sign in sign up
<<
Home , Dendritic cell, Hematopoietic stem cell

Marrow Transplantation (2002) 30, 261–266  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt Dendritic cells recovery after autologous 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, 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 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 presentation, in particular dendritic cell peripheral 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 . 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 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 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 reconstitution, could be IL-12) T helper type 1 (Th1), which produce IL-1 and IFN␥ related to functionally efficient DC. (pro-inflammatory ); 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 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), (n ϭ 15), acute tive controls, with irrelevant isotypic were pre- leukaemias (n ϭ 4) and breast (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, 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 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 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 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 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. The median time to 6/46 HL ϫ 9 ϩ 3/46 MM reach a count >20 10 /l was day 11 (9–30), 4/46 AL day ϩ14 (10–21) and day ϩ12 (10–30), respectively. Not 5/46 BC considering CD34ϩ selected patients 23/46 (50%) had a febrile episode; only in 8/46 (17.3%) was a gram ϩ bacter-

Bone Marrow Transplantation Dendritic cell recovery D Damiani et al 263 Table 2 Graft composition and transplant-related data according to stem cell source

PB not selected (n ϭ 48) PB selected (n ϭ 12) BM (n ϭ 12)

CD34ϩ ϫ 106/kg 2.6 (0.77–4) 2.8 (2–5) 1.07 (0.4–1.87) >1 ϫ 109/l ϩ10 (9–14) ϩ13 (11–15) ϩ12 (10–30) >20 ϫ 109/l ϩ11 (9–30) ϩ14 (10–21) ϩ12 (10–30) G-CSF (days) 7 (6–9) 10 (8–12) 8 (7–13) In patients (days) 19 (16–20) 21 (18–24) 23 (19–26) Red cells unit 3 (2–5) 4 (2–6) 4 (2–7) Platelet apheresis 4 (3–7) 5 (4–9) 6 (5–8)

Table 3 Infectious complications after transplant 10 9 Selected Not selected 8 (%) (%) DC2 normal value 7 DC1 normal value None 5/12 (41.6) 23/46 (50) 6 FUO 5/12 (41.6) 15/46 (32.6) Bacteraemia 2/12 (16.6) 8/46 (17.3) 5 DC1 l Stomatitis (III–IV WHO) 4/12 (33.3) 16/46 (34) m 4 DC2 DC/ 3

2 aemia detected. A stomatitis grade III–IV, was the most frequent toxic side-effect (16/46 patients, 34.7%). In 1 CD34ϩ selected patients 5/12 (41.6%) had a febrile epi- 0 sode; in 2/12 (16.6 %) a gram ϩ bacteraemia was detected. BL 0 7 30 60 90 180 Time (days) A stomatitis grade III–IV, was the most frequent toxic side- effect (4/12 patients, 33.3%). No deaths occurred during Figure 1 DC1 and DC2 recovery in selected patients. Each time point the early post-transplant period (follow-up of 12 months). is expressed as mean value Ϯ 2 standard deviations. BL indicates day We did not test T helper 1 and T helper 2 cell functions before starting conditioning therapy; day 0 indicates day of stem cell infusion (before infusion). on the basis of DC phenotype; we tested DC1 and DC2 in patients who had infection and we found, as expected, low levels of both. 10 9

Dendritic cell reconstitution 8 In all patients DC1 and DC2 cells were already significantly 7 DC2 normal value DC1 DC1 normal value lower than in normal individuals before conditioning ther- 6 apy (DC1 mean/␮l 3.1 Ϯ 1.0 vs mean 6.0 Ϯ 3.3 P ϭ 0.025, DC2

