Biology of Blood and Marrow Transplantation
Volume 14, Issue 2 , Pages 208-219, February 2008

Elevated Numbers of Immature/Transitional CD21− B Lymphocytes and Deficiency of Memory CD27+ B Cells Identify Patients with Active Chronic Graft-versus-Host Disease

  • Hildegard T. Greinix

      Affiliations

    • Department of Internal Medicine I, Bone Marrow Transplantation, Medical University of Vienna, Vienna, Austria
    • Corresponding Author InformationCorrespondence and reprint requests: Hildegard T. GREINIX, MD, Medizinische Universitaet Wien, Klinik fuer Innere Medizin I, Knochenmarktransplantation, Waehringer Guertel 18-20 A-1090 Vienna, Austria. Phone: +43-1-40400-5706 Fax: +43-1-40400 2511.
    • ,
  • David Pohlreich

      Affiliations

    • Department of Internal Medicine I, Bone Marrow Transplantation, Medical University of Vienna, Vienna, Austria
    • ,
  • Michal Kouba

      Affiliations

    • Department of Internal Medicine I, Bone Marrow Transplantation, Medical University of Vienna, Vienna, Austria
    • ,
  • Ulrike Körmöczi

      Affiliations

    • Institute of Immunology, Medical University of Vienna, Vienna, Austria
    • ,
  • Imke Lohmann

      Affiliations

    • Institute of Immunology, Medical University of Vienna, Vienna, Austria
    • ,
  • Karin Feldmann

      Affiliations

    • Department of Internal Medicine I, Bone Marrow Transplantation, Medical University of Vienna, Vienna, Austria
    • ,
  • Christoph Zielinski

      Affiliations

    • Department of Internal Medicine I, Bone Marrow Transplantation, Medical University of Vienna, Vienna, Austria
    • ,
  • Winfried F. Pickl

      Affiliations

    • Institute of Immunology, Medical University of Vienna, Vienna, Austria
    • Corresponding Author InformationWinfried F. PICKL, MD Institut fuer Immunologie, Medizinische Universitaet Wien, Borschkegasse, 8°, A-1090 Vienna, Austria, Phone: +43-1-4277-64945, Fax: +43-1-4277-9649.

Article Outline

Abstract 

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT) and a leading cause of non-relapse mortality (NRM). Currently, biology-based markers are lacking both for diagnosis and for monitoring the activity of cGVHD. Seventy patients who received HSCT were enrolled in a pilot study, including 21 without cGVHD and 49 with active or resolved cGVHD. Evaluations were comprised of clinical parameters including cGVHD severity and infections. Peripheral blood cells were analyzed by multi-parameter flow cytometry. The CD19+ B cell compartment was further subdivided by staining for surface IgD, CD21 and CD27. No significant differences in absolute B, T, and natural killer (NK) cell numbers were observed between the groups with and without cGVHD. However, elevated numbers (>15% of B lymphocytes) of immature/transitional CD19+/CD21- B cells were associated with the occurrence of severe infections (P = .003). Most significantly, all patients with active cGVHD and elevated numbers of CD19+/CD21- B lymphocytes experienced severe infections (P = .00016). The numbers of both non-class-switched and class-switched memory B cells were significantly lower in patients with active cGVHD when compared to patients who never experienced cGVHD (P = .002 and P = .001). Perturbation of circulating B lymphocyte compartments may serve as a novel biomarker for monitoring cGVHD activity and its impact on the immune system. A prospective study on unselected patients assessed serially for B cell reconstitution after HSCT is warranted.

Key Words: chronic GVHD, immature/transitional and memory B lymphocytes

 

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Introduction 

Chronic graft-versus-host disease (cGVHD) is a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT) and occurs in 30% to 80% of patients [1]. It varies in severity and clinical course; cGVHD is the leading cause of nonrelapse mortality (NRM) more than 2 years after transplantation 2, 3. After HSCT, immune reconstitution occurs gradually over time 4, 5. Immune reconstitution is known to be slower for human leukocyte antigen (HLA)-mismatched [6] or T cell depleted grafts [7], and in survivors with cGVHD [8].

The pathophysiology of cGVHD is still poorly understood [9]. The importance of autoreactivity is suggested by clinical manifestations of cGVHD that frequently mimic those of autoimmune diseases [10], and by the finding of auto-antibodies derived from B cells after T helper type 2 (TH2) mediated stimulation and cytokine release [11]. Recently, chimeric anti-CD20-antibody therapy was reported to be effective for resistant cGVHD; this supports the hypothesis that distinct B cells or B cell subsets might be directly pathogenic in cGVHD 12, 13. Another probable mechanism for cGVHD is dysfunctional T cell selection in the thymus inducing autoimmune diseases [14]. Functional asplenia, with elevated numbers of erythrocytes bearing Howell-Jolly bodies (HJ) as the hallmark, was observed in patients with cGVHD [15]. Asplenia also affects homeostasis of memory B lymphocytes circulating in the peripheral blood (PB) [16].

In general, B cell development is a stepwise process. B cell precursors are generated in the bone marrow (BM) and migrate to the periphery at the immature/transitional CD19+/CD21- B cell stage when they are still short-lived and functionally immature [17]. Immature/transitional B cells are transported via the blood vessels to the spleen, where they develop into long-lived mature B cells (CD19+/CD21+). This developmental step is critically dependent on the presence of a mature B cell receptor [18] and on tightly regulated additional survival signals brought about by transmembrane proteins such as B cell activation factor from the tumor necrosis factor family (BAFF) [19]. Mature B cells re-circulate between the lymphoid follicles of spleen and lymph nodes, and they play a major role in the adaptive immune response. Significantly, in the initial phases of infection and before specific antibody production commences, natural antibodies limit the spread of pathogens. Subsequently, the antigen-specific, neutralizing antibody response clears cytopathic infections [20]. In fact, all vaccines that are clinically efficient today are dependent on neutralizing antibody responses (rather than T cell-mediated immunity). This demonstrates the unique importance of B cells and their products for protective immunity [20].

Hence, the in-depth analysis of the composition of major B cell sub-populations within a group of patients who underwent HSCT should provide important insights into the patients' level of clinically relevant protective immunity and thus, their overall immuno-competence. This knowledge would especially be warranted in situations where the post-transplant immune system is compromised by ongoing immune responses, such as in cGVHD.

