Xenoreactive antibodies and latent fibrin formation in VAD and cardiac transplant recipients can confound the detection and measurement of anti-AT1R antibodies.

Autoantibodies to the angiotensin II type 1 receptor (AT1R) are thought to be important in antibody-mediated rejection (AMR), especially in the absence of anti-HLA antibodies. We used a variety of methods to examine the specificity of a commercially available kit designed to quantitate anti-AT1R antibodies. We found that fibrin formation in serum samples from patients awaiting cardiac transplantation with ventricular assist devices (VADs) can produce falsely elevated anti-AT1R values. In addition, absorption studies with a variety of cell lines with or without expression of human AT1R, and those that express xenoantigens, suggest that many of the antibodies detected in the AT1R test system are heterophilic and have reactivity to xenoantigens. Furthermore, we provide data that show that reactivity to the sialic acid Neu5Gc is a common finding among samples that are highest in anti-AT1R levels. We conclude that a common laboratory method for quantitation of anti-AT1R antibodies is nonspecific and overestimates the frequency of true positives. A reevaluation of the role that anti-AT1R antibodies play in allograft function and patient outcomes is warranted.

Flow cytometry assessment of residual melanoma cells in tumor-infiltrating lymphocyte cultures.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.

Biochemical analysis of CTLA-4 immunoreactive material from human blood.

BACKGROUND: CTLA-4 was initially described as a membrane-bound molecule that inhibited lymphocyte activation by interacting with B7.1 and B7.2 molecules on antigen presenting cells. Alternative splicing of mRNA encoding the CTLA-4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. Despite studies finding that people with autoimmune disease more frequently express high levels of sCTLA-4 in their blood than apparently healthy people, the significance of these findings is unclear. METHODS: Molecules isolated from blood using CTLA-4 specific antibodies were analyzed with ligand binding assays, mass spectroscopy, and biochemical fractionation in an effort to increase our understanding of CTLA-4 immunoreactive material. RESULTS: Mass spectroscopy analysis of the molecules recognized by multiple CTLA-4-specific antibodies failed to identify any CTLA-4 protein. Even though these molecules bind to the CTLA-4 receptors B7.1 and B7.2, they also exhibit properties common to immunoglobulins. CONCLUSION: We have identified molecules in blood that are recognized by CTLA-4 specific antibodies but also exhibit properties of immunoglobulins. Our data indicates that what has been called sCTLA-4 is not a direct product of the CTLA-4 gene, and that the CTLA-4 protein is not part of this molecule. These results may explain why the relationship of sCTLA-4 to immune system activity has been difficult to elucidate.

Lack of association between sCTLA-4 levels in human plasma and common CTLA-4 polymorphisms.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an important downregulatory molecule expressed on both T and B lymphocytes. Numerous population genetics studies have documented significant associations between autoimmune diseases and single nucleotide polymorphisms (SNPs) within and around the CTLA-4 region of chromosome 2 in man. Furthermore, circulating levels of a soluble form of CTLA-4 (sCTLA-4) have been reported in a variety of autoimmune mediated diseases. Despite these findings, the relationship between levels of sCTLA-4 protein, mRNA transcript levels, and SNPs within the CTLA-4 region have not been clearly defined. In order to further clarify this relationship, we have tested four different SNPs within the CTLA-4 region among subjects whom are negative (n = 53) versus positive (n = 28) for sCTLA-4. RESULTS: Our data do not support a clear association between sCTLA-4 levels and any of the four SNPs tested. CONCLUSION: The variation in the SNPs tested does not appear to effect sCTLA-4 protein levels, despite reports that they affect sCTLA-4 mRNA.

Multiple Myeloma Vaccination Patterns in a Large Health System: A Pilot Study.

PURPOSE: Common reasons for hospitalization and death in patients with multiple myeloma (MM) are infections. As patients with MM are living longer and are treated with immunomodulatory drugs, there is a need to immunize against vaccine-preventable diseases and ultimately determine the efficacy of these vaccines. We evaluated vaccination practice patterns in MM patients at our health system using electronic medical records and data analytics. METHODS: This institutional review board-approved study retrospectively reviewed patients with MM who visited the health system from May 2012 to May 2014. Data collected included demographics, influenza vaccination (FV) and pneumonia vaccination (PV) history, hospitalization episodes and associated costs, and duration of survival. Patients were considered PV-positive if vaccinated within 5 years prior to study. FV was defined as optimal (two FV in 2012-2014), suboptimal (one FV in 2012-2014) or none (in 2012-2014). RESULTS: Of 411 MM patients, 55% were male and 85% Caucasian. Nearly 58% received PV in the past 5 years. FV was 15% optimal, 52% suboptimal and 33% none. A total of 444 hospitalizations involving 204 patients were observed over 2-year follow-up. More than $23 million was incurred from hospitalizations in the 2-year study period. There was no statistically significant difference in all-cause hospitalization and overall survival by FV and PV status. CONCLUSIONS: Despite recommendations of vaccination in multiple myeloma, our cohort had low rates of influenza and pneumonia vaccination. FV and PV status did not show any significant association with additional hospitalization or overall survival in this pilot study. Future prospective studies are needed to ascertain the immunological and clinical efficacy and effectiveness of these vaccines in immunosuppressed patients.

