Original article. Introduction

Transcription

1 Original article Annals of Oncology 14: , 2003 DOI: /annonc/mdg107 CD34+-selected versus unmanipulated autologous stem cell transplantation in multiple myeloma: impact on dendritic and immune recovery and on complications due to infection D. Damiani 1 *, R. Stocchi 1, P. Masolini 1, A. Michelutti 1, A. Geromin 1, A. Sperotto 1, C. Skert 1, M. Michieli 2, M. Baccarani 3 & R. Fanin 1 1 Division of Haematology, Bone Marrow Transplant Unit, Department of Medical and Morphological Research, University Hospital, Udine; 2 Centro di Riferimento Oncologico (CRO), Aviano; 3 Institute of Haematology and Medical Oncology L. and A. Seràgnoli, University Hospital, Bologna, Italy Received 7 June 2002; revised 20 September 2002; accepted 22 October 2002 Background: Large-scale CD34+ enrichment has been demonstrated a safe method in autologous transplantation for multiple myeloma. However, the high CD34+ enrichment and the consequent plasma cell purging result in concomitant T-cell and dendritic-cell (DC) depletion, theoretically increasing the risk of lifethreatening infections. Patients and methods: We evaluated immunological and dendritic reconstitution in 72 myeloma patients who had undergone CD34+-selected (n = 45) and unmanipulated (n = 27) stem cell transplant, and its correlation with infections. Results: Haematological recovery occurred promptly in all patients. Only a slight delay in platelet recovery to > /l was observed in patients receiving CD34+-enriched graft. Natural killer (NK) cell count recovered in all patients within 2 months and B-cell count had recovered by 6 months post-transplant in both groups. CD3 cells remained lower than normal in both groups. CD8 cells increased above the normal level, reaching a peak at day 90, and lowered to normal level within 1 year post-transplant. CD4 lymphocytes remained <50% of normal, especially in selected patients. In both groups, both DC1 and DC2 counts were already significantly lower than in normal individuals before conditioning therapy. Pre-conditioning levels of DC1 were reached in unmanipulated patients at day 30 and became normal at 6 months. In selected patients, DC1 pre-transplant level was observed at day 60 and was maintained thereafter. DC2 recovery showed a similar trend. In unselected patients, DC2 count increased to pre-conditioning level at haematological recovery and was normal after 1 year. In selected transplants, DC2 increased more slowly than DC1 in the same patients: pre-transplant level was detected at day 90 but was still significantly lower than normal 1 year after transplant. The incidence of infection was similar in both groups. Sepsis had Gram+ aetiology in the majority of cases. After engraftment only viral infections were recorded, mostly due to herpes reactivation, with no difference between groups. Discussion: In spite of a delay in immune recovery, CD34 enrichment is not associated with a significant increase of complications due to infection. Relatively fast NK cell recovery to pre-transplant levels and the presence of functionally efficient DCs can justify the low incidence of infections. Key words: CD34+ selection, infections, multiple myeloma, transplantation Introduction In the last decade, autologous peripheral blood stem cell transplantation (ASCT) has been largely used as a rescue from high-dose therapy in multiple myeloma patients, significantly improving event-free survival [1]. Nevertheless, no myeloma patients have ever been cured by ASCT and relapse occurs in all cases within a few years from transplant [2]. Among factors affecting progression, an important role has been attributed to *Correspondence to: D. Damiani, Division of Haematology, University Hospital, P. le S. Maria della Misericordia, Udine, Italy. Tel: ; Fax: ; daniela.damiani@drmm.uniud.it plasma cells and clonotypic plasma cell precursors contaminating the graft. Because myeloma cells lack uniform expression of B-cell-related antigen, in the past clinical grade devices have been set-up to indirectly purge the graft by CD34+ selection [3]. Large-scale CD34+ enrichment has been demonstrated a safe method and selected CD34+ cells permit fast and durable haematological engraftment [4 6]. However, the high CD34+ enrichment and the consequent plasma cell purging result in concomitant T-cell and dendritic-cell (DC) depletion, theoretically increasing the risk of life-threatening infections. In particular, in myeloma T-cell deficiency, resulting from purging procedures, can worsen B-cell defect typical of this malignancy. For this reason, the addition of autologous T lymphocytes, natural killer (NK) cells and 2003 European Society for Medical Oncology

