Phenotypic changes of γδ T cells in Plasmodium falciparum placental malaria and pregnancy outcomes in women at delivery in Cameroon

Abstract

Introduction: Depending on the microenvironment, γδ T cells may assume characteristics similar to those of Th1, Th2, Th17, regulatory T cells or antigen presenting cells. Despite the wide documentation of the effect of Th1/Th2 balance on pregnancy associated malaria and outcomes, there are no reports on the relationship between γδ T cell phenotype change and Placental Malaria (PM) with pregnancy outcomes. This study sought to investigate the involvement of γδ T cells and its subsets in placental Plasmodium falciparum malaria.

Methods: In a case-control study conducted in Yaoundé, Cameroon from March 2022 to May 2023, peripheral, placental and cord blood samples were collected from 50 women at delivery (29 PM negative: PM- and 21 PM positive: PM+; as diagnosed by light microscopy). Hemoglobin levels were measured using hemoglobinometer. PBMCs, IVBMCs and CBMCs were isolated using histopaque-1077 and used to characterize total γδ T cell populations and subsets (Vδ1⁺, Vδ2⁺, Vδ1^-Vδ2^-) by flow cytometry.

Results: Placental Plasmodium falciparum infection was associated with significant increase in the frequency of total γδ T cells in IVBMC and of the Vδ1⁺ subset in PBMC and IVBMC, but decreased frequency of the Vδ2⁺ subset in PBMC and IVBMC. The expression of the activation marker: HLA-DR, and the exhaustion markers (PD1 and TIM3) within total γδ T cells and subsets were significantly up-regulated in PM+ compared to PM- group. The frequency of total γδ T cells in IVBMC, TIM-3 expression within total γδ T cells and subsets in IVBMC, as well as HLA-DR expression within total γδ T cells and Vδ2⁺ subset in IVBMC were negatively associated with maternal hemoglobin levels. Furthermore, the frequency of total γδ T cells in PBMC and PD1 expression within the Vδ2⁺ subset in CBMC were negatively associated with birth weight contrary to the frequency of Vδ1^-Vδ2^- subset in PBMC and HLA-DR expression within the Vδ2⁺ subset in IVBMC which positively associated with maternal hemoglobin level and birth weight, respectively.

Conclusion: The data indicate up-regulation of activated and exhausted γδ T cells in Plasmodium falciparum placental malaria, with effects on pregnancy outcomes including maternal hemoglobin level and birth weight.

Keywords: HLA-DR; PD1; Plasmodium falciparum; TIM-3; placental malaria; pregnancy outcomes; women; γδ T cells.

Figure 1

Gating strategy of Gamma Delta T cells and different subsets. (A) Samples were initially gated on lymphocytes based on SSC-A/FSC-A gating. (B) Single cells were gated from lymphocytes using FSC-H/FSC-A. (C) T cells were gated from singlets as CD3⁺ cells. (D) Gamma delta T cells were gated from CD3⁺ as TCRγδ⁺. (E) Different subsets of Gamma delta T cells were characterized TCR Vδ1 and TCR Vδ2 markers as Vδ1⁺, Vδ2⁺ and Vδ1^-Vδ2^-.

Figure 2

Frequencies of γδ T cells and different subsets Vδ1⁺, Vδ2⁺, Vδ1^-Vδ2^- in women at delivery. γδ T cells and different subsets were analyzed through multiparametric flow cytometry in PBMC, IVBMC and CBMC of PM- (in blue) and PM+ (in red) women. (A–D) frequency of (A) total TCR γδ⁺ (B) TCR Vδ1⁺ (C) TCR Vδ2⁺ and (D) TCR Vδ1^-Vδ2^-. Frequencies were compared in each group using Mann-Whitney test. PM-, Placental Malaria negative women; PM+, Placental Malaria positive women; PBMC, Peripheral blood mononuclear cell; IVBMC, Intervillous blood mononuclear cell; CBMC, Cord blood mononuclear cell. Each dot represents a single individual.

