Unusual kinetics of white cell clearance in transfused mice

BACKGROUND: Donor white cells (WBCs) in blood transfusions are responsible for complications in recipients, including alloimmunization, graft-versus-host disease (GVHD), and virus transmission and reactivation. The recent use of sequence-specific polymerase chain reaction assays to monitor the kinetics of clearance of donor WBCs in transfused humans and dogs found transient recirculation of donor lymphocytes on Days 3 to 5 after transfusion; this presumably reflected an abortive GVHD reaction to major histocompatibility complex-incompatible recipient cells, after which donor WBCs were cleared to undetectable levels. STUDY DESIGN AND METHODS: This study sought to develop a murine model to further characterize the kinetics and major histocompatibility complex restriction of donor WBC clearance. A sensitive murine Y chromosome- specific polymerase chain reaction assay was developed and applied to serial blood samples collected after transfusions of allogeneic blood to naive inbred, primed inbred, and outbred mice, as well as after transfusions of gamma-radiated blood to naive inbred mice. RESULTS: In inbred mice, both naive and primed to the allogeneic blood donor, transfused WBCs were not cleared to undetectable levels for more than 1 month after transfusion. Transfused outbred mice also showed prolonged donor WBC survival, although at lower levels than inbred mice. There was no evidence of GVHD in either inbred or outbred mice, and gamma radiation had no significant impact on donor WBC persistence. CONCLUSION: These results contrast with the rapid clearance of donor WBCs observed in humans and dogs. The immunologic basis for this discrepancy remains unclear. Caution should be exercised in any extrapolation to humans of conclusions drawn from results in transfused mice.     

Minimum conditions of major histocompatibility complex compatibility and recipient immune compromise required to establish donor white blood cell persistence in a murine transfusion model

BACKGROUND: In some patients multiply transfused to treat severe trauma, white blood cells (WBCs) from a single blood donor can persist for years, constituting up to 5 percent of all circulating WBCs. The immunologic mechanisms responsible for this are not known but, if understood, might allow manipulation of the human immune system to induce microchimerism for a variety of therapeutic purposes. To better characterize these mechanisms, a murine transfusion model was developed with a panel of immunologic knockouts as transfusion recipients. By conducting a systematic series of transfusion experiments, the purpose was to determine which recipient immune cell population, when abrogated, could lead to prolonged survival of donor cells (microchimerism). STUDY DESIGN AND METHODS: Blood was transfused from normal donors to knockout recipients in syngeneic, allogeneic, and xenogeneic settings. Donor WBC survival was evaluated by quantitative polymerase chain reaction, and recipient lymphocyte subsets by fluorescence-activated cell sorting. RESULTS: In the syngeneic setting, donor WBCs persisted in C2ta, RAG-1, and TCR knockout recipients. Allogeneic donor WBCs persisted in RAG-2 and RAG-2/Common gamma knockout recipients. Xenogeneic donor WBCs required RAG-2/Common gamma and RAG-2/Pfp double knockouts to persist. CONCLUSION: It is concluded that donor-recipient major histocompatibility complex (MHC) concordance alone is not sufficient to achieve microchimerism. Further, the degree of recipient immune compromise necessary to achieve persistent microchimerism is directly proportional to the degree of donor-recipient MHC disparity.     

Identification of the blood component responsible for increased susceptibility to gut-derived infection