We performed a pilot trial to analyze leukocyte populations in PB and we focused on the homeostasis of major B cell subpopulations. We studied CD21 negative immature/transitional B lymphocytes 21, 22, which are known to be increased in autoimmune diseases, ie SLE [23], and in primary and secondary immunodeficiencies, such as common variable immunodeficiency [24] and advanced stages of HIV [25]. Furthermore, we analyzed the CD27 positive memory B lymphocyte compartment, which harbors the direct precursors of immunoglobulin secreting B cells 26, 27.

No significant differences in absolute B, T, and natural killer (NK) cell numbers between the groups with and without cGVHD were seen. However, in patients with >15% immature/transitional CD 19+/CD21- B cells, significantly more severe infections after HSCT were observed as compared to patients with low CD19+/CD21- B cell counts. The number of both class-switched and non-class-switched memory B cells was significantly lower in patients with active cGVHD compared to patients never experiencing cGVHD in our pilot study. In summary, B lymphocyte subsets might represent novel biomarkers for the assessment of cGVHD activity and its impact on the immune system seen as increased susceptibility to severe infections. Our preliminary findings should be evaluated prospectively in patients after HSCT.

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Methods 

Trial Conduct 

During February 2005, all patients with active cGVHD from the Outpatient Clinic of the Bone Marrow Transplant (BMT) Facility who had routine follow-up visits were asked participate. Study inclusion criteria were complete multi lineage donor cell engraftment, being at least 100 days after allogeneic HSCT, available data on all follow-up visits since discharge after HSCT, and written informed consent. Of these 26 patients sampled, 4 were later considered to have resolved cGVHD at the time of sampling whereas 22 had active cGVHD. After observing the disturbance in B cell homeostasis another 44 patients were asked to join the study in September 2005 and they participated after signing the informed consent. This group included 13 patients with active cGVHD, 10 with resolved cGVHD, and 21 who had never experienced cGVHD. All of these patients were seen in the Outpatient Clinic of the BMT Facility for routine follow-up visits. During the sampling period of February to September 2005, only the following patients were excluded: those who missed follow-up visits; were unable to understand the germane informed consent; refused to participate in the study; with unmeasurably low B-cell numbers; or with a relapse of hematological disease. A total of 70 patients were enrolled into this pilot study that had been approved by the local institutional review board (IRB). The study was conducted in accordance with the declaration of Helsinki. In addition to the analysis of leukocyte subpopulations, blood counts, C-reactive protein, and serum immunoglobulin levels were assessed during routine follow-up examinations of patients. Examinations were done at least weekly during the first 100 days after HSCT, monthly until day 365 after HSCT, then every 3 months for 2 years, followed by every 6 months thereafter.

In 36 patients, B lymphocyte analyses were repeated once (n=7), twice (n=13), three (n=7), or four (n=9) times 6 to 27 months after the first analysis. Three of these 36 patients had never had cGVHD; 8 patients had resolved cGVHD; and 25 patients had active cGVHD.

The Infection Module of Common Toxicity Criteria, National Cancer Institute [28], was used for retrospective evaluation of the severity of infections according to hospital records dating from HSCT until study entry. Patients were stratified into those with never or only mild infectious episodes (CTC grades 0 to II); and severe ones (CTC grades III to IV) requiring i.v. treatment and unexpected hospitalization or being life-threatening. In addition, in all patients, all infectious episodes during the post-transplant period (from day 0 of HSCT until first B lymphocyte analysis) were collected retrospectively and stratified according to clinically defined bacterial, viral, or fungal infection. Clinically defined infection was an infection that resulted in a change of therapy with systemic antimicrobial agents or suspected infection with supporting clinical or radiographic findings. In cases of bacterial, viral, or fungal infection, appropriate documentation of the organism had to be present.

Patients and Therapeutic Modalities 

Transplant and patient characteristics are shown in Table 1, Table 2. cGVHD was diagnosed according to published criteria 2, 9 and treatment response was assessed according to the NIH Consensus Development Project [29]. For the analysis, resolved cGVHD was defined as the complete resolution of all organ manifestations. The active cGVHD group consisted of patients with partial resolution or progression of organ manifestations despite immunosuppressive therapy. Figure 1 shows the time from HSCT until first assessment of B cell subpopulations. In the group never experiencing cGVHD, there was a median of 48 (range, 5 to 127) months compared to a median of 62 (range, 10 to 150) months in the group with resolved cGVHD; and a median of 42 (range, 8 to 122) months in the group with active cGVHD.

Table 1. Characteristics of all Transplant Recipients and Patient Subgroups according to chronic GVHD∗
All (N=70)Never chronic GVHD (N=21)Chronic GVHD (N=49)
number (percent)
Median age in years {range}46.3 {20-66}47.8 {25-62}45.6 {20-66}
Sex
Male46 (66)12 (57)34 (69)
Female24 (34)9 (43)15 (31)
Diagnosis
Acute leukemia39 (56)12 (57)27 (55)
Chronic myelogenous leukemia13 (19)5 (24)8 (16)
Malignant lymphoma9 (13)1 (5)8 (16)
Other†9 (13)3 (14)6 (12)
Disease status at transplantation ‡
Standard risk48 (69)18 (86)30 (61)
High risk22 (31)3 (14)19 (39)
Conditioning regimen
Myeloablative conditioning46 (66)12 (57)34 (69)
Reduced intensity conditioning24 (34)9 (43)15 (31)
Stem cell donors
Related35 (50)13 (62)22 (45)
Unrelated35 (50)8 (38)27 (55)
HLA-identical67 (96)21 (100)46 (94)
HLA-mismatched3 (4)03 (6)
Stem cell source
Bone Marrow22 (31)10 (48)12 (25)
Peripheral Blood Stem Cells48 (69)11 (52)37 (75)
Median number of CD34 + cells x106 /kg transplanted {range}5.5 {0.7-14.0}4.4 {1.2-8.4}5.4 {0.7-14.0}
Post-transplant immunosuppressive prophylaxis
Cyclosporine only2 (3)1 (5)1 (2)
Cyclosporine-Methotrexate49 (70)12 (57)37 (76)
Cyclosporine-Mycophenolate Mofetil19 (27)8 (38)11 (22)
Acute GVHD
Grade 038 (54)15 (71)23 (47)
Grades I-II16 (23)2 (10)14 (29)
Grades III-IV16 (23)4 (19)12 (24)

∗GVHD indicates graft-versus-host-disease.

†Other diagnosis includes aplastic anemia, myelodysplastic syndrome, chronic lymphocytic leukemia, and myeloma.

