ALBERT

Description: Factors regulating which patients become alloimmunized to red blood cell (RBC) antigens are poorly understood. Using a murine model of transfusion, we recently reported that viral-like inflammation with polyinosinic polycytidylic acid [poly (I:C)] significantly enhances RBC alloimmunization. Herein, we tested the hypothesis that poly (I:C) exerts this effect, at least in part, at the level of antigen-presenting cells (APCs). Using a novel in vivo method, we report that in the noninflamed state, most transfused RBCs were consumed by splenic macrophages, with only trace consumption by splenic dendritic cells (DCs). To a lesser extent, RBCs were also consumed by APCs in the liver. However, unlike soluble antigens, no RBCs were consumed by APCs in the lymph nodes. Inflammation with poly (I:C) induced significant consumption of transfused RBCs by splenic DCs, with a concomitant increase in costimulatory molecule expression. Moreover, this resulted in increased proliferation of CD4+ T cells specific for the mHEL RBC alloantigen. Finally, splenectomy abrogated the enhancing effects of poly (I:C) on RBC alloimmunization. Together, these data provide additional insight into the nature of transfused RBCs as an immunogen and provide a mechanism by which viral-like inflammation enhances alloimmunization to transfused RBCs.

Description: Background: Factors influencing rates of alloimmunization to antigens on transfused red blood cells (RBC alloimmunization) are poorly defined. In particular, the role of the spleen with respect to alloantibody formation is unclear, with conflicting clinical reports in the literature. Moreover, the complexities of multiply mismatched antigens and antigen priming due to transfusions prior to splenectomy make human studies difficult to interpret. To better define the role of the spleen in RBC alloimmunization, we utilized a murine model of transfusion medicine (with the model RBC antigen mHEL (membrane bound hen egg lysozyme)). Methods: Cohorts of splenectomized and non-splenectomized mice (C57BL/6 × B10.BR) were transfused with the human dose equivalent of 1 unit of leukoreduced mHEL RBCs. RBC alloimmunization was assessed by anti-HEL IgG specific ELISA. The role of antigen-specific CD4+ T cells was studied by the adoptive transfer of 1.5 × 106 HEL-specific CFSE-labeled CD4+ T cells from 3A9 TCR transgenic donors. Adoptively transferred cells were visualized using a congenic marker (Thy1.1); enumeration and division of these cells were monitored by flow cytometry in liver, spleen (if applicable), and lymph node preparations. Results: Splenectomy dramatically decreased RBC alloimmunization; 14 of 14 splenectomized mice (from 3 experiments) had undetectable to very low levels of anti-HEL IgG following transfusion with mHEL RBCs (average 6.3 fold less than non-splenectomized mice, 95% C.I. 4.6). Moreover, ten of ten splenectomized mice failed to make detectable levels of anti-HEL IgG even following the adoptive transfer of HEL-specific CD4+ T cells. In comparison, elevating the precursor frequency of HEL-specific CD4+ T cells increased RBC alloimmunization by 10,000 fold in non-splenectomized mice. Proliferation and division of CD4+ T cells were detectable in both spleen and liver preparations of non-splenectomized mice by day 3 following transfusion; in contrast, no expansion nor division of CD4+ T cells was seen in liver nor lymphatic preparations of splenectomized animals. Conclusions: The low level of RBC alloimmunization seen in non-splenectomized mice in this system is limited by existing CD4+ helper T cell responses, as increasing the naive helper T cell precursor frequency dramatically increased RBC alloimmunization. Furthermore, the spleen itself is critical to CD4+ helper T cell function during alloimmunization given that, in splenectomized mice, adoptively transferred HEL-specific CD4+ T cells fail to expand, divide, or stimulate production of detectable alloantibody. Ongoing studies are investigating the phenotype of HEL-specific CD4+ T cells (as effector cells, anergic cells, or regulatory cells) in splenectomized and non-splenectomized mice. These studies have implications for preventing alloimmunization in transfusion-dependent patient populations.