Multiple Myeloma Baseline Immunoglobulin G Level and Pneumococcal Vaccination Antibody Response.

Infections are a major cause of morbidity and mortality in multiple myeloma (MM), a cancer of the immune system. Vaccination clinical efficacy endpoints have not been demonstrated, and there are limited data on surrogate markers of efficacy. This pilot study evaluated sequential immunologic markers after standard pneumococcal vaccination (PV) in patients with MM and non-MM controls. Vaccination was standard for PV (PCV13 or PPV23), with laboratory testing at baseline and at 2, 4, 12 and 24 weeks after vaccination. Immunoglobulin G (IgG) antibodies to pneumococcal antigens were detected by ELISA. Prevaccination total IgG levels and IgG subclass levels were also measured by ELISA. Four of 6 controls responded with at least a 2-fold increase in antibody concentration; only 2 controls had a sustained increase in concentration. Six of 8 patients with MM had at least a 2-fold antibody increase; however, only 2 of these patients showed a sustained increase of antipneumococcal antibody. Response rate differences were not statistically significant in this small pilot, and there was no relationship between responsiveness to PV and initial serum total IgG levels or IgG subclasses at study entry. Future prospective studies are needed to ascertain the immunological and clinical efficacy and effectiveness of various vaccines and vaccination strategies in MM.

Vaccination in Multiple Myeloma: Review of Current Literature.

Multiple myeloma is a cancer of the immune system. Infection is a major cause of morbidity and mortality in patients with multiple myeloma. Some of these infections are preventable by vaccines available to the general population. However, little is known about the clinical effectiveness of these vaccines in patients with multiple myeloma, and the cellular and humoral immune response to vaccination has not been well characterized, especially in conjunction with modern myeloma therapies. The present report reviews the basics of multiple myeloma and the immune system, the available evidence on the immunologic response of patients with multiple myeloma after vaccination, and current practice recommendations regarding specific vaccines. Understanding the immune response to vaccines could help us understand how immuno-oncology-based therapies work in multiple myeloma and provide future directions for research.

Association of the CTLA-4 gene with rheumatoid arthritis in Chinese Han population.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is important for downregulation of T-cell activation, and CTLA-4 gene polymorphisms have been implicated as risk factors for rheumatoid arthritis (RA). Previous studies of the association between the +49 polymorphism of the CTLA-4 gene in RA have provided conflicting results. In order to determine association of the CTLA-4 gene with RA in Chinese Han population, we used denaturing gradient gel electrophoresis (DGGE) to genotype polymorphisms of four SNPs (MH30, +49, CT60 and JO31) of the CTLA-4 gene in 326 RA patients and 250 healthy controls. Furthermore, meta-analysis of all available studies relating +49 polymorphism to the risk of RA was performed to confirm the disease association. Among the SNPs examined, the genotype frequencies of CTLA-4 +49 and CT60 in RA patients differed significantly from controls (P=0.028 and 0.007). In addition, the distribution of four haplotypes constructed by these two SNPs was significantly different between patients and controls (chi(2)=10.58, d.f. =3, P=0.014). The meta-analysis also revealed that in both European and Asian populations, the CLTA-4 +49 G allele was associated with the risk of RA. These results suggested that the CTLA-4 gene might be involved in the susceptibility to RA in the Chinese Han population and both +49 and CT60 of CTLA-4 gene might be the causal variants in RA disease.

CTLA-4 is important in maintaining long-term survival of cardiac allografts.