‡Patients with standard risk were defined as those who received a transplant during the first or second complete remission of acute leukemia or in the first chronic phase of chronic myelogenous leukemia. Patients with high risk were those with myelodysplastic syndrome and a more advanced stage of acute leukemia or chronic myelogenous leukemia than those with standard risk.

Table 2. Characteristics of chronic GVHD Patients
chronic GVHD (N=49)
number (percent)
Onset type of chronic GVHD
De novo24 (49)
Quiescent15 (31)
Progressive10 (20)
Activity of chronic GVHD at study entry
Resolved14 (29)
Active∗35 (71)
Chronic GVHD organ involvement at study entry
Skin20 (41)
Eyes18 (37)
Oral mucosa16 (33)
Liver11 (22)
Lungs11 (22)
Musculosceletal4 (8)
none, 1 or 2 organs35 (71)
> 2 organs14 (29)
Median duration of immunosuppressive therapy for chronic GVHD in months {range}36 (3-104)
Immunosuppressive therapy at study entry
None12 (25)
Cyclosporin/FK50614 (29)
Cyclosporin/FK506 + extracorporeal photoimmunotherapy +/- Steroids9 (18)
Cyclosporin/FK506 + Steroids +/- Mycophenolate mofetil5 (10)
Other†9 (18)

∗Presence of at least 1 diagnostic clinical sign of chronic GVHD as defined by the NIH Consensus 2 and partial response or worsening to therapy as previously defined by Pavletic et al. [29]

†Other include various combinations of above mentioned treatment regimens.

  • View full-size image.
  • Figure 1 

    Time from HSCT until analysis of B cell subpopulations. On the X axis, the 3 patient groups consisting of never cGVHD (no), resolved (res.) and active (act.) cGVHD at the time of first flow cytometric exam are shown; on the Y axis, the months from day 0 of hematopoietic stem cell transplantation (HSCT) until first B cell analysis. Bars represent mean values in each group. P-value between patient groups never cGVHD and active cGVHD was calculated by two-tailed Students t-test.

In the majority of patients, first-line treatment of cGVHD consisted of prednisolone in combination with a calcineurin inhibitor. Patients with cGVHD were under anti-infective prophylaxis in accordance with local guidelines: 1) antibiotic prophylaxis targeting encapsulated bacteria, and 2) Pneumocystis carinii prophylaxis as recommended [30]. In addition, antibiotic prophylaxis before invasive procedures and i.v. immunoglobulins (Ig) in case of hypogammaglobulinemia and recurrent severe infections was given to patients with cGVHD [31].

Isolation of Blood Cells, Immunophenotyping, Flow Cytometry, and Fluorescein in situ Hybridization of Sorted Cells 

Optimal concentrations of directly conjugated monoclonal antibodies (mAbs) were added to 50 μl of whole blood, and incubated for 20 mins at room temperature. Afterwards, 100 μl ADG-lysis solution (An der Grub, Austria) was added, incubated for 10 mins, followed by hypotonic lysis of red blood cells in H2O. Cells were collected by centrifugation; supernatant was discarded; cells were re-suspended in sheath fluid at 4°C (Becton Dickinson, Mountain View, CA) and analyzed by acquiring 5 × 103 cells in the lymphogate for leukocyte subpopulations and 1-3 × 103 CD19+ B cells for B cell subset analysis. Three-color data acquisition was performed on a FACS Calibur flow cytometer, supported by the Cell Quest software (Becton Dickinson). Forward versus side scatter parameters were used to gate on major leukocyte subpopulations. We used mAbs CD3-APC (S4.1), CD16-PE (3G8), CD19-FITC (SJ25-C1), CD21-PE (BU32), CD56-PE (MEM-188), negative control IgG1-APC, and goat affinity purified F(ab‘)2 anti-human IgD antibody from Caltag Laboratories (Burlingame, CA), CD4-FITC (VIT4), CD8-PE (VIT8), CD14-PE (MEM-18), CD45-FITC (VIT200), and negative control mAbs VIAP-FITC and 4H1-A7-PE from An der Grub (Kaumberg, Austria) and CD69-FITC mAb (L78) from Becton Dickinson (San Jose, CA). CD27-FITC (VIT14) was kindly provided by Dr. Majdic. FISH analyses were performed on cytospin preparations of lymphoid subsets in patients after sex-mismatched transplantation as previously described [32].

Statistical Analysis 

Data were evaluated by the Fisher's exact test. Statistical comparisons of numeric data were made using either unpaired or paired Student's t-test. Results were considered statistically significant when P was below .05.

A multiple logistic regression analysis for factors impacting the occurrence of severe infections was performed. In univariate analysis, the variables–“occurrence of cGVHD, conditioning (myeloablative vs reduced-intensity (RIC)), use of antithymocyte globulin, hematopoietic stem cell source (BM vs peripheral blood stem cells [PBSC]), patient age, immunosuppressive therapy (yes or no), steroid use (yes or no), presence of HJ bodies, hypogammaglobulinemia (yes or no), number of CD3+, CD4+, CD8+, CD19+, CD19+/CD21-, CD19+/IgD+/CD27+, CD19+/IgD-/CD27+ cells, CD4/CD8 ratio, number of monocytes and granulocytes at study entry”–were assessed. Covariates with a P-value under .10 were entered into the multiple logistic regression analysis. If absolute values and percent values of a covariate were available as different variables, then these covariates were entered into separate multiple logistic models. If continuous variables showed interaction with another variable, they were centered by the median. All dichotomous variables were dummy-coded by 0 and 1. In the multiple logistic regression analysis, a backward selection was conducted with a removal P-value of .10 calculated with the Wald statistic. The results of the logistic regression analyses are presented as odds ratio (OR) and 95% confidence interval (95% CI). P-values below .05 were considered significant.

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Results 

Characteristics of the Patients and Infectious Complications 

Among our 70 patients, 21 had never experienced cGVHD (never cGVHD); 49 had experienced cGVHD including 14 with complete resolution of cGVHD (resolved cGVHD); and 35 patients had partial organ response or progression of cGVHD (active cGVHD) at study entry as previously defined [29]. All patients showed complete multilineage donor chimerism as determined by standard VNTR analysis a median of 47.6 (range, 5-150) months after HSCT. In addition, FISH analysis confirmed full donor chimerism of sorted B lymphocytes in 6 patients after sex-mismatched transplantation. No significant difference between overall B cell numbers; absolute CD4+ and CD8+ T cell counts; NK cell; monocyte and overall leukocyte counts was evident in the active cGVHD group when compared to the never and resolved cGVHD groups. However, granulocyte counts were significantly elevated in the active and resolved cGVHD groups, which might reflect the use of steroids.

