Tho D. Pham

Effects of Aging, Cytomegalovirus Infection, and EBV Infection on Human B Cell Repertoires

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual’s age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

Single B-cell deconvolution of peanut-specific antibody responses in allergic patients

BACKGROUND The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance. OBJECTIVE We sought to characterize peanut allergen-specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy. METHODS B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones. RESULTS Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase. CONCLUSION Most peanut allergen-binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.

IgH sequences in common variable immune deficiency reveal altered B cell development and selection

Deep sequence analysis of the IgH repertoires of common variable immune deficiency patients highlights phenotypic features of the disorder and potential disease mechanisms. Not immune to memory problems Although elderly patients typically lament loss of memory, Roskin et al. now highlight a different kind of memory problem plus developmental difficulties that bedevils the immune systems of children and young adults with common variable immune deficiency (CVID). These aberrations may drive a variety of outcomes, including impaired antibody responses to foreign antigens, generation of autoimmune responses, and lymphoid cancers. As the most common symptomatic primary immune deficiency, CVID affects nearly one in 25,000 persons, but the biological basis for the constellation of clinical phenotypes remains hazy. Genetic recombination in the V(D)J regions of immunoglobulin (Ig) genes, which occurs in developing lymphocytes during the early stages of B cell maturation, gives rise to the diverse repertoire of antibodies produced by activated B lymphocytes. This so-called Ig gene rearrangement—a seminal component of the adaptive immune system—gives rise to a broad range of amino acid sequences in the antigen-binding regions of Igs, which permits the recognition of antigens from many pathogens and abnormal cells (such as cancer cells) and subsequent activation of the immune response. The authors first used high-throughput genomic DNA sequencing to explore Ig heavy chain gene rearrangements in B cells from CVID patients versus control subjects. CVID patients displayed abnormal VDJ rearrangement and thus abnormal formation of complementarity determining region 3 (CDR3), which are part of the Ig variable chains and central to the ability of B cells to recognize a wide range of antigens. The authors then sorted B cell populations to retrieve, specifically, the naïve and memory B cells. They detected decreased selection against antibodies with long CDR3 regions in CVID memory repertoires and fewer of the antibody gene mutations that accompany the formation of immune memory. The unmutated naïve B cell pool displayed decreased diversity and abnormal clonal expansion. Together, these alterations could explain the immune deficiencies and autoimmune reactions detected in CVID patients, and suggest that CVID phenotypes stem from aberrant generation and selection of B cell repertoires. Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ~1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. The CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity-determining region 3 (CDR3). We observed a decreased selection against antibodies with long CDR3s in memory repertoires and decreased variable gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive from both decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. The CVID patients also exhibited an abnormal clonal expansion of unmutated B cells relative to the controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro–B stage, cell and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients.

Big data modeling to predict platelet usage and minimize wastage in a tertiary care system

Significance In modern hospital systems where complicated, severely ill patient populations are the norm, there is currently no reliable way to forecast the use of perishable medical resources to enable a smart and economic way to deliver optimal patient care. We here demonstrate a statistical model using hospital patient data to quantitatively forecast, days in advance, the need for platelet transfusions. This approach can be leveraged to significantly decrease platelet wastage, and, if adopted nationwide, would save approximately 80 million dollars per year. We believe our approach can be generalized to all other aspects of patient care involving timely delivery of perishable medical resources. Maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care in modern health care systems. However, daily blood product use is difficult to anticipate. Platelet products are the most variable in daily usage, have short shelf lives, and are also the most expensive to produce, test, and store. Due to the combination of absolute need, uncertain daily demand, and short shelf life, platelet products are frequently wasted due to expiration. Our aim is to build and validate a statistical model to forecast future platelet demand and thereby reduce wastage. We have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-mo period. Using a convex statistical formulation, we have found that platelet usage is highly dependent on weekday/weekend pattern, number of patients with various abnormal complete blood count measurements, and location-specific hospital census data. We incorporated these relationships in a mathematical model to guide collection and ordering strategy. This model minimizes waste due to expiration while avoiding shortages; the number of remaining platelet units at the end of any day stays above 10 in our model during the same period. Compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5 to 3.2%. Extrapolating our results to the ∼2 million units of platelets transfused annually within the United States, if implemented successfully, our model can potentially save ∼80 million dollars in health care costs.

High-fat diet induces systemic B-cell repertoire changes associated with insulin resistance

The development of obesity-associated insulin resistance is associated with B-lymphocyte accumulation in visceral adipose tissue (VAT) and is prevented by B-cell ablation. To characterize potentially pathogenic B-cell repertoires in this disorder, we performed high-throughput immunoglobulin (Ig) sequencing from multiple tissues of mice fed high-fat diet (HFD) and regular diet (RD). HFD significantly changed the biochemical properties of Ig heavy-chain complementarity-determining region-3 (CDRH3) sequences, selecting for IgA antibodies with shorter and more hydrophobic CDRH3 in multiple tissues. A set of convergent antibodies of highly similar sequences found in the VAT of HFD mice but not RD mice showed significant somatic mutation, suggesting a response shared between mice to a common antigen or antigens. These findings indicate that a simple high-fat dietary intervention has a major impact on mouse B-cell repertoires, particularly in adipose tissues.

Streamlining a blood center and hospital transfusion service supply chain with an informatics vendor-managed inventory solution: development, implementation, and 3-month follow-up: INFORMATICS VENDOR-MANAGED INVENTORY

The ordering process at Stanford Health Care involved twice‐daily shipments predicated upon current stock levels from the blood center to the hospital transfusion service. Manual census determination is time consuming and error prone. We aimed to enhance inventory management by developing an informatics platform to streamline the ordering process and reallocate staff productivity.

How do I implement an automated screen for high-titer ABO antibody as an inventory management tool for ABO plasma–incompatible platelets?

Plasma volume reduction (PVR) may reduce the risk of hemolysis associated with transfusion of plateletpheresis blood products (PLTs) containing ABO‐incompatible plasma. But PVR may delay PLT issue. In collaboration with our blood donor center we evaluated an automated screen of PLT for high‐titer ABO antibody and to apply PVR to high‐titer PLTs.