l 5 DC2 mean 3.0 Ϯ 1.1 vs 6.7 Ϯ 3.5 P ϭ 0.025). On day 0 m ϩ 4 before stem cell infusion, and on day 7 the mean DC1 DC/ and DC2 number was very low (0.03 Ϯ 0.01 and 0.25 Ϯ 3 0.01 on day 0; 0.06 Ϯ 0.01 and 0.04 Ϯ 0.01/␮l on day ϩ7) 2 in both groups (selected and not selected), but the recovery kinetics were quite different. As shown in Figures 1 and 2, 1 patients who received unmanipulated marrow or peripheral 0 stem cells reached pre-conditioning levels of DC1 and DC2 BL 0 7 30 60 90 180 cells at day ϩ30 (3.9 Ϯ 1.2 and 2.8 Ϯ 1.3, respectively). Time (days) ϩ On the contrary, in patients receiving selected CD34 cells, Figure 2 DC1 and DC2 recovery in unselected patients. Each time point dendritic cells increased slowly and pre-transplant counts is expressed as mean value Ϯ 2 standard deviations. BL indicates day were observed only on day ϩ60. No difference was before starting conditioning therapy; day 0 indicates day of stem cell observed in the kinetics of DC1 and DC2 reconstitution. infusion (before infusion). Nearly ‘normal’ levels of DC1 and DC2 could be observed ϩ Ϯ in the first group of patients from day 180 (6.3 2.3 Immune reconstitution and 5.8 Ϯ 2.0), and were maintained thereafter (6.3 Ϯ 3.0 and 6.8 Ϯ 3.0 at 1 year; 7.3 Ϯ 3.3 and 6.6 Ϯ 3.0 at 3 years The count (defined as the number of cells expressing post transplant). In CD34ϩ selected patients DC1 and DC2 the pan-B CD19 antigen) and the NK cell count (defined counts were lower than normal up to 1 year post transplant by the CD56 expression and the lack of CD3 antigen) rose (4.3 Ϯ 0.2 and 4.3 Ϯ 0.1, respectively). to normal levels after 3 months post transplant in all

Bone Marrow Transplantation Dendritic cell recovery D Damiani et al 264 patients. CD3+ cells rose to normal level after 3 years post Discussion transplant. CD8+ cells rose over the normal level reaching ϩ Ϯ ␮ Ϯ ␮ a peak at day 60 (811 150/ l vs 557 78/ l) and In the past years the availability of high stem cell numbers fell to normal levels within 6 months post transplant in the after peripheral blood mobilisation has extended the use of unselected group, and within 1 year in the selected group. intensification regimens followed by autologous stem cell + CD4 lymphocytes remained less than 50% normal, so that transplantation to very large series of haematological and + + all patients had a low CD4 /CD8 ratio (<1) over the first non-haematological patients. The rapid haematological 6 months post transplant. In particular, the level of naive recovery has significantly reduced mortality during the CD4ϩ/CD45RAϩ cells was persistently low in this period transplant period, but a delay in lymphocyte recovery has (at 6 months, mean 88 Ϯ 18/␮l vs 524 Ϯ 60/␮l in normal been described, and is possibly responsible for long-term individuals, P ϭ 0.000). CD4ϩ/CD45ROϩ memory cells defects in immune reactivity involving both cellular and reached a plateau within the first 90 days post transplant humoral arms of the .1–7 The reduced and after that maintained this level (Figure 3). immune reactivity after stem cell transplantation could also

a b 2000 700

1750 (NV) 600 1500 500 1250 Not selected

l 400 Selected

l m

m 1000 (NV) Selected 300 CD19/

CD3/ 750

500 200 Not selected 250 100

0 0 1 36 121 36 12 Time (months) Time (months) cd 1400 1400

1200 (NV) 1200

1000 1000 l 800 l 800 m m Selected 600 600 CD4/ Not selected CD8/ (NV)

400 400 Not selected Selected 200 200 0 13 6 120 13 6 12 Time (months) e f Time (months) 600 600

500 (NV) 500 (NV) l l

m m 400 400 Not selected 300 300 Selected 200 200 CD45RA/CD4/ CD45RO/CD4/

100 100 Not selected Selected 0 0 1 36 121 36 12 Time (months) Time (months)

Figure 3 Immunological reconstitution in selected (circles) and in unselected (squares) patients of CD3 (a), CD19 (b), CD4 (c), CD8 (d), CD4/CD45RO (e), CD4/CD45RA (f) lymphocyte subsets. NV indicates normal value. Each time point is expressed as mean value Ϯ 2 standard deviations.

