Acute rotavirus infection is associated with the induction of circulating memory CD4+ T cell subsets

Abstract

Strong CD4⁺ T cell-mediated immune protection following rotavirus infection has been observed in animal models, but its relevance in humans remains unclear. Here, we characterized acute and convalescent CD4⁺ T cell responses in children who were hospitalized with rotavirus-positive and rotavirus-negative diarrhoea in Blantyre, Malawi. Children presenting with laboratory-confirmed rotavirus infection had higher proportions of effector and central memory T helper 2 cells during acute infection i.e., at disease presentation compared to convalescence, 28 days post-infection defined by a follow-up 28 days after acute infection. However, circulating cytokine-producing (IFN-γ and/or TNF-α) rotavirus-specific VP6-specific CD4⁺ T cells were rarely detectable in children with rotavirus infection at both acute and convalescent stages. Moreover, following whole blood mitogenic stimulation, the responding CD4⁺ T cells were predominantly non-cytokine producers of IFN-γ and/or TNF-α. Our findings demonstrate limited induction of anti-viral IFN-γ and/or TNF-α-producing CD4⁺ T cells in rotavirus-vaccinated Malawian children following the development of laboratory-confirmed rotavirus infection.

Figure 1

RV-IgA responses in children with rotavirus-positive diarrhoea. RV-IgA was measured from the plasma of the children with laboratory-confirmed rotavirus diarrhoea using a standardized quantitative ELISA. (A) RV-IgA geometric mean concentration (U/mL) for children with rotavirus-positive diarrhoea at acute and convalescent phases of infection. (B) Paired sample analysis for children with rotavirus-positive diarrhoea at acute and convalescent phases of infection. CI confidence interval, GMC geometric mean concentration.

Figure 2

CD4⁺ T subsets in children with or without rotavirus diarrhoea. The cells were stained with the following fluorochrome-conjugated antibodies CD3 PerCP-CY5.5, CD4 APC-CY7, CCR7 AF700, CD45RA FITC, CRTH2 APC, CXCR3 Qdot 605, PD1 PE CY7 and BCL6 PE. Central memory T cells were defined as CD4⁺, CCR7⁺ and CD45RA-, effector memory T cells as CD4⁺, CD45RA⁻ and CCR7⁻, terminally differentiated as CD4⁺, CD45RA⁺ and CCR7⁻ and naïve T cells as CD4⁺, CCR7⁺ and CD45RA⁺. Th1 was defined as CD4⁺ and CXCR3⁺, Th2 as CD4⁺ and CRTH2⁺ and Tfh as CD4⁺ BCL6⁺ and PD1^hi. Central memory Th1 was defined as CD4⁺, CXCR3⁺, CD45RA⁻ and CCR7⁺, effector memory Th1 as CD4⁺, CXCR3⁺, CD45RA⁻ and CCR7⁻, Central memory Th2 was defined as CD4⁺, CRTH2⁺, CD45RA⁻ and CCR7⁺ and effector memory Th1 as CD4⁺, CRTH2⁺ CD45RA⁻ and CCR7⁻. Mann–Whitney U test was used to compare the proportion of the T cells between children with rotavirus-positive and rotavirus-negative at both acute and convalescent phases of infection. Differences after comparisons were considered statistically significant if p-values were less than 0.05. (A) Representative flow cytometry plot from a child with rotavirus diarrhoea peripheral whole blood showing the memory T cell subsets. (B) Proportion of naïve, central memory, effector memory and terminally differentiated T cells for children with rotavirus-negative (in Green) diarrhoea at the acute and convalescent phase of infection and children with rotavirus-positive (in Red) diarrhoea at the acute and convalescent phase of infection. (C) Representative flow cytometry plot from children with rotavirus-positive diarrhoea and rotavirus-negative diarrhoea peripheral whole blood showing Th1 and Th2 cell subsets. (D) Proportion Th1, Th2 and Tfh cells for children with rotavirus-negative (In Green) diarrhoea at the acute and convalescent phase of infection and children with rotavirus-positive diarrhoea (In Red) at the acute and convalescent phase of infection. (E) Proportion of central memory Th1, central memory Th2, effector memory Th1 and effector memory Th2 for children with rotavirus-negative (In Green) diarrhoea at the acute and convalescent phase of infection and children with rotavirus-positive (In Red) diarrhoea at the acute and convalescent phase of infection.

Figure 3

CD4⁺ T stimulations in children with or without rotavirus diarrhoea. The cells were stained with the following fluorochrome-conjugated antibodies CD3 PerCP-CY5.5, CD4 BV 421, CD8 APC-CY7, TNF-α FITC, IFN-γ PE-CF594 and CD69 AF700. Mann–Whitney U test was used to compare the proportion of the T cells between children with rotavirus-positive and rotavirus-negative at both acute and convalescent phases of infection. Differences after comparisons were considered statistically significant if p-values were less than 0.05. (A) Representative FACS plot from children with rotavirus diarrhoea peripheral whole blood showing CD4 ⁺ T cells producing TNF-α and IFN-γ cells from PMA + ION (Mitogen) and VP6 protein responses and the unstimulated control. (B) Proportion of CD4 ⁺ producing TNF-α or IFN-y and both TNF-α and IFN-γ cells for children with rotavirus rotavirus-positive and rotavirus-negative diarrhoea at the acute phase of infection. (C) Proportion of CD4 ⁺ producing TNF-α or IFN-y and both TNF-α and IFN-γ cells for children with rotavirus-positive and rotavirus-negative diarrhoea at the convalescent phase of infection.

Figure 4

CD4⁺ T cells mitogenic cytokine profiles in children with or without rotavirus diarrhoea. The cells were stimulated with either PMA + ION or VP6 protein for 18 hours, with the unstimulated control in all experiments. The cells were stained with the following fluorochrome-conjugated antibodies CD3 PerCP-CY5.5, CD4 BV421, CD8 APC-CY7, TNF-α FITC, IFN-γ PE-CF594 and CD69 AF700. Single producers were either CD4 ⁺ T cells producing TNF-α or CD4 ⁺ T cells producing IFN-γ and double producers were CD4 ⁺ T cells producing TNF-α and IFN-γ. Mann–Whitney U test was used to compare the proportion of the T cells between children with rotavirus-positive and rotavirus-negative at both acute and convalescent phases of infection. Differences after comparisons were considered statistically significant if p-values were less than 0.05. (A) Frequency of CD4 ⁺ T cells producing TNF-α and IFN-γ, activated (CD69⁺) or not activated (CD69⁻) in children with rotavirus-positive and rotavirus-negative diarrhoea at the acute phase of infection. (B) Frequency of CD4 ⁺ T cells producing TNF-α and IFN-γ, activated (CD69⁺) or not activated (CD69⁻) in children with rotavirus-positive and rotavirus-negative diarrhoea at the convalescent phase of infection. (C) Frequency of CD4 ⁺ T cells producing TNF-α and IFN-γ, activated (CD69⁺) or not activated (CD69⁻) in children with rotavirus-positive and rotavirus-negative diarrhoea at the acute and convalescent phases of infection.