2 476 Table 1. Clinical and laboratory characteristics of patients Selected Unmanipulated Patients Sex (M/F) 25/20 16/11 Median age, range (years) 55 (41 65) 56 (43 66) M component IgG IgA 5 6 Micromolecular 2 2 Cy 7 g/m Cy 4 g/m Tumour stage a I 10 3 II 15 5 III Cy, cyclophosphamide; IgA, immunoglobulin A; IgG, immunoglobulin G; M, monoclonal component isotype. expanded DCs has been proposed to overcome the impaired immune function [5, 7]. In this study we compared dendritic and immune reconstitution after CD34+-selected and unmanipulated stem cell transplantation in 72 myeloma patients who had undergone intensification therapy after the same induction protocol, and the incidence and type of infections that occurred in the two groups within the first year of transplant. Patients and methods Seventy-two consecutive patients affected by multiple myeloma who had undergone intensification therapy with autologous stem cell rescue between July 1998 and December 2001 entered the study. The first 27 patients underwent unmanipulated transplant, while the remaining 45 patients underwent selected transplant. Clinical and laboratory characteristics at diagnosis are summarised in Table 1. All patients received four cycles of induction therapy according to the vincristine, doxorubicin, dexamethasome (VAD) scheme. Stem cell collection was performed within 7 months (range 6 14) of the start of chemotherapy using high-dose cyclophosphamide (7 g/m 2 ) or intermediate cyclophosphamide (4 g/m 2 ) according to age (>60 years) or clinical parameters (heart ejection fraction <60% and/or creatinine value >1.4 mg/dl). Granulocyte colony-stimulating factor (G-CSF; 5 µg/kg body wt) was administered from day 4 until collection of a minimum of /kg CD34+ cells. At least two leukapheresis procedures were performed to obtain the minimum CD34+ cell dose required for CD34 selection. A total of 11 (6 13) 10 6 /kg median CD34+ cells were collected. The immunomagnetic system (Clini- Macs; Myltenyi Biotec, GmbH Bergish Gladback, Germany) was used in all patients for CD34+ cell enrichment. After selection, 2.7 ( ) 10 6 /kg median CD34+ cells were obtained. Graft purity after the selection procedure was 97 ± 4.3% (harvest 92%). The percentage of residual T cells (CD3+) was <0.1 ± 0.03% (other cells were B lymphocytes). An unmanipulated aliquot of at least /kg CD34 cells was cryopreserved as back-up in case of CD34+ selection. Plasma cell contamination Plasma cell contamination was evaluated by flow cytometry as described previously [8]. In brief, a total of 10 6 cells were incubated with the monoclonal antibody CD138 and alternatively coupled with CD38 and intracellular κ and λ light chains in 100 µl of a phosphate-buffered saline (PBS)/0.02% saponin solution for 15 min in separate samples. At least CD138/CD38 events were acquired, considering only plasma cells with the same light chain at diagnosis. Before selection, 17 (10 22) 10 4 /kg median plasma cells were collected. Using both CD34 selection methods a maximum of 3-log plasma cell depletion was obtained. Only 1 ( ) 10 4 /kg median plasma cells were infused. Myeloablative therapy Stem cell transplantation was performed at a median of 9 months from the diagnosis (range 6 19 months). The conditioning regimen consisted of busulfan 12 mg/kg orally on days 5, 4 and 3, and melphalan 120 mg/m 2 on day 2. A median of /kg CD34+ cells (range ) were reinfused on day 0. Antibiotic prophylaxis included ciprofloxacin and itraconazole. G-CSF (filgrastim; Neupogen, Amgen) at a dose of 5 µg/kg s.c. was given from day 4, until leucocyte count exceeded /l for three consecutive days. Immune reconstitution Peripheral blood lymphocyte subsets were performed by flow cytometry at diagnosis of disease, before starting the conditioning regimen, on days 30, 60, 90, 180 and 365, 100 µl of peripheral blood anticoagulated with EDTA were incubated at laboratory temperature for 20 min with the following monoclonal antibodies: CD3 FITC, CD19 PE, CD4 PE, CD8 FITC, CD16 PE, CD56 PE, CD45RO FITC and CD45RA FITC. At the end of incubation red cells were lysed by Facs Lysis solution (Becton Dickinson, Bruxelles, Belgium), washed twice and analysed within 1 h. Acquisition and analysis were performed by a FacsCalibur (Becton Dickinson) flow cytometer with the Lysis II software. White blood