Description: Background A critical barrier to progress in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been a lack in understanding regarding why some transplant recipients of HLA-matched transplant grafts develop severe graft-versus-host disease (GvHD) while other recipients have relapse of their cancer without GvHD. Patients who develop a modest degree of acute and/or chronic GvHD have less relapse and optimal survival after allogeneic BMT. Thus, a mechanistic understanding of regulation of donor T-cell activation after allo-HSCT is needed. Using mouse models, Desmarets et al. have shown that pre-transplant leukoreduced RBC transfusions can cause recipient immunization against minor histocompatibility antigens (miHA) and activation and expansion of recipient T-cells that recognize donor miHA, contributing to rejection of subsequent allo-HSCT (Blood. 2009; 114:2315). Preliminary data from our lab suggest that leukoreduced RBC transfusions given concurrently with allo-HSCT can also increase post-transplant activation and expansion of donor T-cells, an effect which may lead to increased GvHD after transplant. Here, we have conducted a retrospective study of post-transplant RBC transfusions and acute GvHD (aGvHD) in allo-HSCT patients. We hypothesized that increased numbers of transfusions during the 30-day post-transplant period would be correlated with increased severity of aGvHD in these patients. Methods We conducted a retrospective analysis of RBC transfusion records and aGvHD data collected for 181 adult allo-HSCT patients who received their transplants at Emory University Hospital (EUH) between 2004 and 2009. Nine patients were excluded who died 〈 50 days post-transplant without developing aGvHD, since this was too early to determine aGvHD occurrence. Of the remaining 172 patients studied (median age 48 yrs at time of transplant, range 18-72), 88 (49%) were male and 84 (51%) were female. Patients had received either matched related HSCT (n=69, 40%) or matched unrelated HSCT (n=103, 60%) for treatment of SAA (n=7), BAL (n=2), ALL (n=18), AML (n=69), hemolytic anemia (n=2), CLL (n=6), CML (n=8), HD (n=5), MDS (n=23), myelofibrosis (n=6), MM (n=7) or NHL (n=19). For pts who developed aGvHD, the onset time ranged from 1 to 139 days post-transplant, with a median of 30 days. No aGvHD (grade 0) was diagnosed in 58 pts (34%), while 37 pts (21%) developed grade 1 aGvHD and 77 pts (45%) developed grade 2-4 aGvHD. The number of ABO matched, irradiated RBC units transfused 0 - 30 days post-transplant was tallied for each patient, ranging from 0 (no transfusions, n=13, 7.6% of pts) to 26 units, with an average of 5.6 and median of 4 units. All transfusions during this timeframe were administered at EUH. The median follow up time was 22 months post-transplant (range, 1.1 – 96.1 months). Results Pts were assigned to two groups, those who developed grade 0-1 aGvHD (n=95, 55%) or grade 2-4 aGvHD (n=77, 45%) within 140 days post-transplant. This study did not include analysis of late-onset aGvHD or chronic GvHD past this time point. Patients with grade 2-4 aGvHD had a higher average number of transfusions 0 - 30 days post-transplant compared with patients having grade 0-1 aGvHD (6.5 vs. 4.9 units, p = 0.02). Receiver-operator characteristics (ROC) analysis showed that a cutoff value of 〉 4 transfusions 0 - 30 days post-transplant had 56% sensitivity and 65% specificity to predict development of grade 2-4 aGvHD. When tested by logistic regression in a multivariate model, this cutoff value had a highly significant correlation with grade 2-4 aGvHD, with an odds ratio of 2.83 and p value = 0.0024. Other covariates including patient age, gender, and type of transplant (related vs. unrelated) were not significantly associated with aGvHD outcome. Conclusion Our retrospective analysis identified a significant positive correlation between the number of post-transplant RBC transfusions and severity of aGvHD after allo-HSCT. Additional studies are planned to determine whether RBC transfusions 0 - 30 days post-transplant stimulate allo-reactive T-cells via allo-antigen presentation or by otherwise promoting inflammation, and if one or both of these mechanisms contribute to increased GvHD. If so, it may be possible to develop strategies for optimization of RBC transfusion practices to reduce the risk of severe aGvHD after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p

Description: Transfusion of red blood cells (RBCs) into patients with anti–donor RBC antibodies (crossmatch-incompatible transfusion) can result in lethal antibody-mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival or adversely affecting the transfusion recipient. In an effort to elucidate the mechanistic details of this process, we describe the first animal model of nonhemolytic antibody-induced RBC antigen loss. RBCs from transgenic mHEL mice express surface hen egg lysozyme (HEL) as a transmembrane protein. Transfusion of mHEL RBCs into mice immunized with HEL results in selective loss of HEL antigen from donor RBCs without affecting other blood group antigens or reducing the circulatory life span of the transfused RBCs. While this process does not require the presence of a spleen, it requires both anti-RBC immunoglobulin G (IgG) antibodies and the FcγIII receptor. These studies provide mechanistic insight into the phenomenon of antigen loss during incompatible transfusion in humans.

