Immunologic recovery after hematopoietic cell transplantation with nonmyeloablative conditioning

Experimental Hematology 31 (2003) 941–952

Immunologic recovery after hematopoietic cell transplantation with nonmyeloablative conditioning Michael Marisa,b, Michael Boeckha,b, Barry Storera,b, Monja Dawsona, Kristen Whitea, Michael Kenga, Brenda Sandmaiera,b, David Maloneya,b, Rainer Storba,b, and Jan Storeka,b a

Fred Hutchinson Cancer Research Center, Seattle, Wash., USA; bUniversity of Washington, Seattle, Wash., USA (Received 13 May 2003; revised 10 June 2003; accepted 23 June 2003)

Objective. We studied immune reconstitution in 51 recipients of HLA-identical hematopoietic cellular transplant (HCT) after nonmyeloablative conditioning compared to a reference group of 67 recipients after myeloablative conditioning. Methods. Nonmyeloablative conditioning consisted of 2 Gy total-body irradiation ⫾ fludarabine and postgrafting cyclosporine and mycophenolate mofetil. All patients received G-CSF–mobilized peripheral blood mononuclear cells. Patients were followed with serial assessments of lymphocyte subset counts, antibody levels, virus-induced lymphoproliferation, and limiting-dilution assays for cytomegalovirus (CMV) T helper (TH) cells. Rates of infections over the first year after transplant were calculated. Results. During the first 180 days, absolute lymphocyte subset counts were similar (except higher total and memory B cell counts on day 80 in nonmyeloablative patients). At 1 year, however, total and naı¨ve CD4 counts, and naı¨ve CD8 counts, were higher in myeloablative patients. The levels of antibodies were similar at all time points and after vaccinations. The function of CD4 cells assessed by virus-induced lymphoproliferation was similar. However, the absolute counts of CMV TH cells were higher at days 30 and 90 (p ⫽ 0.002 and p ⫽ 0.0003, respectively) after nonmyeloablative conditioning. The rates of definite infections were lower for nonmyeloablative patients during the first 90 days, but were higher later. The higher number of CMV-specific T cells days 30 and 90 after nonmyeloablative HCT coincided with a lower rate of CMV infections during that time. Conclusion. The immunity of nonmyeloablative HCT recipients appears better than the immunity of conventional HCT recipients early, but not late, after HCT. 쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc.

Conventional myeloablative allogeneic HCT is accepted therapy for young, otherwise healthy patients with a variety of malignant and nonmalignant diseases of hematopoiesis. Many groups of investigators have extended the benefits of HCT to patients considered ineligible for conventional HCT with the use of reduced-intensity conditioning regimens [1– 7]. These regimens, generally, derive therapeutic benefit from graft-vs-tumor (GVT) responses. The degree of immunologic impairment after reducedintensity conditioning has not been extensively studied [8– 10]. It is reasonable to expect that the intensity of cytotoxic conditioning and degree of peritransplant host T-cell depletion may effect the incidence of infections and immunologic

Offprint requests to: Michael Maris, M.D., Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., D1-100, P.O. Box 19024, Seattle, WA 98109-1024; E-mail: [email protected]

reconstitution [1,4,11–13]. The immunologic reconstitution after very-low-intensity conditioning with 2 Gy of totalbody irradiation (TBI) with or without fludarabine followed by postgrafting immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) has not been defined. Very-low-intensity nonmyeloablative conditioning may lend to better immune reconstitution and lower risk of opportunistic infections relative to intensive conditioning for several reasons. First, after nonmyeloablative conditioning, there is a low risk of severe and protracted neutropenia, directly reducing the risks of opportunistic bacterial and fungal infections [4]. Second, important mucosal and dermal barriers remain intact after nonmyeloablative conditioning, greatly reducing the risk of bacterial translocation into soft tissues and the bloodstream. Finally, host T cells (mixedchimerism) [4,14] that survive nonmyeloablative conditioning may continue to be immunologically competent, thereby

0301-472X/03 $–see front matter. Copyright 쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 1 0. 10 1 6 / S0 3 0 1- 4 7 2X ( 0 3) 0 0 20 1 - 7

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M. Maris et al. / Experimental Hematology 31 (2003) 941–952

preventing infectious complications from impaired cellular immunity. Several retrospective studies have compared infectious complications after very-low-intensity nonmyeloablative conditioning. One study defined a delayed onset of bacterial infections and skewing of bacterial infections from typical gut-related species to central catheter-associated species in nonmyeloablative compared to conventional transplant recipients [15]. These findings were consistent with the lower incidence of neutropenia and lack of mucositis in the nonmyeloablative patient group. Another study observed a delay in the onset of cytomegalovirus (CMV) infections after nonmyeloablative hematopoietic cellular transplant (HCT) and this was presumed to be related to better surveillance of CMV from persistent CMV-specific host T cells within the first 3 months after transplant [14]. Both studies showed that later after HCT, the overall incidence of bacterial and CMV infections became similar, usually in the context of late-onset acute graft-vs-host disease (GVHD) and the initiation of corticosteroids. This suggested that the early immunologic protective benefits of nonmyeloablative conditioning could be abrogated by GVHD and/or the treatment of GVHD. The current study was performed to describe immune functions over the first year after HLA-matched sibling HCT using nonmyeloablative conditioning with 2 Gy total-body irradiation with or without 90 mg/m2 fludarabine and postgrafting CSP and MMF. The focus was on laboratory parameters of immunity that have previously been associated with increased propensity to develop infections after HCT, particularly CD4 T cell counts [16–18], B cell counts [19], or IgG2 levels [20,21]. Enumeration of CMV-specific T helper (TH) cells was performed to determine whether the persistence of host CMV-specific T cells could account for the observed delay in onset of CMV infections. These data were then compared to data collected from a reference group of patients who received HLA-matched sibling HCT after myeloablative conditioning. Finally, infectious complications were enumerated and compared for both studied cohorts.