All patients with cGVHD and 4 patients without cGVHD (but within the first year after unrelated donor HSCT) were under antibiotic prophylaxis targeting encapsulated bacteria and Pneumocystis carinii. Whereas no bacterial, viral, and fungal infection were observed beyond 12 months of HSCT in patients never experiencing cGVHD, markedly more of these infectious episodes were documented in patients with active cGVHD. These included clinically defined upper respiratory tract infection (n=19), pneumonia (n=7), clostridium difficile gastroenteritis (n=3), E. coli cystitis (n=2), cytomegalovirus (CMV) reactivation (n=3), herpes simplex viral infection (n=6), candida albicans stomatitis (n=3), candida glabrata gastroenteritis (n=2), and aspergillus flavus pneumonia (n=1). Patients with active cGVHD also had markedly more infectious episodes of any kind throughout their follow-up when assigned to the subgroup with severe infections (CTC grades III-IV). Between patients never experiencing cGVHD, ones with resolved cGVHD, and ones with active cGVHD, no significant differences were observed concerning the incidence of severe infectious complications after HSCT.

Immature/Transitional CD19+/CD21- B Lymphocytes are Significantly Increased in Active cGVHD Patients with Severe Infections and Multiple Infectious Episodes 

Whereas patients of the never cGVHD group and healthy donors (data not shown) had low numbers of immature/transitional circulating CD19+CD21- B cells (7.4% +/- 7.5%), this compartment was considerably enlarged among active cGVHD patients (7.4% +/- 7.5% vs 11.1% +/- 11.9% and 20.4 vs. 34.8 cells/μL) (Table 3 and Figure 2, P=.074). Most interestingly, in the overall patient sample, elevated numbers (>15%) of immature/transitional CD19+CD21- B lymphocytes were significantly associated with occurrence of severe infections (P=.003). When we applied the mean value, plus one standard deviation of never cGVHD (ie 15% as the cut-off level), the subgroup analysis revealed that all patients (9/9) with active cGVHD and more than 15% of circulating CD19+CD21- B lymphocytes had experienced severe infections (CTC grades III-IV). In contrast, only 7 of 26 patients (27%) with active cGVHD and ≤15% of circulating CD19+CD21- B lymphocytes had a history of severe infections. The observed association between high numbers of CD19+/CD21- B cells and severe infectious complications among active cGVHD patients was highly significant (P = .00016). In contrast, in patients in the never or resolved cGVHD group, no significant association between high CD19+CD21- B lymphocyte numbers and severe infections was observed (Table 3).

Table 3. High numbers of immature CD21- B lymphocytes in chronic GVHD correlate with increased susceptibility to severe infections
CD19+/CD21- B lymphocytes in subgroups of patients
No. of patientsPercentage of CD19+Absolute cell number/μL
All patients7010.629.3
Never cGVHD217.420.4
Resolved cGVHD1414.129.0
Active cGVHD3511.134.8
P = 0.678†P = 0.112†
Number (%) of patients with infectious complications
No. of patientsInfections CTC grades 0-II∗Infections CTC grades III-IV∗
All patients7041 (59)29 (41)
Never cGVHD2113 (62)8 (38)
Resolved cGVHD147 (50)7 (50)
Active cGVHD3519 (54)16 (46)
P = 1. 0†
Severe infectious complications correlate with elevated numbers of CD19+/CD21- B lymphocytes
No. of patientsInfections CTC grades 0-II∗ no. (%)Infections CTC grades III-IV∗ no. (%)
All patients >15% CD19+/21-153 (20)12 (80)
All patients ≤ 15% CD19+/21-5536 (66)19 (34)
P = .003
Active cGVHD patients >15% CD19+/21-90 (0)9 (100)
Active cGVHD ≤ 15% CD19+/21-2619 (73)7 (27)
P = .00016
Never and resolved cGVHD >15% CD19+/21-63 (50)3 (50)
Never and resolved cGVHD ≤ 15% CD19+/21-2917 (59)12 (41)
P = 1.0
All Patients ‡ H-J bodies +154 (27)11 (73)
All patients H-J bodies -5335 (66)18 (34)
P = .009

∗Infection Module, National Cancer Institute - Common Toxicity Criteria (CTC) Manual. [28]

†P value showing no significant difference between active- and never+resolved cGVHD subgroups.

‡2 patients with splenectomy before HSCT were excluded from the analysis.

  • View full-size image.
  • Figure 2 

    Phenotype of circulating B cells after HSCT. Whole blood cells were stained with the indicated fluorochrome conjugated mAbs. Subsequently, red blood cells were lysed and the remaining nucleated cells were analyzed by flow cytometry. CD19+ peripheral blood B lymphocytes were gated and the differential expression of surface IgD, CD27 and CD21 was assessed in healthy donors (HD) and patients with active (active cGVHD) or without (no cGVHD) cGVHD. Results are expressed as two-parameter dot-plots and are representative for the individuals within the same group. Numbers indicate the percentage of positive cells in the respective area.

In addition, significantly more clinically defined (P = .0049) and fungal (P = .0057) infectious episodes were observed in patients with active cGVHD, and more than 15% of circulating CD19+CD21- B lymphocytes 1 to 24 months and 1 to 36 months (P = .036 and P = .039) after HSCT compared to active cGVHD patients with ≤15% of circulating CD 1+9 CD21- B lymphocytes.

The significance of elevated CD19+CD21- B lymphocytes as an independent factor for susceptibility to severe infections in active cGVHD patients was confirmed in logistic regression analysis with an OR of 1.495 (95% CI 1.118-1.999, P = .007). Of note, steroid use was not more frequent in patients with high CD19+CD21- B lymphocyte numbers or severe infections (P = .16 and P = .11).

When we analyzed the expression of the activation marker CD69, no increase was observed in any patient in the study.