INTRODUCTION: CTLA-4 is a negative regulatory molecule upregulated on activated T cells; however, its role in induction and maintenance of transplant tolerance is not well understood. METHODS: The characteristics and effects of a novel mouse anti-rat CTLA-4 antibody (Ab) (242B58) were examined using fluorescence-activated cell sorter, mixed lymphocyte reaction, enzyme-linked immunospot, signaling studies, and a rat model of cardiac transplant tolerance induced by administration of anti-CD28 Ab and cyclosporine. RESULTS: The anti-CTLA4 Ab was shown to bind to CTLA-4 but not prevent subsequent binding of B7 to CTLA-4. Binding to CTLA-4 did not result in phosphorylation of early cytoplasmic tyrosine kinases, suggesting that this is not a signaling Ab. However, its in vitro function was compatible with antagonization of the effects of CTLA-4, thereby increasing T-cell proliferation and interferon-gamma production in mixed lymphocyte reaction and enzyme-linked immunospot assays, respectively. Administration of 242B58 to animals treated with anti-CD28 Ab and cyclosporine either at the time of transplantation or various time-points up to 33 days posttransplantation did not result in immediate rejection, but rather caused a delayed severe acute allograft rejection at approximately 45 days posttransplant. CONCLUSIONS: Our results seem to be a reflection of the unique properties of the 242B58 Ab, which does not antagonize B7 binding to CTLA-4 and affect its ability to out-compete CD28 for B7 binding. It does, however, seem to interfere with CTLA-4 signaling, suggesting that competition for B7 may be important in induction of tolerance, but signaling through CTLA-4 is more important in maintaining a tolerant phenotype.

Basal and adrenocorticotropin-stimulated corticosterone in the neonatal rat exposed to hypoxia from birth: modulation by chemical sympathectomy.

We previously demonstrated that 7-d-old rat pups exposed to hypoxia from birth exhibit ACTH-independent increases in corticosterone associated with an increase in steroidogenic acute regulatory (StAR) and peripheral-type benzodiazepine receptor (PBR) proteins. The purpose of the present study was to determine whether this increase in corticosterone could be attenuated by chemical sympathectomy induced with guanethidine treatment. Rat pups were exposed to normoxia or hypoxia from birth and treated with vehicle or guanethidine and studied at 7 d of age. Hypoxia per se resulted in an increase in plasma corticosterone without a change in plasma ACTH. Guanethidine treatment attenuated the increase in basal corticosterone in hypoxic pups but did not attenuate ACTH-stimulated corticosterone production. This effect was specific as basal and ACTH-stimulated aldosterone was not affected. Guanethidine also attenuated the increase in StAR protein induced by hypoxia. Neither the effect of hypoxia nor that of guanethidine could be explained by changes in the levels of adrenal tyrosine hydroxylase, StAR, or P450scc mRNA, adrenal tyrosine hydroxylase immunohistochemistry, or adrenal catecholamine content. We conclude that chemical sympathectomy normalizes basal corticosterone levels but has no effect on ACTH-stimulated corticosterone levels in 7-d-old rats exposed to hypoxia from birth. The mechanism of the effect of guanethidine to normalize hypoxia-stimulated basal corticosterone remains to be identified, although StAR protein may be an important mediator. This ACTH-independent increase in corticosterone may be a mechanism by which the neonate can increase circulating glucocorticoids necessary for survival while bypassing the hyporesponsiveness of the neonatal hypothalamic-pituitary-adrenal axis.

Alloimmune induction of endothelial cell-derived interferon-gamma-inducible chemokines.

BACKGROUND: The interaction between host lymphocytes and endothelial cells on the transplanted organ is believed to play an important role in acute and chronic graft rejection. Trafficking and recruitment of lymphocytes to the site of inflammation is known to be controlled by several cytokines and chemokines. It is unclear whether endothelial cells themselves can be a source of inflammatory chemoattractant molecules on alloimmune induction. METHODS: Using a semiquantitative polymerase chain reaction method, the authors analyzed the expression of chemokine mRNA coding for interferon (IFN)-gamma-induced protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in a pool of human aortic endothelial cells. Both of these chemokines are known to be induced by IFN-gamma. Endothelial cell-derived chemokine mRNA was assayed at rest, after IFN-gamma activation, and after co-culture with allogeneic peripheral blood mononuclear cells (PBMC) from normal blood donors with and without a monoclonal antibody to IFN-gamma. Finally, protein release into the media was assayed using an enzyme-linked immunosorbent assay to IP-10. RESULTS: Mig and IP-10 were expressed in human endothelial cells both after IFN-gamma treatment and after PBMC co-culture. Furthermore, the expression of both of these endothelial cell-derived chemokines was dependent on IFN-gamma because PBMC-induced expression was blocked with anti-IFN-gamma. IP-10 levels in the endothelial cell supernatant increased from a baseline of 13.4+/-10.8 pg/mL to 299.5+/-13.4 pg/mL (P<0.0001) with exposure to PBMC and was likewise inhibited by anti-IFN-gamma A-b (33.8+/-17.8 pg/mL). CONCLUSIONS: Vascular endothelial cells are capable of producing inflammatory chemokines when activated and potentially serve to amplify the allogeneic response.

Practical value of identifying antibodies to cryptic HLA epitopes in cardiac transplantation.