Patients, materials, and methods Patients Among recipients of nonmyeloablative conditioning at the Fred Hutchinson Cancer Research Center between December 1997 and November 2001, fifty-one patients consented to participate in an institutional review board–approved study to evaluate immunologic recovery. Fifty patients had hematologic malignancies and one had metastatic melanoma. All 51 patients received G-CSF-mobilized peripheral blood mononuclear cells (G-PBMC) from HLAmatched related donors. The nonmyeloablative conditioning consisted of 2 Gy TBI with or without 90 mg/m2 fludarabine (Table 1). This was followed by postgrafting immunosuppression of mycophenolate mofetil for 28 days and cyclosporine until day 35 or 56 with subsequent taper. Fludarabine was added to the nonmyeloablative regimen on May 2, 2000 after a 17% graft rejection was

observed [4]. All subsequent patients received fludarabine in addition to TBI conditioning except most recipients of planned tandem autologous HCTs followed by nonmyeloablative HCTs, since graft rejection was found not to occur in this setting. The characteristics of the patients are shown in Table 1. Results were analyzed through day 365 after HCT. Patients were removed from analysis at times of graft rejection, relapse of underlying malignancy, or donor lymphocyte infusions due to the confounding effects of these variables on immunologic recovery. As reference, we used data from recipients of conventional HLA-matched sibling myeloablative HCT who were transplanted between July 1996 and December 1999 and whose immune recovery was described previously [22]. The myeloablative regimens consisted of high-dose chemotherapy alone or in combination with total-body or total-marrow irradiation. Patients given nonmyeloablative or myeloablative conditioning were similar for most but not all characteristics (Table 1). Quantitation of mononuclear cell (MNC) subsets Blood specimens were drawn from recipients after HCT on about day 30 (median, day 35 for nonmyeloablative and day 32 for myeloablative, respectively), day 80 (median, day 91 and day 79, respectively), day 180 (median, day 187 and day 188, respectively) and day 365 (median, day 375 and day 376, respectively). MNC subsets were enumerated as previously described [22]. Briefly, MNCs were separated by density gradient (Ficoll) centrifugation. The MNCs were stained with fluorochrome-conjugated monoclonal antibodies and subjected to three-color flow cytometry. Each absolute MNC subset count was calculated as the percentage of the MNC subset multiplied by the absolute mononuclear cell count divided by 100. The absolute MNC count consisted of the sum of the absolute lymphocyte count and absolute monocyte count determined by the clinical hematology laboratory. MNC subsets in the hematopoietic cell graft were calculated using the absolute nucleated-cell count in the graft multiplied by the percentage of the MNC subset among total nucleated cells in the graft divided by 100. IgG levels Standard nephelometry was used to determine total IgG levels. Total IgG2 levels were determined by ELISA using kits from the Binding Site (Birmingham, UK). Polio 1 Ig levels and Streptococcus pneumoniae IgG levels (total IgG for 23 pneumococcal serotypes) were determined by ELISA as described [23]. Measurements of Poliomyelitis 1 Ig levels and S. pneumoniae IgG levels were also made 4 weeks after immunization with pneumococcal nonconjugated polysaccharide vaccine (typically Pnu-Immune, Wyeth/Lederle, Philadelphia, PA, USA, 0.5 mL intramuscularly) and formalin-inactivated poliovirus vaccine (IPOL, Pasteur/Connaught, Swiftwater, PA, USA 0.5 mL subcutaneously) at 1 year. Lymphoproliferation assays Proliferation of lymphocytes upon stimulation with CMV, herpes simplex virus (HSV), and varicella zoster virus (VZV) was measured as previously described [24–26]. Briefly, MNCs separated by Ficoll centrifugation were suspended at a concentration of 2 × 106 cells/mL in RPMI with 10% human serum albumin, glutamine (4 mM), penicillin (100 U/mL), streptomycin (0.1 mg/mL), and amphotericin B (250 ng/mL). One hundred µL of peripheral

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Table 1. Characteristics of patients

Patients’ median (range) age at Tx, y Donors’ median (range) age at Tx, y Patients’ gender (m/f) Donor-patient histocompatibility HLA-A-, B-, and DR-matched sibling HLA-A-, B-, and DR-matched child/parent Disease/disease stage at Tx∗ Good risk Poor risk First Tx CMV serostatus before Tx Donor positive/ recipient positive Donor negative/ recipient positive Donor positive/ recipient negative Donor negative/ recipient negative Unknown or equivocal HSV serostatus before Tx Patient positive Patient negative Unknown or equivocal VZV serostatus before Tx Patient positive Patient negative Unknown or equivocal Patients postsplenectomy Tx conditioning Low-dose TBI (2 Gy) Low-dose TBI (2 Gy) and FLU High-dose chemotherapy High-dose chemotherapy and high-dose TBI (12 Gy) High-dose chemotherapy and high-dose TMI (900 Gy) GVHD prophylaxis CSP/MMF CSP/MTX Acute GVHD Grade 0–1 (%) Grade 2–4† (%) Chronic GVHD diagnosed before day 365 None or clinically limited Clinically extensive‡ Not applicable (relapse or death by day 100) Chimerism status Full chimera by day 80 (⬎90% marrow cells of donor origin) Partial chimera by day 80 (⬍90% marrow cells of donor origin) Unknown Relapse or DLI between days 0 and 365|| Death without relapse or DLI between days 0 and 365 Glucocorticoid treatment between days 0 and 365 Day 0–30 Day 31–90 Day 91–365 CMV prophylaxis Ganciclovir if pp65-antigenemia positive Infusion of ex vivo–expanded CMV-specific T cells¶ Viral prophylaxis with acyclovir/valacyclovir/famciclovir beyond standard# Yes No Unknown/ death before day 30

Nonmyeloablative conditioning (n ⫽ 51)

Myeloablative conditioning (n ⫽ 67)

