Bone Marrow Transplantation (2002) 30, 261–266 2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt Dendritic cells Dendritic cell recovery after autologous stem cell transplantation D Damiani1, R Stocchi1, P Masolini1, A Michelutti1, A Sperotto1, A Geromin1, C Skert1, M Cerno1, M Michieli3, M Baccarani2 and R Fanin1 1Chair and Division of Haematology, Bone Marrow Transplant Unit, Department of Medical and Morphological Research, University Hospital, Udine, Italy; 2Institute of Haematology and Medical Oncology ‘L and A Sera`gnoli’, University Hospital, Bologna, Italy; and 3Centro di Riferimento Oncologico (CRO), Aviano, Italy Summary: haematologic malignancies and solid tumours. It is now well-recognised that there is persistent immunosuppression There is persistent immunosuppression not only in allo- not only in allogeneic but also in autologous stem cell trans- geneic but also in autologous stem cell transplantation plantation because humoral and cellular immunity may take because humoral and cellular immunity may take a year a year or more to return to normal, with increased risk of or more to return to normal, with increased risk of infectious complications.1 Functional reconstitution of the infectious complications. This immune defect may also lymphoid compartment after autologous bone marrow/ involve antigen presentation, in particular dendritic cell peripheral blood HSCT depends either on the presence of (DC) function. We evaluated DC subset reconstitution adequate numbers of antigen-specific T and B cells in the in 58 patients who underwent bone marrow (BM) or transplant or the maturation of lymphoid progenitors and peripheral blood (PB) autologous haematopoietic stem perhaps multipotent stem cells into antigen-specific T and cell transplantation (HSCT). In all patients DC type 1 B cells within thymic and bone marrow microenviron- (DC1) and DC type 2 (DC2) were already significantly ments. The early post-transplant period is characterised by lower than in normal individuals before conditioning a reduction of CD4+ T cells (due to a persistently low level therapy (DC1/␮l 3.1 ؎ 1.0, DC2/␮l 3.0 ؎ 1.1). On day of naive CD4+/CD45RA+ T cells) and by elevated numbers and day ؉7 the mean DC1 and DC2 numbers were of CD8+ T cells. The number of B cells is also reduced.2–7 0 very low in both groups. Patients who received unman- This immune defect may also involve antigen presentation. ipulated marrow or peripheral blood stem cells reached On this subject, it has been suggested that the patients’ anti- pre-conditioning levels of DC1 and DC2 cells on day gen presenting cells (APC) are abnormal either numerically ؉20. In patients receiving selected CD34 cells, DC or functionally after transplant.8 Dendritic cells (DC) are increased slowly and pre-transplant counts were the most powerful APC in inducing T cell activation. They ;observed only on day ؉60. Nearly ‘normal’ levels of originate from pluripotent stem cells in the bone marrow DC1 and DC2 could be observed in the first group from then, they migrate from peripheral blood to tissues, where .day ؉180, and were maintained thereafter; in CD34ϩ they achieve the capacity to capture and process antigens selected patients DC1 and DC2 counts remained lower In lymphoid organs DC present these processed antigens to than normal. Our data emphasise that circulating anti- and activate T cells.9 gen presenting cells (APC) recover quickly. It remains Recently, two subsets of DC have been identified in per- to be determined if DC frequency in PB reflects their ipheral blood. DC type 1 (lineage negative, HLA-DR posi- tissue function. The relatively low incidence of infections tive and CD11c positive) are myeloid and activate (through in patients undergoing autologous transplantation, interaction between CD80 and CD28, in the presence of despite defective lymphocyte reconstitution, could be IL-12) T helper type 1 (Th1), which produce IL-1 and IFN␥ related to functionally efficient DC. (pro-inflammatory cytokines); DC type 2 (lineage negative, Bone Marrow Transplantation (2002) 30, 261–266. HLA-DR positive and CD123 positive) may be lymphoid doi:10.1038/sj.bmt.1703637 and activate (through interaction between CD86 and CD28, Keywords: dendritic cell; autologous stem cell transplan- in the presence of IL-10) T helper type 2 (Th2), which tation; immunological recovery produce IL-4 and IL-10 (anti-inflammatory cytokines).8,10–12 In this paper we evaluated the kinetics of lymphocyte and dendritic cell subset reconstitution in 58 patients undergo- ing bone marrow or peripheral blood autologous HSCT and High-dose chemotherapy followed by haemopoietic stem its relationship with infectious complications. cell transplantation (HSCT) is increasingly used for various Patients and methods Correspondence: Dr D Damiani, Division of Haematology, University Hospital, P le S Maria della Misericordia, 33100 Udine, Italy The kinetics of lymphocyte and dendritic cell reconstitution Received 26 September 2001; accepted 1 May 2002 were studied in 58 