It has previously been reported that the transfusion of allogeneic whole blood increases sepsis-related mortality and decreases the ability of the host to kill bacteria that have translocated from the intestinal tract. To determine which blood component contributes to this adverse effect, the impact of the transfusion of white cells (WBCs), red cells (RBCs), and plasma on microbial translocation, bacteria killing, and mortality rate was studied. Blood from C3H/HeJ mice was separated into WBCs, RBCs, and plasma, and these fractions were transfused to Balb/c mice. Controls received sterile saline. Five days after transfusion, all Balb/c mice underwent a 20-percent burn and gavage with 1 × 10(10) Escherichia coli labeled with 14C-glucose. Mortality was observed for 10 days. Four additional groups, receiving the same treatment as above, were sacrificed 4 hours after the burn, and mesenteric lymph nodes, liver, kidney, and blood were harvested aseptically. For each tissue, quantitative colony counts, radionuclide counts, and percentage of translocated bacteria that remained alive were calculated. By radionuclide counts, no difference was observed in the degree of 14C E. coli translocation among the groups. In contrast, the percentage of viable bacteria and the mortality rate were significantly higher in the group receiving allogeneic WBCs than in all other groups (p < 0.05). It is concluded that WBCs are the component in transfused blood that has an adverse effect on host resistance to gut- derived infection.     

输注脾细胞建立输血相关的移植物抗宿主模型

目的探讨输注同种脾细胞建立小鼠输血相关的移植物抗宿主(TA-GVHD)模型及其特点。方法分离C57BL/6(H-2b)脾细胞,分别经静脉输注给Balb/c(H-2d)裸鼠与F1代(C57BL/6×Balb/c H-2b/d),建立输血相关的移植物抗宿主模型。活体染料羧基荧光素乙酰乙酸(5,6-carboxyfluorescein diacetate succini midyl ester,CFSE)标记供者脾细胞,通过流式细胞仪分析供者细胞在受者体内的增殖;免疫组化分析供者脾细胞survivin的表达;检测Balb/c裸鼠体内抗供者抗体及IL-10浓度;分析皮肤、小肠、肝脏的病理变化。结果输注同种脾细胞成功建立急性TA-GVHD模型。受者脾脏及外周可以检测到标记后增殖的供者细胞。小鼠的病理改变主要出现在肝脏,浸润细胞早期表达survivin。小肠及皮肤未见明显的病理改变。TA-GVHD裸鼠第7天IL-10、抗-Balb/c显著升高。结论输注同种脾细胞可以建立TA-GVHD模型,并具有简单、快速、稳定的特性,为诊断、治疗GVHD提供了良好的模型。     

Effect of blood transfusion on survival in a mouse bacterial peritonitis model

Allogeneic blood transfusions can result in alloimmunization or immunosuppression. A previous study demonstrated a deleterious effect of allogeneic blood transfusion on tumor growth in mice that was dependent, in part, on the dose of tumor cells with which the host animal was inoculated. The current study examined the effect of a similar allogeneic blood transfusion protocol on survival in a mouse bacterial peritonitis model. C57Bl/6J mice were transfused with 0.2 mL of heparinized fresh whole blood from C57Bl/6J (syngeneic) or Balb/c (allogeneic) mice. Transfusions were given on Days -10 and -7. On Day 0, mice were injected intraperitoneally with 10(7) Escherichia coli. Survival at Day 7 was 61 percent in the allogeneic blood transfusion group and 55 percent in the syngeneic blood transfusion group (p = 0.52). Experiments using different strains of mice, different transfusion protocols, and different doses of bacteria also failed to demonstrate an effect of allogeneic blood transfusion on survival. The results demonstrate that blood transfusion does not influence survival after a septic challenge with bacteria. The data obtained in the present study, together with those obtained in the tumor model, suggest that the mechanisms by which the allogeneic blood transfusion impedes host defense against bacterial infections is different from the mechanisms involved in tumor growth.     

WBC-reduced blood transfusions and clinical outcome in children with acute lymphoid leukemia