52 49 33/18

43 40.5 44/23

51 0

65 2

46 5 29

36 31 67

21 10 8 12 0

16 19 14 18 0

36 10 5

46 15 6

0.93

44 5 2 2

62 3 2 3

0.50 0.88

23 28 0 0 0

0 0 27 36 4

⬍0.001

51 0

0 67

⬍0.001

18 (35%) 33 (66%)

25 (37%) 42 (63%)

0.82

10 29 12

16 37 14

0.84

28 7 16 18 10 39 5 22 30

48 1 18 12 14 41 24 31 28

0.03 0.86 0.08 ⬍0.001 0.17 0.09

49 2

63 4

0.61

48 3 0

25 34 8

⬍0.001

p-value ⬍0.001 ⬍0.001 0.91

0.13 ⬍0.001 ⬍0.001

0.24

0.005

(continued)

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M. Maris et al. / Experimental Hematology 31 (2003) 941–952

Table 1. (continued)

Bacterial/pneumocystis/fungal prophylaxis with sulfamethoxazole and trimethoprim and/or fluconazole/itraconazole beyond standard∗∗ Yes No Not applicable (early relapse or death) or unknown Prophylaxis with intravenous immunoglobulin†† Median no. of CD34 cells infused (× 106 cells/kg) Median no. of CD4 cells infused (× 108 cells/kg) Median no. of CD8 cells infused (× 108 cells/kg) Median no. of CD20 cells infused (× 108 cells/kg)

Nonmyeloablative conditioning (n ⫽ 51)

Myeloablative conditioning (n ⫽ 67)

p-value

26 10 16 4 10.4 2.59 1.21 0.930

55 0 12 10 7.4 1.71 0.73 0.575

⬍0.001 0.23 ⬍0.001∗∗∗ ⬍0.001∗∗∗ ⬍0.001∗∗∗ 0.005∗∗∗

Values are numbers (percentages) of patients unless otherwise indicated. The significance of differences between patients receiving nonmyeloblative and myeloablative conditioning was assessed by either t-test (age and neutrophil engraftment) or log rank test (all other variables). Tx indicates transplantation; y, years; CMV, cytomegalovirus; HSV, herpes simplex virus; VZV, varicella zoster virus; TBI, total-body irradiation; Gy, Gray; FLU, fludarabine, TMI, total-marrow irradiation; GVHD, graft-vs-host disease; CSP, cyclosporine; MMF, mycophenolate mofetil; MTX, methotrexate. ∗Good risk was defined as the presence of chronic myelogenous leukemia (CML) in first chronic or accelerated phase, acute leukemia in first remission, refractory anemia, or myelofibrosis. Poor risk was defined as the presence of CML in blast crisis, acute leukemia beyond first remission, refractory anemia with excess blasts, lymphoma, or multiple myeloma. † Typically treated with prednisone (1–2 mg/kg per day orally for 10–14 days with subsequent tapering during 50 days). ‡ Typically treated with prednisone (0.5–1.0 mg/kg given orally every other day) with cyclosporine (6 mg/kg given orally) for at least 9 months. || For patients with CML, detection of bcr/abl transcript by reverse transcriptase polymerase chain reaction in the absence of cytogenetic or hematologic manifestations of CML was not considered a relapse. ¶ Values were typically 1 × 109/L CD4 cells/m2 and 2 × 109 CD8 cells/m2 of body surface area. # According to standard practice all patients received acyclovir (800 mg twice a day given orally) for at least 30 days. Patients were given acyclovir, valacyclovir, or famciclovir between days 30 and 365 and are included here. ∗∗According to standard practice all patients received prophylactic sulfamethoxazole and trimethoprim until day 180 after transplantation (800 mg sulfamethoxazole and 160 mg trimethoprim given orally twice a day, given Monday and Tuesday) and fluconazole until day 75 (400 mg given every other day). After the end of the routine bacterial/pneumocystis/fungal prophylaxis, some patients or physicians chose to continue prophylactic antibiotics; the patients are counted here. Typically, these were patients with clinically extensive chronic GVHD who were treated with immunosuppressive drugs, sulfamethoxazole and trimethoprim (800 mg and 160 mg, respectively, given orally twice a day every Monday and Tuesday), penicillin (500 mg given orally twice a day every day), and rarely fluconazole or itraconazole. †† Typically, 200 mg/kg was given weekly before day 100 after transplantation, and 500 mg/kg was given monthly after day 100. ∗∗∗The reason for the significant difference in the numbers of cells infused is that the myeloablative patients typically receive one apheresis collection whereas the nonmyeloablative patients received two apheresis collections.

blood MNC suspension was placed into wells of 96-well roundbottomed plates. CMV, HSV, and VZV were grown in sheep foreskin, and viral antigens were then prepared by sonication and heat inactivation of virus. At day 0, CMV, HSV, or VZV antigens at 10 µg/mL were added to triplicate wells containing MNCs. The plates were incubated at 37⬚C in humidified 5% carbon monoxide (CO2) environment for 4 days. Two µCi of tritium thymidine were added to each well 18 to 24 hours prior to harvesting. The relative uptake of thymidine (∆ cpm [counts per minute]) was calculated by subtracting the cpm of cells exposed to medium alone from the mean cpm of cells exposed to antigen. As an index of function of specific TH cells, ∆ cpm per 1000 CD4 T cells was calculated. Precursors of CMV-specific TH cells were enumerated by limiting-dilution assay (LDA) as follows. One hundred µL of peripheral blood mononuclear cells (PBMC) were plated in 96well round-bottom plates in 8 serial twofold dilutions starting from 105 cells per well. For each dilution level, 24 replicates received 50 µL CMV antigen and 12 replicates received 50 µL mock antigen (same preparation and dilution as CMV antigen with uninfected human foreskin fibroblasts). Each well also received 104 gamma irradiated (3300 rad) autologous PBMC (in 50 µL) to serve as