Memory B Lymphocyte (CD19+/CD27+) Numbers are Significantly Lower in Patients with Active cGVHD 

To assess the homeostasis of mature B cell subsets in cGVHD, we examined CD19+ B cell memory subpopulations (non-class-switched IgD+ cells and class-switched IgD- cells) [33] and stratified patients according to cGVHD activity and infectious complications. We found significantly lower relative and absolute numbers of non-class-switched CD27+IgD+ memory B cells (Figure 3A and Table 4) in patients with active cGVHD compared to patients who never experienced cGVHD (2.8 versus 4.7%, P = .002; 12.0 versus 22.1 cells/μL, P=.047). Similar results were obtained for class-switched CD27+/IgD- memory B cells in patients with active cGVHD compared to patients who never experienced cGVHD (3.1 versus 6.5%, P = .001; 10.1 versus 27.2 cells/μL, P = .023) (Figure 3B and Table 4).

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  • Figure 3 

    Significantly lower numbers of memory B lymphocytes in patients with active cGVHD. Whole blood cells were stained for the expression of CD19, IgD, and CD27 as exemplified in Figure 3. After HSCT, patients were assigned to various groups: 35 patients with active (act.–partial response or progression) and 14 with resolution (res.–complete response) of cGVHD according to published criteria. Another 21 patients had never experienced cGVHD (no) until study entry. In patients analyzed serially, only the first time point was included in this figure. Diamonds represent the percentage of non-class-switched (A) and class- switched (B) memory B lymphocytes for each patient. Statistical analysis was performed by the paired Student's t-test, and P values are indicated. The 2 outliers in A have been excluded from calculations.

Table 4. Low numbers of memory B cells (CD27+/IgD+ and CD27+/IgD-) in active chronic GVHD patients
CD27+∗3% of CD27+/CD19+
Percentage of CD19+Absolute cell number /mLNo. of patients (%)
IgD+IgD-IgD+IgD-IgD+IgD-Ratio between CD21- and CD27+IgDall
Never cGVHD (N=21)4.76.522.127.26 (29)2 (9)0.85 ± 1.14
Resolved cGVHD (N=14)4.87.710.917.52 (14)2 (14)1.25 ± 0.83
Active cGVHD (N=35)2.83.112.010.123 (65)25 (71)1.99 ± 1.52
P Value.002†.001†.047†.023†.012‡<.001‡.0046††

∗Percentages as calculated from the overall numbers of CD 19+ B cells.

††P Value was calculated by comparing the ratios of the indicated B-lymphocyte subsets in the subgroups of active and never chronic GVHD.

†P Values were calculated comparing percentages and absolute numbers of B lymphocyte subsets in the subgroups of active and never cGVHD.

‡P Values were calculated by comparing the number of patients in the subgroups of active and never cGVHD.

When applying the mean value of 3 percent as the cut-off level for low memory B cells, as observed in the active cGVHD group, 65% (23/35) and 71% (25/35) of patients with active cGVHD fulfilled these criteria. This was in significant contrast to only 29% (6/21) and 9% (2/21) of patients who never experienced cGVHD (Table 4).

Moreover, the ratio formed between immature/transitional B lymphocytes and memory B cell subsets confirms the discrimination of the 3 groups of GVHD patients (Table 4). No significant correlation between incidence and severity of infectious complications of patients (with or without active cGVHD activity) and low memory B cell numbers was evident. In addition, steroid use was not more frequent in active cGVHD patients with decreased CD19+CD27+IgD+ or CD19+CD27+IgD- B lymphocytes (P = 1.0 and P = .66).

Serial Analyses 

Among the 36 patients monitored serially both for cGVHD activity and homeostasis of B cell subsets, 25 patients had active cGVHD at first assessment of B cell subpopulations. Twelve of them did not respond to immunosuppressive therapy during the study period and, in some of these patients, CD21-/CD27+IgDall ratios remained markedly elevated (Figure 4A) compared to the groups with no cGVHD or partial overall response (defined as improvement in a measure for at least one organ without progression in measures for any other organ) [29]. In the 13 patients with active cGVHD achieving partial overall response during the study period, a decrease in CD21-/CD27+IgDall ratios was observed as shown in Figure 4B. The 11 patients without cGVHD at study entry remained low with their CD21 -/CD27+IgDall ratio and had no chronic GVHD activity during their time in the study (Figure 4C).

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  • Figure 4 

    Serial analyses of B cell subpopulations. On the X-axis, the time on study in months calculated from first assessment of B cell subsets is shown; on the Y-axis, the ratio formed between immature/transitional B lymphocytes and memory B cells is shown. A represents patients with active cGVHD not responding or progressing during study (n=12); B shows patients with active cGVHD achieving a partial overall response during study (n=13); and C shows patients without cGVHD (n=11).

Other Predictors of Infectious Complications are Only Weakly Associated with Active cGVHD 

A hallmark of cGVHD is functional asplenia with the possible appearance of HJ bodies in the circulation [15]. Therefore, we correlated the presence of HJ bodies in blood smears with the patients' susceptibility to severe infections. In all eligible patients, the presence of HJ bodies correlated to some degree with susceptibility to severe infections (Table 3, P = .009). Although HJ bodies were more frequent in patients with active cGVHD (11/35; 31%) than in the other two groups (4/33, 12%), no significant correlation between the presence of HJ bodies and infectious complications was observed in either of the cGVHD subgroups (P = .065 and P = .28).

Immunoglobulins are the major effector molecules of B lymphocytes and Ig serum levels can be taken as surrogate-markers for B lymphocyte function. Hypogammaglobulinemia is not uncommon after HSCT [33]. Indeed, 13 of 70 patients (19%) had low IgG serum levels (< 7g/L) including 9 of 35 (26%) with active cGVHD. Both in the overall group of patients and in the subgroup with active cGVHD, hypogammaglobulinemia did not significantly correlate with incidence and severity of infections. This clearly implies that an elevated number of immature/transitional CD19+/CD21- B lymphocytes is a stronger predictor for susceptibility to infection than is hypogammaglobulinemia.

Multiple logistic regression analysis revealed a mild significant association between a decreased CD4/CD8 ratio and severe infections (OR 0.308, 95%CI 0.106-0.895, P = .031) in the overall patient group, however, this phenomenon was not from cellular deficiency. Instead, it reflected the disproportional increase of CD8 T cell counts as observed in active cGVHD patients. Along those lines, no significant differences of CD4/CD8 ratios were observed between active cGVHD patients with or without severe infections. In addition, neither granulocyte counts nor CD4+ and CD8+ T lymphocyte counts, NK cell, monocyte and leukocyte counts correlated with severity of infections in these patients.

