Priming to Mycobacterial Antigen In Vivo Using Antigen‐Pulsed Antigen Presenting Cells Generated In Vitro is Influenced by the Dose and Presence of IL‐4 in APC Cultures

Abstract

Antigen presenting cells (APC) similar to immature dendritic cells can be generated in vitro from bone marrow precursors. The authors have compared the yield, the phenotype and the function of murine bone marrow cells cultured for 7 or 11 days in either granulocyte macrophage colony stimulating factor alone (GM BMAPC) or in combination with interleukin‐4 (GM/IL‐4 BMAPC). The results showed that GM/IL‐4 BMAPC expressed the highest levels of MHC Class 2 molecules, CD86 /B7‐2 and CD80/B7‐1 co‐stimulatory molecules and the lowest levels of F4/80 macrophage marker. However, when these APC were pulsed with BCG culture filtrate antigen or PPD they were not correspondingly more effective at stimulating activated T lymphocytes in vitro or priming naive T lymphocytes in vivo. Also, in contrast to GM BMAPC, high backgrounds recorded following injections of GM/IL‐4 BMAPC without antigen were not consistently reduced by lowering the dose and irradiating the cells prior to administration. The authors conclude that the degree of maturity of BMAPC varies with culture conditions and that this may be an important consideration where BMAPC are to be used in vivo in immunotherapeutic regimens.

Influence of Interleukin‐4 on the Phenotype and Function of Bone Marrow‐Derived Murine Dendritic Cells Generated Under Serum‐Free Conditions

“…The expression of these molecules is of course of fundamental importance in the presentation of antigen and the delivery of signals to T cells. Other groups have observed that, in the presence of FCS, DC cultured in the absence of IL‐4 also express suboptimal levels of MHC class II, CD40, CD80 and CD86 when compared with DC cultured in the presence of IL‐4 [7, 12–16, 18], although we did not find this to be the case in this study.…”

“…Although we, in this study, did not explore the ability of DC cultured in FCS to take up antigen, previous studies have reported that DC that are generated in the presence of IL‐4 are less efficient at taking up FITC–Dextran [7, 12, 15] or mannosylated BSA [13] via the mannose receptor than DC cultured in the absence of IL‐4. It is possible that serum has an effect on the levels of mannose receptor expression, which would affect the uptake of antigens.…”

Mycobacterial antigens induce apoptosis in human purified protein derivative‐specific αβ T lymphocytes in a concentration‐dependent manner

“…In vivo naı¨ve T lymphocytes can be sensitized against bacillary antigens by antigen‐presenting cells delivering Mtb‐PPD antigens 10–14 . Peripheral blood lymphocytes from 11 tuberculin‐negative, healthy individuals were cocultured with autologous DC and 400 µg/ml Mtb‐PPD (MLC).…”

“…To address this question Mtb‐PPD‐specific T lymphocytes were generated using autologous dendritic cells (DC) loaded with bacillary antigens. As the most potent antigen‐presenting cells, 8,9 DC are capable of taking up and processing not only whole mycobacteria but also Mtb‐PPD, and are known to deliver Mtb‐PPD and other bacillary antigens to naı¨ve T lymphocytes resulting in a Th1 immune response in vivo and in vitro 10–14 . Herein we report that on re‐exposure to bacillary antigens Mtb‐PPD‐specific γδ + , αβ + CD4 + , and αβ + CD8 + T lymphocytes become reactive and apoptotic via Fas‐dependent cytolytic mechanisms in an antigen‐concentration‐dependent manner.…”

Blockade of B7‐2, Not B7‐1, Inhibits Purified Protein Derivative‐Primed T‐Lymphocyte Responses But Fails to Influence the Proportion of Th1 Versus Th2 Subsets

“…Antigen-presenting cell source. A modified method of Inaba et al [23], for the isolation of bone marrow precursors, was used [25]. Briefly, the bone marrow was flushed from femurs and tibias with cDMEM þ 5% FCS, the red blood cells were lysed with ammonium chloride and the remaining cells were washed four times.…”