antigen-presenting cells. After 5 days of incubation at 37⬚C, 5% CO2, the wells were pulsed with 0.6 µCi 3H and harvested after 18 to 24 hours. Wells were scored positive if the cpm were greater than 3 × mean cpm in the corresponding mock wells. The CMV-specific precursor frequency was determined using the X2 minimization technique [27] with a computer program written by L. Siviuek (provided by C. Orosz, both Ohio State University, Columbus, OH, USA) [28]. Enumeration of infections Definite infection was defined as the microbiological identification of a pathogen from culture from a sterile or nonsterile site (if judged to be clinically pathogenic), or by histologic or immunohistochemical techniques. The only exception to this definition was dermatomal zoster, in which the clinical diagnosis was considered sufficient to classify the infection as definite. Definite infections could be symptomatic or asymptomatic (e.g., bacteremia, viremia, or fungemia). Data from some of the patients reported here were also included in the analysis of infections in nonmyeloablative HCT by Junghanss et al. [14,15].

M. Maris et al. / Experimental Hematology 31 (2003) 941–952

Clinical infection was defined as illness with signs and symptoms consistent with an infection, but with no microorganism identified. Presumed infections of oral cavity, gastrointestinal tract, conjunctivae, and upper respiratory tract were not included because these syndromes could not be reliably distinguished from GVHD or allergies. Fevers of unknown origin were considered clinical infections if fever of greater than 38.5⬚C responded within 3 days to antibiotic therapy. Radiological documentation was required for pneumonia and sinusitis to be included as clinical infections. Infections with one microorganism in two noncontiguous organs were counted as two infections. The upper respiratory tract was considered contiguous to the paranasal sinuses and lungs. The paranasal sinuses and the lungs were not considered contiguous. An organ infection in the context of bacteremia, viremia, and fungemia was considered as a single infection. Severe infections were considered those identified and/or acutely treated while hospitalized. Nonsevere infections were considered those identified and treated as an outpatient. Infection rates were evaluated at three time periods: early, days 0–30; intermediate, days 31–90; and late, days 91–365. The reason these time points were chosen was that between days 0 and 30 the nonmyeloablative HCT recipients have higher neutrophil counts than recipients of myeloablative HCTs [29]. Between days 31 and 90, 10 to 100 (median 60) percent of T cells can be of donor origin after nonmyeloablative HCT [4]. There were no significant changes in microbial resistance patterns, hospital infection rates, or antibiotic usage during the time periods that the nonmyeloablative and myeloablative conditioning recipients were transplanted except that prophylactic acyclovir administration was extended for 1 year posttransplant in 2000 as reflected in Table 1. Death associated with a definite infection was defined as 1) autopsy findings consistent with infection and detection of pathogen from an autopsy specimen, or 2) death after a definite infection that was judged to have caused death directly or indirectly. Chimerism analyses Chimerism analyses were performed in marrow nucleated cells and flow sorted peripheral blood CD3⫹ T cells and CD33⫹ myeloid cells. Percentages of donor-host chimerism for recipients of sexmismatched HCT were evaluated by fluorescent in situ hybridization (FISH) for X- and Y-chromosomes [30], while those for recipients of sex-matched HCT were based upon polymerase chain reaction– based amplification of variable number tandem repeat (VNTR) sequences unique to donors and hosts [31]. Quantitation of chimerism using VNTR was by visual inspection of silver-stained agarose gels with a standard error of 1 to 5%. Statistical analysis The significance of differences between nonmyeloablative and myeloablative conditioning HCT recipients in MNC-subset counts, serum Ig levels ∆ cpm at each time point, and other continuous variables was tested by the Mann-Whitney rank sum test. Differences in categorical variables were evaluated with the Chi-squared test. The number of infections was treated as a Poisson random variable. Regression models were fit with the SAS Genmod (SAS Institute, Cary, NC, USA) procedure using the log of the number of days at risk as a fixed predictor (offset). All p-values (except those from the Mann-Whitney) are based on likelihood-ratio statistics, and are two-sided.

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Results MNC subsets Figure 1 shows the results of enumeration of MNC subsets after nonmyeloablative and myeloablative HCT. Similar total B cell counts and IgD⫹ (naı¨ve) B cell counts were found for both patient groups except at day 80 after HCT, when significantly higher total B cell (p ⫽ 0.01) and IgD⫹ B cell (p ⫽ 0.016) counts were noted among the nonmyeloablative patients. IgD⫺ (memory) B cells were similar at early time points, but a trend toward higher cell counts was found at day 365 for the myeloablative recipients (p ⫽ 0.063). There were no differences in B cell subset counts in nonmyeloablative patients who did or did not receive fludarabine. There were no statistical differences in total CD4 cell counts observed among the two patient groups, but at day 365 there tended to be higher CD4 counts in the myeloablative group (p ⫽ 0.07) (Fig. 1). The counts of naı¨ve (CD45 RAhigh) CD4 T cells appeared to be higher in nonmyeloablative patients on day 30 (p ⫽ 0.085), but were higher in myeloablative patients at day 365 (p ⫽ 0.037). The numbers of CD4 memory/effector T cells (CD45 RAlow/⫺) were not significantly different in the two groups. There were higher CD4 T cell counts on day 30 in nonmyeloablative patients who did not receive fludarabine compared to those who did (median 374/µL vs 180/µL, respectively; p ⫽ 0.044), but there were no differences at other time points. Consistent with this finding were higher CD4 subset counts only at day 30 for CD28⫹ (p ⫽ 0.075), CD28⫺ (p ⫽ 0.035), and CD45 RAlow/⫺ cells (p ⫽ 0.055) for patients who did not receive fludarabine compared to those who did. Cell subsets found not to be significantly different at any time point after transplant included total CD8 T cells, CD11ahigh (memory/effector) CD8 T cells, CD28⫹ CD8 T cells, CD28⫺ CD8 T cells, CD28⫹ CD4 T cells, CD28⫺ CD4 T cells, natural killer cells, and monocytes. Similar to naı¨ve CD4 T cells, naı¨ve CD8 T cells (CD11alow) were higher in myeloablative patients at day 365 (p ⫽ 0.01). Cell subsets found not to be significantly different at any time point after transplant between nonmyeloablative conditioning recipients who did and did not receive fludarabine included total CD8 T cells, CD11ahigh (memory/effector) CD8 T cells, CD28⫹ CD8 T cells, CD28⫺ CD8 T cells, CD45 RAhigh CD4 T cells, natural killer cells, and monocytes. Multivariate analyses of CD4 and CD8 counts after HCT were performed, adjusting for patient age and CD4 or CD8 cell doses in the G-PBMC grafts (Table 2). The multivariate analysis resulted in a less significant trend towards higher CD4 counts in myeloablative patients at day 365 (p ⫽ 0.13), but a more significant trend developed for higher CD8 counts (p ⫽ 0.06) in these patients. Serum antibody levels The levels of total IgG, total IgG2, and antibodies specific for S. pneumoniae and poliomyelitis virus in patients not