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Discussion 

GVHD selects the lymphoid system as a target organ [11] leading to lymphoid hypocellularity and atrophy as characteristic histologic hallmarks of severe GVHD. The primary cause of NRM associated with cGVHD is infection. T and B lymphocyte deficiencies are thought to play a role in the susceptibility of transplant recipients to infections [34]. Here, we demonstrate a severe disturbance of B cell homeostasis in a group of patients with active cGVHD seen as significantly elevated numbers of immature/transitional CD21- B cells and low CD27+ memory B cell counts. Whereas severe infectious complications were not significantly more frequent in all patients with active cGVHD, the patients with more than 15% of circulating immature/transitional CD19+/CD21- B lymphocytes (ie those having a CD19+/CD21- percentage that lies above the mean plus 1 standard deviation of never cGVHD) in the subgroup of active cGVHD had significantly more severe infections [28]. In addition, significantly more clinically defined and fungal infectious episodes up to 36 months after HSCT were observed in patients with active cGVHD and elevated immature/transitional CD19+/CD21- B lymphocyte numbers. Of note, supportive care and anti-infectious strategies were similar in the subgroups studied. Logistic regression analysis confirmed elevated immature/transitional CD19+/CD21- B lymphocyte counts as independent risk factors for severe infections in active cGVHD.

Storek et al [34] reported a significant association of low B cell and monocyte counts on day 80 with an increased rate of infections between days 100 and 365 after allogeneic marrow transplantation. In our study, no significant differences in CD4+ and CD8+ lymphocyte counts, NK cell, monocyte and overall B lymphocyte numbers were seen between patients with or without severe infections. Since the majority of our patients, however, were analyzed more than 1 year after HSCT, our patient cohort is not comparable and the role of immune re-constitution on HSCT recipient defense against microorganisms cannot be evaluated in our pilot study. Following allogeneic HSCT, grafted donor-derived precursor cells must recapitulate the ontogeneic process of B-lymphopoiesis. An early post-transplant period of severely depressed blood B cell counts is followed by a progressive normalization of B-cell numbers within the first year after HSCT 35, 36. However, the development of acute GVHD (aGVHD) or cGVHD after HSCT is associated with a prolonged B-cell-mediated humoral immunodeficiency with decreased or absent marrow B-cell precursors 5, 37, 38. Thus, Storek et al [5] observed an 18-fold lower number of B cell precursors on day 365 after HSCT in patients with extensive cGVHD.

To date, reports on the reconstitution of B cell subpopulations after allogeneic HSCT are sparse. Recently, Cuss et al [39] observed a 4- to 5-fold increase in immature/transitional B cell numbers and below 5% memory B cells compared to normal donors in patients at 2-3 mos after HSCT. In the follow-up period, immature/transitional B cell numbers declined, whereas memory B cell counts increased, indicating that in post-transplant patients immature/transitional B cells were generated early and were replaced over time by memory B cells. Similarly, Avanzini et al [40] assessing B lymphocyte reconstitution in children observed a slow recovery of memory B cells up to 24 months after HSCT. Immature/transitional B lymphocytes lack CD21 and classical activation or memory markers such as CD69 [23] and CD27, indicating that these cells most probably have not responded to antigens recently. They typically co-express CD10 and high levels of CD38. CD21 is exclusively expressed on mature B cells, where it forms a signal-transduction complex that also includes CD19, CD81, and CD225. Thus, CD21-negativity serves as an excellent marker for immature/transitional B cells 21, 22. Whereas Cuss et al [39] observed replacement of immature/transitional B cells by memory B cells during immune reconstitution in HSCT patients, in our active cGVHD subgroup circulating memory B lymphocytes remained low and the expansion of immature/transitional B cells was prominent. These findings indicate an impairment of the immune system, which is reminiscent of other primary or acquired immunodeficiencies and distinct autoimmune diseases. Along those lines, Warnatz et al [41] reported an elevation of immature/transitional CD21- B cells and low class-switched memory B cell counts in patients with common variable immunodeficiency. A high percentage of CD21- B cells has been observed in the circulation of HIV-infected individuals with advanced disease [25] and in SLE patients [23]. It remains to be shown whether the observed disturbance of the B cell compartments is caused by direct interference with B cell function or if it is secondary to subtle changes within distinct T cell compartments. Undoubtedly, the development of memory B cells is closely linked to the germinal center (GC) reaction, which takes place in secondary lymphoid organs upon encounter with antigen. The finding that significantly fewer memory B cells are present in cGVHD patients might argue for a disturbed GC reaction in cGVHD. Several possible reasons for a disturbed GC reaction are known. Mutations in the CD40 or CD40 ligand gene (CD154) 42, 43; defects of the tumor necrosis factor alpha (TNF-α) family and its receptors [44]; an impaired expression of co-stimulatory molecules such as CD86 [45] or ICOS [46]: all interfere significantly with the formation and function of GCs. Thus, a concerted action between T and B cells within the reticular network of lymph nodes is essential for the transition of a primary into a secondary follicle. Currently, however, we do not have any indications that one of the above cited gene defects are causative in the immunopathology of cGVHD. Apart from non-functional receptor-ligand pairs, deterioration of whole cellular subsets might influence distinct B cell compartments in an individual. Both essential and HIV-induced CD4 lymphocytopenia were found to be associated with the expansion of immature B lymphocytes, a phenomenon that is directly correlated with elevated serum interleukin-7 (IL-7) levels 25, 47. As pointed out by the authors, it was, however, difficult to determine whether the expansion of immature B cells was a direct response to the increased levels of IL-7 or an indirect effect that emanated from the loss of CD4 T cells. Evidence for a direct effect of IL-7 on B cells comes from the observation that IL-7 treatment of cancer patients leads to a clear increase in immature progenitor B cells within the bone marrow [48]. In our pilot study, there were no significant differences in the CD4 cell counts between patients with cGVHD and those without and resolved disease. It will be interesting, therefore, to examine IL-7 serum levels in our patient sample and to correlate them with immature B cell numbers in the future.

