Direct T cell-tumour interaction triggers TH1 phenotype activation through the modification of the mesenchymal stromal cells transcriptional programme.

BACKGROUND: Mesenchymal stromal cells (MSCs) are heterogeneous cells with immunoregulatory and wound-healing properties. In cancer, they are known to be an essential part of the tumour microenvironment. However, their role in tumour growth and rejection remains unclear. To investigate this, we co-cultured human MSCs, tumour infiltrating lymphocytes (TIL), and melanoma cells to investigate the role of MSCs in the tumour environment. METHODS: Mesenchymal stromal cells were co-cultured with melanoma antigen-specific TIL that were stimulated either with HLA-A*0201(+) melanoma cells or with a corresponding clone that had lost HLA-A*0201 expression. RESULTS: Activated TIL induced profound pro-inflammatory gene expression signature in MSCs. Analysis of culture supernatant found that MSCs secreted pro-inflammatory cytokines, including TH1 cytokines that have been previously associated with immune-mediated antitumor responses. In addition, immunohistochemical analysis on selected markers revealed that the same activated MSCs secreted both the TH1 cytokine (interleukin-12) and indoleamine 2,3 dioxygenase (IDO), a classical immunosuppressive factor. CONCLUSIONS: This study reflected that the plasticity of MSCs is highly dependent upon microenvironment conditions. Tumour-activated TIL induced TH1 phenotype change in MSCs that is qualitatively similar to the previously described immunologic constant of rejection signature observed during immune-mediated, tissue-specific destruction. This response may be responsible for the in loco amplification of antigen-specific anti-cancer immune response.

The relationship between alloimmunization and posttransfusion granulocyte survival: experience in a chronic granulomatous disease cohort.

BACKGROUND: The efficacy of granulocyte transfusions in patients with HLA alloimmunization is uncertain. A flow cytometric assay using dihydrorhodamine 123 (DHR), a marker for cellular NADPH oxidase activity, was used to monitor the differential survival of transfused oxidase-positive granulocytes in alloimmunized patients with chronic granulomatous disease (CGD). STUDY DESIGN AND METHODS: Ten patients with CGD and serious infections were treated with daily granulocyte transfusions derived from steroid and granulocyte-colony-stimulating factor-stimulated donors. The proportion of neutrophils with intact oxidase activity was quantitated by DHR fluorescence on samples drawn before and 1 hour after transfusion. The incidence of acute transfusion reactions was correlated with the results of DHR fluorescence and biweekly HLA serologic screening assays. RESULTS: Eight of 10 patients experienced acute adverse reactions in association with granulocyte transfusions. Four had only chills and/or fever, and four experienced respiratory compromise; all eight exhibited HLA alloimmunization. Mean (± SD) oxidase-positive cell recovery was 19.7 ± 17.4% (n = 15 transfusions) versus 0.95 ± 1.59% (n = 16) in the absence and presence of HLA allosensitization, respectively (p < 0.01). Greater than 1% in vivo recovery of DHR-enhancing donor granulocytes was strongly correlated with lack of HLA alloimmunization. CONCLUSION: The ability to detect DHR-positive donor granulocytes by flow cytometry is strongly correlated with absence of HLA alloimmunization and lack of acute reactions to granulocyte transfusions in patients with CGD. If HLA antibodies are present and the survival of donor granulocytes is low by DHR analysis, transfusions should be discontinued, avoiding a therapy associated with high risk and unclear benefit.

The immunologic aspects of poxvirus oncolytic therapy.

The concept of using replicating oncolytic viruses in cancer therapy dates to the beginning of the twentieth century. However, in the last few years, an increasing number of pre-clinical and clinical trials have been carried out with promising preliminarily results. Novel, indeed, is the suggestion that viral oncolytic therapy might not operate exclusively through an oncolysis-mediated process but additionally requires the "assistance" of the host's immune system. Originally, the host's immune response was believed to play a predominant obstructive role against viral replication, hence limiting the anti-tumor efficacy of viral vectors. Recent data, however, suggest that the immune response may also play a key role in promoting tumor destruction in association with the oncolytic process. In fact, immune effector pathways activated during oncolytic virus-induced tumor rejection seem to follow a similar pattern to those observed when the broader phenomenon of immune-mediated tissue-specific rejection occurs in other immune-related pathologies. We recently formulated the "Immunologic Constant of Rejection" hypothesis, emphasizing commonalties in transcriptional patterns observed when tissue-destruction occurs: whether with a favorable outcome, such as in tumor rejection and pathogen clearance; or a destructive one, such as in allograft rejection or autoimmunity. Here, we propose that a similar mechanism induces clearance of virally infected tumors and that such a mechanism is primarily dependent on innate immune functions.