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Figure 1. Median MNC-subset counts in transplant recipients on days 30, 80, 180, and 365 after nonmyeloablative (black) and myeloablative (gray) conditioning. Error bars indicate the 25th to 75th percentiles. Stars indicate a significant difference (p ⫽ ⬍0.05). Our normal values are shown as horizontal lines (thick solid line for the 10th and 90th percentiles and broken line for the median). Days after transplant are shown on all x-axes. On all y-axes, values for cell counts are per microliter of blood. The numbers of nonmyeloablative and myeloablative transplant recipients studied are given in Table 2.

given intravenous immunoglobulin after HCT were similar between the nonmyeloablative and myeloablative HCT recipients at all time points, including 4 weeks after immunization with pneumococcal nonconjugated polysaccharide vaccine at 1 year (Fig. 2). In addition, there were no differences after transplant in total IgG2 and antibodies specific for S. pneumoniae and poliomyelitis virus in nonmyeloablative patients who received fludarabine compared to those who did not. The only exception was lower day-30 S. pneumoniae and poliomyelitis virus antibodies in nonmyeloablative patients who did not receive fludarabine (p ⬍ 0.05 for

both), which may have been due to a disproportionate number of multiple myeloma patients with recent autologous transplants in that patient cohort. Lymphoproliferation upon CMV, HSV, and VZV stimulation T helper cell function was evaluated as ∆ cpm per 1000 CD4 T cells. We focused on patients with latent or active infections as no lymphoproliferative responses are expected from uninfected individuals. Thus, CMV (HSV, VZV)-specific lymphoproliferations were evaluated in CMV (HSV, VZV)seropositive patients. Donor serostatus was not evaluated,

M. Maris et al. / Experimental Hematology 31 (2003) 941–952 Table 2. Univariate and multivariate analysis of absolute CD4 and CD8 counts, CMV lymphoproliferation per 1000 CD4 cells, and absolute CMV TH cell number after HLA-matched sibling transplantation after nonmyeloablative and myeloablative conditioning Medians NM/MA CD4 count day 30 day 80 day 180 day 365 CD8 count day 30 day 80 day 180 day 365 CMV∆ cpm‡ day 30 day 80 day 365 CMV TH‡ day 30 day 80 day 365

NM

p-value MA

Unadj∗

Adj†

24/25 23/40 10/26 17/30

262 150 216 261

186 162 339 432

0.12 0.66 0.20 0.07

0.44 0.56 0.22 0.13

24/45 23/40 10/26 17/30

124 105 193 215

84 134 605 440

0.30 0.53 0.15 0.19

0.70 0.20 0.19 0.06

10/22 9/21 4/14

290 361 516

120 366 683

0.22 0.50 0.96

0.53 0.58 0.21

15/13 12/15 10/13

73,300 165,000 175,000

700 12,400 78,300

0.002 0.0003 0.08

⬍0.0001 ⬍0.0001 0.21

NM-nonmyeloablative; MA-myeloablative; Unadj-unadjusted; Adj-adjusted; ∆ cpm-delta counts per minute; Th-T helper cell. ∗Wilcox rank-sum test (Mann-Whitney) † regression analysis of ranks, adjusted for CD4 or CD8 cell dose and patient age ‡ excludes donor CMV⫹, recipient CMV⫺ or donor CMV⫺, recipient CMV⫺

except for CMV, in which case it was documented that the percentages of seropositive donors were similar in the myeloablative (45%) and nonmyeloablative groups (56%). The helper function to viral stimulation was similar in nonmyeloablative and myeloablative patients except for

Figure 2. Median IgG levels and antigen-specific antibody levels in recipients of nonmyeloablative (black) and myeloablative (gray) conditioning. Error bars indicate the 25th to 75th percentiles. Days (D) after transplant are shown on all x-axes. Day 30 and day 395 IgG2 levels were not studied. Normal adult reference (2.5th to 97.5th percentile) ranges are shown in the thick horizontal lines in each graph. For total IgG2, the numbers of nonmyeloablative and myeloablative patients studied were 29 and 25, respectively, on day 30, 31 and 27 on day 80, 11 and 32 on day 180, and 17 and 21 on day 365. For Streptococcus pneumoniae IgG, the numbers of recipients studied were 28 and 27, respectively, on day 30, 30 and 39 on day 80, 11 and 36 on day 180, 17 and 31 on day 365, and 8 and 21 on day 395. For polio Ig, the numbers of recipients studied were 26 and 26, respectively, on day 30, 26 and 38 on day 80, 7 and 33 on day 180, 17 and 33 on day 365, and 10 and 22 on day 395. The black arrows indicate vaccinations for S. pneumoniae and polio virus, respectively. No statistical difference was found at any time point for the levels studied.