To our knowledge, this is the first report indicating a correlation of disturbed B cell homeostasis and susceptibility to infections in cGVHD. Monitoring the frequency of immature/transitional as well as memory B cells may provide a means of assessing the immunocompetence of individuals after HSCT. Interestingly, immature/transitional B cells appear to represent a checkpoint where auto-reactive B cells are removed from the peripheral population [49]. Aberrations at this stage of B cell development (which are possibly also promoted directly by high levels of BAFF [19]) may contribute to the appearance of circulating auto-reactive B cells as has been shown in mouse models for SLE [50] and collagen-induced arthritis [51]. Aberrations at this stage of B cell development might even directly contribute to the pathogenesis of cGVHD 52, 53. Further investigation of immature/transitional B cells could improve our knowledge of how alterations to the processes of B cell reconstitution after HSCT may precipitate immunodeficiency or autoimmunity. Such knowledge might also lead to strategies for enhancing the differentiation of immature B cells into mature effector cells in vivo, which might alleviate the B cell deficiency of such individuals.

To date, a limited number of biomarkers for cGVHD have been evaluated [54] despite the clear need for assessment tools that are dynamic and allow direct investigation of effector cells of the immune system. Our preliminary data obtained from the studied patients serially support a trend towards normalization of B cell homeostasis in patients with decreasing cGVHD activity whereas ongoing active cGVHD was associated with continuous B cell disturbance in the circulation. Thus, an expansion of immature/transitional B cells combined with low memory B cell numbers could serve as novel biomarkers both for cGVHD activity, and for its impact on the immune system. We are aware of the limitations of our retrospective study, including the potential impact of concomitant immunosuppressive therapies on prolongation of immunodeficiency and the patient heterogeneity with regard to duration of cGVHD and follow-up times. Therefore, a prospective study of consecutive patients after HSCT that assesses both time to immune reconstitution and the impact of cGVHD on the immune system is highly warranted.

In conclusion, flow cytometric evaluation of B lymphocyte subsets allows assessment of activity of cGVHD and the perturbation of the circulating B lymphocyte compartments can help to identify cGVHD patients with severe immunodeficiency seen as increased susceptibility to severe infections.

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Acknowledgments 

We thank Dr. Majdic, Mr. Gruze, and Kueng from the Institute of Immunology of the Medical University of Vienna, Austria, for providing mAbs, technical help, and help with artwork, respectively. We thank Drs. Kalhs, Steinberger, Pfistershammer, Schmetterer, and Lawitschka for critical review of the manuscript. This work was supported by European Commission Grant QLK-CT-2002-01936 Transeurope and by MCRTN-CT-2004-5 12253.

H.T.G. and W.F.P. designed the research study, analyzed and interpreted the data, and co-authored the manuscript; D.P. and M.K. performed the clinical research, collected and analyzed data; U.K. and I.L. performed the flow cytometric analyes; K.F. performed the chimerism analyses; C.Z. interpreted the data and contributed to the manuscript. The authors declare no competing financial interests.