BACKGROUND: WBC reduction offers a variety of benefits to patients requiring multiple transfusions during induction therapy for childhood acute lymphoid leukemia (ALL), including reductions in febrile transfusion reactions, HLA alloimmunization, and CMV transmission. One potential benefit is a reduction in the deleterious effects of transfusion immunomodulation. In the surgical setting, transfusion immunomodulation has been linked to increases in postoperative infections and decreases in host cellular immunity that are mitigated by WBC reduction of transfused blood. STUDY DESIGN AND METHODS: A retrospective review was conducted of the medical records of 68 consecutive children undergoing induction therapy for newly diagnosed ALL from 1988 through 1995, a period whose midpoint is 1991, the year WBC reduction was introduced in this hospital. RESULTS: WBC reduction of platelet and RBC transfusions was associated with fewer days with fever (mean, 5.7 days [no WBC reduction] and 2.1 days [WBC reduction]; p = 0.012) and days with positive microbial cultures (mean, 1.5 [no WBC reduction] and 0.71 [WBC reduction]; p = 0.0055). There were more high-risk ALL patients in the group receiving WBC-reduced transfusions. CONCLUSION: Allogeneic WBCs in transfused blood may cause impairment of host defenses against microbial infection during induction therapy for childhood ALL.     

Transfusion-related acute lung injury due to granulocyte-agglutinating antibody in a patient with paroxysmal nocturnal hemoglobinuria

BACKGROUND: Transfusion-related acute lung injury (TRALI) is usually reported after the transfusion of blood components from donors with white cell (WBC) antibodies, but only very rarely if the patient has these antibodies. The pathogenesis of TRALI is not fully understood. Not all transfusion recipients develop TRALI, even though WBC antibodies are present in the donor or the recipient. CASE REPORT: A patient with paroxysmal nocturnal hemoglobinuria (PNH) who developed TRALI after the transfusion of non-WBC-reduced red cells is described. Granulocyte-agglutinating anti-5b was detected in his serum, and the crossmatch with the donor granulocytes was positive. The patient also developed a severe exacerbation of hemolysis with renal failure; serologic results excluded an immune hemolytic posttransfusion reaction. The patient recovered from both events after about 1 week. CONCLUSION: Granulocyte-agglutinating antibodies present in the recipient play an important role in TRALI, and also other factors may contribute to its pathogenesis. The reaction between the PNH patient's antibody (anti-5b) and transfused WBCs was found not only to be responsible for the respiratory distress but also to have triggered, through the innocent-bystander mechanism of complement activation, an intensive hemolysis, which was very likely a contributing factor in the development of TRALI.     

Transfusion-related immunomodulation by platelets is dependent on their expression of MHC Class I molecules and is independent of white cells

BACKGROUND: Transfusion‐related immunomodulation (TRIM) has been correlated with the presence of white cells (WBCs) in blood transfusions, but the role of components such as platelets (PLTs) in mediating TRIM has not been extensively examined. We designed a murine PLT transfusion model to study whether leukoreduced PLTs mediate TRIM effects. STUDY DESIGN AND METHODS: CBA recipient mice were administered four weekly transfusions of either fresh (4 hr) or aged (24 and 72 hr) donor leukoreduced PLTs from allogeneic BALB/c mice and then transplanted with skin grafts from donor‐matched mice. TRIM was measured by comparing the times to graft rejection and these were correlated with immunoglobulin G (IgG) antibody development measured by flow cytometry. RESULTS: Compared with nontransfused control recipients, four transfusions of fresh, extremely leukoreduced (<0.05 WBCs/mL), allogeneic PLTs significantly (p < 0.002) reduced the recipient's ability to reject donor‐matched skin grafts (survival >49 days compared with <14 days in nontransfused controls) despite the presence of high‐titered serum IgG donor antibodies. In contrast, however, aged PLTs or fresh PLTs devoid of MHC Class I molecules were unable to affect skin graft survival nor stimulate antibody production. The PLT age‐related inability to induce TRIM was shown to be due to loss of PLT‐associated MHC Class I molecules; soluble supernatant MHC molecules that were transfused were unable to induce TRIM. CONCLUSION: These results suggest that fresh PLTs can induce TRIM independently of WBCs due to their MHC antigen expression whereas aging results in loss of MHC and ability to mediate TRIM. The findings support the concept that either active MHC removal from fresh PLTs or passive removal by, for example, storage, may reduce any deleterious effects of TRIM in transfusion recipients.     