Description: Red Blood Cells (RBCs) units which can be stored for up to 42 days in the US undergo biochemical and morphological changes known as the storage lesion. The clinical significance of these changes is unclear. Results from 〉55 observational studies have produced conflicting results: some are negative while others report associations between transfusion of longer-stored RBCs and mortality, infections, lung injury, deep vein thrombosis, multiple organ failure, and a decrease in tissue oxygenation. Recent advances are shedding some light on this controversy. While some elements of the RBC storage lesion such as pH and cationic changes and decreases in adenosine triphosphate and 2,3-diphosphoglycerate are well known, the recent application of "omics" technologies is revealing complex changes in metabolites, proteins, and lipids during storage. RBCs storage causes dysregulations in several metabolic (e.g., glycolytic) pathways which vary with unit processing, additive solution, storage period, and blood donor characteristics. Longer-stored RBCs demonstrate decreased antioxidant activity and impaired energy metabolism. Kinases and proteolytic enzymes become activated which affect Band 3 and structural proteins and result in remodeling of the RBCs' cytoskeleton; leading to increasing osmotic fragility and shedding of microparticles in the supernatant. The timing and extent of these changes need to be further elucidated; some appear to occur immediately (e.g., reduction in S-nitrosohemoglobin) while most appear after 2 weeks. These changes lead one to question the safety and efficacy profiles of longer-stored RBC transfusions. Animal models have recently evaluated potential consequences and possible mechanisms that could underlie adverse events in "susceptible" hosts. Two major hypotheses have been corroborated by animal studies. The first relates to the potential inhibition of Nitric Oxide (NO)-mediated vasodilatory effects as a result of NO scavenging by excess cell-free hemoglobin or because of a loss of RBC-mediated hypoxic vasodilation. The second is based on the fact that transfusion of a 42-day old RBC unit provides a large iron bolus to the mononuclear phagocyte system. Such a bolus can result in acute increases in non-transferrin bound iron (NTBI) which can cause oxidative damage and potentiate bacteria proliferation. Both the NO and Iron hypotheses appear at play in a study in septic canines that showed that transfusion of 42-day RBCs resulted in increases in cell free hemoglobin, NTBI, and plasma labile iron resulting in increased shock, lung injury, and mortality. However, two recent clinical trials in 377 premature infants and 2430 intensive care patients, respectively, did not demonstrate differences in outcomes following transfusion of

Description: Background : Recipients of hematopoietic stem cell transplantation (HSCT) can be severely immunocompromised, predisposing to opportunistic infections including cytomegalovirus (CMV). While adoptive transfer of ex vivo expanded donor antiviral T-cells can prevent viral disease, this approach is not frequently used clinically because cell culture and selection procedures are lengthy, labor-intensive, and expensive. We describe an alternative approach. A single inoculation of a strongly immunogenic, but safe, live-attenuated, Listeria-based vaccine (Lm-MCMV) can rapidly drive extensive in vivo expansion of anti-murine CMV (MCMV) CD8+ T-cells during immune reconstitution following HSCT. Methods : The Lm-MCMV vaccine is derived from a genetically defined Listeria monocytogenes ΔactAΔinlB vaccine strain (Brockstedt et al. PNAS in press) that is attenuated by 4-logs in a mouse virulence assay, as compared to wild-type Listeria. Lm ΔactAΔinlB was engineered to express and secrete the MCMV H-2b immunodominant peptide HGIRNASFI within an ovalbumin scaffold. C57BL/6 (H-2b; CD45.2) HSCT recipients were conditioned with 11 Gy irradiation on day -1, and injected with 5x106 T cell depleted (TCD) C57BL/6 bone marrow cells on day 0. Selected mice simultaneously received 3x107 splenocytes from PepBoy (H-2b; CD45.1) donors that were either naive or immunized with 1x107 colony forming units (cfu) Lm-MCMV on day -7. On day +21, selected groups were vaccinated with 1x107 cfu Lm-MCMV, and sacrificed up to 14 days later. MCMV-specific CD8+ T-cells were quantified by intracellular cytokine and tetramer staining. Residual viable Lm-MCMV vaccine in the spleen, liver, and brain of BMT recipients was measured by plating on BHI agar media. Results : No mortality occurred following vaccination. Viable