cell (WBC) counts were determined using an automated cell counter (Coulter Miami, Florida, USA). Total lymphocyte count was determined by flow cytometry after incubating a sample of whole peripheral blood with the CD14PE/CD45FITC (Becton Dickinson) antibodies. The absolute number of lymphocytes was calculated by multiplying the percentage of CD14-/CD45+ cells by the total WBC count. The absolute number of cells in any given lymphocyte population was calculated by multiplying the percentage of positive cells for each lymphocyte marker by the absolute lymphocyte number. Dendritic reconstitution Dendritic recovery was evaluated in 50 of 72 patients at diagnosis of disease, before starting the conditioning regimen, before stem cell infusion (day 0) and post transplant at the same time as lymphocyte counts. DC1 (CD11c+, myeloid origin) and CD2 (CD123+, lymphoid origin) subsets have been identified using a three-colour assay on lysed whole blood to minimise selective loss. For each test, 100 µl of blood were incubated with 10 µl of the human leucocyte antigen (HLA)-DR per CP (BD) with 20 µl of a mixture of lineage-related antibodies in fluorescein isothiocyanate (FITC; Lineage cocktail 1 FITC, BD), including monoclonal antibodies CD3, CD14, CD16, CD19, CD20 and CD56, and with 10 µl of the CD11c or CD123 antibodies. At the end of a 15-min incubation, red cells were lysed as described above, samples were washed twice and immediately analysed. A minimum of events were acquired for each experiment. Dendritic cells express high levels of HLA-DR and lack lineage-related antigens. After gating lineage negative events, the two DC1 and DC2 subsets were identified in the high HLA-DR-expressing population, on the basis of their high CD11c or CD123 intensity. Negative controls, with irrelevant isotypic antibodies, were prepared

3 477 in each experiment, as appropriate. The absolute number of DCs was calculated from the WBC count multiplied by the proportion of each subpopulation among the WBCs, as determined by flow cytometric analysis. Normal controls Circulating DCs and lymphocyte subsets of 10 healthy donors were evaluated to obtain normal reference values. Data analysis Mean differences at different times between patients and normal controls were assessed by the Mann Whitney rank sum test. Results Transplant-related data according to stem cell type (unmanipulated versus selected) is summarised in Table 2. Haematological recovery occurred promptly in all patients; only a slight delay in platelet recovery to > /l was observed in patients receiving CD34-enriched graft (P = 0.02), which resulted in longer hospital stays (P = 0.02). No deaths occurred during the early posttransplant period. Immune reconstitution Data on lymphocyte reconstitution are shown in Table 3. Data related to time before conditioning regimen are not shown but were similar to those at diagnosis of disease. NK cell count (defined by the CD56 expression and the lack of CD3 antigen) recovered promptly in all patients and normal levels were observed within 2 months of transplant in both groups. B-cell count (defined as the number of cells expressing the pan-b CD19 antigen) increased to normal levels after 6 months post-transplant in both groups. CD3 cells reached a plateau at day 365 and remained lower than normal in both groups, but especially in selected groups (selected versus unselected; P = ). CD8 cells increased above the normal level, reaching a peak at day 90 (mean 1492 ± 320/µl versus 557 ± 78/µl, P = 0.01) and lowered to normal level within 1 year post-transplant in both groups. CD4 lymphocytes remained <50% of normal, so that all patients, especially selected patients, had a low CD4/CD8 ratio (<1) during the first year post-transplant. In particular, there was a persistently low level of naïve CD4+/CD45RA+ cells during this period (at day 365, mean 53 ± 3.6/µl in selected patients and 85 ± 6 in unmanipulated patients versus 524 ± 60/µl in normal individuals; P = and 0.015, respectively). When we compared the CD4+/ CD45RA+ value between the two groups, we found a statistical difference (P = ). CD4+/CD45RO+ memory cells reached a plateau within the first 90 days post-transplant and maintained this level thereafter (mean 263 ± 103/µl and 380 ± 100/µl in selected and unmanipulated patients, respectively). Dendritic reconstitution Circulating DCs were evaluated in 50 of 72 patients at the same time as lymphocytes, during the first year after transplant. Both DC1 and DC2 counts were already