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better function in response to HSV stimulation in myeloablative patients at day 365 (p ⫽ 0.01) (Fig. 3). This difference may perhaps be due to a greater number of the nonmyeloablative patients being on acyclovir late posttransplant. The quantity of CMV-specific TH precursors was evaluated by LDA and was expressed as the number of CMVspecific TH cells/L of blood (Fig. 4). This number was significantly higher for nonmyeloablative patients at day 30 (p ⫽ 0.002) and day 80 (p ⫽ 0.0003), with a trend towards higher numbers on day 365 (p ⫽ 0.08). Insufficient data points were available to make a valid comparison at day 180. Higher numbers of CMV-specific TH precursors were also seen when the CMV-seropositive patient and CMVseronegative donor combinations were evaluated. However, this was statistically significant only at day 80 (p ⫽ 0.002). There were no differences after transplant in numbers of CMV-specific TH precursors in nonmyeloablative patients who received fludarabine compared to those who did not at any time point. Multivariate analyses of CMV TH function per 1000 CD4 cells and CMV TH precursor counts were performed adjusting for patient age and CD4 cell dose in the G-PBMC graft (Table 2). On multivariate analysis, there was no difference in CMV TH function after nonmyeloablative compared to myeloablative HCT. However, the multivariate analysis confirmed significantly higher numbers of CMV TH-specific cell numbers after nonmyeloablative HCT at days 30 and 80, but the trend towards higher CMV TH-specific cell numbers at day 365 was lost (p ⫽ 0.21). Collectively, any improved CMV immune function early after nonmyeloablative HCT was the result of higher CMV-specific T cell numbers rather than better function of individual CMVspecific T cells. It should be noted that median day of onset of GVHD for patients who underwent CMV analysis was

Figure 3. Results of lymphoproliferation assays using stimulation with CMV, HSV, and VZV per 1000 CD4⫹ cells. The symbols are the same as in Figure 1. Only HSV- and VZV-seropositive recipients were included in the analysis of HSV- and VZV-induced proliferation. In the CMV analysis, CMV⫹ recipients with CMV⫹ or CMV⫺ donors were included. The numbers of nonmyeloablative and myeloablative conditioning recipients studied for CMV were 11 and 27, respectively, on day 30, 12 and 21 on day 80, 0 and 18 on day 180 (data points were excluded), and 6 and 14 on day 365. For HSV, the numbers of nonmyeloablative and myeloablative conditioning recipients studied were 16 and 37, respectively, on day 30, 15 and 29 on day 80, 4 and 22 on day 180, and 12 and 16 on day 365. For VZV, the numbers of nonmyeloablative and myeloablative conditioning recipients studied were 20 and 31, respectively, on day 30, 18 and 33 on day 80, 5 and 32 on day 180, and 13 and 25 on day 365.

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Figure 4. CMV T helper (TH) cell precursors per liter of blood. The symbols are the same as in Figure 1. Stars indicate a significant difference (p ⬍ 0.05). Only CMV-seropositive recipients with CMV-seropositive or CMV-seronegative donors were included in the analysis (A). The numbers of nonmyeloablative and myeloablative conditioning recipients studied were 18 and 13, respectively, on day 30, 16 and 15 on day 80, and 12 and 13 on day 365. (B): Only CMV-seropositive recipients with CMV-seronegative donors were included in the analysis. The numbers of nonmyeloablative and myeloablative conditioning recipients studied in this analysis were 4 and 7, respectively, on day 30, 5 and 8 on day 80, and 3 and 8 on day 365.

day 21 in patients given myeloablative conditioning and day 35 for patients given nonmyeloablative conditioning. This 2-week difference in the onset of GVHD may have affected the ability of the myeloablative conditioning recipients to expand CMV-specific T cells. Infections The overall (days 0–365) rates of total definite infection were greater in patients given myeloablative (0.62/100 days) compared to nonmyeloablative (0.52/100 days) conditioning (p ⫽ 0.03). As shown in Table 3, myeloablative recipients had higher rates of definite infections than nonmyeloablative recipients during the early (days 0–30, p ⫽ ⬍0.0001) and intermediate (days 31–90, p ⫽ 0.009) time periods. Conversely, nonmyeloablative patients had higher rates of definite infections at the later time point (days 91–365, p ⫽ 0.0004). The differences were caused by higher rates

of bacterial, viral, and fungal infections between days 0 and 30 and higher rates of viral and fungal infections between days 31 and 90 in the myeloablative HCT recipients. The nonmyeloablative HCT recipients had higher rates of late (days 91–365) bacterial infections. It should be noted that the myeloablative HCT recipients did have higher rates of late clinical infections (those where no definite organism was found) so that total infection rates in the late period were similar (Table 3). The infection rates were compared between nonmyeloablative conditioning recipients that did and did not receive fludarabine (Table 4). There were no statistical differences in the rates of viral, fungal, and bacterial infections at the early (days 0–30), intermediate (days 31–90), and late time periods (days 91–365), with the only exception being a large difference in rates of bacterial infections at the later time point (p ⬍ 0.0001). To provide a clinical correlation to the in vitro studies of CMV TH cell numbers, the rates of CMV infections were compared. Significantly lower rates of CMV infections were identified between days 31 and 90 (0.22/100 days vs 0.03/100 days, p ⫽ 0.03) after nonmyeloablative HCT, but the rates were not different between days 0 and 30 and days 91 and 365. Deaths between days 0 and 365 associated with definite infection diagnosed between days 0 and 365 occurred in 5 patients (10%) given nonmyeloablative and 8 patients (12%) after myeloablative conditioning (hazard ratio ⫽ 0.76, 95% CI 0.2–2.3; p ⫽ 0.63). Chimerism results Peripheral blood chimerism was evaluated in all nonmyeloablative patients and not in myeloablative transplant patients. Figure 5 shows the average percent donor chimerism at days 28, 56, 84, 180, and 365 for all patients (A) and those who had LDA performed (B). All nonmyeloablative patients engrafted with mixed T-cell chimerism at day 28 except for three patients who showed “full-donor” chimerism (⬎95% T-cell chimerism). No patients had graft rejection. Bone marrow chimerism was compared at 3 months posttransplant for nonmyeloablative and myeloablative conditioning recipients. There was lower marrow chimerism among nonmyeloablative compared to myeloablative patients at day 80 (p ⫽ 0.005) (Table 1).

