Human cord blood CXCR5+ CD4 T cells: association with in utero exposure and antibody response to Plasmodium falciparum.

Ambe Lionel Neba (lionelambe@yahoo.com)
Microbiology, parasitology, haematology and infectious diseases, The University of Yaoundé I
July, 2015
 

Abstract

Introduction
In malaria-endemic regions such as sub-Saharan Africa, foetuses may be exposed in utero to malaria parasite products from their infected mothers. Though the foetus has long been considered to be immunologically hypo-responsive to in utero aggression, recent studies have shown the contrary. Functional T and B cells have been identified in the human foetus as early as the 12th week of gestation. Cytokines and immunoglobulins have also been found to be produced by cord blood T and B cells respectively, in response to Plasmodium falciparum (Pf) exposure. Whether or not these responses are specific or could lead to the generation of long-term immune memory and long-lived plasma cells that produce high affinity antibodies is not well known. However, T follicular helper cells located in the germinal centre help B cells to differentiate into plasma cells and memory B cells. The foetus not having matured lymphoid follicles is thus not capable of producing these cells. Recently, CXCR5-positive (CXCR5+) CD4 T cells, also called circulatory T-follicular helper cells (cTFH), have been identified in adult peripheral blood to help B cells in antibody production. This raises the question on whether cTFH cells are also produced in utero and are involved in prenatal antibody responses.
With this in mind, we carried out a research study aimed at determining if CXCR5+ CD4 T cells are present in umbilical cord blood and if they are associated with in utero exposure and antibody responses to Pf antigens.
Methods
To attain our objectives, we designed a cross-sectional study involving pregnant women at delivery and their new-born babies from the maternities in the Yaoundé Central Hospital and the Nkolbisson Health District. HIV positive women and women who delivered by Caesarean Section or who refused to participate were excluded. Blood was collected at delivery from the umbilical cord and intervillous space (IVS) of the placenta of women who had active malaria confirmed by RDT or a history of malaria from the twenty secondth week of pregnancy. Blood smear microscopy and nested polymerase chain reaction (PCR) were used for malaria diagnosis and speciation. The MagPix Multiplex Analyte Platform (MAP) assay was used to quantify IgM and IgG, to the malarial antigens AMA1, EBA175 and MSP1, in cord blood plasma and in cord blood mononuclear cell (CBMC) culture supernatants respectively. Real-Time quantitative PCR was used to quantify CXCR5 gene expression in CD4+T cells isolated from CBMC. Data were analyzed using GraphPad Prism 5.0. The study was approved by the Cameroon National Ethics Committee for Research on Humans and we submitted our research protocol to the Ethics Committee of the Faculty of Medicine and Biomedical Sciences, University of Yaoundé 1.
Results:
A total of 30 paired maternal and cord blood (CB) samples and 6 control non-pregnant adult peripheral blood samples were used. 20% of the women were placental malaria-positive by microscopy with parasites detected in IVS blood and/or placental impression smears. However, 40% of the women were positive for P. falciparum by PCR. No parasites were detected by microscopy in CB smears, but 16.7% were positive for malaria by PCR. None of the cord blood plasma tested positive for antimalarial IgM, while 16.7% of the CBMC culture supernatants tested positive for antimalarial IgG to all three malarial antigens.
We detected CXCR5 expression in CD4 T cells in 66.7% of the CB samples with 20% having CXCR5 expression levels greater than the least expressing adult sample and 3.3% having expression levels comparable to the median expression of all adult samples. The CXCR5 relative gene expression was significantly higher in neonates born to mothers with placenta malaria (p=0.016) and treatment of cultured CBMC with 10µg/mL of MSP-1 antigen, increased CXCR5 gene expression (compared to expression in paired untreated CBMC) in 55% of the CB samples. The expression of CXCR5 in CB CD4 T cells did not correlate with anti-malarial IgG titres in CBMC culture supernatant.
Conclusion and Recommendations
We concluded as follows: CXCR5+ CD4 T cells are produced in utero and are detectable in cord blood; The abundance of CXCR5-expressing cord blood CD4 T cells was significantly associated with placental malaria; A direct link was not found between CXCR5 expression in cord blood and in utero anti-malarial antibodies in our study population. From these, we do recommend the following: To further evaluate and characterize circulating TFH in cord blood using accessory but supplementary markers of TFH such as PD-1, BCL6, ICOS and CD45RO; Employ complementary techniques such as Flow Cytometry to enumerate CXCR5+ CD4 T cells in cord blood; Carry out a longitudinal study to determine if high CXCR5 expression in cord blood CD4 T cells at birth influences the acquisition of immunity to malaria during childhood.

Key words: CXCR5, in utero exposure, antibody production


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