Selective accumulation of Th2-skewing immature erythroid cells in developing neonatal mouse spleen

Abstract

Environmental factors likely regulate neonatal immunity and self-tolerance. However, evidence that the neonatal immune system is suppressed or deviated is varied depending on the antigen and the timing of antigen exposure relative to birth. These disparate findings may be related to the availability of the appropriate antigen presenting cells but also point to the possibility of homeostatic changes in non-lymphoid cells in the relevant lymphoid tissues. Here we show that, while leukocytes are the most abundant cell population present in spleen during the first 4-5 days after birth, a massive accumulation of nucleated immature erythroid population in the spleen takes places on day 6 after birth. Although the relative frequency of these immature erythorid cells slowly decreases during the development of neonates, they remain one of the most predominant populations up to three weeks of age. Importantly, we show that the immature erythroid cells from neonate spleen have the capacity to modulate the differentiation of CD4 T cells into effector cells and provide a bias towards a Th2 type instead of Th1 type. These nucleated erythroid cells can produce cytokines that participate in the Th2/Th1 balance, an important one being IL-6. Thus, the selective accumulation of immature erythroid cells in the spleen during a specific period of neonatal development may explain the apparent differences observed in the type(s) of immune responses generated in infants and neonates. These findings are potentially relevant to the better management of immune deficiency in and to the design of vaccination strategies for the young.

Keywords: Neonatal immunity; T lymphocytes.; erythrocytes.

Figure 1

Presence of a non-lymphoid population of cells in neonatal spleen after 6 days of birth. A. Flow cytometric analysis of CD44 and CD45 in spleens from adult mice and neonates at different periods of time after birth. Numbers represent the percentage of cells in each quadrant. B. Giemsa staining of cytospun spleen cells from adult mice and day-8 and day-17 neonates. Arrows point to large cells with less condensed nuclei. Data shown are representative of three experiments.

Figure 2

Phenotype of Ter119+ immature erythroid cells in neonatal spleen. A. Flow cytometry analysis for Sca1, C-kit, B220 and CD34 in spleen from day-7 neonates. Numbers represent the percentage of cells in each quadrant. B. Flow cytometry analysis for Ter119 together with CD45, Ly6G/C (Gr1), CD11b, B220 and TCRβ. Numbers represent the percentage in each gated population. Data from A and B are representative of three experiments. C. Expression of fetal and adult hemoglobin in Ter119+ cells isolated from the liver of three day-17 fetuses (fetal liver) and from spleens of three day-6 neonates (neonatal spleen) by RT-PCR using total RNA. Values are expressed as fold induction relative to the expression in fetal liver Ter119 cells.

Figure 3

The accumulation of immature erythroid cells compromises the representation of T and B cells in neonate spleens. (A, C, D, E and F) Flow cytometry analysis to determine the relative percentage of Ter119+ cells (A), CD4 T cells (C), CD8 T cells (D), B cells (B220) (E) and neutrophil/granulocytes (Gr1) (F) in spleens from mice at different days after birth. Each bar in represents the mean of two mice and are representative of 2 experiments. (B) Relative percentages of Ter119+ cells in spleen from mice at 7, 9, 11 and 21 days after birth. Error bars represent the standard error of the mean of at least four mice per group. Asterisk denotes a statistically significant (p <0.05) decrease in the percentage of Ter119+ cells between day 7 and day 9, and between day 9 and 21. Overall significance is at p<0.0001. Analyses were performed by ANOVA with Newman-Keuls Multiple comparisons test.

Figure 4

The presence of Ter119+ cells during the differentiation of CD4 T cells provides a Th2 bias. A. Purified adult CD4 T cells (10⁶ cells) were activated with anti-CD3 and anti-CD28 Abs for 3 days in the absence (-) or the presence of 1.5 x10⁵ or 3x 10 ⁵ Ter119+ cells from day-7 neonatal spleens. Cells were washed and equal number was restimulated with anti-CD3 Ab alone for 24 h. Levels of IL-2, IFNγ, and IL-4 in supernatant were determined by ELISA. B. CD4 T cells were activated and restimulated as in (A) using 1.5 x10⁵ Ter119+ cells. Relative levels of IL-2, IFNγ and IL-4 mRNA were determined by real time RT-PCR. Fold induction relative to CD4 cells activated in the absence of Ter119 cells is shown. Data shown is representative of two experiments.

Figure 5

Neonate immature erythroid cells produce IL-6 during the co-culture with CD4 T cells. A. Relative IL-4 mRNA levels in adult CD4 T cells activated with anti-CD3 and anti-CD28 Abs in the absence (-) or presence (Ter119) of day-7 neonatal spleen Ter119+ cells (3x10⁵) for 3 days, determined by real time RT-PCR. B. Purified adult CD4 T cells were activated with anti-CD3 and anti-CD28 Abs in the absence (-) or presence of the indicated number of neonatal spleen Ter119+ cells for 2 or 3 days and IL-6 production in the supernatant was determined by ELISA. C. Day-6 neonatal spleen Ter119+ cells were incubated in medium alone. Purified adult CD4 T cells were activated with anti-CD3 and anti-CD28 Abs. After 24 h, relative levels of IL-6 mRNA (left panel) in both cultures were determined by real time RT-PCR, and IL-6 production in the culture supernatants (right panel) was determined by ELISA. D. Adult WT CD4 T cells were activated with anti-CD3 and anti-CD28 Abs in the presence of Ter119+ cells isolated from WT (WT Ter119) or IL-6 KO (KO Ter119) day 6-neonates. After 24 h, relative levels of IL-6 mRNA (left panel) in both co-cultures were determined by real time RT-PCR, and IL-6 production in the co-culture supernatants (right panel) was determined by ELISA. E. Adult CD4 T cells were activated in the absence (-) and presence of Ter119+ cells isolated from WT or IL-6 KO neonates for three days. Cells were washed and restimulated with anti-CD3 for 24 h and the relative IL-4 mRNA levels were determined by real time RT-PCR. Data shown is the mean of duplicate wells for each assay performed.