Rapid clearance of transfused murine red blood cells is associated with recipient cytokine storm and enhanced alloimmunogenicity

BACKGROUND: Fourteen‐day stored red blood cells (RBCs) containing an RBC‐specific transgenic antigen (HOD) induce a recipient proinflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes. STUDY DESIGN AND METHODS: HOD RBCs were treated by two distinct methods known to lead to rapid posttransfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells before transfusion, and RBC recovery, recipient cytokine response, and recipient anti‐HOD alloimmunization response were measured after transfusion. RESULTS: Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours posttransfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine‐ or heat‐treated RBCs had elevated circulating levels of keratinocyte‐derived chemokine/CXCL‐1, monocyte chemoattractant protein‐1, and interleukin‐6 after transfusion. Furthermore, phenylhydrazine‐ or heat‐treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1‐ and 10.3‐fold enhancement, respectively, of anti‐HOD alloimmunization magnitude by flow cytometric crossmatch. CONCLUSIONS: Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid posttransfusion clearance, with a recipient proinflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes and suggest that human donor RBCs with favorable posttransfusion clearance profiles may be less immunogenic.     

Depletion of white cells from platelet concentrates with a new adsorption filter

Removal of white cells (WBCs) from platelets may reduce alloimmunization to WBC antigens, prevent febrile reactions, and improve platelet increments in multiply transfused patients receiving HLA-matched platelets. A new surface-modified fibrous polyester filter was evaluated; it requires no special processing of pooled platelet concentrates and can be used at the patient's bedside. The studies were designed to measure WBC removal, platelet function, in vitro platelet recovery, and in vivo platelet survival. WBC mean removal was 99.8 percent +/- 0.56 (n = 37) when a pool similar in volume to 6 platelet concentrates was tested. The mean number of residual WBCs after filtration was 5.6 x 10(5). In vitro mean platelet recovery was 86.9 percent for a pool size of 6 units (n = 37). Clot retraction and platelet aggregation were unaffected by filtration. Survival studies of 111Indium-labeled platelets done with filtered autologous platelets showed no reduction in the normally expected survival. These studies indicated that the filter efficiently removes WBCs without substantially decreasing platelet number, survival, or function. This device offers the potential of considerably improving platelet transfusion therapy.     

Managing recalls and withdrawals of blood components

Donor centers are issuing a growing number of recalls and market withdrawals to hospital transfusion services about blood components. More than 1 in 2,000 units were recalled in the late 1990s in the United States. The most common reason for these notices from donor centers is postdonation donor information. Most of these units had been transfused, and many present a "risk of a risk" (ie, a problem might have been present that might have affected the recipient). A few regulations and standards address recalls in general terms, but transfusion services generally have wide discretion in the management of specific common recall problems. The Food and Drug Administration (FDA) is now including posttransfusion evaluations in its guidelines for emerging infectious threats to the blood supply. We suggest that hospital transfusion services should have standard operating procedures for managing recalls and that the hospital transfusion committee and the quality management program should provide local input or oversight. Using the FDA's categories of donor center biological product deviations, we provide recommendations to consider for when to notify the recipient's physician, after postdonation information is received about a previously transfused blood component. More study of this important everyday issue in transfusion medicine is highly desirable.     

Recipient inflammation affects the frequency and magnitude of immunization to transfused red blood cells