BACKGROUND: Identification of antibodies to human leukocyte antigens (HLA) by single antigen bead arrays has led to the common practice of virtual crossmatching. However, inappropriate assignment of anti-HLA specificities can lead to false-positive virtual crossmatching, resulting in the decline of potentially crossmatch-negative organ offers. In this study we describe identification of antibodies to cryptic HLA present on denatured forms of HLA on single antigen bead array and provide a reassessment of calculated panel-reactive antibody (CPRA) based on elimination of false-positive reactions due to antibodies to cryptic HLA epitopes. METHODS: Sera from 96 patients with positive HLA antibodies detected on a standard single antigen bead platform were tested under denaturing conditions and with a new single antigen bead product (iBeads; One Lambda, Inc., Canoga Park, CA) to identify antibodies to cryptic HLA vs. native HLA. Flow cytometry crossmatching and complement-fixation assays were performed to assess clinical relevance. RESULTS: Antibodies to cryptic HLA were present in approximately 21% of patients on our waiting list for cardiac transplantation. These antibody responses were not associated with factors commonly thought to be associated with antibody responses to HLA such as age, gender, transfusions or presence of circulatory support. CONCLUSIONS: Antibodies to cryptic HLA can be reliably identified by iBeads technology, and usually do not fix complement nor produce positive flow cytometry crossmatches. Identification and removal of antibodies to cryptic HLA from the panel of unacceptable antigens may have dramatic and meaningful effects on CPRA and virtual crossmatch strategies.

Autoantibodies targeting tumor-associated antigens in metastatic cancer: Sialylated IgGs as candidate anti-inflammatory antibodies.

In addition to the well-established effector functions of IgGs, including direct cytotoxicity and antibody-dependent cellular cytotoxicity, some populations of IgGs may exert anti-inflammatory effects. Here, we describe a population of antibodies that form in the natural course of metastatic cancer and contain glycans that terminate with sialic acid. We demonstrate that both the titer of these antibodies and their level of sialylation are relatively stable throughout the progression of metastatic melanoma. The sialylation pattern of these antibodies somehow correlates with their specificity for tumor-associated antigens, as IgGs targeting several antigens associated with infectious agents are relatively poor of sialic acid. We also show that some antibodies targeting the melanoma-associated antigen NY-ESO-1 bind to the human C-type lectin CD209 (DC-SIGN). We propose that these antibodies are candidate anti-inflammatory antibodies. The presence of anti-inflammatory antibodies in cancer patients may explain, at least in part, why tumors persist and spread in the host despite strong tumor-specific humoral responses. The elucidation of the cellular and molecular pathways involved in the induction of anti-inflammatory antibodies specific for tumor-associated antigens and their function may yield important insights into how tumors evade immune detection and progress.

A mechanistic study of immune system activation by fusion of antigens with the ligand-binding domain of CTLA4.

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4-Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4-Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.

Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone, StAR, and PBR.

The adrenocortical response to hypoxia may be a critical component of the adaptation to this common neonatal stress. Little is known about adrenal function in vivo in hypoxic neonates. The purpose of this study was to evaluate adrenocortical responses to ACTH in suckling rat pups exposed to hypoxia from birth to 5-7 days of age compared with normoxic controls. We also evaluated potential cellular controllers of steroidogenic function in situ. In 7-day-old pups at 0800, hypoxia from birth resulted in increased basal (12.2 +/- 1.4 ng/ml; n = 12) and ACTH-stimulated (94.0 +/- 9.4 ng/ml; n = 14) corticosterone levels compared with normoxic controls (basal = 8.3 +/- 0.5 ng/ml; n = 11; stimulated = 51.3 +/- 3.8 ng/ml; n = 8). This augmentation occurred despite no significant difference in plasma ACTH levels in normoxic vs. hypoxic pups before (85 +/- 4 vs. 78 +/- 8 pg/ml) or after (481 +/- 73 vs. 498 +/- 52 pg/ml) porcine ACTH injection (20 microg/kg). This effect was similar in the afternoon at 6 days of age and even greater at 5 days of age at 0800. The aldosterone response to ACTH was not augmented by exposure to hypoxia from birth. Adrenocortical hypoxia-inducible factor (HIF)-1alpha mRNA was undetectable by RT-PCR. Steroidogenic acute regulatory (StAR) protein in adrenal subcapsules (zona fasciculata/reticularis) was augmented by exposure to hypoxia; this effect was greatest at 5 days of age. Peripheral-type benzodiazepine receptor (PBR) protein was also increased at 6 and 7 days of age in pups exposed to hypoxia from birth. We conclude that hypoxia from birth results in an augmentation of the corticosterone but not aldosterone response to ACTH. This effect appears to be mediated at least in part by an increase in controllers of mitochondrial cholesterol transport (StAR and PBR) and to occur independently of measurable changes in endogenous plasma ACTH. The augmentation of the corticosterone response to acute increases in ACTH in hypoxic pups is likely to be an important component of the overall physiological adaptation to hypoxia in the neonate.