Rachael P. Jackman

Exosomes from red blood cell units bind to monocytes and induce proinflammatory cytokines, boosting T-cell responses in vitro

Extracellular vesicles (EVs) are small, double membrane vesicles derived from leukocytes, platelets, and cells of other tissues under physiological or pathological conditions. Generation of EVs in stored blood is thought to be associated with adverse effects and potentially immunosuppression in blood transfusion recipients. We measured the quantity and cells of origin for EVs isolated from stored red blood cell (RBC) units and tested whether they had any effects on T-cell-mediated immune responses. Mixing peripheral blood mononuclear cells (PBMCs) with EVs resulted in secretion of proinflammatory cytokines and chemokines and increased survival of unstimulated PBMCs. EVs augmented mitogen-induced CD4(+) and CD8(+) T-cell proliferation in an antigen-presenting cell (APC)-dependent manner. We demonstrated that EVs interacted primarily with monocytes and induced proinflammatory cytokine secretion. We also showed that the exosome fraction of EVs and not larger microvesicles was responsible for induction of TNF-α production by monocytes. Furthermore, blockade of CD40 or CD40L accessory molecules largely neutralized the EV augmentation of T-cell responses, implying a role for cell-cell interaction between T cells and EV-activated monocytes. Contrary to our hypothesis, the data demonstrate that EVs isolated from RBC units increase the potency of APCs and boost mitogen-driven T-cell proliferative responses.

Low-level HLA antibodies do not predict platelet transfusion failure in TRAP study participants

In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of 530 participants became refractory to platelet transfusions without evidence of HLA or human platelet antigen (HPA) antibodies. We used a more sensitive bead-based assay to detect and quantify HLA antibodies and a qualitative solid-phase enzyme-linked immunosorbet assay for HPA to determine whether low-level antibodies could predict refractoriness in longitudinal panels from 170 lymphocytotoxicity assay (LCA)(-) and 20 LCA(+) TRAP participants. All TRAP recipients who previously tested LCA(+) were HLA antibody(+), using the bead-based system. Levels of HLA or HPA antibodies did not predict refractoriness among LCA(-) recipients, although higher levels of HLA antibodies were associated with refractoriness among LCA(+) recipients. These data demonstrate that weak to moderate HLA antibody levels detectable by modern binding assays are not associated with platelet refractoriness.

Distinct roles of trauma and transfusion in induction of immune modulation after injury

BACKGROUND: Trauma and transfusion can both alter immunity, and while transfusions are common among traumatically injured patients, few studies have examined their combined effects on immunity.

Transfusion-associated microchimerism: The hybrid within

Microchimerism, the coexistence of genetically disparate populations of cells in a receptive host, is well described in both clinical and physiological settings, including transplantation and pregnancy. Microchimerism can also occur after allogeneic blood transfusion in traumatically injured patients, where donor cells have been observed decades after transfusion. To date, transfusion-associated microchimerism (TA-MC) appears confined to this clinical subset, most likely due to the immune perturbations that occur after severe trauma that allow foreign donor cells to survive. Transfusion-associated microchimerism appears to be unaffected by leukoreduction and has been documented after transfusion with an array of blood products. The only significant predictor of TA-MC to date is the age of red cells, with fresher units associated with higher risk. Thus far, no adverse clinical effect has been observed in limited studies of TA-MC. There are, however, hypothesized links to transfusion-associated graft vs host disease that may be unrecognized and consequently underreported. Microchimerism in other settings has gained increasing attention owing to a plausible link to autoimmune diseases, as well as its diagnostic and therapeutic potential vis-a-vis antenatal testing and adoptive immunotherapy, respectively. Furthermore, microchimerism provides a tool to further our understanding of immune tolerance and regulation.

Effects of blood sample age at time of separation on measured cytokine concentrations in human plasma.