Lm-MCMV, which peaked the day after immunization in the liver (avg = 6.1x104 cfu) and spleen (avg = 1.4x105 cfu) of HSCT recipients, were completely cleared within 5 days of inoculation. Viable Listeria were not isolated from brain. Without vaccination at day +21, none of the HSCT recipients had detectable anti-MCMV CD8+ T cells. In contrast, immunization with Lm-MCMV at day +21 lead to marked antiviral T-cell expansion in all groups. HGIRNASFI-specific CD8+ T-cells averaged 2.4% of total CD8+ T-cells in vaccinated TCD marrow recipients. Levels were significantly higher in recipients of TCD marrow plus naive (7.1%) or immune (19.0%) donor splenocytes. Conclusions : Immunization of BMT mice with live-attenuated Lm-MCMV was safe. All HSCT recipients survived immunization and rapidly cleared viable Listeria. A single Lm-MCMV immunization during the period of immune reconstitution following HSCT was also effective, driving extensive antiviral T-cell expansion. In HSCT recipients of TCD marrow alone, vaccination produced levels of HGIRNASFI-specific CD8+ T-cells (2.4%) comparable to that seen with adoptive immunotherapy. Co-transplantation of naive or immune donor splenocytes lead to significantly higher levels of antiviral CD8+ T-cells following vaccination (7.1% and 19%, respectively). This approach could represent a broadly applicable alternative to adoptive immunotherapy. Future studies will focus on optimizing vaccination strategies, including use in allogeneic transplantation.

Description: Background: GvHD and opportunistic infections are the major causes of morbidity and mortality in cancer patients treated with allogeneic BMT. In allogeneic BMT patients, donor derived T-cells help eradicate residual cancer and fight against opportunistic infections but they also cause the major deleterious effects, including GvHD which is the result of host allo-antigens recognition by the donor T-cells. Moreover, donor T-cells also play a critical role in promoting stem cell engraftment, encouraging rapid recovery of cellular immunity, and decreasing the probability of disease relapse. Thus, to establish a therapeutically useful adoptive immunotherapy using donor T-cells, separation of the beneficial anti-opportunistic infection and anti-tumor effects of donor T-cells from the deleterious GvHD effect are highly desirable. We previously showed that amotosalen-treated splenocytes rescued recipients from a lethal dose of MCMV administered on day 0 in experimental parent to F1 allogeneic bone marrow transplant (BMT). To model early post-transplant CMV reaction, in this study, we investigated the anti-viral immune responses and GvHD activity of treated donor T-cells after infecting allogeneic BMT recipients with a lethal dose of MCMV on 7 days post transplant. Methods: Using a parent to F1 mouse BMT model, splenocytes (3×106 untreated or 10×106 amotosalen-treated) harvested from the MCMV immunized C57BL/6 donors were transplanted along with 5×106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 (C57BL/6 × Balb/C) recipients. Recipient mice were infected i.p. with a sublethal dose (5×104 pfu per mouse) of MCMV 7 days after transplant. Flow cytometry was used to quantitate T cell chimerism (in recipient spleen and thymus) and MCMV-peptide specific tetramer+ CD8+ T-cells. Serum IFN-γ and TNF-α were determined by ELISA. Liver viral load was determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: MCMV infection in recipients of amotosalen-treated splenocytes did not cause any mortality whereas recipients of untreated splenocytes had 40% early mortality due to acute GvHD. Like the recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes effectively cleared MCMV from their liver within 10 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with donor spleen- and host-derived MCMV peptide specific tetramer+ CD8+ T cells that proliferated following day 7 post MCMV infection. Significantly higher numbers of host derived CD4−CD8− (DN) TCRαβT-cells appeared in the spleen with peak on day 3 post MCMV infection among recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes. Lower levels of serum IFN-γ and TNF-α and preservation of thymic function were also noted in the recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes following MCMV infection. Conclusion: Adoptive immunotherapy with amotosalen-treated T cells is an ideal therapeutic approach that facilitates early hematopoietic engraftment, anti-viral donor immune reconstitution and preserves early post-transplant host immunity leading to protection from lethal viral infection without causing aGvHD.

