Limits...
Quantification of newly produced B and T lymphocytes in untreated chronic lymphocytic leukemia patients.

Motta M, Chiarini M, Ghidini C, Zanotti C, Lamorgese C, Caimi L, Rossi G, Imberti L - J Transl Med (2010)

Bottom Line: T-lymphocyte subsets were analyzed by six-color flow cytometric analysis.Data comparison was performed by two-sided Mann-Whitney test.This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology, Spedali Civili, Piazzale Spedali Civili 1, 25123, Brescia, Italy.

ABSTRACT

Background: The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes.

Methods: The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test.

Results: KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECs+ cells was preserved. Furthermore, the observed increase of CD4+ lymphocytes could be ascribed to the accumulation of CD4+ cells with effector memory phenotype.

Conclusions: The decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease.

Show MeSH

Related in: MedlinePlus

KRECs and TRECs determination in increasing concentrations of non-tumoral DNA into DNA from a lymphoblastoid B-cell line. DNA extracted from two healthy controls with either high (filled symbols) or low (open symbols) number of KRECs (circles) and TRECs (diamonds) was diluted into DNA extracted from a lymphoblastoid B-cell line, in order to obtain decreasing concentration of tumoral DNA. Straight line: regression line for KRECs; dotted-line: regression line for TRECs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2991330&req=5

Figure 1: KRECs and TRECs determination in increasing concentrations of non-tumoral DNA into DNA from a lymphoblastoid B-cell line. DNA extracted from two healthy controls with either high (filled symbols) or low (open symbols) number of KRECs (circles) and TRECs (diamonds) was diluted into DNA extracted from a lymphoblastoid B-cell line, in order to obtain decreasing concentration of tumoral DNA. Straight line: regression line for KRECs; dotted-line: regression line for TRECs.

Mentions: To exclude the potential confounding effect of tumor DNA derived from monoclonal B cells on the quantification of KRECs and TRECs, genomic DNA from PBMC of 2 healthy donors with high and low number of KRECs and TRECs was serially diluted into DNA of a human lymphoblastoid cell line to obtain final concentrations of normal lymphocyte DNA ranging from 3% to 100%. While KRECs and TRECs were undetectable in 100% tumor DNA, the amount of KRECs/106 and TRECs/106 cells of both donors showed a linear change, being detected even at concentration as low as 3% of normal DNA (Figure 1), suggesting that the presence of high number of blasts in CLL patient samples should not bias the assay results.


Quantification of newly produced B and T lymphocytes in untreated chronic lymphocytic leukemia patients.

Motta M, Chiarini M, Ghidini C, Zanotti C, Lamorgese C, Caimi L, Rossi G, Imberti L - J Transl Med (2010)

KRECs and TRECs determination in increasing concentrations of non-tumoral DNA into DNA from a lymphoblastoid B-cell line. DNA extracted from two healthy controls with either high (filled symbols) or low (open symbols) number of KRECs (circles) and TRECs (diamonds) was diluted into DNA extracted from a lymphoblastoid B-cell line, in order to obtain decreasing concentration of tumoral DNA. Straight line: regression line for KRECs; dotted-line: regression line for TRECs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2991330&req=5

Figure 1: KRECs and TRECs determination in increasing concentrations of non-tumoral DNA into DNA from a lymphoblastoid B-cell line. DNA extracted from two healthy controls with either high (filled symbols) or low (open symbols) number of KRECs (circles) and TRECs (diamonds) was diluted into DNA extracted from a lymphoblastoid B-cell line, in order to obtain decreasing concentration of tumoral DNA. Straight line: regression line for KRECs; dotted-line: regression line for TRECs.
Mentions: To exclude the potential confounding effect of tumor DNA derived from monoclonal B cells on the quantification of KRECs and TRECs, genomic DNA from PBMC of 2 healthy donors with high and low number of KRECs and TRECs was serially diluted into DNA of a human lymphoblastoid cell line to obtain final concentrations of normal lymphocyte DNA ranging from 3% to 100%. While KRECs and TRECs were undetectable in 100% tumor DNA, the amount of KRECs/106 and TRECs/106 cells of both donors showed a linear change, being detected even at concentration as low as 3% of normal DNA (Figure 1), suggesting that the presence of high number of blasts in CLL patient samples should not bias the assay results.

Bottom Line: T-lymphocyte subsets were analyzed by six-color flow cytometric analysis.Data comparison was performed by two-sided Mann-Whitney test.This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology, Spedali Civili, Piazzale Spedali Civili 1, 25123, Brescia, Italy.

ABSTRACT

Background: The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes.

Methods: The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test.

Results: KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECs+ cells was preserved. Furthermore, the observed increase of CD4+ lymphocytes could be ascribed to the accumulation of CD4+ cells with effector memory phenotype.

Conclusions: The decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease.

Show MeSH
Related in: MedlinePlus