ABSTRACT Measurement of peripheral blood cytokines and other immunomodulatory proteins is a useful and popular tool for assessing human immune responses to a wide range of assaults. A common challenge in this work is obtaining fresh, high-quality samples and limiting the time between blood collection and the separation of plasma or serum from cells. In this study we sought to determine the effect of sample age at the time of processing on the measured levels of 41 soluble immune mediators. Two cohorts were examined: healthy lab donors and trauma patients, who have significant immune perturbation. Whole-blood samples were aliquoted, and plasma was isolated, at days 0, 1, 2, and 3 after collection. Multiplexing techniques were used to measure protein concentrations, and general estimating equations were used to determine if there was a significant change over time. Over the 3-day period examined, only 15 of the 41 proteins showed no significant change in either cohort. Among the remaining proteins both increases and decreases were observed, with changes ranging from 2.4% per day to 325% per day. Proteins with significant changes in one cohort did not always show significant changes in the other group. These results support the need to separate plasma or serum from whole blood as quickly as possible and/or to standardize the length of time to processing within a given study of peripheral blood protein concentrations. When this is not possible, care should be taken to account for differences due to sample age.

Immune modulation and lack of alloimmunization following transfusion with pathogen‐reduced platelets in mice

Transfusion of allogeneic blood products can lead to alloimmunization, impacting success of subsequent transfusions and solid organ transplants. Pathogen reduction using riboflavin and ultraviolet B (UVB) light has been shown to eliminate the immunogenicity of white blood cells (WBCs) in vitro through down regulation of surface adhesion molecules, effectively blocking cell–cell conjugation and direct presentation. We sought to determine if this loss of immunogenicity is extended in vivo where indirect presentation of allogeneic antigens can occur.

Understanding loss of donor white blood cell immunogenicity after pathogen reduction: mechanisms of action in ultraviolet illumination and riboflavin treatment.

BACKGROUND: Donor white blood cells (WBCs) present in transfusion products can lead to immune sequelae such as production of HLA antibodies or graft‐versus‐host disease in susceptible transfusion recipients. Eliminating the immunogenicity of blood products may prove to be of clinical benefit, particularly in patients requiring multiple transfusions in whom allosensitization is common. This study examines a method of pathogen reduction based on ultraviolet light illumination in the presence of riboflavin. In addition to pathogens, WBCs treated with this system are affected and fail to stimulate proliferation of allogeneic peripheral blood mononuclear cells (PBMNCs) in vitro.

Leukoreduction and ultraviolet treatment reduce both the magnitude and the duration of the HLA antibody response

Both leukoreduction and ultraviolet (UV) light treatment of blood products have been shown to reduce the incidence of HLA antibody development in recipients, but the impact of these treatments on the magnitude and persistence of the antibody response is less clear.

Reduced alloimmunization in mice following repeated transfusion with pathogen‐reduced platelets

Allogeneic transfusion can result in alloimmunization, leading to platelet (PLT) refractoriness and rejection of solid organ transplants. Previously we demonstrated that pathogen reduction using UV light and riboflavin (UV + R) eliminates the immunogenicity of white blood cells (WBCs) in vitro, blocks alloimmunization from transfusion in mice, and results in reduced ex vivo cytokine responses to subsequent untreated transfusions. We sought to determine if repeated transfusion with pathogen‐reduced PLT‐rich plasma (PRP) would eventually cause breakthrough alloimmunization or enhanced tolerance.

C1q-binding anti-HLA antibodies do not predict platelet transfusion failure in Trial to Reduce Alloimmunization to Platelets study participants

In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of 530 subjects became clinically refractory (CR) to platelets (PLTs) without lymphocytotoxicity assay (LCA)‐detectable anti‐HLA antibodies. The LCA only detects complement‐binding antibodies and is less sensitive than newer assays. Utilizing a more sensitive bead‐based assay that does not distinguish between complement‐binding versus non–complement‐binding antibodies, we have previously shown that while many LCA‐negative (LCA–) patients do have anti‐HLA antibodies, these low‐ to moderate‐level antibodies do not predict refractoriness. As complement can contribute to PLT rejection, we assessed if previously undetected complement‐binding antibodies account for refractoriness among LCA– patients.