Discussion The major goal of this study was to describe the immunity of recipients of nonmyeloablative conditioning and to ascertain whether it markedly differed from the immunity in patients given myeloablative conditioning. Interestingly, many of the parameters studied, such as the total and subset counts of T cells, proliferative ability of T cells to viral stimulation, and antibody levels, were similar after either type of transplant. The exceptions were higher counts of total and naı¨ve

M. Maris et al. / Experimental Hematology 31 (2003) 941–952

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Table 3. Infection rates from days 0 through 30, 31 through 90, and 91 through 365 in patients given nonmyeloablative and myeloablative conditioning Days 0–30

Days 31–90

Days 91–365

Infections/100d∗

Infections/100d∗

Infections/100d∗

MA N Definite infections Definite severe infections Definite nonsevere infections Viral infections CMV Bacterial infections Fungal infections Total definite infections Clinical infections Severe clinical infections Nonsevere clinical infections Total infections

NMA

67

p

51

MA

NMA

55

p

51

MA 50

NMA

p

41

2.86 0.00 0.42 0.05 2.08 0.16 2.65

0.52 0.39 0.07 0.00 0.59 0.00 0.65

⬍.0001 0.002 0.03 0.28 0.0001 0.06 ⬍.0001

0.45 1.37 0.67 0.22 0.67 0.13 1.47

0.41 0.65 0.21 0.03 0.52 0.03 0.76

0.83 0.005 0.006 0.03 0.44 0.19 0.009

0.10 0.09 0.06 0.01 0.10 0.02 0.18

0.33 0.12 0.08 0.02 0.29 0.07 0.45

0.0001 0.48 0.60 0.35 0.001 0.04 0.004

0.36 0.16 3.17

0.20 0.07 0.92

0.35 0.42 ⬍.0001

0.13 0.16 1.76

0.38 0.10 1.23

0.05 0.54 0.10

0.05 0.25 0.48

0.09 0.07 0.56

0.27 0.0006 0.20

d-day, CMV-cytomegalovirus, MA-myeloablative, NMA-nonmyeloablative. ∗Number of infections/100 days means the number of infections in all recipients of peripheral blood mononuclear cells divided by the number of days at risk for each time period multiplied by 100.

B cells for nonmyeloablative patients on day 80 (perhaps due to the later onset of GVHD in HCT recipients after nonmyeloablative conditioning [32] as B lymphopoiesis is inhibited by GVHD and/or GVHD treatment [33]) and higher counts of naı¨ve CD4 and naı¨ve CD8 counts in the myeloablative patients on day 365. The significance of the few differences in absolute lymphocyte subset counts is unclear. The lower naı¨ve T cell counts may reflect low counts of recent thymic emigrants [18]. The lower naı¨ve CD4 counts may be explained by the older age and/or later onset of corticosteroid use between days 91 and 365 in the nonmyeloablative patients studied. Age has previously been shown to influence the tempo of recovery of naı¨ve CD4 T cells [34] and glucocorticoid treatment may inhibit thymopoiesis [35]. The most important finding was that nonmyeloablative patients had significantly higher numbers of CMV-specific

helper T cells and lower rates of CMV disease in the context of mixed T-cell chimerism. (Complete chimerism in lymphocyte lineages is typically observed within 30 days after myeloablative conditioning [36,37].) The higher numbers of CMV-specific helper T cells were also found when CMVseropositive patients with CMV-seronegative donors were analyzed. In this donor-recipient combination, the CMVspecific T cells may be mostly of host origin, supporting the hypothesis that these cells survived nonmyeloablative conditioning. The implication of this finding is that persistent host T cells may exist for other pathogens resulting in better overall T-cell immunity for nonmyeloablative HCT recipients. This may be especially true for anti-viral T cells given the much lower viral infection rates early (days 0–90) after nonmyeloablative HCT. Better host immunity after nonmyeloablative conditioning through higher numbers of antigen-specific host immune

Table 4. Infection rates from days 0 through 30, 31 through 90, and 91 through 365 in patients given nonmyeloablative conditioning with or without fludarabine Days 0–30

Days 31–90

Infections/100d∗

N Viral infections Bacterial infections Fungal infections Total definite infections

Days 91–365

Infections/100d

Infections/100d

No Flu

Flu

p

No Flu

Flu

p

No Flu

Flu

p

23 0.14 0.72 0.0 0.87

28 0.0 0.48 0.0 0.48

0.21 0.53 –† 0.34

23 0.22 0.67 0.0 0.90

28 0.19 0.38 0.06 0.64

0.84 0.27 0.27 0.42

19 0.10 0.10 0.04 0.24

20 0.06 0.58 0.12 0.76

0.53 ⬍.0001 0.19 0.0007

d-day, CMV-cytomegalovirus, MA-myeloablative, NMA-nonmyeloablative, No Flu-no fludarabine in HCT conditioning, Flu-fludarabine as part of HCT conditioning. ∗Number of infections/100 days means the number of infections in all recipients of peripheral blood mononuclear stem cells of marrow or blood stem cells divided by the number of days at risk for each time period multiplied by 100. † No events to analyze.