significantly lower than normal in individuals at diagnosis of disease and before conditioning therapy in both groups (DC1: mean 2.4 ± 1.1 versus 6.0 ± 1, P = 0.03; DC2: mean 2.9 ± 0.1 versus 6.7 ± 1, P = 0.01). At day 0 (just before stem cell infusion) mean circulating DC1 were 0.28 ± 0.01 and mean DC2 were 0.97 ± 0.1 in both groups. Preconditioning levels of DC1 were reached in patients receiving unmanipulated stem cells on day 30, at the same time as haematological recovery, and increased to normal reference values 6 months after transplant (mean 5.5 ± 1). Patients transplanted with selected CD34+ cells showed slower DC1 recovery: pretransplant level was observed on day 60 (2.5 ± 0.2) and was maintained thereafter. Normal range was not reached during the period of study. Similarly, in unselected patients DC2 levels increased to pre-conditioning level at haematological recovery and were normal after 1 year. In manipulated transplants, DC2 levels increased more slowly than DC1 levels in the same patients: pre-transplant level was detected on day 90 and 1 year after transplant was still significantly lower than normal (mean 4 ± 1). In unmanipulated patients, both DC1 and DC2 levels reached normal value after 1 year post-transplant, while in selected patients they did not (Table 3). Table 2. Transplant-related data Selected Unmanipulated P Time DX to ASCT (months) 9 (6 19) 8 (6 20) NS Neutrophils > /l 13 (11 15) 11 (10 17) NS Platelets > /l 24 (13 170) 17 (12 75) 0.02 G-CSF (days) 11 (7 24) 10 (7 27) NS Inpatients (days) 25 (18 36) 17 (13 29) 0.02 Red cells (units) 4 (2 6) 3 (1 5) NS Platelets (units) 5 (4 9) 4 (3 8) NS Stomatitis III IV a 19/45 (42%) 11/27 (40%) NS ASCT, autologous stem cell transplantation; DX, diagnosis; G-CSF, granulocyte colonystimulating factor; NS, not significant. a World Health Organization classification.

4 478 Table 3. Lymphocyte and dendritic reconstitution in selected and in unmanipulated patients CD3 CD4 CD8 CD19 NK CD4/45RO CD4/45RA DC1 DC2 Selected Onset 1340 ± ± ± ± ± ± ± ± ± 1 Day ± ± ± ± ± 30 9 ± 12 7 ± ± ± 0.1 Day ± ± ± ± ± ± ± ± ± 0.8 Day ± ± ± ± ± ± ± ± ± 0.9 Day ± ± ± ± ± ± ± ± ± 1.2 Day 365 a 887 ± ± ± ± ± ± ± ± ± 1 N 1522 ± ± ± ± ± ± ± 60 6 ± ± 1 Unmanipulated Onset 1340 ± ± ± ± ± ± ± ± ± 1 Day ± ± ± 75 8 ± ± ± ± ± ± 0.1 Day ± ± ± ± ± ± ± ± ± 0.8 Day ± ± ± ± ± ± ± 4 3 ± ± 0.9 Day ± ± ± ± ± ± ± ± ± 1.2 Day 365 a 1150 ± ± ± ± ± ± ± ± ± 1 N 1522 ± ± ± ± ± ± ± 60 6 ± ± 1 NK, natural killer cells; onset, value related to the same patients at diagnosis of disease. N = reference value in normal individuals (n = 10). a At day 365, analysis was performed in 68 of 72 patients for lymphocytes and in 46 of 50 patients for dendritic cells. At day 180, all the patients were studied (72 of 72 for lymphocytes and 50 of 72 for dendritic cells). Infections Table 4 summarises infections according to stem cell source. A trend towards a higher frequency of infections was observed in the group of patients receiving manipulated graft. No difference was observed with regard to aetiological agents. Sepsis had Gram+ aetiology in the majority of cases: only one Gram sepsis was recorded in the group of selected transplants. After engraftment, only viral infections were recorded, mostly due to herpes reactivation, with no difference between groups (P = 0.001). No bacterial infections were documented. Only one fungal infection (zygomycosis) occurred in a patient receiving CD34+-selected stem cells, causing death 7 months after transplant. Discussion Because of low extra-haematological toxicity and very low transplant-related mortality, ASCT has been extensively used in multiple myeloma patients <70 years old with significant improvement of event-free survival [1, 2]. Nevertheless, relapse inevitably occurs in all patients within a few years of transplant. To explain progression, an important role has been attributed to myeloma cells contained in the apheresis, and on this basis several purging methods have been used in the past years in an attempt to decontaminate the graft [3]. In particular, CD34+ cell selection has been demonstrated as able to significantly reduce apheresis contamination without negatively affecting the haematological reconstitution ability [4 6]. However, as a consequence of the enrichment procedure, there is loss of mature lymphocytes and of circulating antigen-presenting cells that could impair immune reactivity and so increase complications due to infection. In this study we have compared the kinetics of transplant dendritic