Kappa-deleting Recombination Excision Circles Research Articles

Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients

The anti-α4 monoclonal antibody natalizumab inhibits lymphocyte extravasation into the central nervous system and increases peripheral T and B lymphocytes in multiple sclerosis patients. To investigate whether the lymphocyte accumulation was due to a higher lymphocyte production, an altered homeostasis, or a differential transmigration of lymphocyte subsets through endothelia, T-cell receptor excision circles and kappa-deleting recombination excision circles were quantified before and after treatment, T-cell receptor repertoire was analyzed by spectratyping, and T- and B-lymphocyte subset migration was studied using transwell coated with vascular and lymphatic endothelial cells. We found that the number of newly produced T and B lymphocytes is increased because of a high release and of a low propensity of naïve subsets to migrate across endothelial cells. In some patients this resulted in an enlargement of T-cell heterogeneity. Because new lymphocyte production ensures the integrity of immune surveillance, its quantification could be used to monitor natalizumab therapy safety.

The Kinetics of Early T and B Cell Immune Recovery after Bone Marrow Transplantation in RAG-2-Deficient SCID Patients

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT.

Idiopathic CD4+ T lymphopenia without autoimmunity or granulomatous disease in the slipstream of RAG mutations

AbstractA girl presented during childhood with a single course of extensive chickenpox and moderate albeit recurrent pneumonia in the presence of idiopathic CD4+ T lymphocytopenia (ICL). Her clinical condition remained stable over the past 10 years without infections, any granulomatous disease, or autoimmunity. Immunophenotyping demonstrated strongly reduced naive T and B cells with intact proliferative capacity. Antibody reactivity on in vivo immunizations was normal. T-cell receptor-Vβ repertoire was polyclonal with a very low content of T-cell receptor excision circles (TRECs). Kappa-deleting recombination excision circles (KRECs) were also abnormal in the B cells. Both reflect extensive in vivo proliferation. Patient-derived CD34+ hematopoietic stem cells could not repopulate RAG2−/−IL2Rγc−/− mice, indicating the lymphoid origin of the defect. We identified 2 novel missense mutations in RAG1 (p.Arg474Cys and p.Leu506Phe) resulting in reduced RAG activity. This report gives the first genetic clue for ICL and extends the clinical spectrum of RAG mutations from severe immune defects to an almost normal condition.

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

BackgroundThe 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.MethodsThe 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.ResultsKRECs 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.ConclusionsThe 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.

Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation

A major problem in the field of stem cell transplantation is the difficulty to monitor the efficacy of immune reconstitution. By modifying the widely used method of measuring T-cell receptor excision circles (TRECs) and the recently proposed kappa-deleting recombination excision circles (KRECs) assay, we set up a duplex Real-Time PCR that allowed the simultaneous quantification of newly produced T and B cells in children with primary immunodeficiency undergone to transplantation. We found that lymphocyte recovery involves the mobilization of both new T and B cells from production and maturation sites, and that the increase of TRECs and KRECs can be or strictly associated or independent one from the other. Some patients showed a "lymphocyte rebound" which is followed by a progressive decrease of newly produced T and B lymphocytes starting about 2years after transplantation. In other patients, TRECs and KRECs number remained very low for the entire period of study.

Quantification of Recent Bone Marrow B-Cell and Thymic T-Cell Emigrants in Chemotherapy Naïve Patients with Chronic Lymphocytic Leukemia.

