Differential CD4 T Regulatory Cell Phenotype Induced by Andes Hantavirus Glycoprotein

Abstract

Hantavirus cardiopulmonary syndrome (HCPS) caused by Andes orthohantavirus (ANDV) in South America is a public health threat due to the significant rate of mortality and the lack of a specific treatment. Interestingly, the virus does not produce cytopathic effect, thereby the strong antiviral immune response is suspected to contribute to pathogenesis, hence is important to understand the balance between protective and harmfully immunity. CD4⁺ T regulatory cells (Treg) are essential to control an exacerbated immune response. In human ANDV infection, little is known about CD4⁺ Treg cells, which may be involved in control immunopathology associated to the infection. In this report, we characterize the phenotype of memory CD4⁺ Tregs in a HCPS survivor's cohort. Based on the expression of CXCR3, CCR4, and CCR6, we identified different Th-like Treg populations in ANDV survival's PBMCs. In addition, the effect of ANDV-glycoprotein virus like particles (VLP) was determined. We demonstrated that memory CD4⁺ Treg from HCPS present a specific phenotype, showing higher frequency of PD-1 compared to healthy donors (HD). In addition, it was observed a decrease in the frequency of Th1-like memory CD4⁺ Treg in HCPS, important to highlight that this signature could be preserved even years after resolution of infection. Moreover, to gain insight in the mechanism involved, we evaluated whether ANDV-glycoprotein (GP) VLP could modulate CD4⁺ Treg. Interestingly, ANDV-GP VLP induced a decrease in the frequency of CXCR3 (Th1-like) and an increase in CCR4 (Th2-like) memory CD4⁺ Treg in both HD and HCPS PBMCs, indicating that ANDV-GP could specifically act over CXCR3 and CCR4 in CD4⁺ Treg. This report contributes to the study of human CD4⁺ Treg cells in ANDV infection.

Keywords: ANDV-GP; CD4 Treg; CXCR3; HCPS; Th-like Treg; Th1/Th2; VLPs.

Figure 1

Increase frequency of memory CD4⁺ Treg PD-1⁺ on HCPS survivors. (A) Gating strategy of a representative sample showing CD4⁺, CD4⁺CD45RO⁺, CD127^low/−CD25⁺, CD127^low/−CD25⁺ FoxP3⁺, memory Treg PD1⁺, and memory Treg CTLA-4⁺ cells. (B) Frequency of total CD4⁺ T cells gated in live PBMC. (C) Frequency of memory CD4⁺ T cells CD4⁺CD45RO⁺. (D) Frequency of memory CD4 Treg (CD4⁺CD45RO⁺CD25⁺CD127^low/−FoxP3⁺). (E) Frequency of memory Treg PD-1⁺ (left) and memory Treg CTLA-4⁺ (right). Data represented as dots (HD n = 15) and squares (HCPS n = 16), indicating the mean ± SD. **P = 0.0021 by unpaired Student's t-test.

Figure 2

HCPS survivors present a decrease on memory Th1-like Treg. (A) Gating strategy of a representative sample showing the CD4⁺T cells, CD127^low/−CD25⁺ Treg cells, memory Treg CD45RA⁻CCR4⁺, and finally the memory Th-like Treg subpopulations, specifically Th1-like Treg (CXCR3⁺CCR6⁻), Th2-like Treg (CXCR3⁻CCR6⁻), and Th17-like Treg (CXCR3⁻CCR6⁺). (B) Frequency of memory Treg CD45RA⁻CCR4⁺ in HD and HCPS. (C) Frequency of Th1-like, Th2-like, and Th17-like memory Treg in HD and HCPS. Data represented as dots (HD n = 13) and squares (HCPS n = 7), indicating the mean ± SD. **P = 0.003 by unpaired Student's t-test.

Figure 3

ANDV-GP virus like particles induces a Th2-like phenotype on memory CD4⁺ Treg cells. (A) ANDV-GP VLPs are detected in CD4⁺ T cells. PBMC were incubated with VLPs and detected by flow cytometry using an anti-ANDV-GP Qdot655 conjugated antibody (left). Purified CD4⁺ T cells were incubated with VLP and ANDV-GP was detected with anti-ANDV-GP Qdot655 conjugated antibody and visualized in CD4⁺CXCR3⁺ cells by confocal microscopy, arrows indicate the intracellular ANDV-GP staining (right). Following the same gating strategy of Figure 2 was evaluated the effect of ANDV-GP VLP on Th-like Treg frequency. (B) Frequency of total Treg (CD4⁺CD127^low/−CD25⁺) in HCPS and HD PBMCs after VLP overnight stimulation follow by analysis at day 7 of cell culture. (C) Frequency of memory CD4⁺ Treg CD45RA⁻CCR4⁺ in HCPS and HD in mock and VLP stimulus. (D) Frequency of Th-like memory CD4⁺ Treg CCR4⁺ in HCPS (above panel) and HD (below panel) in mock and VLP conditions. Data for each individual is represented as a line connecting mock and VLP conditions (HD n = 7; HCPS n = 7), *P < 0.05; **P < 0.001, ****P < 0.0001 by paired Student's t-test.

Figure 4

ANDV-GP induces Th1/Th2 cytokine expression on memory CD4⁺ T cells. Frequency of memory CD4⁺ T cells expressing the following cytokines (IFNγ, IL-5, IL-10, and IL-17) in HD stimulated with VLP in presence of anti-CD3/CD28 beads or mock as control after 3 days in cell culture (n = 4). Each line represents a subject sample. *P < 0.05; **P < 0.001, by paired Student's t-test.

Figure 5

ANDV-GP modulates CD4⁺ Treg phenotype by down regulating CXCR3. Three healthy donors were used to isolate CD4⁺ Treg from PBMCs, stimulated with VLP or no-treated (mock) and analyzed after 3 days in cell culture. (A) Mean fluorescence intensity (MFI) of T-bet, GATA3, and RORγt that allows the detection of Th1-like Treg, Th2-like Treg, and Th17-like Treg, respectively. (B) Frequency and expression measured by MFI of CXCR3 on the T-bet⁺ CD4⁺Treg population. Each line represents a subject sample (n = 3). *P < 0.05, by paired Student's t-test.