Bone Marrow Transplantation Dendritic cell recovery D Damiani et al 265 be explained by defective antigen presentation involving age can contribute to explaining the delayed recovery in dendritic cells. With the aim of better understanding the our myeloma group. DC1/DC2 ratio was close to 1 at each kinetics of post-transplant immune reconstitution we have time in both groups suggesting that CD34−dim dendritic pre- evaluated dendritic cell recovery in 58 patients who cursors are lost by the selection procedure, but equal underwent stem cell transplantation with unmanipulated lymphoid and myeloid dendritic potential is maintained by peripheral blood or bone marrow stem cells or with highly the residual stem cell pool. purified, positively selected, CD34+ cells. A group of 15 In conclusion, as previously reported by several normal donors was employed as a reference population. authors,1–7 our data confirm the delayed post-transplant Very little data on normal dendritic cell frequency in the immune recovery, in particular concerning ‘naive’ CD4 peripheral blood are available. Fearnley et al13,14 developed lymphocytes, but emphasise that circulating antigen a flow cytometric method suitable for routine evaluation of presenting cells are quickly recovered. It remains to be peripheral blood DC concentrations by using the mono- determined if DC frequency in peripheral blood reflects clonal CMRF44. By applying this method on 103 their tissue function. Shiobara et al18 showed that defects normal individuals they found counts ranging between 3 in Ig secretion after allogeneic bone marrow grafting are and 17 ϫ 106/l. No difference was found on segregating due primarily to defective T and B cell function and that the reference population by sex and age. However it must the accessory function is intact in most patients be emphasised that they used, as the reference marker, an studied. So the relative low infections incidence in patients activation antigen whose intensity of expression could be undergoing autologous transplantation, despite the defec- influenced by the culture system. Savary et al15 showed that tive lymphocyte reconstitution, could be related to the pres- in normal individuals lin−DRϩ dendritic cells represent 0.75 ence of functionally efficient DC. Ϯ 0.044% PBMC. More recently Galy et al16 evaluated lin−HLA-DRϩ CD11cϩ cells in five normal volunteers Ϯ counting 0.14 0.19% of MNC, corresponding to an Acknowledgements absolute count of 3.8 Ϯ 4.8 cells/␮l. Arpinati et al17 evalu- ated DC1 and DC2 subsets in peripheral blood of normal Programmi di Ricerca di Rilevante Interesse Nazionale-Cofinan- stem cell donors finding values overlapping ours. Our ziamento 2000. patients, studied before transplant, showed significantly lower DC levels in the blood than healthy volunteers, prob- ably as a consequence of previous chemotherapy. However our unpublished data on peripheral blood DC obtained in References patients with haematological malignancies during staging procedures, show that low concentrations of both blood 1 Guillame T, Rubinstein DB, Symann M. Immune reconsti- dendritic subsets are already detectable at the onset of the tution and after autologous hematopoietic disease. It remains to be determined if this is an intrinsic stem cell transplantation. Blood 1998; 5: 1471–1490. patient characteristic, able to weaken the immune response 2 Anderson K, Soiffer R, DeLage R et al. T-cell depleted auto- to malignancies, or the effect of the higher blood efflux as logous bone marrow transplantation therapy: analysis of immune deficiency and late complications. Blood 1990; 76: a consequence of recruitment into the tumour. Different to 235–241. what is observed in the conventional allogeneic setting (our 3 Keever C, Small T, Flomenberg N et al. Immune reconsti- data submitted for publication), at the end of the condition- tution following bone marrow transplantation: comparison of ing regimen (day 0) and at day ϩ7 low concentrations of recipients of T-cell depleted marrow with recipients of con- dendritic cells could be detected, perhaps as an effect of ventional marrow grafts. Blood 1989; 73: 1340–1351. the less myeloablative conditioning regimen. The persist- 4 Atkinson K, Hansen J, Rainer S et al. T-cell subpopulations ence of host dendritic cells can play an important role in identified by monoclonal antibodies after human marrow inducing tolerance and chimerism when a non-myeloabl- transplantation. Helper inducers and cytotoxic-suppressor sub- ative regimen is used for allogeneic transplantation. In our sets. Blood 1982; 59: 1292–1301. series of patients dendritic cell recovery, at the pre-trans- 5 Lum L. The kinetics of immune reconstitution after human marrow transplantation. Blood 1987; 69: 369–375. plant level, occurred nearly as promptly as the haematolog- 6 Bomberger C, Singh-Jairam M, Rodey G et al. Lymphoid ical recovery, but is delayed in myeloma patients receiving ϩ reconstitution after autologous PBSC transplantation with highly purified CD34 cells. In this cohort of patients pre- FACS-sorted CD34ϩ hematopoietic progenitors. Blood 1998; transplant counts are reached only 3 months after transplan- 91: 2588–2600. tation and normal levels were never observed. Analogously, 7 Steingrimdottir H, Gruber A, Bjorkholm M et al. Immune Savary et al15 found uniformly low DC concentrations in reconstitution after autologous trans- breast cancer patients 1–5 months after stem cell transplan- plantation in relation to underlying disease, type of high-dose tation. Conversely Fearnley et al14 could not point out dif- therapy and infectious complications. Haematologica 2000; ferences among patients according to graft source or CD34 85: 832–838. selection, also observing a complete DC recovery 60 days 8 Di Nicola M, Lemoli RM. Dendritic cells: specialized antigen 16 presenting cells. Haematologica 2000; 85: 202–207. after autograft. Likewise Galy et al obtained normal DC 9 Bachereau T, Steinman RM. Dendritic cells and the control levels at day 42 post-CD34 selected transplantation. Differ- of immunity. Nature 1998; 392: 245–252. ent previous treatment regimens and mobilisation protocols, 10 Lanzavecchia A, Sallusto F. The instructive role of dendritic CD34 enrichment procedures (Isolex vs CliniMacs), differ- cells on T cell responses: lineages, plasticity and kinetics. ent degrees of bone marrow infiltration and finally higher Curr Opin Immunol 2001; 13: 291–298.