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M. Maris et al. / Experimental Hematology 31 (2003) 941–952

Figure 5. Mean percent donor chimerism after nonmyeloablative HCT. The symbols indicate average donor T-cell chimerism at individual time points. The error bars indicate standard deviations at each time point. The numbers of nonmyeloablative conditioning recipients studied overall were 50 on day 28, 45 on day 56, 44 on day 84, 42 on day 180, and 27 on day 365 (A). Only CMV-seropositive (R⫹) recipients with CMVseropositive or CMV-seronegative donors (D⫺) who had any LDA assay performed were included in the chimerism analysis in (B). The numbers of R⫹/D⫺ recipients studied were 26 on day 28, 24 on day 56, 24 on day 84, 25 on day 180, and 16 on day 365.

cells rather than improved function of residual host immune cells is supported by two observations. First, the proliferation of T cells to CMV, HSV, and VZV stimulation per 1000 CD4 T cells was the same after nonmyeloablative or myeloablative HCT, suggesting equivalent CD4 T cell function after either HCT. Second, much higher numbers of CMV-specific T cells were seen early after nomyeloablative HCT even after statistical adjustment for the higher CD4 cell dose and older patient age. Thus, the higher numbers of CMV-specific helper T cells rather than improved function of CMV-specific T cells may have translated into fewer CMV infectious complications between days 0 and 90. Confounding this conclusion was the earlier use of corticosteroids in the myeloablative HCT group, which could partially account for the difference of CMV infection rates during the early post-HCT period [32]. The persistence of larger

numbers of CMV-specific T helper cells after nonmyeloablative HCT may help explain the delayed onset of CMV disease reported by Junghanss et al. [14], and the importance of host T cells to prevent CMV infection is supported by recent allogeneic transplant studies in the mouse model [38]. Reductions in the rate of infectious complications occurred during the first 3 months but not later after nonmyeloablative HCT. The improved immunity in the first 3 months is most likely due to three major factors that benefit recipients of nonmyeloablative conditioning. These include 1) a lack of mucositis, 2) reduction in severity and duration of neutropenia, and 3) higher number of antigen-specific helper T cells (presumably due to the coexistence of host and donor cells early posttransplant) in the nonmyeloablative patients. In the late HCT period, the rates of infections declined in both patient groups, but more rapidly for the myeloablative patients (Table 3). This could be due to the increasing number of nonmyeloablative patients studied who were treated with glucocorticoids during the late time period. The fact that the number of definite infections in the late period was higher in the nonmyeloablative patients is of importance. It indicates that nonmyeloablative patients remain at risk for serious viral, bacterial, and fungal infections during the first year after HCT. Therefore, close follow-up and aggressive evaluation of symptoms or suspicious radiologic findings in these patients are imperative [15,39]. Within the recipients of nonmyeloablative conditioning, we evaluated whether there were any differences in immunologic parameters and infections between patients that did or did not receive fludarabine conditioning. Interestingly, most parameters were not statistically different, with the exception of CD4 counts and CD4 subset counts at day 30. This may not be too surprising given the more profound depleting affect that fludarabine has on CD4 cells [40]. However, much higher rates of late bacterial infections were observed in the fludarabine-treated cohort, which is not explained by differences in T cell or B cell counts, or antibody levels at the last time point. The higher rate of infections in fludarabine-treated patients has been previously observed in a larger nonmyeloablative patient cohort and has been associated with higher rates of nonrelapse mortality, possibly related to the use of fludarabine in the nonmyeloablative conditioning [41]. The influence of fludarabine on transplant and immunologic outcomes using nonmyeloablative conditioning is the focus of a current randomized trial. It should be noted that the present study was not designed to address the influence of fludarabine on immunologic outcome. This is the first report of immune reconstitution after very-low-intensity conditioning. Other groups of investigators have published results of immune reconstitution studies [8,9] or infection rates [10] after more myelosuppressive reduced-intensity regimens. The study by Savage showed delayed reconstitution of naı¨ve T cells and inability to generate neo-antigen antibody response in the older (nonpediatric)

M. Maris et al. / Experimental Hematology 31 (2003) 941–952

patients given a T cell–depleted PBMC product after reduced-intensity conditioning with cyclophosphamide and fludarabine [8]. The delayed reconstitution of naı¨ve CD4 cell subsets in older patients was consistent with the results of the present study. The study by Morecki evaluated immune reconstitution after fludarabine and busulfan conditioning with antithymocyte globulin [9]. This study found generally intact gross T and B cell subset percentages, intact responses to mitogenic stimulation, and lower number of infections in the reduced-intensity group relative to a conventional HCT group. However, an uncontrolled retrospective series evaluating infectious complications using essentially the same fludarabine, busulfan, and antithymocyte globulin regimen raised concerns that infectious complications may be frequent with this regimen [10], which could be because the conditioning was intense. It should be emphasized that the current study does not represent a comparison of nonmyeloablative and myeloablative patients truly matched for all demographic and clinical characteristics. This would be impossible at present as allogeneic HCTs after nonmyeloablative conditioning at our center are generally reserved for elderly patients or patients with organ dysfunction who are not candidates for myeloablative regimens. However, we have controlled for age in multivariable statistical models of CD4 and CD8 counts, T helper function to CMV stimulation, and number of CMVspecific TH cells. The modeling did not account for the differences in the onset of GVHD and temporal use of corticosteroids between the two groups, which may affect a number of outcomes in this analysis. It should be noted that less-acute GVHD and lower corticosteroid use early posttransplant were observed in a previous study of wellmatched cohorts of HCT patients after nonmyeloablative compared to conventional conditioning [32], suggesting that similarities in the incidence of GVHD observed in the present study may be due to differences in variables such as age, underlying malignancy (and treatment), and stage of disease. If in the future a randomized trial comparing myeloablative to nonmyeloablative conditioning is undertaken, immune reconstitution should be studied in order to confirm or refute the results of this study.

Acknowledgments This work was supported in part by grants AI46108, HL36444, K23CA92058, CA15704, CA18029, and CA78902 from the National Institutes of Health, DHHS, Bethesda, MD, USA. The authors would like to thank Debbie Bassuk for data management and Terry Stevens-Ayers, Patrick Sudour, and Jeremy Smith for assistance with laboratory evaluations.

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