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. 2017 Aug 17;2(16):e93739.
doi: 10.1172/jci.insight.93739.

Neonatal and adult recent thymic emigrants produce IL-8 and express complement receptors CR1 and CR2

Marcin L Pekalski  1   2 , Arcadio Rubio García  1   2 , Ricardo C Ferreira  1   2 , Daniel B Rainbow  1   2 , Deborah J Smyth  2 , Meghavi Mashar  2 , Jane Brady  2 , Natalia Savinykh  2 , Xaquin Castro Dopico  2 , Sumiyya Mahmood  2 , Simon Duley  2 , Helen E Stevens  2 , Neil M Walker  2 , Antony J Cutler  1   2 , Frank Waldron-Lynch  2 , David B Dunger  3 , Claire Shannon-Lowe  4 , Alasdair J Coles  5 , Joanne L Jones  5 , Chris Wallace  2   6 , John A Todd  1   2 , Linda S Wicker  1   2
Affiliations

Neonatal and adult recent thymic emigrants produce IL-8 and express complement receptors CR1 and CR2

Marcin L Pekalski et al. JCI Insight. .

Abstract

The maintenance of peripheral naive T lymphocytes in humans is dependent on their homeostatic division, not continuing emigration from the thymus, which undergoes involution with age. However, postthymic maintenance of naive T cells is still poorly understood. Previously we reported that recent thymic emigrants (RTEs) are contained in CD31+CD25- naive T cells as defined by their levels of signal joint T cell receptor rearrangement excision circles (sjTRECs). Here, by differential gene expression analysis followed by protein expression and functional studies, we define that the naive T cells having divided the least since thymic emigration express complement receptors (CR1 and CR2) known to bind complement C3b- and C3d-decorated microbial products and, following activation, produce IL-8 (CXCL8), a major chemoattractant for neutrophils in bacterial defense. We also observed an IL-8-producing memory T cell subpopulation coexpressing CR1 and CR2 and with a gene expression signature resembling that of RTEs. The functions of CR1 and CR2 on T cells remain to be determined, but we note that CR2 is the receptor for Epstein-Barr virus, which is a cause of T cell lymphomas and a candidate environmental factor in autoimmune disease.

Keywords: Autoimmunity; Immunology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Gene expression profiling of 4 naive CD4+ T cell subsets identify age-related molecular signatures.
(A) Gating strategy defining human naive CD4+ T cells; naive T cells were further stratified by CD31 and CD25. Representative examples (from n = 391; 371, 15, and 5 from cohorts 1–3, respectively; see Methods for details) of naive CD4+ T cells. (B) The proportion of naive CD4+ T cells as a function of age (color coding shown above graph). (C) Volcano plot of differences in gene expression (microarray platform) between CD31+CD25− and CD31–CD25− naive CD4+ T cells; red and blue symbols for genes with higher and lower, respectively, expression in CD31+CD25− naive CD4+ T cells (n = 20, cohort 1).
Figure 2
Figure 2. CR2 marks the most naive CD4+ T cell subset.
(A) Representative examples of CR2 expression in naive T cell subsets. (B) Percentage CR2+ cells in each subset and frequency of CR2+ cells in the CD31+CD25− naive CD4+ T cell subset as a function of age (from n = 389; 371, 15, and 3 from cohorts 1–3, respectively). Significance determined by paired t test. (C) Representative sorting strategy for CD31+CD25− naive CD4+ T cells identified as CR2−, CR2lo, and CR2hi (donors 1–4). For donors 5–7, the CR2+ gate is a combination of low- and high-CR2-expressing cells. Sorted cells were assessed for signal joint T cell receptor rearrangement excision circles (sjTRECs) (n = 7; 1 and 6 donors from cohorts 1 and 3, respectively).
Figure 3
Figure 3. Higher complement receptor 2 (CR2) expression by human naive CD4+ T cells during de novo reconstitution.
(A) Treatment and sampling time points of multiple sclerosis (MS) patients. (B) Frequency of CD31+CD25− naive CD4+ T cells expressing CR2 in MS patients before (baseline, BL) and 12 months after lymphocyte depletion with anti-CD52 (Campath). Significance determined by paired t test. (C) CR2 expression on CD31+CD25− naive CD4+ T cells from 2 patients before and at various times during reconstitution; time points from 6 additional patients are shown in Supplemental Figure 3C.
Figure 4
Figure 4. CR2+ naive CD4+ T cells have a unique molecular signature.
(A) Volcano plots of differences in gene expression (NanoString platform) between CR2+ versus CR2− naive CD4+ T cells (gating strategy shown as insert) ex vivo and after activation (anti–CD3/CD28). Genes expressed at a higher or lower level in CR2+ cells have red or blue symbols, respectively. Underlined genes have lower expression after activation. Genes in boxes are from the RNA-seq platform (n = 4 adult donors from cohorts 1 and 3). (B) Ex vivo CR1 protein expression on CR2+ and CR2− cells (n = 34, age range 0–67, cohorts 1–3, paired t test). Red and gray histograms gated on CR1+ and CR1− cells, respectively. (C) Representative histograms and compiled frequencies of cytokine production following activation of CR2+ and CR2− cells sorted from CD31+CD25− naive CD4+ T cells (n = 3, age range 30–44, cohort 3, paired t test). (D) Representative histograms of IL-8 production from isolated CD4+ T cells following activation with PMA and ionomycin. Compiled data of percentage IL-8+ cells out of naive (CD45RA+) CD4+ T cells (unpaired t test, n = 3 cord bloods from cohort 3, 4 multiple sclerosis [MS] patients 6 to 9 months after treatment, 3 MS patients >10 years after treatment. Correlation of percentage IL-8+ cells (following activation) and percentage CR2+ cells (assessed prior to activation) in the MS patients (n = 7).
Figure 5
Figure 5. CR2 and CR1 are coexpressed on a subset of central memory CD4+ T cells.
(A) Gating strategy and CR2 expression. Correlation of CR2 expression on central memory versus naive CD4+ T cells (B) and CR2+ central memory CD4+ T cells versus age (C) (n = 389; 371, 15, and 3 from cohorts 1–3, respectively). (D) Gating example of central memory cells sorted by CR2 expression, and gene expression analysis (NanoString). Color coding is described in Figure 4. (E) FACS analysis and compiled data of CR1 and CR2 coexpression (n = 34, age range 0–67, cohorts 1–3, paired t test). Red and gray histograms gated on CR1+ and CR1− cells, respectively. (F) Example and compiled data of IL-8 production from sorted and activated CR2+ and CR2− memory CD4+ T cells (n = 3, age range 30–44, cohort 3, paired t test).
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