A preliminary comparison of the prevalence of transfusion reactions in recipients of platelet components from donors with and without human leucocyte antigen antibodies.

BACKGROUND: Human leucocyte antigen (HLA) antibodies have been implicated in transfusion-related acute lung injury, but the probability that the transfusion of a blood component containing HLA antibodies will cause a reaction is not known. This study compared the prevalence of reactions associated with the transfusion of platelet components with and without HLA antibodies. STUDY DESIGN AND METHODS: This retrospective study tested 96 consecutive apheresis platelet donors for HLA class I and II antibodies. Matched control donors without HLA antibodies were selected and records were reviewed to determine the proportion of components from each group that caused reactions. In addition, all apheresis platelet donors involved with two or more reactions were identified and tested for HLA class I antibodies. RESULTS: Five of the 96 donors had antibodies to class I or class II antigens and, of these, four had components transfused. The prevalence of reactions to components from these four donors with HLA antibodies and the 12 matched control donors without antibodies was similar (three reactions to 167 transfusions or 1.8% vs. three to 295 or 1.0%, respectively, P = 0.32). A retrospective review of the transfusion records from all platelet donors found that components from 22 caused two or more reactions and three (13.6%) had antibodies to HLA class I compared to 4.2% of the consecutively selected donors (P = 0.12). None of the patients experienced transfusion-related acute lung injury. CONCLUSION: Reactions associated with transfusion of apheresis platelets containing HLA antibodies are unusual.

Patient-specific transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-associated mortality. The inadvertent transfusion of neutrophil antibodies can cause pulmonary transfusion reactions and TRALI. However, not all patients transfused with neutrophil antibodies experience transfusion reactions. A 22-year-old man with severe aplastic anaemia (SAA) experienced TRALI after a platelet transfusion. The donor was found to be alloimmunized to human neutrophil antigen (HNA)-3a, an antigen expressed by neutrophils from approximately 90% of Caucasians. Eleven other platelet components from this donor were transfused prior to this event and two caused reactions: one chills and one TRALI. Both episodes of TRALI occurred in the same male patient with SAA. The fact that one patient experienced TRALI following both exposures to anti-HNA-3a from the same donor whereas nine other recipients did not adds evidence to the observation that patient factors make a significant contribution to neutrophil antibody-mediated transfusion reactions.

Cryopreserving and deglycerolizing sickle cell trait red blood cell components using an automated cell-processing system.

RBC components with rare phenotypes are sometimes required for patients with sickle cell disease, and these rare components can often be found among donors with sickle cell trait. Cryopreserving RBC components from sickle cell trait donors requires a modified deglycerolization method to preserve the integrity of the RBCs. This study evaluated the feasibility of using an automated cell-processing system to cryopreserve and deglycerolize sickle cell trait donor RBC components. CP2D/AS-3 RBC components were collected from three donors with sickle cell trait. Each component was processed with an automated cell-processing system (ACP 215, Haemonetics Corp., Braintree, MA) and cryopreserved within 6 days of collection. The components were stored at -65 degrees C or less for at least 2 days and were deglycerolized using the automated cell-processing system's standard procedure. Before cryopreservation and after deglycerolization, several variables were measured. Deglycerolization resulted in recovery of 43.0, 76.5, and 67.5 percent of RBCs from the three sickle-cell-trait donor components compared with 80 percent or greater for all six control components. A small, dark red, jelly-like mass was noted in the bowl of the disposable set after deglycerolization of each of the three RBC sickle cell trait components. The osmolalities of all three sickle cell trait components were less than 400 mOsm/kg, but only one of the three was acceptable for a 14-day outdate. Freezing and deglycerolization of sickle cell trait donor RBC components with the automated cell-processing system resulted in recovery of some RBCs, but a decrease in RBC recovery was problematic. Modifications of the procedure are needed for processing sickle cell trait donor RBC components.

Rapid, single-subject genotyping to predict red blood cell antigen expression.

