CD69 expression after antigenic stimulation

Abstract

We read with interest the paper by Maino et al. (1) which evaluated whether the expression of CD69 on lymphocytes might replace standard tritiated thymidine incorporation as a measure of lymphocyte activation and proliferation (LPA). We have used LPA as a screen after vaccination to determine whether further T cell studies (such as cytokine evaluation) are warranted, and were enthused by the possibility that an equivalent to proliferation could potentially be found as early as 4-6 h after obtaining whole blood or after thawing frozen peripheral blood mononuclear cells (PBMC). Since the majority of the results in that paper were obtained using mitogens, we carried out experiments to determine whether such rapid indicators of activation could be obtained in antigen-specific systems.We evaluated the response of PBMC (fresh and frozen) from 5 normal volunteers to antigens (candida 20 pg/ml, tetanus toxoid 5 flocculation units (LFU)/ml, and herpes simplex 10 pg/ml), as well as mitogens [phytohemagglutinin (PHA) 5 pg/ml, pokeweed mitogen 2.5 pg/ml, and concanavalin A 5 pg/ml]. Staining procedures followed the recommendations of the manufacturer. Equivalent percent rises in CD69+ values (corrected for unstimulated background fluorescence) were obtained whether the analysis included all acquired events or a lymphocyte subset defined by a light scatter gate.We confirmed the results of Maino et al. (l), observing that PHA was able to induce CD69 responses reproducibly within 30 min after addition to the cells, and other mitogens induced CD69 responses within 4 h, as previously published (Fig. 1 ). However, despite stimulating PBMC from five individuals with antigens which were shown to give excellent proliferation in simultaneous LPA experiments, no early (4-6 h) CD69 expression was seen. By extending the stimulation to 3 d, reproducible CD69 expression was seen in samples that also showed high proliferation (Fig. 1 ). Maximal CD69 expression was observed at 6 d for four different antigens in repeat experiments.Thus, the measurement of CD69 expression by flow cytometric methods could provide useful information regarding the cellular subsets which are activated by antigens or mitogens, and would avoid the radioactive waste generated by LPA. However, the rapid response seen with PHA and other mitogens does not appear evident in the context of antigen-specific stimulation.

Gamma radiation induces CD69 expression on lymphocytes

“…Enhanced CD69 expression may be observed in situations unrelated to radiation, i.e., Krowka et al showed that although PMA induces 95% of cells to express CD69, minimal proliferation takes place (24). Conversely, antigen or allogeneic stimulation induces only a small percentage of cells to express CD69 but induces extensive proliferation (6,19). In sum, our results add to the mounting evidence that one cannot generalize that enhancement of expression of CD69 in disease states is indicative of proliferative activity.…”

“…5A). A number of workers have suggested the use of CD69 expression as a marker for antigen and mitogen induced proliferation (19,27,28). Our data demonstrate that cells, stimulated by PHA, express CD69 and proceed to proliferate.…”

“…CD69, a Ca 2ϩ -dependent (C-type) lectin (15), is one of the earliest lymphocyte surface markers detected after mitogen or antigen stimulation (9,13,19,20,26,27). The biological function of the molecule is not defined.…”

Detection of Intracellular Antigen‐Specific Cytokines in Human T Cell Populations

“…By using a gating strategy that employed intracellular staining of activated cells using CD69, they were able to show that up to 0.5% of such cells produced IFN-g. However, since surface expression of CD69 does not occur after antigen stimulation until ∼48 h [27], these investigations were likely measuring ongoing effector responses in contrast to the overall T cell memory pool. In our case, in which we wish to show the potential memory immune response after vaccination, the level of ongoing stimulation is likely too small to measure directly in effector cell populations.…”

Gamma radiation induces CD69 expression on lymphocytes

“…CD69, a Ca 2ϩ -dependent (C-type) lectin (15), is one of the earliest lymphocyte surface markers detected after mitogen or antigen stimulation (9,13,19,20,26,27). The biological function of the molecule is not defined.…”