Immune responses in vitro can be effected as either primary or secondary responses. The former make use of leukocytes from naive (nonimmunized) animals, the latter from animals previously immunized with the antigen in question. Both types of response require the same basic components of lymphocytes, monocytes and intercellular communications, including cytokine activity (Table 1).

Cellular requirements

As with responses in vivo, B lymphocytes react directly with the antigen for which they are specific, but complete differentiation into antibody-producing plasma cells requires immunological help from helper T (Tn) lymphocytes (Figure 1). A few antigens can stimulate B lymphocytes independently of TH lymphocytes, but the majority of antigens are TH

lymphocyte dependent. Antigen-stimulated precursor cytotoxic T (Tc) lymphocytes also require immunological help from TH lymphocytes (Figure 1). In contrast to the B lymphocyte, both TH and T< lymphocytes require antigen to be processed and presented in peptide form associated with major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (Figures 1 and 2). When the peptide is associated with MHC class II, Tn lymphocytes can be stimulated; when associated with MHC class I, Tc lymphocytes can be stimulated (Figure 2). Monocytes, the related dendritic cells and, to a lesser degree, macrophages appear to be the main antigen-presenting cells. B lymphocytes can also present antigen, but apparently only to memory T lymphocytes; the monocytic antigen-presenting cells present to both naive and memory T lymphocytes. Accessory-cells, such as endothelial cells and fibroblasts, play important additional roles in intercellular signaling (Figure 3), and are important for maintaining viability in a culture.

Cytokine requirements

The cytokines of the in vitro immune response are linked to the TH lymphocytes and monocytes which produce them (Figure 1). Table 2 shows examples of those which have been most extensively studied and applied to in vitro immune responses. Whether exogenous cytokines are to be applied, or only the stimulation of the cytokine-producing cells within a culture, will depend on the type of in vitro immune response under study, and the objectives of the experiment. Nonetheless, it is the cognate interactions between helper cells and effector cells, or their precursors, which are essential for optimum immune responses in vitro.

Antigenic requirements

Antigen requirements in vitro will not necessarily relate to those in vivo, and are normally determined by experimentation. Immunogenicity in vivo is not a guarantee that an in vitro response can be stimulated. Generally, the more complex an antigen - in terms of structure and topography - the more efficient will be the induction of an immune response (see below under the topics of initiation of primary and secondary immune responses in vitro).

Table 1 The basic requirements for in vitro immune responses


Conventional sources

Monocytic APC

Dendritic cell APC B lymphocytes

T lymphocytes

Endothelial cells Fibroblast accessory cells Cytokines


Lymphoid organs, blood

Lymphoid organs, blood, skin Lymphoid organs, blood

Lymphoid organs, blood

Lymphoid organs, tissues/organs containing microvascular capillaries Lymphoid organs, lung, skin

Recombinant biotechnology, mitogen-actlvated mononuclear cell cultures

As required for the study

Antigen presentation; intercellular signaling;

endogenous cytokine production Antigen presentation; intercellular signaling Antigen-specific antibody production; antigen presentation to memory TH lymphocytes (secondary immune responses) Antigen-specific immunological help (TH lymphocytes, via their cytokine production); endogenous cytokine production (TH lymphocytes); antigen-specific cytotoxicity (Tc lymphocytes) Intercellular signaling; endogenous cytokine production

Intercellular signaling; endogenous cytokine production

Lymphocyte proliferation and differentiation; antigen processing (activity depends on the cytokine or mixture of cytokines) Stimulation of the lymphocyte proliferation, either directly (for B lymphocytes) or indirectly after processing and presentation by APC (for T lymphocytes)

APC, antigen-presenting cell.

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