and immune recovery in two groups of myeloma patients undergoing autologous transplantation with unmanipulated and CD34-selected stem cells. The first 27 patients underwent unmanipulated transplant, while the remaining 45 patients underwent selected transplant. Early and late complications due to infection were also evaluated. Haematological recovery occurred quickly in all patients and only a few days delay in platelet recovery was observed compared to unmanipulated transplant. This could be explained by a selective loss during the selection procedure of committed megakaryocytic precursors, possibly expressing CD34 antigen at low intensity. Lymphocyte subsets were similar to normal at diagnosis and before starting conditioning therapy. Conversely, a delay in immune recovery was observed after transplant, especially in selected patients. In this group, only NK cells quickly reached normal range within 8 weeks after transplant, as observed after unfractioned transplant. These data are in agreement with previous studies in which rapid NK cell reconstitution was observed, with recovery of high cytotoxicity ability and graft versus leukaemia effect [9, 10]. Conversely, Divinè et al. [11] found persistence of altered NK cell subsets. Recovery kinetics of B cells, CD3 and CD8 lymphocytes was not significantly different between groups. B-cell count normalised 6 months after transplant in both groups, and previous studies demonstrated that count normalisation seems to correspond to function normalisation [12], even if lower serum immunoglobulin level concentrations were found after CD34-

5 479 Table 4. Complications caused by infection Selected (%) Unmanipulated (%) None 16/45 (35.5) 15/27 (55.6) Fever 29/45 (65) 12/27 (44) FUO 22/29 (76) 10/12 (83) Gram+ 6/29 (20) 2/12 (16.6) Staphylococcus aureus 1 1 Staphylococcus coagulasi neg. 4 0 Streptococcus α haemoliticus 1 0 Propionibacterium acnes 0 1 Gram 1/29 (4) 0/12 (0) Pseudomonas picketti 1 0 Fungal 1/45 (2) 0/27 (0) Zygomicosis 1 0 Viral 13/45 (29) 8/27 (29) Herpes virus 12 6 Varicella zoster virus 1 1 Cytomegalovirus 0 1 FUO, fever of unknown origin. selected transplant. CD8 cell levels rapidly increased above normal values but returned to reference range within 12 months. On the contrary, as previously observed by Vescio et al. [13], both CD4 and especially CD4 naïve recovery were impaired, with significantly lower counts in CD34-selected patients over the observation period. CD4 memory cells reached a plateau on day 90 and remained stable thereafter, even at lower levels in the selected group. Taken together, these data confirm the hypothesis that T-cell reconstitution after stem cell transplantation depends on expansion of peripheral lymphocyte progenitors contained in the graft or surviving after conditioning therapy, and justify the persistent depression in CD34-selected patients in which reconstitution occurs by maturation from stem cell progenitor through a thymus-independent pathway. Moreover, the absence of thymus function explains the reduction of clonal diversity and the very low CD4/CD45RA cell counts in patients who have undergone T-cell-depleted transplant [14, 15]. We also evaluated the kinetics of dendritic recovery. It must be emphasised that, at diagnosis and before transplant was performed, dendritic cell levels were already significantly lower than normal. So, whether it is a consequence not only of previous chemotherapy but also of an intrinsic characteristic of patients able to impair immune response to malignancies remains to be clarified. Dendritic recovery at pre-transplant level was obtained quickly in unfractioned transplants and was significantly delayed in selected transplants. Similarly, normal references were reached only by unmanipulated grafts. A possible explanation could be the loss of more mature committed dendritic progenitors with low CD34 cell expression during the selection procedure. Despite impaired immune recovery, we did not observe a high incidence of infection complications and no significant difference was observed between groups. In particular, life-threatening infections were very rare: a Gram bacteria leading to invasive fungal infection was recorded in only one patient. During aplastic phase the majority of infections were associated with a fever of unknown origin and responded well to antibiotic therapy. Among documented infections, a predominance of Gram+ bacteria was observed, probably related to insertion of a central venous catheter. In general, the fast