 response to signal
Specific and nonspecific target-cell killing
Signal, identity of responder cell
Signal, cell population, culture conditions
Reagent specificity, identity of producer cell
Monoclonal antibody and accuracy of the gating strategy
Specificity depends on signal and detection system
Antigen/antibody - may require antigenic stimulation Depends upon target and effector cell
Biochemical or monoclonal antibody assay or gene activation of responder cell
Radioisotopic tracer incorporation measures DNA synthesis after cell culture with activator; DNA-binding dyes
ELISA and ELISPOT use antibody/antigen; intracellular cytokine use monoclonal antibodies
Monoclonal antibodies coupled to fluorochromes as enzyme-labelled antibody reaction, specific activation
Recombinant antigens, monoclonal antibody, limiting-dilution methods Specificity of target-cell killing, relative strength/restriction measured
Flow-cytometric assay of CD69
Microtitre culture Whole blood
Mononuclear cells isolated by density gradient Purified cell populations ELISA: cytokine level ELISPOT can identify secreting-cell frequency
Intracellular detection can identify producer cell Flow cytometry
Detection of interferon secretion ELISPOT
Flow-cytometric detection of activated cell
ATP production by specific cells
ELISPOT Chromium release ELISPOT Flow cytometry
ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme linked immune spot; RIA, radioimmune assay.
In addition to assessment of relative percentages of T-cells, B-cells and NK cells, immunophenotyping for activation-antigen expression (e.g. CD69), coex-pression of critical molecules involved in cell-cell interaction (e.g. CD28), T-cell receptor (TCR) changes and percentages of naive and memory cells may be informative. Functional studies should be carried out on fresh anticoagulated blood whenever possible (or blood stored at room temperature in the dark for under 24 h) before mononuclear cells are isolated. When blood is being sent by air or transported to a distant laboratory, it is extremely important to include a control specimen drawn in parallel to serve as an internal standard for the shipping process. In addition, the type of tube chosen to draw the blood is important. Lithium heparin- or ethylenediamine tetra-acetic acid (EDTA)-containing tubes cannot be used for functional studies. Sodium heparin (preservative-free) or acid citrate dextrose (ACD) tubes should be used and consistency of tube type is important. There may be differences between venous and arterial blood. The question of when the blood should be drawn is important. In general, most data have been obtained with blood drawn in the morning and there are circadian effects on hormones and immune-cell phenotypes that may influence results. When this cannot be done, it is helpful to continue to maintain a uniformity of drawing time for an individual subject or group. Concurrent control blood must be drawn to ensure that technical performance standards are met. It is important that positive and negative (normal range and abnormal range) controls be included. Double-baseline studies - as a minimum, before and after intervention is undertaken - are recommended.
Studies of immune function usually start with a general assessment of mononuclear response in vitro to a mitogen, to another non-specific activator or to antigen, as discussed above. These methods are generally based on assay of cell division at the peak of response following microtitre plate culture for several days. Culture methods profoundly affect results, and conditions need to be optimized according to the kinetics of the response. Responses measured under most conditions favour T-cell proliferation, as the T-cell is the most prevalent lymphocyte in peripheral blood. The elicited composite response is highly quantitative when radioactive tracers - usually thymidine - are used. Recently, whole-blood methodology has been introduced as an alternative ex vivo method that can reflect potential response in vivo (Sottong et al., 2000); this method correlates with the level of DNA synthesis found when isolated mononuclear cells are cultured under optimal standard conditions. Comparative studies have also shown that there is a significant correlation between the whole-blood method and isolated mononuclear cells for cytokine production (Yaqoob et al., 1999). Some laboratories have replaced thymidine incorporation assays with a combination of cell-surface marker-induction assays and a measurement of the percentage of cells in various phases of the cell cycle following activation. Dyes have been developed that stably integrate into the membranes of live lymphocytes, such that, with each successive division, the amount of dye per cell is decreased. Fluorescence can be used to measure the number of cell divisions. Other assays based on whole blood measure early responses of cells selected through adherence to magnetic beads to which monoclonal antibodies recognizing cells of particular interest are attached. Assessment is achieved by an assay of adenosine triphosphate (ATP) production by the luciferin/luciferase reaction (Sottong et al., 2000). Assays such as this may provide accurate assessment of in vivo response in vitro. This method may be combined with a quantitative measure of specific lymphocyte subsets by flow cytometry for examination of response per cell.
Other approaches use measurement of cytokine response, receptor up-regulation or activation antigen to assess initial immune response, rather than the secondary response of cells recruited in the amplified reaction. Also, in vivo regulation of the immune response can be assessed through evaluation of unstimu-lated levels of secreted products when the producer-cell source of these products and normal levels are known. Methods measuring early events in T lymphocyte activation may or may not correlate with cell division, since cell division is only one aspect of the immune response. One of the earliest events that occurs following T-cell activation is the rapid increase in intracellular free calcium. This is followed by a change in pH and changes in the membrane potential. All of these effects can be measured by flow cytometry, using functional probes. Following T-cell activation via CD3/TCR or via CD2 (the alternate T-cell activation pathway), the first measurable surface marker induced is CD69. This marker is a disul-phide-linked homodimer that is present on 20-30% of normal thymocytes, but which is not expressed on resting peripheral-blood lymphocytes. CD69 reaches peak levels within 18-24 h and declines if the stimulus is removed. Using flow cytometry, it is possible to measure increase in CD69 expression on specific lymphocyte subsets. It is apparent that CD69 induction is not part of the pathway leading to cell division, as induction of CD69 can occur without subsequent cell proliferation. A good way to measure CD69 expression is to consider the relative expression of this marker on the subpopulation of interest, as this removes the confounding effect of subpopulation size. Other cell-surface markers appear on activated T-cells at variable times following activation, including CD25 (the a chain of the interleukin-2 (IL-2) receptor) and the transferrin receptor CD71 (both within 24-48 h) and human leucocyte antigen (HLA)-DR (after 48 h).
Finally, statistical evaluation is crucial to all of the studies described here. This includes evaluation of both the internal and the study-group controls. Studies of certain types may be suitable for the collection and banking of specimens prior to assay, such as cytokine supernatants. This may be helpful in giving a homogeneous data set with a low coefficient of variation, as long as controls and experimental specimens are run simultaneously. Good design is often based on internal cross-checks, which can be developed from the working hypothesis and which allow for different elements in the same pathway to be considered.
In summary, the emerging field of nutritional immunology has benefited from the evolution of cellular and molecular immunology. New approaches have provided a strong foundation for experimental design and offer a choice of analytical methods for approaching hypothesis testing. The key to any specific investigation is the identification of clear questions and the choice of relevant and practical methods. These methods then need to be tested in a pilot study, before launching the investigation. The use of an integrated design, including biostatistical considerations and complementary assays, is important in the development of meaningful data and of critical knowledge.
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