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References 

  1. Lee SJ, Vogelsang G, Gilman A, et al. A survey of diagnosis, management, and grading of chronic GVHD. Biol Blood Marrow Transplant. 2002;8:32–39
  2. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant. 2005;11:945–956
  3. Socie G, Stone JV, Wingard JR, et al. Long-term survival and late deaths after allogeneic bone marrow transplantation. Late Effects Working Committee of the International Bone Marrow Transplant Registry. N Engl J Med. 1999;341:14–21
  4. Atkinson K. Reconstruction of the haemopoietic and immune systems after marrow transplantation. Bone Marrow Transplant. 1990;5:209–226
  5. Storek J, Joseph A, Espino G, et al. Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation. Blood. 2001;98:3505–3512
  6. Roberts MM, To LB, Gillis D, et al. Immune reconstitution following peripheral blood stem cell transplantation, autologous bone marrow transplantation and allogeneic bone marrow transplantation. Bone Marrow Transplant. 1993;12:469–475
  7. Small TN, Avigan D, Dupont B, et al. Immune reconstitution following T-cell depleted bone marrow transplantation: effect of age and posttransplant graft rejection prophylaxis. Biol Blood Marrow Transplant. 1997;3:65–75
  8. Barrett J. Improving outcome of allogeneic stem cell transplantation by immunomodulation of the early post-transplant environment. Curr Opin Immunol. 2006;18:592–598
  9. Sullivan KM, Blume KG, Forman SJ, Applbaum FR. Graft versus host disease. In: Thomas'Hematopoietic Cell Transplantation. Malden, MA: Blackwell Publishers; 2004;p. 635–664
  10. Tivol E, Komorowski R, Drobyski WR. Emergent autoimmunity in graft-versus-host disease. Blood. 2005;105:4885–4891
  11. Hakim FT, Mackall CL. The immune system: effector and target of graft-versus-host disease. In:  Deeg HJ,  Burakoff SJ editor. Graft-vs. -Host Disease. New York: Marcel Dekker, Inc.; 1997;p. 257–289
  12. Cutler C, Miklos D, Kim HT, et al. Rituximab for steroid-refractory chronic graft-versus host disease. Blood. 2006;108:756–762
  13. Ratanatharathorn V, Ayash L, Reynolds C, et al. Treatment of chronic graft-versus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant. 2003;9:505–511
  14. Teshima T, Reddy P, Liu C, et al. Impaired thymic negative selection causes autoimmune graft-versus-host disease. Blood. 2003;102:429–435
  15. Kalhs P, Panzer S, Kletter K, et al. Functional asplenia after bone marrow transplantation. A late complication related to extensive chronic graft-versus-host disease. Ann Intern Med. 1988;109:461–464
  16. Kruetzmann S, Rosado MM, Weber H, et al. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J Exp Med. 2003;197:939–945
  17. Chung JB, Silverman M, Monroe JG. Transitional B cells: step by step towards immune competence. Trends Immunol. 2003;24:343–349
  18. Carsetti R, Rosado MM, Wardemann H. Peripheral development of B cells in mouse and man. Immunol Rev. 2004;191:179–191
  19. Mackay F, Silveira PA, Brink R. B cells and the BAFF/APRIL axis: fast-forward on autoimmunity and signalling. Curr Opin Immunol. 2007;19:327–336
  20. Zinkernagel RM, Hengartner H. Protective ‘immunity’ by pre-existent neutralizing antibody titers and preactivated T cells but not by so-called ‘immunological-memory’. Immunol Rev. 2006;211:310–319
  21. Burrows PD, Cooper MD. B cell development and differentiation. Curr Opin Immunol. 1997;9:239–244
  22. Tedder TF, Fearon DT, Gartland GL, et al. Expression of C3b receptors on human B cells and myelomonocytic cells but not natural killer cells. J Immunol. 1983;130:1668–1673
  23. Sims GP, Ettinger R, Shirota Y, et al. Identification and characterization of circulating human transitional B cells. Blood. 2005;105:4390–4398
  24. Warnatz K, Wehr C, Drager R, et al. Expansion of CD19(hi)CD21(lo/neg) B cells in common variable immunodeficiency (CVID) patients with autoimmune cytopenia. Immunobiology. 2002;206:502–513
  25. Malaspina A, Moir S, Ho J, et al. Appearance of immature/transitional B cells in HIV-infected individuals with advanced disease: correlation with increased IL-7. Proc Natl Acad Sci U S A. 2006;103:2262–2267
  26. Maurer D, Fischer GF, Fae I, et al. IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset. J Immunol. 1992;148:3700–3705
  27. Maurer D, Holter W, Majdic O, Fischer GF, Knapp W. CD27 expression by a distinct subpopulation of human B lymphocytes. Eur J Immunol. 1990;20:2679–2684
  28. Cancer Therapy Evaluation Program: Common Toxicity Criteria 1999: Bethesda, MD: National Cancer Institute (Accessed July 17, 2006, at http://ctep.cancer.gov/forms/CTCManual_v4_10-4-99.pdf).
  29. Pavletic SZ, Lee SJ, Socie G, et al. Chronic graft-versus-host disease: implications of the National Institutes of Health consensus development project on criteria for clinical trials. Bone Marrow Transplant. 2006;645–651
  30. Rizzo JD, Wingard JR, Tichelli A, et al. Recommended screening and preventive practices for long-term survivors after hematopoietic cell transplantation: joint recommendations of the European Group for Blood and Marrow Transplantation, the Center for International Blood and Marrow Transplant Research, and the American Society of Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2006;12:138–151
  31. Couriel D, Carpenter PA, Cutler C, et al. Ancillary therapy and supportive care of chronic graft-versus-host disease: national institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: V. Ancillary Therapy and Supportive Care Working Group Report. Biol Blood Marrow Transplant. 2006;12:375–396
  32. Keil F, Kalhs P, Chen X, et al. Hematopoietic donor chimerism and graft-versusmyeloma effect in relapse of multiple myeloma after allogeneic bone marrow transplantation. Ann Hematol. 1999;78:376–379
  33. Perreault C, Giasson M, Gyger M, et al. Serum immunoglobulin levels following allogeneic bone marrow transplantation. Blut. 1985;51:137–142
  34. Storek J, Espino G, Dawson MA, et al. Low B-cell and monocyte counts on day 80 are associated with high infection rates between days 100 and 365 after allogeneic marrow transplantation. Blood. 2000;96:3290–3293
  35. Storek J, Ferrara S, Ku N, et al. B cell reconstitution after human bone marrow transplantation: recapitulation of ontogeny?. Bone Marrow Transplant. 1993;12:387–398
  36. Leitenberg D, Rappeport JM, Smith BR. B cell precursor bone marrow reconstitution after bone marrow transplantation. Am J Clin Pathol. 1994;102:231–236
  37. Storek J, Witherspoon RP, Webb D, et al. Lack of B cells in marrow transplant recipients with chronic graft-versus-host disease. Am J Hematol. 1996;52:82–89
  38. Sanchez-Garcia J, Serrano J, Gomez P, et al. The impact of acute and chronic graft-versus-host disease on normal and malignant B-lymphoid precursors after allogeneic stem cell transplantation for B-lineage acute lymphoblastic leukemia. Haematologica. 2006;91:340–347
  39. Cuss AK, Avery DT, Cannons JL, et al. Expansion of functionally immature transitional B cells is associated with human-immunodeficient states characterized by impaired humoral immunity. J Immunol. 2006;176:1506–1516
  40. Avanzini MA, Locatelli F, Dos Santos C, et al. B lymphocyte reconstitution after hematopoietic stem cell transplantation: functional immaturity and slow recovery of memory CD27+ B cells. Exp Hematol. 2005;33:480–486
  41. Warnatz K, Denz A, Drager R, et al. Severe deficiency of switched memory B cells (CD27(+)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. Blood. 2002;99:1544–1551
  42. Korthauer U, Graf D, Mages HW, et al. Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature. 1993;361:539–541
  43. errari S, Giliani S, Insalaco A, et al. Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM. Proc Natl Acad Sci U S A. 2001;98:12614–12619
  44. Endres R, Alimzhanov MB, Plitz T, et al. Mature follicular dendritic cell networks depend on expression of lymphotoxin beta receptor by radioresistant stromal cells and of lymphotoxin beta and tumor necrosis factor by B cells. J Exp Med. 1999;189:159–168
  45. Han S, Hathcock K, Zheng B, et al. Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers. J Immunol. 1995;155:556–567
  46. Grimbacher B, Hutloff A, Schlesier M, et al. Homozygous loss of ICOS is associated with adult-onset common variable immunodeficiency. Nat Immunol. 2003;4:261–268
  47. Malaspina A, Moir S, Chaitt DG, et al. Idiopathic CD4+ T lymphoctopenia is associated with increases in immature/transitional B cells and serum levels of IL-7. Blood. 2007;109:2086–2088
  48. Rosenberg SA, Sportes C, Ahmadzadeh M, et al. IL-7 administration to humans leads to expansion of CD8+ and CD4+ but a relative decrease of CD4+ T-regulatory cells. J Immunother. 2006;29(3):13–319
  49. Wardemann H, Yurasov S, Schaefer A, et al. Predominant autoantibody production by early human B cell precursors. Science. 2003;301:1374–1377
  50. Gross JA, Johnston J, Mudri S, et al. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature. 2000;404:995–999
  51. Zhang M, Ko KH, Lam QL, et al. Expression and function of TNF family member B cell activating factor in the development of autoimmune arthritis. Int Immunol. 2005;17:1081–1092
  52. Yurasov S, Wardemann H, Hammersen J, et al. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med. 2005;201:703–711
  53. Samuels J, Ng YS, Coupillaud C, et al. Impaired early B cell tolerance in patients with rheumatoid arthritis. J Exp Med. 2005;201:1659–1667
  54. Schultz KR, Miklos DB, Fowler D, et al. Toward biomarkers for chronic graft-versus host disease: National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: III. Biomarker Working Group Report. Biol Blood Marrow Transplant. 2006;12:126–137

PII: S1083-8791(07)00539-3

doi:10.1016/j.bbmt.2007.10.009

Biology of Blood and Marrow Transplantation
Volume 14, Issue 2 , Pages 208-219, February 2008

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