Bone Marrow Transplantation Dendritic cell recovery D Damiani et al 266 11 Reid CD. The dendritic cell lineage in haemopoiesis. Br J blood of normal donors and cancer patients. Cancer Immunol Haematol 1997; 96: 217–233. Immunother 1998; 45: 234–240. 12 Tanaka J, Asaka M, Imamura M. T-cell co-signalling mol- 16 Galy A, Rudraraju S, Baynes R, Klein J. Recovery of lympho- ecules in graft versus host disease. Ann Haematol 2000; 79: cytes and dendritic cell subsets after autologous CD34ϩ cell 283–290. transplantation. Bone Marrow Transplant 2000; 25: 1249– 13 Fearnley DB, McLellan AD, Mannering SI et al. Isolation of 1255. human blood dendritic cells using the CMRF-44 monoclonal 17 Arpinati M, Green CL, Heimfeld S et al. -colony antibody: implications for studies on antigen-presenting cell stimulating factor mobilizes T helper 2-inducing dendritic cell. function and immunotherapy. Blood 1997; 89: 3708–3716. Blood 2000; 95: 2484–2489. 14 Fearnley DB, Whyte LF, Carnoutsos SA et al. Monitoring 18 Shiobara S, Whiterspoon RP, Lum LG, Storb R. Immunoglob- human blood dendritic cell numbers in normal individuals and ulin synthesis after HLA-identical marrow grafting: the role in stem cell transplantation. Blood 1999; 93: 728–736. of peripheral blood in the regulation of in vitro 15 Savary AC, Grazziutti ML, Melichar B et al. Multidimen- immunoglobulin secretion stimulated by pokeweed mitogen. sional flow-cytometric analysis of dendritic cells in peripheral J Immunol 1984; 132: 2850–2856.

Bone Marrow Transplantation