Genotyping is useful to predict the expression of those RBC antigens for which antisera are difficult to obtain and to determine the probable phenotype of highly transfused patients, and it can be used to test stored DNA when a blood sample is not available. This study assessed a sequence-specific primer (SSP)-based genotyping system for blood group alleles suitable for the rapid testing of a small number of samples and assessed the use of stored whole blood. Genomic DNA was isolated from fresh and 1- and 2-week-old stored blood from 20 donors with known ABO and Rh phenotypes and was used for ABO, RHD, and RHCE genotyping using SSPs. The amplicons were analyzed using gel electrophoresis and a novel microfluidic on-chip electrophoresis system. Analysis of DNA from fresh and 1- and 2-week-old blood by SSP and gel electrophoresis yielded the correct ABO, RHD, and RHCE type in all samples, but with DNA from 2-week-old stored blood the amplicons were more difficult to visualize. Analysis of the same samples with the SSP on-chip electrophoresis assay correctly typed all samples except for one RHCE typing discrepancy of a fresh sample and one RHCE typing discrepancy of a 2-week-old sample. Analysis of amplicons by on-chip electrophoresis required one tenth the DNA that gel electrophoresis did and could be completed within 30 minutes compared with 2 hours with gel electrophoresis. Amplicons were also more readily visualized with on-chip electrophoresis. Fresh and 1- and 2-week-old samples could be ABO and RH genotyped with SSP. Analysis using on-chip electrophoresis was easier and more rapid than that using gel electrophoresis, but test reliability was slightly more variable.

Persistence of recipient plasma cells and anti-donor isohaemagglutinins in patients with delayed donor erythropoiesis after major ABO incompatible non-myeloablative haematopoietic cell transplantation.

Delayed donor erythropoiesis and pure red-cell aplasia (PRCA) complicate major-ABO mismatched non-myeloablative allogeneic stem-cell transplantation. To characterize these events, we analysed red-cell serology and chimaerism in lymphohaematopoietic lineages, including plasma cells and B cells, in 12 consecutive major-ABO incompatible transplants following cyclophosphamide/fludarabine-based conditioning. Donor erythropoiesis was delayed to more than 100 days in nine (75%) patients including six (50%) who developed PRCA. During PRCA, all patients had persistent anti-donor isohaemagglutinins and recipient plasma cells (5-42%), while myeloid and T cells were completely donor in origin. In contrast, B-cell chimaerism was frequently full-donor when significant anti-donor isohaemagglutinins persisted. Four patients with early mixed haematopoietic chimaerism and the prolonged presence of anti-donor isohaemagglutinins and recipient plasma cells developed delayed-onset (>100 days post-transplant) red cell transfusion dependence and PRCA after myeloid chimaerism converted from mixed to full donor. These findings confirm that donor-erythropoiesis is impacted by temporal disparities in donor immune-mediated eradication of recipient lymphohaematopoietic cells during major-ABO incompatibility and suggest that plasma cells are relatively resistant to graft-versus-host haematopoietic effects.

Neutrophil and platelet antibodies in autoimmune lymphoproliferative syndrome.

BACKGROUND AND OBJECTIVES: Autoimmune lymphoproliferative syndrome (ALPS), is an inherited disorder characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly, accumulation of T-cell receptor (TCR)-alphabeta+ CD4- CD8- T cells (double-negative T cells) and autoimmunity. We investigated the incidence and nature of neutrophil and platelet antibodies in patients with ALPS. MATERIALS AND METHODS: Sera from 26 patients with ALPS were tested for neutrophil antibodies by granulocyte immunofluorescence, granulocyte agglutination and monoclonal antibody immobilization assays of granulocyte antigens, and for platelet antibodies using a solid-phase antibody-detection system. RESULTS: Neutrophil antibodies were detected in 46% of patients with ALPS. Antibody specificity could be defined in eight of the 12 patients with neutrophil antibodies. Among these eight patients, four had antibodies directed against more than one antigen. Overall, 14 antibodies directed to specific antigens were identified: three were directed to the HNA-1a antigen of FcgammaRIIIb; two to the HNA-1b antigen of Fcgamma-RIIIb; two to epitopes common to all FcgammaRIIIb molecules; four to the HNA-2a antigen of the NB1 glycoprotein; and three to neutrophil beta2 integrins. Platelet antibodies were detected in 35% of patients with ALPS. No antibody specificities were identified among the platelet antibodies. There was no association between the detection of neutrophil antibodies and a history of clinical neutropenia, or between the detection of platelet antibodies and a history of clinical thromobocytopenia. CONCLUSIONS: Neutrophil and platelet antibodies are important markers of ALPS, but do not always cause clinical cytopenias. The specificities of neutrophil antibody were similar to those found in children with autoimmune neutropenia but without ALPS.

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d.

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.

Assessment of the relative number of copies of the gene encoding human neutrophil antigen-2a(HNA-2a), CD177, and a homologous pseudogene by quantitative real-time PCR.

