We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.

No abstract

Although autoantibodies have been used for decades as diagnostic and prognostic markers in type 1 diabetes (T1D), further analysis of developmental abnormalities in B cells could reveal tolerance checkpoint defects that could improve individualized therapy. To evaluate B cell developmental progression in T1D, immunophenotyping was used to classify circulating B cells into transitional, mature naïve, mature activated, and resting memory subsets. Then each subset was analyzed for the expression of additional maturation-associated markers. While the frequencies of B cell subsets did not differ significantly between patients and controls, some T1D subjects exhibited reduced proportions of B cells that expressed transmembrane activator and CAML interactor (TACI) and Fas receptor (FasR). Furthermore, some T1D subjects had B cell subsets with lower frequencies of class switching. These results suggest circulating B cells exhibit variable maturation phenotypes in T1D. These phenotypic variations may correlate with differences in B cell selection in individual T1D patients.

Background: Flow cytometry is used to monitor lymphocyte subsets in both the clinical and research settings. An understanding of the degree of inter-and intrasubject variability of these populations is critical for data interpretation.Methods: Peripheral blood lymphocytes of 18 healthy adults were analyzed on two separate occasions using a multicolor flow cytometric panel with B, T, and NK cell markers. Variability was calculated using the coefficient of variation and compared between and within individuals using agglomerative clustering.Results: Each subject appears to have B and T cell subset profiles that are stable over the two time points, but differ from the profiles of other subjects. Thus, the range of measurements for a particular B or T cell subset is larger between subjects and narrower for an individual. In addition, the level of variability correlates inversely with the size of the lymphocyte subset. When lymphocyte profiles are analyzed by agglomerative clustering, replicate samples from the same individual tend to cluster. When single samples from different individuals are analyzed, individuals appear to cluster into different subgroups.Conclusions: Variability of lymphocyte subsets is usually greater between individuals than within a single individual and each person appears to have a characteristic profile of lymphocyte subsets. These results underscore the importance of obtaining a baseline value for each subject when investigating the impact of a treatment on lymphocyte subsets over time. These results also highlight the potential utility of cluster analysis as a tool for immune subset profiling and biomarker discovery.

There are few studies of the developmental changes in B-cell subsets in children. Recent data from adult populations demonstrate that alterations to B-cell subsets have functional consequences and can be helpful diagnostically. Comparable studies in children have been hindered by the lack of normative data and by significant changes with age. This study evaluated B-cell subsets by 4-color flow cytometry in 47 children of different ages. The use of a 4-color platform is compatible with broad use in clinical laboratories. We found that there are rapid changes in the B-cell compartment in infancy and early childhood. Total B-cell numbers decline early in life, and this correlates with a decline in transitional B cells and naïve B cells. The decline is most rapid between 1 and 5 years of age, with a slower decline later in childhood. In contrast, nonswitched and switched memory B cells both increase during the 1st 5 years of life. The decline in B-cell numbers did not occur until after 1 year of age, suggesting that the period after birth is a unique developmental window. These data provide a reference set for further studies on B-cell dysfunction in pediatric disorders. The changes occurring in early childhood document the need for age-related assessments and serve to underscore the B-cell-specific kinetics of immunologic development in humans.

Intrathecal triple chemotherapy (ITT) with hydrocortisone, methotrexate, and cytarabine is commonly used in treatment of pediatric acute leukemias. While prolonged systemic administration of corticosteroids is known to suppress the hypothalamic-pituitary-adrenal axis, there have been no reports describing this effect following administration of ITT. We present an infant with relapsed acute myelogenous leukemia who developed clinically significant central adrenal axis suppression following six doses of ITT over 3 weeks, proven by corticorelin stimulation test. As multiple pediatric leukemia protocols incorporate ITT, particularly in infants, we feel that ITT should be considered as a potential source of adrenal axis suppression.

Manual gating of bivariate plots remains the most frequently used data analysis method in flow cytometry. However, gating is operator-dependent and cumbersome, particularly with the increasing complexity of modern multicolor immunophenotyping data. A method that can remove operator bias, enable systematic and thorough analysis of complex high-dimensional data, correlate temporal changes in different subsets and lead to biomaker discovery is needed. Here we apply such a method, called cytometric fingerprinting (CF), to data obtained on peripheral blood B cells from an adult patient with type-1 diabetes who underwent pancreatic islet transplantation. We establish that CF can be used to analyze longitudinal trends in immunophenotypic data, and show that results from CF are comparable to those obtained with traditional gating methods. Both methods reveal the appearance of transitional B cells and subsequent accumulation of more mature B cells following immunosuppression and transplantation. This pattern is consistent with a temporally ordered process of B cell auto-reconstitution. We also show the comparative efficiency of fingerprinting in recognizing relative changes in B cell subsets with respect to time, its ability to couple the data with statistical methods (agglomerative clustering) and its potential to define novel subsets.

No abstract