Description: Background: Hemolysis is not the inevitable result of transfusing crossmatch incompatible blood, even for clinically significant antigens. Less-well appreciated is the phenomenon of “antigen-loss” where the offending antigen is stripped off of RBCs, leaving the RBCs intact with a normal circulatory lifespan. Recently, we described a novel murine model of antigen-loss during transfusion of crossmatch incompatible RBC. mHEL mice express the model antigen Hen Egg Lysozyme (HEL) as a RBC cell surface protein. Transfusion of mHEL RBC into recipients immunized against HEL results in only minimal hemolysis, with the majority of RBC undergoing non-hemolytic loss of the mHEL antigen. In the current report, we isolated a panel of novel monoclonal anti-HEL antibodies to investigate the properties of antibodies that can cause antigen-loss. Methods: BALB/c mice were hyper-immunized with HEL, resulting in a polyclonal IgG anti-HEL response. Splenic fusions with myeloma cells were performed and monoclonal antibodies were isolated. To test the ability of the monoclonal antibodies to induce antigen-loss, recipient mice were infused with the antibodies and then transfused with mHEL RBC labeled with a fluorescent dye. RBC survival, antibody binding to the transfused RBC and mHEL surface antigen levels were monitored by flow cytometry. Results: A panel of 7 monoclonal antibodies that showed strong binding to mHEL RBC were tested. No antigen-loss was observed when monoclonal antibodies were infused alone, despite strong binding of the antibodies to transfused RBC. This was not due to slowed kinetics, as no antigen-loss was observed even after 21 days. In contrast, infusing a mixture of the 7 monoclonal antibodies caused complete antigen-loss within 4 days, similar to polyclonal antiserum. Each combination of 2 antibodies was tested (21 conditions). (14/21) combinations caused greater than 99% antigen-loss by 4 days, whereas no antigen-loss was observed in the other 7 combinations. To determine the relative epitope specificity of the monoclonal antibodies, binding competition studies were carried out. Of the 7 antibodies, 4 completely blocked each other (Group 1 – 5B9.A1, 4B7.B1, 8E12.D10, and GD7). In contrast, 2 antibodies (Group 2 – 2F4 and 6F7.D8) did not block any antibodies from group 1. Preincubation with 2F4 blocked binding of 6F7.D8 but 6F7.D8 did not block 2F4 (defined as partial identity). The seventh antibody, 6F7.D8, had partial identity to group 1, but did not block antibodies from group 2. A comparison of the groups revealed that antigen-loss occurred in all cases where two antibodies bound to different epitopes (including partial identity), but not when antibodies bound the same epitope. Discussion: The current findings demonstrate that antigen-loss of mHEL only occurs when at least two antibodies, which recognize different epitopes, simultaneously bind the mHEL antigen. These data suggest mechanisms of lattice formation, involving crosslinking of multiple antigens by different antibodies. Ongoing mechanistic studies are assessing this hypothesis. A practical implication of these findings is that the likelihood of antigen-loss in a given patient may correlate to the multiplicity of epitopes of a blood group antigen recognized by a given patient’s antibody response.