Human neutrophil antigen-2a (HNA-2a; NB1) is located on the 58-64 kD NB1 glycoprotein (GP) and is encoded by the gene CD177. Searches of human genome databases have revealed that a pseudogene highly homologous to exons 4-9 of CD177 is located adjacent to CD177 on chromosome 19. The purpose of this study was to document the presence of the pseudogene and determine whether the polymorphic expression of NB1 GP is due to CD177 gene deletions and duplications. Genomic DNA was isolated from leukocytes of 12 subjects. The number of copies of exon 2 of CD177, an exon that is unique to this gene, and the number of copies of exon 9, an exon that is found in both CD177 and the pseudogene, was assessed with quantitative real-time PCR. The ratio of the number of copies of sequences homologous to CD177 exon 9 to the number of copies of exon 2 was 1.5 or greater in 7 of the 12 subjects, suggesting that both CD177 and the homologous pseudogene were present. The ratio of exon 9 to exon 2 in the other 5 subjects ranged from 1 to 1.25, suggesting that the pseudogene was not present in these subjects. However, results of assays were variable and we could not exclude the possibility that all subjects carried the pseudogene. These studies confirmed the presence of the pseudogene homologous to CD177, but quantitative real-time PCR was not precise enough to detect CD177 duplications or deletions.

A novel method using formamide for the elution of antibodies from erythrocytes.

BACKGROUND AND OBJECTIVES: Accurate identification of antibodies that sensitize red blood cells (RBCs) involves dissociating them from RBCs using an in vitro elution method that does not alter their antigen-binding properties, and analysis of the eluates against a panel of RBCs. MATERIALS AND METHODS: A method was developed that allowed efficient RBC antibody elution. Human polyclonal anti-D was used to sensitize Rh-positive RBCs, and known antigen-antibody disruptive reagents were tested using these RBCs. The best reagent conditions were optimized. Eluates made were tested and compared to results obtained with a glycine-acid-based commercial elution kit to determine efficacy. Patient samples that were positive with direct antiglobulin tests (DATs), and in vitro commercial antisera-sensitized RBCs representing clinically significant antibodies, were used for evaluating the new method. RESULTS: The formamide method was efficient at removing antibodies from RBCs. The patient samples with a positive DAT had antibodies recovered with the same specificity when compared to the acid-based technique. The length of preparation time was similar for both formamide and acid-based methods. Results of testing the eluates made from reagent RBCs sensitized with commercial antisera were distinct with antigen-positive and -negative erythrocytes. CONCLUSIONS: The formamide method compares well with acid techniques and may be an alternative choice of elution method.

In vitro RBC exposure to Plasmodium falciparum has no effect on RBC antigen expression.

Severe malarial anaemia is a leading cause of death in African children younger than 3 years of age who are infected with Plasmodium falciparum. The pathogenesis of this anaemia is not understood. The purpose of this study was to determine if P. falciparum induces changes in RBC membranes that contribute to the immune destruction of RBCs. RBCs were collected from healthy subjects and tested using standard haemagglutination assays for 45 antigens representing 21 blood group systems/collections before and after exposure to P. falciparum, strain FVO. Lectins were used to determine whether crypt or neoantigens were expressed on the RBC membrane. Polybrene was used to detect changes in sialic acid. RBCs were cultured in vitro with and without the parasite, and blinded serologic studies were completed. CD35 (complement receptor 1), CD55 (decay-accelerating factor), CD59 (membrane inhibitor of reactive lysis) and CD47 (integrin-associated protein) flow cytometric assays were compared for infected and uninfected RBCs. The percentage of parasitaemia was determined using Giemsa-stained thin blood films. Two (Ch, Lub) of the 45 antigens had differing strengths of agglutination between infected and uninfected RBCs, but these differences were resolved with a second source of antisera. Forty-three antigens showed no significant differences in the strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no significant differences. P. falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. The pathogenesis of the haemolytic episode that occurs in these children remains unclear.

Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures.

