Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D+ repl; n=28) or T-cell-depleted (D+ depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D+ depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D-) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D+ depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D- patients.

Validation of cord blood split products prepared by an automated method.

OBJECTIVES: In this study, we studied whether the contents of the two compartments of automatically processed cord blood (CB) units are comparable with respect to cell counts and viability and therefore suitable for clinical therapy. BACKGROUND: CB-derived stem cells are increasingly used for allogeneic transplantation. Many centres prepare the transplants by automated methods allowing to split the product into two portions. METHODS: CB was collected at different sites in Germany and transported to a single centre for processing. Before and after cryopreservation laboratory analyses were performed to compare the quality of the two CB segments. RESULTS: In total, 33 products were processed [mean collection volume: 18·6 ± 1·2 mL (range 15·2-20·2 mL) segment A; mean: 4·7 ± 0·3 mL (range 4·2-5·2 mL) segment B]. CD34+ cell counts, viability of CD34+ cells and many other haematological parameters showed a good comparibility between the two segments. However, lymphocyte counts and results of clonogenic assays were significantly different between the two segments of the split product. CONCLUSION: We conclude that the preparation of the cord blood unit by the automated process results in a homogenous distribution of stem and progenitor cells. However, our findings show that the clonogenic capacity differs between the two segments.

Perturbations of peripheral B lymphocyte homoeostasis in children with systemic lupus erythematosus.

OBJECTIVE: To investigate the distribution of peripheral B cell subpopulations of children with active and inactive systemic lupus erythematosus (SLE) compared with healthy controls. METHODS: Peripheral B cell subpopulations of 11 children with SLE (6 with active and 5 with inactive disease) and 14 age matched normal healthy children were analysed. Active disease was diagnosed in children with a flare of SLE, who received treatment by i.v. cyclophosphamide or i.v. methylprednisolone pulse to control the disease. Additionally, the peripheral B cells of the children with SLE were compared with those of 13 consecutive patients with adult onset SLE. RESULTS: No major difference was found in the frequency and total number of CD27(-)/CD19(+) naïve B cells and CD27(+)/CD19(+) memory B cells between patients with active and inactive lupus and healthy controls, but there was a significant increase in CD27(high) expressing plasma blasts in patients with active SLE. These cells coexpress CD38(+), HLA-DR(dim), surface Ig(low) and lack the expression of CD20 but are clearly positive for intracellular Ig, indicative of early plasma cells. Most CD138(+) cells coexpress CD27(high)/CD19(+). The enhanced frequency of peripheral plasma blasts in children with active SLE is consistent with previous findings in adult patients with SLE, whereas a relative predominance of CD27(+) memory B cells was only identified in the adult patients. CONCLUSIONS: Profound abnormalities in the distribution of B cell compartments are more pronounced in older patients with SLE, but an enhanced frequency and cell number of peripheral plasma blasts is characteristic of both diseases during active stages. Thus detection of CD27(high) plasma blasts significantly correlates with active lupus in both children and adults.

[Cytometric analyses in systemic autoimmune diseases]. / Zytometrische Analysen bei systemischen Autoimmunerkrankungen.

A number of autoantibodies play a significant role in collagen vascular diseases and represent diagnostic markers of some of these entities. Despite increasing knowledge of these serological findings, data are limited about potential disturbances of precursor cells that finally lead to the autoantibody producing plasma cells. Recent evidence of disturbed B cell homeostasis indicates that the peripheral B cell compartments in systemic lupus erythematodes (SLE) and Sjögren's syndrome are characteristically different to normal. Although the identification of autoreactive B cells in peripheral blood is still subject of ongoing studies, the differences in B cell subsets add to the understanding of the immunopathogenesis of these diseases and may provide new diagnostic clues and therapeutical avenues of these entities.

Immunoglobulin Vlambda light chain gene usage in patients with Sjögren's syndrome.

OBJECTIVE: To determine whether patients with Sjögren's syndrome (SS) have abnormalities in Ig Vlambda and Jlambda gene usage, differences in somatic hypermutation, defects in selection, or indications for perturbations of B cell maturation. METHODS: Individual peripheral B cells from SS patients were analyzed for their Vlambda gene usage by single-cell polymerase chain reaction amplification of genomic DNA and compared with those from normal controls. RESULTS: Molecular differences from controls in Vlambda-Jlambda recombination were identified that were reflected by findings in the nonproductive Vlambda repertoire of the patients, including enhanced rearrangement of Vlambda10A and Jlambda2/3 gene segments. In addition, a number of abnormalities in the productive repertoire were identified, indicating disordered selection. A greater usage of 4 Vlambda genes (2A2, 2B2, 2C, and 7A), representing 56% of all productive Vlambda rearrangements, was observed, suggesting positive selection of these genes. Overutilization of Jlambda2/3 and underutilization of Jlambda7 in both nonproductive and productive Vlambda rearrangements of SS patients compared with controls suggested decreased receptor editing in SS. The mutational frequency did not differ from that in controls, and positive selection of mutations into the productive V gene repertoire was found, similar to that in controls, although mutational targeting toward RGYW/WRCY motifs, typically found in controls, was not found in SS patients. CONCLUSION: Disturbed regulation of B cell maturation with abnormal selection, defects in editing Ig receptors, and abnormal mutational targeting may contribute to the emergence of autoimmunity in SS.

Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus.

In patients with active systemic lupus erythematosus (SLE), a marked B lymphocytopenia was identified that affected CD19(+)/CD27(-) naive B cells more than CD19(+)/CD27(+) memory B cells, leading to a relative predominance of CD27-expressing peripheral B cells. CD27(high)/CD38(+)/CD19(dim)/surface Ig(low)/CD20(-)/CD138(+) plasma cells were found at high frequencies in active but not inactive SLE patients. Upon immunosuppressive therapy, CD27(high) plasma cells and naive CD27(-) B cells were markedly decreased in the peripheral blood. Mutational analysis of V gene rearrangements of individual B cells confirmed that CD27(+) B cells coexpressing IgD were memory B cells preferentially using V(H)3 family members with multiple somatic mutations. CD27(high) plasma cells showed a similar degree of somatic hypermutation, but preferentially employed V(H)4 family members. These results indicate that there are profound abnormalities in the various B cell compartments in SLE that respond differently to immunosuppressive therapy.

Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG.

Recent studies in mice have indicated that the long-lasting specific antibody responses seen after vaccination are probably due to the existence of long-lived plasma cells. Therefore, because the maintenance of humoral immunity does not necessarily reflect continuous restimulation of long-lived memory B cells, the question arises as to what degree antibody immunity, as determined by measuring serum immunoglobulin titers against a particular antigen, and memory B cell immunity, as determined by counting circulating memory B cells with specificity for that same antigen, correlate. Here, using a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate and characterize antigen-specific memory B cells, we show for tetanus toxin C-fragment in blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B in blood of wasp venom-allergic donors undergoing an immune therapy with wasp venom, that there is no statistically significant linear correlation between the frequencies of circulating antigen-specific IgG-bearing memory B cells and the serum titers of antigen-specific IgG. This lack of a statistically significant linear correlation is in accordance with the idea that B memory cells and plasma cells represent independently controlled forms of immunological memory.