Activated CD4+ T cells and CD14hiCD16+ monocytes correlate with antibody response following influenza virus infection in humans

Abstract

The failure to mount an antibody response following viral infection or seroconversion failure is a largely underappreciated and poorly understood phenomenon. Here, we identified immunologic markers associated with robust antibody responses after influenza virus infection in two independent human cohorts, SHIVERS and FLU09, based in Auckland, New Zealand and Memphis, Tennessee, USA, respectively. In the SHIVERS cohort, seroconversion significantly associates with (1) hospitalization, (2) greater numbers of proliferating, activated CD4⁺ T cells, but not CD8⁺ T cells, in the periphery during the acute phase of illness, and (3) fewer inflammatory monocytes (CD14^hiCD16⁺) by convalescence. In the FLU09 cohort, fewer CD14^hiCD16⁺ monocytes during early illness in the nasal mucosa were also associated with the generation of influenza-specific mucosal immunoglobulin A (IgA) and IgG antibodies. Our study demonstrates that seroconversion failure after infection is a definable immunological phenomenon, associated with quantifiable cellular markers that can be used to improve diagnostics, vaccine efficacy, and epidemiologic efforts.

Keywords: antibody; cellular immunity; humoral immunity; immune correlate; infection; influenza; monocytes; mucosal immunity; respiratory virus; seroconversion.

Graphical abstract

Figure 1

Characteristics of seroconverters (SCs) and non-seroconverters (non-SCs) in the SHIVERS cohort (A–C) Distribution of (A) age, (B) time-to-first-sera (days), and (C) first sera hemagglutination-inhibition (HAI) titers in the SCs (red, n = 21) and non-SCs (blue, n = 45) in the SHIVERS cohort. Serology profile of SCs (red, n = 7) and non-SCs (blue, n = 9) in the subcohort with HAI titers <40 within 14 days of enrollment that were selected for further analysis of their cellular immune profile. (D–F) HAI titer (D), (E) neuraminidase inhibition (NAI) titer, and (F) influenza-specific IgG titer. Dashed line in (C)–(E) indicates a titer of 40 and in (F) a titer of 100. ∗∗∗∗p < 0.0001, by 1-way analysis of variance, with Bonferroni corrections applied for multiple comparisons using log-transformed data. Serum samples were tested in single wells at least twice for HAI and NAI assays, while samples were run in duplicate wells in the ELISA assays. Lines and error bars indicate the median and interquartile range.

Figure 2

Peripheral CD4⁺ T cell profile in the SCs (red, n = 7) and non-SCs (blue, n = 9) during the acute and convalescence phases (A and B) Frequency (A) and (B) number of total CD3⁺CD4⁺ T cells during the acute and convalescence phases. (C–F) Frequency of proliferating (Bcl2⁻ and Ki67⁺) and activated (HLA-DR⁺CD38⁺) CD4⁺ T cells, during the (C and E) acute phase and (D and F) the convalescence phase, respectively. Cells were stimulated overnight with either media (unstimulated), pooled influenza peptides corresponding to the matrix (M1), nucleoprotein (NP), and PB1 protein, or the non-specific mitogen, phytohemagglutinin (PHA). ∗p < 0.05 and ∗∗p < 0.01, ∗∗∗p < 0.001, by 1-way analysis of variance, with Bonferroni corrections applied for multiple comparisons. Lines and error bars indicate the median and interquartile range.

Figure 3

Profile of the monocyte subpopulations and myeloid dendritic cells (mDCs) in the peripheral blood of the SCs (red, n = 7) and non-SCs (blue, n = 9) during the acute and convalescence phases (A) Gating strategy to identify the monocyte subpopulations based on the relative expression levels of the markers CD14 and CD16: “classical” monocytes are CD14^hi/CD16⁻ (red), “inflammatory” monocytes are CD14^hi/CD16⁺ (blue), and “patrolling” monocytes are CD14^lo/CD16⁺ (orange). (B and C) Percentages of each monocyte subpopulations (per total monocytes) during (B) acute phase and (C) convalescence phase in the SC and non-SC groups. (D and E) Percentage of (D) CD14^hi/CD16⁺ and (E) CD14^lo/CD16⁺ cells in the acute and convalescence time points samples. (F and G) Number (F) and (G) percentage of CD14⁻CD11⁺ mDCs in the SCs and non-SCs during the acute and convalescence phases. ∗p < 0.05 and ∗∗p < 0.01, by 1-way analysis of variance, with Bonferroni corrections applied for multiple comparisons. Lines and error bars indicate the median and interquartile range.

Figure 4

Immune responses in the nasal airways of participants in the FLU09 cohort (A and B) Influenza-specific (A) IgA and (B) IgG titers in the nasal washes collected on days 0, 7, 9, and 28 post-enrollment of SCs (red, n = 10) and non-SCs (blue, n = 9). SCs were defined as individuals with IgA titers >40 at any time points during the study, whereas non-SCs were individuals who did not. (C) Percentages of each monocyte subpopulation (per total monocytes) in the nasal airways of SCs and non-SCs collected within 1 week of enrollment. The monocyte populations are identified based on the relative expression levels of markers CD14 and CD16 as CD14^hi/CD16⁻ (classical), CD14^hi/CD16⁺ (inflammatory), and CD14^lo/CD16⁺ (patrolling) monocytes. ∗p < 0.05, by 1-way analysis of variance, with Bonferroni corrections applied for multiple comparisons. Samples were run in duplicate wells in the ELISA assays. Lines and error bars indicate the median and interquartile range.