BACKGROUND: Detection of immunoglobulin or complement bound to RBCs by using the DAT is valuable in the diagnosis of immune-mediated hemolytic anemia. Traditionally, the DAT has been performed by tube agglutination using anti-IgG or anti-C3d. The purpose of this study was to compare the tube agglutination DAT to gel microcolumn, affinity microcolumn, and flow cytometric DATs on RBCs coated in vitro and on patient RBC samples. STUDY DESIGN AND METHODS: RBCs from 84 patients were assessed by tube agglutination DAT, one gel microcolumn DAT, and two affinity microcolumn DATs. One affinity microcolumn assay was unmodified and one was modified by the addition of polyspecific antiglobulin or anti-IgG as a secondary antibody. RBCs from 15 of the 84 patients underwent analysis by flow cytometry with fluorescence-labeled anti-IgG. The assays were also compared by using D+ RBCs sensitized with serially adjusted concentrations of anti-D. RESULTS: Both tube agglutination and gel microcolumn DATs were positive in 49 patient samples; both assays were negative in 20 samples, and the results were discordant in 15. Gel microcolumn DATs were more likely than were tube agglutination DATs to detect IgG on RBCs. Affinity microcolumn DATs were less likely than gel microcolumn or tube agglutination DATs to detect IgG on RBCs. Flow cytometry results were the same as gel microcolumn results in 12 of 15 patient samples and the same as tube agglutination results in 13 of 15. Tube agglutination and both affinity microcolumn assays reacted with RBCs coated with anti-D that was diluted 1-in-100. The gel microcolumn and flow cytometry assays reacted with RBCs coated with anti-D diluted 1-in-400. There was no correlation between tube agglutination and gel microcolumn DATs in detecting bound C3d. CONCLUSION: Detection of IgG bound to RBCs was not consistent with the methods described. Gel microcolumn DATs were more sensitive than tube agglutination and affinity microcolumn DATs. Given the varied results of these assays, reference laboratories should not rely on a single method for DATs. More comprehensive testing should be performed when the tube agglutination DAT is negative in a patient with suspected immune-mediated hemolytic anemia. Further comparisons are necessary to determine the proficiency of flow cytometric assays.

Delayed donor red cell chimerism and pure red cell aplasia following major ABO-incompatible nonmyeloablative hematopoietic stem cell transplantation.

Delayed donor red cell engraftment and pure red cell aplasia (PRCA) are well-recognized complications of major ABO-incompatible hematopoietic stem cell transplantation (SCT) performed by means of myeloablative conditioning. To evaluate these events following reduced-intensity nonmyeloablative SCT (NST), consecutive series of patients with major ABO incompatibility undergoing either NST (fludarabine/cyclophosphamide conditioning) or myeloablative SCT (cyclophosphamide/high-dose total body irradiation) were compared. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that significantly delayed donor erythropoiesis is (1) common following major ABO-incompatible NST and (2) associated with prolonged persistence of host antidonor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis.

Administration of G--CSF plus dexamethasone produces greater granulocyte concentrate yields while causing no more donor toxicity than G--CSF alone.

BACKGROUND: G-CSF with or without dexamethasone is becoming the standard agent for mobilizing granulocytes for transfusion. The purpose of this study was to determine if the toxicities of G--CSF with or without dexamethasone are offset by greater collection yields and to define the minimum interval that should separate sequential collections. STUDY DESIGN AND METHODS: Twenty donors were studied on three occasions. They were given either dexamethasone (8 mg, by mouth) plus a placebo injection, G--CSF (5 microg/kg, given subcutaneously) plus placebo capsules, or G--CSF plus dexamethasone. Granulocytes were collected by apheresis. A donor symptom survey was administered, and cell counts and blood chemistries were assessed before collection and 1, 2, 7, 14, 21, 28, and 35 days after collection. RESULTS: More granulocytes were collected when G--CSF was given than when dexamethasone was given (41.1 +/- 20.4 x 10(9) vs. 21.0 +/- 10.0 x 10(9); p<0.001), but the use of G--CSF plus dexamethasone produced the greatest yields (67.1 +/- 22.0 x 10(9); p<0.002). When the donors were given dexamethasone alone, 58 percent experienced at least one symptom, compared to 85 percent of those given G--CSF and 75 percent of those given G--CSF plus dexamethasone. In all three regimens, platelet counts fell 19 percent to 24 percent after collection and remained below baseline for 7 to 14 days. Granulocyte counts returned to baseline within 3 to 7 days, but, in all three regimens, a mild granulocytopenia occurred 21 days after collection. With each of the regimens, blood chemistries changed, but the changes were mild and most returned to baseline within 7 days; however, changes in albumin, bilirubin, and AST persisted until 28 days after collection. CONCLUSION: These results support the use of G--CSF plus dexamethasone in granulocyte donors. G--CSF plus dexamethasone resulted in greater granulocyte yields than either agent alone and was associated with donor symptoms and changes in blood cell counts and chemistries similar to those seen with G--CSF alone or dexamethasone alone. Granulocytes can be safely collected a second time after a 7-day interval; however, for regular donors, it may be best to separate collections by 4 weeks.