Figure 3

Heatmap representing the correlation between the γδ T cells phenotype and different parasitemias. The correlation between γδ T cells profile in PBMC, IVBMC, CBMC and different parasitemias was determined using Sperman’s rank Order correlation test. %iRBC Imp, parasitemias from placenta tissue impression smear; Para_Per, Peripheral blood parasitemias; Para_Pla, Placental blood parasitemias; PBMC, Peripheral blood mononuclear cell; IVBMC, Intervillous blood mononuclear cell; CBMC, Cord blood mononuclear cell.

Figure 4

MFI of HLA-DR within γδ T cells and Vδ1⁺, Vδ2⁺, Vδ1^-Vδ2^- subsets in women at delivery. Mean fluorescence intensity (MFI) of HLA-DR within γδ cells and different subsets were analyzed through multiparametric flow cytometry in PBMC, IVBMC and CBMC of PM- (in blue) and PM+ (in red) women. (A–D) Mean fluorescence intensity of HLA-DR within (A) total TRC γδ⁺ (B) TRC Vδ1⁺ (C) TRC Vδ2⁺ and (D) TRC Vδ1^-Vδ2 Mean fluorescence intensities were compared in each group using Mann-Whitney test. PM-, Placental Malaria negative women; PM+, Placental Malaria positive women; PBMC, Peripheral blood mononuclear cell; IVBMC, Intervillous blood mononuclear cell; CBMC, Cord blood mononuclear cell. Each dot represents a single individual.

Figure 5

MFI of PD1 within γδ T cells and Vδ1⁺, Vδ2⁺, Vδ1^-Vδ2^- subsets in women at delivery. Mean fluorescence intensity (MFI) of PD1 within γδ T cells and different subsets were analyzed through multiparametric flow cytometry in PBMC, IVBMC and CBMC of PM- (in blue) and PM+ (in red) women. (A–D) Mean fluorescence intensity of PD1 within (A) total TCR γδ⁺ (B) TCR Vδ1⁺ (C) TCR Vδ2⁺ and (D) TCR Vδ1^-Vδ2 Mean fluorescence intensities were compared in each group using Mann-Whitney test. PM-, Placental Malaria negative women; PM+, Placental Malaria positive women; PBMC, Peripheral blood mononuclear cell; IVBMC, Intervillous blood mononuclear cell; CBMC, Cord blood mononuclear cell. Each dot represents a single individual.

Figure 6

MFI of TIM-3 within γδ T cells and Vδ1⁺, Vδ2⁺, Vδ1^-Vδ2^- subsets in women at delivery. Mean fluorescence intensity (MFI) of TIM-3 γδ T cells and different subsets were analyzed through multiparametric flow cytometry in PBMC, IVBMC and CBMC of PM- (in blue) and PM+ (in red) women. (A–D) Mean fluorescence intensity of TIM-3 within (A) total TCR γδ⁺ (B) TCR Vδ1⁺ (C) TCR Vδ2⁺ and (D) TCR Vδ1^-Vδ2 Mean fluorescence intensities were compared in each group using Mann-Whitney test. PM-, Placental Malaria negative women; PM+, Placental Malaria positive women; PBMC, Peripheral blood mononuclear cell; IVBMC, Intervillous blood mononuclear cell; CBMC, Cord blood mononuclear cell. Each dot represents a single individual.

Figure 7

Heatmap representing the correlation between the γδ T cells phenotype and pregnancy outcomes. The correlation between γδ T cells profile in PBMC, IVBMC, CBMC and pregnancy outcomes was determined using Sperman’s rank Order correlation test. Hb, Hemoglobin level; PM-, Placental Malaria negative women; PM+, Placental Malaria positive women; PBMC, Peripheral blood mononuclear cell; IVBMC, Intervillous blood mononuclear cell; CBMC, Cord blood mononuclear cell.

Figure 8

Heatmap representing the correlation between the γδ T cells phenotype and mother age, gestational age, parity. The correlation between γδ T cells profile in PBMC, IVBMC, CBMC and gestational age, parity was determined using Sperman’s rank Order correlation test. PM-, Placental Malaria negative women; PM+, Placental Malaria positive women; PBMC, Peripheral blood mononuclear cell; IVBMC, Intervillous blood mononuclear cell; CBMC, Cord blood mononuclear cell.