Marta Felippo

A flow cytometry evaluation of anti-FVIII antibodies: correlation with ELISA and Bethesda assay

Summary.  In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme‐linked immunosorbent assay (ELISA) that detects both inhibitory (I‐Ab) and non‐inhibitory (NI‐Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I‐Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII‐m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control‐m). Captured anti‐FVIII antibodies were detected using anti‐human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I‐Ab, <0.5 BU mL−1); 13 PI (patients with I‐Ab, 1.1–8200 BU mL−1). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII‐m and control‐m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI‐Ab by FC, and two of them developed high levels of I‐Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 μL of plasma or serum is required especially making it useful for paediatric patients.

Common Variable Immunodeficiency and Circulating TFH

CD4+ T follicular helper cells (TFH) were assessed in adult patients with common variable immune deficiency (CVID) classified according to the presence of granulomatous disease (GD), autoimmunity (AI), or both GD and AI (Group I) or the absence of AI and GD (Group II). TFH lymphocytes were characterized by expression of CXCR5 and PD-1. TFH were higher (in both absolute number and percentage) in Group I than in Group II CVID patients and normal controls (N). Within CXCR5+CD4+ T cells, the percentage of PD-1 (+) was higher and that of CCR7 (+) was lower in Group I than in Group II and N. The percentages of Treg and TFH reg were similar in both CVID groups and in N. TFH responded to stimulation increasing the expression of the costimulatory molecules CD40L and ICOS as did N. After submitogenic PHA+IL-2 stimulation, intracellular expression of TFH cytokines (IL-10, IL-21) was higher than N in Group I, and IL-4 was higher than N in Group II. These results suggest that TFH are functional in CVID and highlight the association of increased circulating TFH with AI and GD manifestations.

Inhibition of normal natural killer cytotoxicity by sera from hemophilic patients

In this study we searched for circulating antibodies or other serum factors that could account for the natural killer (NK) defect observed in hemophiliacs (He) infected with the human immunodeficiency virus (HIV). We analyzed the effect of negative or positive sera for HIV from He on normal NK activity. We showed that sera from He interfered with normal NK cytotoxicity. The inhibitory activity was higher in HIV+ sera and increased as the HIV disease progressed. HIV- sera also inhibited NK function, although to a lesser extent than HIV+, and it was probably due to isoimmunization through replacement treatment with plasma-derived concentrates. For each individual, no direct correlation was found between NK inhibition (NK-INH) of sera and the NK activity of He peripheral blood mononuclear cells (PBMC). Furthermore, He serum was poorly inhibitory on autologous PBMC. Preincubation of allogenic effector or target cells with He sera revealed that the inhibitory effect was the result of the reaction with these cells. A positive correlation was found by comparing NK-INH of whole He sera with the serum levels of circulating immune complexes. When the NK-INH assay was performed using the same concentration of DEAE-purified IgG from N, HIV- or HIV+, we found that HIV+ AIDS IgG was more inhibitory than the others.

Effect of lymphokines on natural killer cytotoxicity in patients with high risk of developing the acquired immune deficiency syndrome

Regulation of natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) from patients with the acquired immune deficiency syndrome (AIDS) and from individuals at high risk of developing AIDS (R-AIDS) was studied. The response of untreated PBMC to the interferon inducer polyinosinic polycytidilic acid (Poly I:C) was lower in AIDS and R-AIDS than in normal controls and PBMC from R-AIDS were more susceptible to stimulation with lymphokine rich supernatants (Con A-SN, PHA-SN, lectin free IL-2) than AIDS and normal controls. To determine the role of the different T lymphocyte subsets in the regulation of NK activity, PBMC were selectively treated with monoclonal non-cytotoxic anti-Leu 2a and anti-Leu 3a antibodies and then stimulated with lymphokine rich supernatants. These results indicate that the effect of crude supernatants was the combination of opposite effects. Leu 2a-blocked R-AIDS-PBMC enhanced NK cytotoxicity when exposed to IL-2 rich supernatants whereas Leu 3a-blocked R-AIDS-PBMC suppressed the cytotoxic reaction.

HIV infection and natural killer cytotoxicity in hemophilic patients

In this study we analyzed the ability of peripheral blood mononuclear cells (PBMC) from hemophilic patients (He) with negative or positive serology for the human immunodeficiency virus (HIV), to increase natural killer (NK) cytotoxicity upon stimulation with physiological and non physiological agents. Purified interleukin-2 (IL-2), the interferon (IFN)-inducer polyinosinic polycytidylic acid (PIC), recombinant alpha- and gamma-IFN and the protein kinase activator phorbol myristate acetate (PMA) were used as stimulatory agents. The NK functional response was correlated with the presence of PBMC bearing phenotypic markers of activated cells (IL-2 receptor, IL-2R) and of different NK cell maturation stages. Our results demonstrate that NK effector cells with slight lytic activity (Leu 7+ CD16-) predominated in HIV+ He patients. On the other hand the occurrence of IL-2R positive cells was similarly high in both HIV+ and HIV- individuals and was probably more related to chronic replacement treatment with Factor VIII or Factor IX concentrates than to HIV infection. The ability to respond to physiological NK regulators such as IL-2 and IFNs, or to the IFN-inducer PIC was impaired in HIV+ He, especially in HIV+ LAS individuals, suggesting that the inability of these cells to increase NK cell activity after appropriate induction was due to an intrinsic defect. Since phosphoinositide turnover and subsequent protein kinase C activation are thought to be part of the physiological mechanism of NK cytotoxicity, we studied the effect of PMA on PBMC from each group of patients. The ability to respond to PMA was lost only in PBMC from HIV+ LAS patients, indicating that impairment of the NK lytic mechanism progresses as the disease gets worse.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased lymphocyte viability after non-stimulated peripheral blood mononuclear cell (PBMC) culture in patients with X-linked lymphoproliferative disease (XLP)

Survival of lymphocytes after prolonged culture was studied in two asymptomatic XLP patients. Viability of XLP PBMC after 30 days of non-stimulated culture was higher than that of normal controls (N), mainly due to the persistence of CD8 memory lymphocytes. IFNgamma high CD8 T lymphocytes remained higher in XLP than in N after 30 days. The number of perforin+ CD8 lymphocytes was markedly reduced after 30 days in XLP and in N. Increased viability was not related to CD127, PD-1, CD27, or CD62L expression. Concerning B lymphocytes, memory CD27+ CD19+ cells prevailed over CD27- cells after 30 days in both XLP and N, with far more surviving cells in XLP. In N, few CD19+ B lymphocytes were viable after prolonged culture. In XLP, these cells were also IgD+, IgM+ and EBNA2+. These results demonstrate that IFNgamma-positive memory CD8 T cells persist in XLP after prolonged culture in association with a subset of viable memory CD27+ B cells expressing latent EBV antigens. The survival advantage of XLP cells might be related to increased frequency of extranodal lymphoma in XLP patients.