Ob

Mononuclear eel culturo r coB ┬╗nrtchod

Enriched tor T or B lymphocytes

7-10 days Anlfcody incjisuremcnt

3-4 days

ProMeratlon measurement.1 hybndoma generation

Figure 7 The secondary immune response system in vitro, showing that antigen preprocessing by APCs may be employed, but is not essential. The mononuclear cell preparations can be from lymphoid organs or blood, and enrichment of particular populations may also be employed.

Antigen processing and presentation

Despite the more efficient capacity of lymphocytes to respond during a secondary immune response in vitro, preactivation of T lymphocytes is a commonly employed procedure. Cultures may simply be mononuclear cells, or alternatively enriched populations. With enrichment, a source of APCs must be ensured: irradiated mononuclear cells or enriched dendritic cells can be employed (Figure 7), although the capacity of memory B lymphocytes to function as APCs should also be considered. In experiments employing enrichment, antigen is often applied 3-6 days before B lymphocytes are added (Figure 7); fresh antigen may be unnecessary, dependent on the antigen and characteristics of the memory lymphocytes in the culture. When nonenriched mononuclear cell cultures are employed, antigen is added at the initiation of the experiment (Figure 7). Measurement of secondary immune responses in vitro often employs T lymphocyte proliferation or antibody production by activated B lymphocytes (Figure 7).

Choice and use of antigen

Secondary immune responses in vitro have been applied to studies employing antigens of, for example, viruses, bacteria and peptides of known sequence or structure. The applicability of the procedures is evident from the studies on peptide antigens, which have been applied to analyses of B lymphocyte and, in particular, T lymphocyte responses. Not all peptide antigens are equally immunogenic. Certain may require costimulation with interleukin 2 (IL-2), followed by restimulation with the peptide antigen before lymphocyte proliferation is detectable. Similar restrictions may apply to certain more complex antigens, but will depend on both the antigen and the capacity of the constituent mononuclear cells in an in vitro culture to respond.

Additional applications particular to secondary immune responses in vitro

Secondary immune responses in vitro have also been applied to studies on intercellular communications.

Cultures of mononuclear cells have been employed for analyses of lymphocyte/monocyte and lymphocyte/lymphocyte interactions. Cocultures of mononuclear and endothelial cells are often used to study the role of the endothelial cells. Both types of culture, as well as those with enriched subpopulations or cell lines, have assisted in the identification of adhesion molecule and cytokine activity. Cultures employing enriched subpopulations have been particularly useful in identifying the roles of these subpopulations in the immune response, especially with respect to intercellular communications and interactions.

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