BACKGROUND: Most alloantigens on transfused red blood cells (RBCs) are weakly immunogenic, with only a 2 to 6 percent overall immunization rate even in patients receiving multiple transfusions. Although recipient genetics may contribute to responder and/or nonresponder status, in most cases HLA type does not predict humoral response to RBC antigens. In contrast, rates of alloimmunization do correspond to the underlying disease status of transfusion recipients, suggesting that acquired host factors may play an important role. In this context, it was hypothesized that the inflammatory status of a transfusion recipient would influence immunization to transfused RBCs. STUDY DESIGN AND METHODS: A novel murine model for alloimmunization to RBC antigens was developed with the mHEL mouse, which expresses hen egg lysozyme (HEL) as a model blood group antigen. Leukoreduced mHEL RBCs were transfused into wild-type recipient mice, and anti-HEL responses were monitored. To test the stated hypothesis, some recipient animals were injected with polyinosinic polycytidylic acid (poly(I:C)), a synthetic double-stranded RNA molecule that induces viral-like inflammation. RESULTS: Similar to the immunogenicity of most RBC antigens in humans, transfusion of mHEL RBCs into uninflamed mice was only a weak immunogen. In contrast, poly(I:C)-treated mice had a significant increase in both the frequency and the magnitude of alloimmunization to the mHEL antigen. CONCLUSIONS: These findings demonstrate that recipient inflammation with poly(I:C) significantly enhances humoral immunization to transfused alloantigens in a murine model. Moreover, these data suggest that the inflammatory status of human transfusion recipients may regulate the immunogenicity of transfused RBCs.     

A prospective,randomized clinical trial of universal WBC reduction

BACKGROUND: Recipient exposure to allogeneic donor WBCs results in transfusion complications for selected populations of recipients. Whether or not WBC reduction should be universally applied is highly controversial. STUDY DESIGN AND METHODS: In a general hospital, a randomized, controlled clinical trial of conversion to universal WBC reduction was conducted. Patients (11%) with established medical indications for WBC-reduced blood were not eligible. All other patients who required transfusion were assigned at random to receive either unmodified blood components or stored WBC-reduced RBCs and platelets. Analysis for each patient was restricted to the first hospitalization. RESULTS: All eligible patients (n = 2780) were enrolled. Three specified primary outcome measures were not different between the two groups: 1) in-hospital mortality (8.5% control; 9.0% WBC-reduced; OR, 0.94 [95% CI, 0.72-1.22]; p = 0.64); 2) hospital length of stay (LOS) after transfusion (median number of days, 6.4 for control and 6.3 for WBC-reduced; p = 0.21); and 3) total hospital costs (median, $19,500 for control and $19,200 for WBC-reduced, p = 0.24). Secondary outcomes (intensive care LOS, postoperative LOS, antibiotic usage, and readmission rate) were not different between the two groups. Subgroup analysis based on patient age, sex, amount of blood transfused, or category of surgical procedure showed no effect of WBC reduction. Patients who received WBC-reduced blood had a lower incidence of febrile reactions (p = 0.06). CONCLUSION: A beneficial effect of conversion from selective to universal WBC reduction was not demonstrated.     

Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood samples. Viral Activation Transfusion Study

BACKGROUND: Previous methods for processing whole blood (WB) for nucleic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that involves the freezing of WB for quantitative polymerase chain reaction (PCR) analyses was evaluated. STUDY DESIGN AND METHODS: Controlled studies were conducted in which paired fresh and frozen WB preparations were analyzed. The integrity of WBCs in the frozen WB samples was first assessed by flow cytometry using CD45 fluorescence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a double-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentrates. Y-chromosome-specific PCR was used to quantitate male WBCs in five female WB samples spiked with serial dilutions of male WBCs and in serially collected frozen WB samples from four females transfused with male blood components. RESULTS: By flow cytometry, all major WBC subpopulations in frozen-thawed WB were quantitatively recovered and immunologically intact, although they were nonviable. HLA-DQ-A PCR quantitation of a dilution series from 8 to 16,700 per mL of WBCs spiked into double-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (r2 = 0.98, p<0.0001 for both), without significant difference between observed and expected values (p>0.05). Y- chromosome-specific PCR results in female WB samples spiked with male WBCs were not significantly different in fresh and frozen preparations over a 3 log10 range of male cells. The results of WBC survival studies on frozen WB samples were consistent with previous observations in fresh blood samples. CONCLUSION: Direct freezing of WB enables subsequent recovery of WBCs for quantitative PCR analyses, with results comparable to those of fresh preparations. This protocol should facilitate wider implementation of nucleic acid-based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in transfusion and transplant recipients.     