neutrophil recovery accelerated by G-CSF administration can account for the absence of infection-related mortality. Mucositis due to an alkylating-based conditioning regimen can explain the high frequency of herpes simplex infections. Reactivation of herpes zoster occurred in 22 cases during the first year. Similar complications were recorded by Dreger et al. [16], without a significant difference between patients receiving CD34-enriched or unfractioned graft, and by Vescio et al. [13] in a series of multiple myeloma cases. In contrast, Friedman et al. [17] reported a significant increase in the incidence of bacterial infections in lymphoma patients transplanted with CD34-enriched cells compared with unmanipulated peripheral blood stem cells within the first year after transplantation, suggesting that the reduced number and function of T lymphocytes may affect the activation and maturation of B cells. In conclusion, CD34 enrichment is not associated with a significant increase of infections, perhaps due to the effect of the relatively fast NK cell recovery and to the presence of functionally efficient DCs. The impact of impaired immune reconstitution on long-term outcome and the actual advantage of CD34+ selection on transplant outcome remains to be evaluated. Acknowledgements Supported by Programmi di ricerca di rilevante interesse nazionale- Cofinanziamento References 1. Samson D. High-dose therapy in multiple myeloma. Curr Opinion Haematol 1996; 3: Bjorkstrand B. European Group for Blood and Marrow Transplantation Registry. Studies in multiple myeloma. Semin Hematol 2001; 38: Gazitt Y, Reading CC, Hoffman R et al. Purified CD34+ Lin Thy+ stem cells do not contain clonal myeloma cells. Blood 1995; 86: Lemoli RM, Fortuna A, Motta MR et al. Concomitant mobilisation of plasma cells and haemopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumour cells. Blood 1996; 87: Schiller G, Vescio R, Freytes C et al. Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 1995; 86: Gupta D, Bybee A, Cooke F et al. CD34+-selected peripheral blood progenitor cell transplantation in patients with multiple myeloma: tumour cell contamination and outcome. Br J Haematol 1999; 104: Galy A, Rudraraju S, Baynes R, Klein J. Recovery of lymphocytes and dendritic cell subsets after autologous CD34+ cell transplantation. Bone Marrow Transplant 2000; 25:

6 Damiani D, Grimaz S, Michieli M et al. Flow cytometry evaluation of plasma cells contaminating leukapheresis pre and post CD34+ positive selection. Br J Haematol 1999; 105: Fegan C, Thomas H, Bailey-Wood R et al. In vitro LAK (lymphokine activated killer) activity following autologous peripheral blood stem cell is significantly greater than that following autologous bone marrow and allogeneic bone marrow transplantation. Bone Marrow Transplant 1995; 16: Scheid C, Pettengell R, Ghielmini M et al. Time-course of the recovery of cellular immune function after high-dose chemotherapy and peripheral blood progenitor cell transplantation for high-grade non- Hodgkin s lymphoma. Bone Marrow Transplant 1995; 15: Divinè M, Boutolleau D, Delfau-Larue MH et al. Poor lymphocytes recovery following CD34+-selected autologous peripheral blood stem cell transplantation for non-hodgkin s lymphoma. Br J Haematol 1999; 105: Nachbaur D, Kropshofer G, Heitger A et al. Phenotypic and functional lymphocyte recovery after CD34+-enriched versus non-t cell-depleted autologous peripheral blood stem cell transplantation. J Hematother Stem Cell Res 2000; 9: Vescio R, Schiller G, Stewart AK et al. Multicenter phase III trial to evaluate CD34+-selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood 1999; 93: Heitger A, Winklehner P, Obexer P et al. Defective T-helper cell function after T-cell depleting therapy affecting naïve and memory populations. Blood 2002; 99: McDonald HR, Blanc C, Lees RK, Sordat B. Abnormal distinction of T cell subsets in athymic mice. J Immunol 1986; 136: Dreger P, Viehmann K, von Neuhoff N et al. Autografting of highly purified peripheral blood progenitor cells following myeloablative therapy in patients with lymphoma: a prospective study of the long-term effects on tumour eradication, reconstitution of haematopoiesis and immune recovery. Bone Marrow Transplant 1999; 24: Friedman J, Lazarus HM, Koc ON. Autologous CD34+ enriched peripheral blood progenitor cell (PBPC) transplantation is associated with higher morbidity in patients with lymphoma when compared to unmanipulated PBPC transplantation. Bone Marrow Transplant 2000; 26: