Highlights of the 33rd annual scientific meeting of the Association of Medical Laboratory Immunologists (AMLI).

The annual meeting of the Association of Medical Laboratory Immunologists (AMLI) was convened virtually over the month of August. Prior to the emergence of the COVID-19 pandemic, AMLI's scientific committee had chosen the following topics as the focus of its 2020 meeting: Histocompatibility Testing and Transplant Immunology; Secondary Immunodeficiency and Immunotherapy Monitoring; ANA Update; and Emerging Infectious Diseases and New Algorithms for Testing. Given the central role of the discipline in the evaluation of the host response to infection, it was apt to add a separate session on antibody testing for SARS-CoV-2 infections to the original program. The current report provides an overview of the subjects discussed in the course of this meeting.

Expression and role of a2 vacuolar-ATPase (a2V) in trafficking of human neutrophil granules and exocytosis.

Neutrophils kill microorganisms by inducing exocytosis of granules with antibacterial properties. Four isoforms of the "a" subunit of V-ATPase-a1V, a2V, a3V, and a4V-have been identified. a2V is expressed in white blood cells, that is, on the surface of monocytes or activated lymphocytes. Neutrophil associated-a2V was found on membranes of primary (azurophilic) granules and less often on secondary (specific) granules, tertiary (gelatinase granules), and secretory vesicles. However, it was not found on the surface of resting neutrophils. Following stimulation of neutrophils, primary granules containing a2V as well as CD63 translocated to the surface of the cell because of exocytosis. a2V was also found on the cell surface when the neutrophils were incubated in ammonium chloride buffer (pH 7.4) a weak base. The intracellular pH (cytosol) became alkaline within 5 min after stimulation, and the pH increased from 7.2 to 7.8; this pH change correlated with intragranular acidification of the neutrophil granules. Upon translocation and exocytosis, a2V on the membrane of primary granules remained on the cell surface, but myeloperoxidase was secreted. V-ATPase may have a role in the fusion of the granule membrane with the cell surface membrane before exocytosis. These findings suggest that the granule-associated a2V isoform has a role in maintaining a pH gradient within the cell between the cytosol and granules in neutrophils and also in fusion between the surface and the granules before exocytosis. Because a2V is not found on the surface of resting neutrophils, surface a2V may be useful as a biomarker for activated neutrophils.

Immune etiology of recurrent pregnancy loss and its diagnosis.

Recurrent Spontaneous Abortion of Immunological Origin (RSAI) is currently diagnosed by the occurrence of 2-3 consecutive miscarriages of unknown origin. The psychological trauma incurred by these events is a serious ailment which may be potentially avoided if a method of analysis is derived which may forecast these events and in turn prevent them from occurring. This review intends to examine studies of recurrent spontaneous abortion (RSA) which use laboratory diagnosis and also studies of RSA that do not use laboratory diagnosis. We believe that when laboratory results are incorporated into the diagnosis of RSA/RSAI that treatment is highly successful whereas the absence of laboratory results severely hinders the effectiveness of treatment. It is worth noting that correlating treatment versus outcome is imprudent because of the multiple variables involved in patient cases. It is not imprudent, however, to say that incorporation of laboratory data is essential when diagnosing RSA/RSAI.

Plasma components affect accuracy of circulating cancer-related microRNA quantitation.

Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concentrations in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids.

Women with multiple implantation failures and recurrent pregnancy losses have increased peripheral blood T cell activation.

PROBLEM: We aim to determine whether peripheral blood T cell activation is associated with repeated implantation failures or recurrent pregnancy losses (RPLs). METHOD OF STUDY: Women with a history of repeated implantation failure (n = 18) or RPLs (n = 17) comprise the study group. Normal fertile women (n = 11) are included as controls. Proportion of activated peripheral blood T cells (CD69(+), CD154(+)) and Th1/Th2 cell ratios are measured by flow cytometric analysis. RESULTS: Proportions (%) of CD4(+)/154(+) of CD4(+) and CD8(+)/154(+) of CD8(+) cells were significantly higher in study group than those of controls. Proportions (%) of CD3(+)/69(+) of CD3(+) cells and CD8(+)/69(+) of CD8(+) cells were significantly increased in study group compared to controls. Proportion (%) of CD4(+)/69(+) cells significantly correlated with % CD4(+)/154(+) cells (P = 0.003). Activated cytotoxic T cells (CD8(+)/154(+), CD8(+)/69(+)) inversely correlated with INF-gamma/IL-10 producing CD3(+)/4(+) T cell ratios. Proportion of activated CD3(+)/8(+)/69 and CD3(+)/8(+)/154(+) cells was inversely correlated with IFN-gamma/IL-10 expressing CD3(+)/4(+) T cell ratios. CONCLUSION: Women with MIFs or RPLs have increased T cell activation in peripheral blood lymphocytes, and T cell suppressor activation seems to be associated with decreased Th1 immunity. Further studies on T cell activation may elucidate molecular mechanisms controlling Th1 effectors.

The a2 isoform of vacuolar ATPase is a modulator of implantation and feto-maternal immune tolerance in early pregnancy.

In mammalian reproduction, two immunologically disparate entities, the mother and her fetus, co-exist in close proximity and mutually tolerate each other. The maternal immune system plays a major contributing role in the reproductive outcome. A coordinated set of immunological events takes place between the maternal and fetal cells to ensure fetal survival. Among these, cytokines secreted by proximal maternal immune cells as well as fetal trophoblast cells play a major role in feto-maternal tolerance. In this review, we describe the role of the vacuolar ATPase (and more specifically the a2 isoform, a2V-ATPase) in controlling the expression of these vital cytokines. a2V-ATPase is a key enzyme that controls the acidification of intracellular vesicles and the extracellular environment, processes that play a major role in cellular function. The localization of a2V-ATPase in tissues and immune cells of the reproductive tract which are essential for pregnancy will be described. Information will be provided on the role of a2V-ATPase on aspects of cell development in pregnancy, from fertilization to implantation and fetal growth. Particular emphasis will be placed on the role of a2V-ATPase in (a) regulating parts of the cytokine network at the implantation site and (b) attenuating the potentially harmful maternal immune response against trophoblast cells.

Correlation between natural cytotoxicity receptors and intracellular cytokine expression of peripheral blood NK cells in women with recurrent pregnancy losses and implantation failures.

PROBLEM: Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. We investigated whether women with recurrent pregnancy losses (RPLs) and implantation failures have aberrant correlation between NCRs and intracellular cytokine expression of NK cells. METHOD OF STUDY: Peripheral blood NK cells (CD56(dim) and CD56(bright)) were analyzed for NCRs (NKp46, NKp44 and NKp30) and cytokine expression (TNF-alpha, IFN-gamma, IL-4, IL-10) using flow cytometry in RPL (n = 22), implantation failures (n = 23) or controls (n = 15). RESULTS: In type 1 cytokine studies, CD56(bright)/NKp30(+) cells in controls (r = 0.696, P < 0.05) were positively correlated with CD56(bright)/IFN-gamma(+)/TNF-alpha(+) cells. CD56(bright)/NKp46(+) cells in implantation failures (r = -0.76, P < 0.01) were negatively correlated with CD56(bright)/IFN-gamma(+)/TNF-alpha(-) cells. RPL did not have any correlation. In type 2 cytokine studies, CD56(+)/NKp46(+) cells (r = 0.758, P < 0.01) and CD56(+)/NKp30(+) cells (r = 0.637, P < 0.05) were positively correlated with CD56(bright)/IL-4(+)/IL-10(+) cells in controls. CD56(+)/NKp30(+) cells in implantation failures (r = -0.778, P < 0.05) were negatively correlated with CD56(bright)/IL-10(+)/IL-4(+) cells. There were no correlations in RPL. CONCLUSION: Recurrent pregnancy losses and implantation failures have lack of, or negative correlation between NCRs and intracellular cytokines expression. This observation suggests that excessive pro-inflammatory cytokine expression in NK cells in RPL and implantation failures may be exerted through the NCRs or interruption of signal transduction processes.

Intracellular cytokine expression of peripheral blood natural killer cell subsets in women with recurrent spontaneous abortions and implantation failures.

OBJECTIVE: To investigate the cytokine expression by peripheral blood natural killer (NK) cells of women with recurrent spontaneous abortion (SAB) or implantation failures. DESIGN: Prospective cohort study. SETTING: University clinic. PATIENT(S): Twenty-five women with recurrent SAB, 20 women with implantation failures, and 15 healthy controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cytokine expression (interferon-gamma, tumor necrosis factor [TNF]-alpha, interleukin [IL]-4, IL-5, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor [GM-CSF]) in NK cells and their subsets (CD56(dim) and CD56(bright)). RESULT(S): Proportion (percentage) of CD56(bright)/interferon-gamma(+)/TNF-alpha(+) cells was significantly higher in women with recurrent SAB and implantation failures as compared with that of healthy controls. Proportion of CD56(bright)/IL-4(+)/IL-10(+) cells was very low (<2%) in all groups but was significantly lower in women with recurrent SAB than that of controls. The TNF-alpha/GM-CSF expressing CD56(bright) cell ratio was significantly higher in women with recurrent SAB and implantation failures than in controls. CONCLUSION(S): Natural killer-1 shift in peripheral blood NK cells was identified in nonpregnant women with recurrent SAB and implantation failures. Tumor necrosis factor-alpha/GM-CSF expressing CD56(bright) cell ratio can be applicable for the diagnosis of recurrent SAB or implantation failures. Further studies are needed as to whether cytokine expression of NK cells during pregnancy can affect pregnancy outcome.

Novel role for the N-terminus domain of the a2 isoform of vacuolar ATPase in interleukin-1beta production.

Interleukin-1beta (IL-1beta) is a mediator cytokine that is released by macrophages and epithelial cells in pregnancy and tumorigenesis before antigen recognition. a2V-ATPase is a protein expressed during pregnancy and tumorigenesis and has a novel role in immune regulation. It is expressed as a 70 kDa molecule in intracellular vesicles. Upon cell stimulation it migrates to the surface followed by the cleavage of a 20 kDa portion (a2 N-terminus domain, a2NTD). This study aimed to determine whether a2NTD could induce IL-1beta production in immune cells. Peripheral blood mononuclear cells (PMBC) were stimulated with a2NTD and analyzed for cytokine gene expression by gene arrays. Supernatants were analyzed for IL-1beta by enzyme-linked immunosorbent assay, and cells were analyzed for intracellular expression of IL-1alpha, IL-1beta, and TNF-alpha by flow cytometry. When PBMC were cultured with a2NTD, there was a 2.5-fold increase in IL1A and IL1B gene expression and no induction of TNF gene expression. There was a 72-fold increase in IL-1beta in supernatants of PBMC cultured with a2NTD. Finally, there was a 204-fold increase in intracellular expression of IL-1beta in monocytes incubated with a2NTD. These results indicate a regulatory role for a2NTD in IL-1 cytokine production and suggest a unique role for this molecule in inflammation.

The N-terminus domain of the a2 isoform of vacuolar ATPase can regulate interleukin-1beta production from mononuclear cells in co-culture with JEG-3 choriocarcinoma cells.

PROBLEM: a2V-ATPase is the a2 isoform of vacuolar ATPase and is expressed in human trophoblast cells. a2V-ATPase resides as a 70-kDa molecule in intracellular vesicles. Upon cell stimulation, it migrates to the surface as a 50-kDa molecule, after a 20-kDa portion [N-terminus domain of the a2V-ATPase (a2NTD)] is cleaved and secreted to the extracellular environment. Previous studies showed that a2NTD-regulated cytokine production from stimulated T cells. The aim of this study was to determine if a2NTD can regulate cytokine production from immune cells that were in contact with JEG-3 cells. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMC) from females were co-cultured with JEG-3 cells in the presence or absence of a2NTD, and supernatants were analyzed by enzyme-linked immunosorbent assay for interleukin (IL)-1beta. Additionally, PBMC cultured with JEG-3 cells, in the presence or absence of a2NTD, were analyzed for cytokine gene expression by gene arrays. RESULTS: There was an increased secretion of IL-1beta and a decrease in type I and II IL-1 receptors (IL1RA and IL-1R2) gene expression in PBMC that were co-cultured with JEG-3 cells in the presence of a2NTD. CONCLUSION: These data suggest a role for a2NTD in the regulation of IL-1beta pro-inflammatory cytokine production at the fetal-maternal interface.

Expression of natural cytotoxicity receptors and a2V-ATPase on peripheral blood NK cell subsets in women with recurrent spontaneous abortions and implantation failures.

PROBLEM: Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. a2V-ATPase is expressed on subsets of PBMC and regulates the extracellular environment, which facilitates NK cytotoxicity or cytokine secretion. In this study, we aim to investigate the expression of NCRs and a2V-ATPase in peripheral blood NK cells of women with recurrent spontaneous abortions (RSA) or implantation failures. METHOD OF STUDY: Peripheral blood NK cells (CD56(dim) and CD56(bright) were analyzed for the expression of NCRs (NKp46, NKp44 and NKp30) and a2V-ATPase using 3-color flow cytometry in women with RSA (n=24), implantation failures (n=19) or normal healthy women (n=13). RESULTS: CD56+/NKp46+ cells were markedly decreased (P<0.05) and CD56(bright)/a2V-ATPase+ cells were significantly increased (P<0.05) in women with RSA as compared to those of normal controls. In women with RSA or implantation failures, expression of NKp46, NKp44, NKp30, and a2V-ATPase on CD56(bright) NK cells was significantly up-regulated as compared with those of CD56(dim) NK cells. CONCLUSION: The differential expression of NCRs and a2V-ATPase in NK cell subsets may suggest dysregulation of NK cytotoxicity and cytokine production in women with RSA and implantation failures.

An in vitro coculture model to study cytokine profiles of natural killer cells during maternal immune cell-trophoblast interactions.

OBJECTIVES: The cytokine milieu at the implantation site plays a role in human pregnancy. Th2 cytokines, such as interleukin (IL)-4 and IL-10, stimulate growth and development of placenta, whereas Th1 cytokines, such as tumor necrosis factor-alpha (TNF-alpha), are associated with pregnancy complications. Natural killer (NK) cells predominate at the implantation site. The aim of the present study is to investigate cytokine expression in NK cells when they are in close contact with JEG-3 trophoblast-like cells using an in vitro coculture model. METHODS: Female peripheral blood mononuclear cells (PBMCs) were cocultured with JEG-3 cells for 24 hours. PBMCs were harvested from the cocultures and stimulated with 25 ng/mL phorbol myristate acetate and 1 micromol/mL ionomycin in the presence of 2 micromol/mL monensin. NK cells were analyzed by flow cytometry for intracellular TNF-alpha, interferon-gamma (IFN-gamma), and IL-4 and IL-10 cytokines. Controls were PBMCs cultured without JEG-3 cells. RESULTS: The proportion of CD56+/TNF-alpha(+) NK cells was significantly decreased when they were in coculture with JEG-3 cells (26.1%) as compared to without JEG-3 cell coculture (40.8%) (P < .05). There was no difference in the proportion of CD56(+) NK cells expressing intracellular IFN-gamma, IL-4, and IL-10. Down-regulation of CD56+/TNF-alpha(+) NK cell levels was dependent on direct cell-to-cell contact between NK cells and JEG-3 cells. The expression of human leukocyte antigen (HLA)-G on trophoblast cell lines did not affect CD56+/TNF-alpha(+) NK cell levels under these experimental conditions. CONCLUSION: We report that JEG-3 cells induce down-regulation of intracellular CD56+/TNF-alpha(+) NK cell levels. It is speculated that trophoblasts may secure themselves from NK cell cytotoxicity via this mechanism.

Expression of killer immunoglobulin-like receptors on peripheral blood NK cell subsets of women with recurrent spontaneous abortions or implantation failures.

PROBLEM: Decidual natural killer (NK) cells express inhibitory receptors (killer immunoglobulin-like receptors, KIRs), which bind to ligands on trophoblast cells (human leucocyte antigen, HLA-C). This interaction appears to block NK cytotoxicity against trophoblast cells. In this study, we investigated the expression of inhibitory and activating receptors in peripheral blood NK cells of women with recurrent spontaneous abortion (RSA) or implantation failures. METHOD OF STUDY: CD56(dim)/CD16(+), CD56(bright)/CD16(-) NK cells and CD56(+)/CD3(+) NKT cells of women with RSA or in vitro fertilization (IVF) failures and normal controls were analyzed for the expression of CD158a, CD158b inhibitory KIRs or CD161-activating receptors, by flow cytometric analysis. RESULTS: CD158a and CD158b inhibitory receptor expression by CD56(dim)/CD16(+) and CD56(bright)/CD16(-) NK cells were significantly decreased, and CD161-activating receptor expression by CD56(+)/CD3(+) NKT cells was significantly increased in women with implantation failures when compared with normal controls. CONCLUSIONS: An imbalance between inhibitory and activating receptor expression was found in NK cells of women with implantation failures. This imbalance may explain the adverse reproductive outcome.

The placental immunomodulatory cytokine regeneration and tolerance factor is also expressed by both human cycling and early pregnant endometrium.

PROBLEM: Regeneration and tolerance factor (RTF) has been recently suggested to contribute to the control of fetal-ablating immunity at the maternal-fetal interface through the induction of T helper 2 (Th2)-dominated response. The protein consists of a membrane-associated domain and an extracellular portion which is proteolitically cleaved to yield a soluble peptide. In humans, it has been shown to be expressed by invading cytotrophoblasts and decidual lymphoid cells, to be increased on peripheral blood B lymphocytes during a normal gestation and on circulating natural killer cells during unsuccessful pregnancies. However, the expression of RTF in other cell types and, specifically, in non-hematopoietic maternal cells of the human uterus has not been characterized in detail. Thus, we have specifically studied the expression and modulation of the cytokine in human endometrium obtained in different phases of the cycle and in early pregnancy. METHODS: The 20 kDa extracellular domain of RTF has been localized by immunohistochemical method and Western blot analysis. Levels of RTF messenger RNA (mRNA) in basal and stimulated conditions have been evaluated by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: The extracellular domain of RTF could be detected in both the glandular epithelium and stroma with diffuse distribution in both cycling endometrium and first trimester decidua. Both cycling and pregnant endometrium expressed the gene for RTF but mRNA levels resulted significantly increased in secretory phase-endometrial stromal cells when compared to proliferative phase samples. Inflammatory cytokines, interleukin-1beta and tumour necrosis factor alpha were able to directly increase endometrial RTF mRNA expression. CONCLUSION: These results indicate that RTF is constitutively expressed at endometrial and decidual level and its up-regulation during the secretory phase of the cycle may be relevant in mediating some immune-related aspects of uterine physiology.