Abstract 2353Poster Board II-330 BackgroundChronic lymphocytic leukemia (CLL) results in accumulation of mature, malignant, monoclonal B cells in blood, lymph nodes, spleen, liver, and bone marrow. Patients with CLL have fundamental defects in both humoral and cell-mediated immunity that significantly impact on its clinical course. It is still not known if these immune abnormalities include a decrease of new B- and T-cell mobilization from their production sites. During early lymphocyte development and differentiation, occurring in bone marrow and in thymus, B- and T-cells undergo rearrangements of B-cell and T-cell receptor genes whereby specific chromosomal sequences are excised to produce episomal DNA products identified respectively as T-cell receptor excisional circles (TRECs) and Kappa-deleting recombination excision circles (KRECs). Being stable circular fragments of DNA, TRECs and KRECs do not replicate during the mitotic process required for cell proliferation and, therefore, they are diluted out by cell divisions and are lost when the cells die. Since T-cells leaving the thymus are 70% TRECs+ and KRECs are present randomly in about 50% of neo-produced normal B-cells, the quantification of TRECs and KRECs allows a good estimation of the thymic and bone marrow output. AimTo investigate the extent of neo-synthesis of normal B and T cells in untreated patients with CLL. Patients and MethodsTwelve previously untreated CLL patients were enrolled in this study. M:F ratio was 5:1; median age was 66; 7 patients had mutated IgVH genes, while 5 patients had unmutated IgVH genes; all patients were stage A Binet; FISH analysis including del(13q), del(11q), del(17p) and trisomy 12 detected 13q deletion in 6 patients while the other 6 patients presented no abnormalities. TRECs and KRECs were measured in mononuclear cells isolated from peripheral blood by duplex quantitative Real-Time PCR. This new assay, based on the use of a standard curve prepared with a plasmid containing fragments of TRECs, KRECs and of a reference gene, allows to quantify neo-produced B and T lymphocytes. T-cell subpopulations were determined by flow cytometry as follows: recent thymic emigrants (RTE) as cells with CD4+CD45RA+CCR7+CD31+ phenotype, regulatory T cells (Treg), as CD4+CD25int/highCD127low, RTE-Treg as Treg expressing CCR7+CD31+CD25int/high markers, effector memory (TEM) and central memory (TCM) T cells as lymphocytes displaying CD4+CD45RA-CCR7- and CD4+CD45RA-CCR7+ phenotype. ResultsThe overall number of CD4+ lymphocytes was increased in chemotherapy-naïve patients with CLL (1585/μl vs 953/μl; p=0.02). TRECs were significantly higher in CLL patients compared to age-matched healthy controls (2.9/ml vs 1.4/ml; p=0.04), while the proportion of RTE was lower (24.9% vs 32.4%; p=0.02). No significant differences were observed in the percentage of Treg and RTE-Treg as well as of TCM. On the contrary, the percentage of TEM was higher (22.4% vs 10.4%; p=0.01) in CLL patients. The number of KRECs was lower in CLL patients than in controls (3.8/ml vs 7.4/ml; p=0.004). No correlation between IgVH gene mutational status, Ig levels and KRECs was found. ConclusionsThe neo-synthesis of normal T, with the exception of Treg, and B cells is reduced in patients with CLL compared to controls, even in a good prognosis, chemotherapy-naïve subset. The increased number of TRECs+ cells and TEM together with low RTE could be ascribed to the increased number of circulating CD4+ lymphocytes which do not appear to be recently mobilized from the thymus (CD31- cells) or to undergo peripheral expansion, but to accumulate in the peripheral blood. The low number of KRECs compared to normal controls could be interpreted as a consequence of the abnormal leukemic B-cell expansion which may impair normal B cell neo-synthesis. The level of KRECs did not seem to correlate to the mutational status of IgVH genes or the Ig level but a larger number of patients is needed to confirm these preliminary data and to establish whether the analysis of TRECs and KRECs may help clarify the complex immune abnormalities in CLL. Disclosures:No relevant conflicts of interest to declare.

Evidence for B cell antigen receptor (BCR)‐driven, BAFF‐facilitated positive selection within a unique population of late transitional B cells

In addition to negative selection, several lines of evidence suggest that positive selection may also play an important role in shaping the naïve mature B cell repertoire.We have characterized a late transitional (T2) B cell subset with a distinct phenotypic, functional, and gene expression profile. Adoptive transfer experiments identify this subset as an immediate precursor of both follicular mature (FM) and marginal zone (MZ) B cells. Intriguingly, in contrast to the largely quiescent early transitional (T1) and FM B cells, a high percentage of T2 B cells are actively cycling as demonstrated by cell cycle analysis, measurement of kappa‐deleting recombination excision circles (KRECS) and in vivo BrdU labeling. Proliferation of this subset is BAFF‐dependent, consistent with the highest levels of BAFF‐R expression among all splenic B cells. Finally, M167 BCR idiotypic‐specific B cells are first selected by self‐antigen within this subset leading, ultimately, to preferential enrichment within the MZ compartment.In summary, our data indicate that late transitional B cells represent a bipotent precursor for mature splenic B cells; and can function as a target for BCR‐driven, BAFF‐facilitated homeostatic signaling. These combined findings suggest that this small pool of cells may operate at a previously unknown selection point important for establishment of the mature B repertoire. Funded by NIH 5RO1HD37091

Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion

The contribution of proliferation to B lymphocyte homeostasis and antigen responses is largely unknown. We quantified the replication history of mouse and human B lymphocyte subsets by calculating the ratio between genomic coding joints and signal joints on kappa-deleting recombination excision circles (KREC) of the IGK-deleting rearrangement. This approach was validated with in vitro proliferation studies. We demonstrate that naive mature B lymphocytes, but not transitional B lymphocytes, undergo in vivo homeostatic proliferation in the absence of somatic mutations in the periphery. T cell–dependent B cell proliferation was substantially higher and showed higher frequencies of somatic hypermutation than T cell–independent responses, fitting with the robustness and high affinity of T cell–dependent antibody responses. More extensive proliferation and somatic hypermutation in antigen-experienced B lymphocytes from human adults compared to children indicated consecutive responses upon additional antigen exposures. Our combined observations unravel the contribution of proliferation to both B lymphocyte homeostasis and antigen-induced B cell expansion. We propose an important role for both processes in humoral immunity. These new insights will support the understanding of peripheral B cell regeneration after hematopoietic stem cell transplantation or B cell–directed antibody therapy, and the identification of defects in homeostatic or antigen-induced B cell proliferation in patients with common variable immunodeficiency or another antibody deficiency.