consecutive patients affected by NHL Dendritic cell recovery D Damiani et al 262 (n ϭ 28), HL (n ϭ 6), multiple myeloma (n ϭ 15), acute tive controls, with irrelevant isotypic antibodies were pre- leukaemias (n ϭ 4) and breast cancer (n ϭ 5) who received pared in each experiment, as appropriate. The absolute intensification therapy and autologous stem cell rescue number of dendritic cells was calculated from the WBC (HSCT) from January 1997 to December 2000. Patients count multiplied by the proportion of each subpopulation with NHL received the BAVC (cytosine-arabinoside, eto- among the WBC, as determined by flow cytometric analy- poside, cyclophosphamide and carmustine) conditioning sis. Data were expressed as DC1 and DC2 mean number regimen, patients with HL received the BEAM (cytosine- Ϯ 2 standard deviation per microliter of PB. arabinoside, etoposide, melphalan and carmustine) con- ditioning regimen, myeloma patients received busulphan Immune reconstitution and melphalan, patients with acute leukemia received bus- ulphan and cyclophosphamide and breast cancer patients Peripheral blood lymphocyte subsets were analysed by flow received etoposide, carboplatinum and iphosphamide. A cytometry before starting the conditioning regimen, at time median of 2.6 ϫ 106/kg (range 0.77–4) CD34ϩ cells were 0, before stem cell infusion, at time ϩ30, ϩ60, ϩ90, ϩ240, infused in peripheral blood HSCT (n ϭ 46) and 1.07 ϫ ϩ360, ϩ3 years. One hundred ␮l of peripheral blood anti- 106/kg (range 0.4–1.87) in bone marrow HSCT (n ϭ 12). coagulated with EDTA were incubated at laboratory tem- Twelve of 15 myeloma patients received highly purified perature for 20 min with the following monoclonal anti- CD34ϩ stem cells (median 2.8 ϫ 106/kg, range 2–5), bodies: CD3FITC, CD19PE, CD4PE, CD8FITC, CD16PE, obtained after immumomagnetic separation by CliniMacs CD56PE, CD45ROFITC, CD45RAFITC. At the end of (Miltenyi Biotec, Bergisch Gladbach, Germany). Antibiotic incubation red cells were lysed by Facs Lysis solution (BD, prophylaxis included ciprofloxacin and itraconazole. G- Bruxelles, Belgium), washed twice and analysed within 1 h. CSF (filgrastim, Neupogen, Amgen, Thousand Oaks, CA, Acquisition and analysis were performed with a FacsCali- USA) at a dose of 5 ␮g/kg was administered from day bur (BD) flow cytometer with Lysis II software. WBC cell ϩ4, until the leukocyte count exceeded 2 ϫ 109/l for 3 counts were determined using an automated cell counter consecutive days. Patients’ characteristics are summarised (CELL DYN 3200). in Table 1. Total lymphocyte count was determined by flow cytome- try by incubating a sample of whole blood with the Dendritic cell reconstitution CD14PE/CD45FITC (BD) antibodies. The absolute number of lymphocytes was calculated by multiplying the percent- Dendritic cell recovery was evaluated before starting the age of CD14−/CD45ϩϩ by the total WBC. The absolute conditioning regimen, at day 0 before stem cell infusion, number of cells in any given lymphocyte population was at day ϩ7 and at the same points as lymphocytes. DC1 calculated by multiplying the percentage of positive cells (CD11cϩ, myeloid origin) and DC2 (CD123ϩ, lymphoid for each lymphocyte marker by the absolute lymphocyte origin) subsets were identified by using a three-colour flow number. Data were expressed as mean number Ϯ 2 cytometric assay on lysed whole blood to minimise selec- standard deviations per microliter of PB. tive loss. For each test 100 ␮l of blood were incubated with ␮ ␮ 10 l of the HLA-DR-PerCP (BD), with 20 l of a mixture Normal controls of lineage-related antibodies FITC (lineage cocktail 1 FITC, BD), including the following monoclonal antibodies: Circulating dendritic cell and lymphocyte subsets of 15 CD3, CD14, CD16, CD19, CD20 and CD56, and with 10 healthy donors were evaluated as described above, to obtain ␮l of the CD11c-PE or the CD123-PE antibodies. At the normal reference values. end of a 15 min incubation, red cells were lysed as described above, samples were washed twice and immedi- Data analysis ately analysed. A minimum of 50 000 events was acquired for each experiment. Dendritic cells express high levels of Mean differences at different times between patients and HLA-DR and lack lineage-related antigens. After gating normal controls were assessed by the Mann–Whitney U lineage-negative events, the two DC1 and DC2 subsets test. were identified in the high HLA-DR expressing population, on the basis of their high CD11c or CD123 intensity. Nega- Results Table 1 Patient characteristics Transplant-related data are summarised in Tables 2 and 3. Not selected Selected Haematological recovery occurred promptly in all patients, without any differences between patients receiving bone Patients 46 12 marrow or peripheral blood stem cells. The median time to Sex (M/F) 28/18 7/5 reach a neutrophil count >1 ϫ 109/l was day ϩ10 (9–14) Median age 40 (16–58) 55 (41–63) in PB patients, day ϩ13 (11–15) in PB selected patients Disease 28/46 NHL 12/12 MM and day ϩ12 (10–30) in BM patients.