Absence of activation of CMV by blood transfusion to HIV-infected, CMV-seropositive patients

BACKGROUND: The Viral Activation Transfusion Study (VATS) afforded an opportunity to determine whether blood transfusions, and in particular exogenous WBCs, "activate" CMV replication in HIV-infected, CMV-seropositive patients, and whether such patients can be superinfected by additional strains of CMV transmitted via blood transfusions. STUDY DESIGN AND METHODS: A total of 531 patients were randomized to receive either WBC-reduced (WBCR) or non-WBC-reduced (NWBCR) RBC units. Plasma CMV PCR assays were performed before transfusion and weekly after transfusion for 4 weeks. NWBCR cases with evidence of possible reactivation and/or superinfection were further studied for donor viremia by DNA PCR of frozen retention segments and new genotype acquisition using gB envelope sequence analysis of pre- and posttransfusion recipient specimens. RESULTS: VATS patients received a median of two RBC units during their initial transfusion. Whether positive or negative for CMV DNA at baseline, there were no significant treatment-arm differences in the percentage of patients who had positive qualitative CMV PCR or increases in CMV viral load at follow-up. Of 50 recipients randomized to NWBCR RBC and meeting criteria for possible CMV superinfection, 25 had sufficient CMV DNA load in a baseline and one or more viremic follow-up sample to permit comparison of gB genotypes. Only two recipients showed genotype shifts. Of 125 WBC pellets prepared from the seropositive units transfused into these 50 cases, only 1 tested weakly PCR positive for CMV DNA (insufficient copy number for genotyping). CONCLUSION: There was no evidence of "activation" of CMV by blood transfusion. Among the NWBCR RBC recipients, there was little evidence of possible transmission of new CMV strains. Hence, the current policy for transfusion support of HIV-infected patients, which allows transfusion of CMV-antibody-positive blood to CMV-seropositive patients, is appropriate.     

新型外周血CD34+细胞动员剂--抗CD49d单克隆抗体的实验研究

为了探讨外周血干细胞动员的新途径，用抗CD49d单克隆抗体和rhG-CSF及二者联合给小鼠皮下注射，动态观察小鼠外周血的白细胞总数和CD34^ 细胞数的变化，并将各种动员方法所获得的干细胞分别进行干细胞移植。结果发现，给予动员剂后小鼠的外周血白细胞总数和CD34^ 细胞比例明显升高，以rhG-CSF和抗CD49d单克隆抗体联合给药效果最佳。移植后各组小鼠均获造血重建，以联合动员组造血恢复速度最快。结论：抗CD49d单克隆抗体能有效动员小鼠外周血干细胞，与rhG-CSF具有协同作用。     

High-level long-term white blood cell microchimerism after transfusion of leukoreduced blood components to patients resuscitated after severe traumatic injury

BACKGROUND: Long-term white blood cell (WBC) microchimerism (MC), of at least 2 years, has been reported in trauma patients receiving fresh nonleukoreduced (non-LR) blood. It is unknown, however, whether this occurs with LR blood products that are nearly devoid of WBCs. Twenty-seven patients transfused with LR and non-LR blood products were studied after severe traumatic injury. A secondary aim was to explore donor-recipient mixed lymphocyte reactivity in vitro. STUDY DESIGN AND METHODS: To quantify MC, allele-specific real-time polymerase chain reaction assays were developed targeting HLA Class II sequence polymorphisms. Extensive validation showed that these assays reliably detect a single copy of target sequence in a complex allogeneic background without false positivity. RESULTS: At a median follow-up of 26 months (range, 24-39 months), long-term MC was observed in 3 of 20 patients (15%) who received non-LR blood products and 2 of 7 (29%) who received LR blood products. The maximum MC ranged from 0.40 to 4.90 percent of circulating WBCs and appeared, by Class II genotype analysis, to be attributable to a single donor. CONCLUSION: It is concluded that robust levels of long-term MC, apparently traceable to a single donor, occur at similar frequency despite leukoreduction of transfused blood products. Exploratory analysis of donor-recipient mixed lymphocyte reactivity suggests that long-term MC may require a state of bidirectional tolerance before transfusion.     

