Immature Dendritic Cells Generated from Cryopreserved Human Monocytes Show Impaired Ability to Respond to LPS and to Induce Allogeneic Lymphocyte Proliferation

<div><p>Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.</p></div

Phenotypic analyses of frh- and crp-imdDCs.

<p>Frh-imdDCs (black), crp-imdDCs (blue) and isotype-matched controls (red) were analyzed for the expression of CD11c/CD14 and CD11c/HLA-DR. (A) Representative dot blot analyses for the expression of CD11c/CD14 and CD11c/HLA-DR from one blood donor. (B) Data from six independent cultures of frh- (circles) and crp-imdDCs (squares). Data was analyzed by Student’s <i>t</i> test and the values shown are the means ± SDs of six individual donors. (C and D) Expression of IL-4 (CD124) and GM-CSF (CD116) receptors on CD11c⁺ cells during monocytes differentiation to imdDCs. Frh-monocytes (circles) and crp-monocytes (squares) were induced to differentiate to imdDCs and growth factors receptors were analyzed at 0, 4 and 7 days post-differentiation (dpd). Data was analyzed using one-way ANOVA followed by a Bonferroni test; values represent means ± SDs of six individual donors. *<i>p</i>≤0.05, frh: freshly obtained, crp: cryopreserved, imdDCs: immature monocyte derived dendritic cells.</p

Maturation of frh- and crp-imdDCs after stimulation with LPS.

<p>Expression of CD40, CD80, CD83 and CD86 on frh- and crp-imdDCs after treatment with LPS (1 µg/mL) at 0 (imdDCs), 6, 24 and 48 h. Histogram of one individual donor (representative of six). frh: freshly obtained, crp: cryopreserved, imdDCs: immature monocyte derived dendritic cells.</p

Cryopreservation interferes with the allogeneic lymphoproliferative response.

<p>(A) Representative histogram of autologous and allogeneic lymphocyte proliferation of one donor (frh-imdDCs (black), crp-imdDCs (blue) and Lyn-stained lymphocytes without DC stimulation (red)). (B) Frh- (circles) and crp-imdDCs (squares) were co-cultured with autologous (negative control) or allogeneic lymphocytes stained with CellTracker™ Green CMFDA in a 1∶10 ratio of imdDCs:lymphocytes. After five days of co-culture lymphocytes proliferation was analyzed by flow cytometry. Data was analyzed by Student’s <i>t</i> test and the values shown are the means ± SDs of six individual donors. ***<i>p</i>≤0.001. frh: freshly obtained, crp: cryopreserved, imdDCs: immature monocyte derived dendritic cells, Lyn: stained lymphocytes without DC stimulation, Aut lyn: autologous lymphocytes, All lyn: allogeneic lymphocytes.</p

Apoptosis of frh- and crp-imdDCs.

<p>Analysis of apoptosis (Annexin V⁺/PI^-) in frh- (circles) and crp-imdDCs (squares) at 0, 6, 24 and 48 h. Data was analyzed by Student’s <i>t</i> test and the values shown are the means ± SDs of six individual donors. *<i>p</i>≤0.05.</p