Alloimmunization to transfused platelets requires priming of CD4+ T cells in the splenic microenvironment in a murine model

BACKGROUND: Alloantibodies are a clinically significant sequelae of platelet (PLT) transfusion, potentially rendering patients refractory to ongoing PLT transfusion support. These antibodies are often IgG class switched, suggesting the involvement of CD4+ T‐cell help; however, PLT‐specific CD4+ T cells have not been visualized in vivo, and specifics of their stimulation are not completely understood. STUDY DESIGN AND METHODS: A murine model of alloimmunization to transfused PLTs was developed to allow in vivo assessment and characterization of CD4+ T cells specific for PLT major histocompatibility complex (MHC) alloantigen. PLTs were harvested from BALB/c mice, filter leukoreduced, and transfused into C57BL/6 recipients. PLT‐specific CD4+ T‐cell responses were visualized by using a T‐cell receptor transgenic mouse that detects peptide from donor MHC I presented on recipient MHC II. Antibody responses were determined by indirect immunofluorescence using BALB/c donor targets. RESULTS: C57BL/6 recipients of BALB/c leukoreduced PLT transfusions produced BALB/c antibodies, with proliferation of antigen‐specific CD4+ T cells seen in the spleen but not lymph nodes or liver. Depletion of recipient CD4+ cells or splenectomy independently abrogated the alloantibody response. CONCLUSION: We report a novel model to study antigen‐specific CD4+ T cells during alloimmunization to PLT transfusion. The presented data support a critical role for CD4+ T‐cell help in the humoral response to PLT transfusion and establish the spleen as a required microenvironment for effective CD4+ T‐cell priming against donor PLT–derived MHC I.     

The production of red blood cell alloantibodies in mice transfused with blood from transgenic Fyb-expressing mice

BACKGROUND: A murine model would be useful to identify which immune mechanisms could be manipulated to treat or prevent red blood cell (RBC) alloimmunization in patients who become sensitized to multiple or widely expressed antigens. STUDY DESIGN AND METHODS: Transgenic mice (B6CBAF1/J-Tg-Fy(b)) expressing the human Fy(b) antigen of the Duffy (Fy) blood group were donors. Recipient B6CBA-F1 mice received four weekly intravenous (IV) transfusions: either 0.3 mL of washed buffy coat-depleted RBCs or 0.3 mL of RBCs with spleen cells. Titers of immunoglobulin M (IgM) and immunoglobulin G (IgG) were measured in recipient serum samples by flow cytometry with RBCs from donor mice as target cells. Recipient serum samples were also tested against human RBCs of various Fy phenotypes. Additionally, RBC survival studies were performed in alloimmunized mice utilizing biotin-labeled Fy(b) transgenic mouse RBCs. RESULTS: B6CBA-F1 mice receiving washed buffy coat-depleted RBCs first made IgM, followed by IgG alloantibodies to transgenic mouse Fy(b)-positive RBCs. Recipients of Fy(b)-positive RBCs mixed with spleen cells also produced IgM and IgG alloantibodies, but at a slower rate than recipients of washed buffy coat-depleted RBCs. Serum samples showed specificity for Fy3, Fy(b), and Fy6. Decreased survival of transfused RBCs was evident at 24 hours after transfusion. CONCLUSIONS: It is possible to elicit the formation of anti-Fy alloantibodies by IV transfusion in mice that lack Fy antigens. The transfusion of RBCs alone was adequate to stimulate alloantibody production in B6CBA-F1 recipient mice. The survival of transfused Fy(b)-positive RBCs is diminished in sensitized mice. This model will be useful in further studies of RBC alloimmunization.