Cloning and functional testing of rhesus macaque (Macaca mulatta) IL-9 and IL-33

Abstract

Background: IL-9 and IL-33 can profoundly influence immune responses. As a necessary first step toward defining their impact in the rhesus macaque model, we confirmed their endogenous expression and sequence identity and generated expression vectors for the recombinant expression of rhesus IL-9 and IL-33.

Methods: RT-PCR and Sanger sequencing was used to define the expression and sequences for rhesus IL-9 and IL-33. The resulting recombinant cytokines were tested by ELISA and proliferation assays.

Results: Full-length rhesus IL-9 and the mature form of rhesus IL-33 share 78% and 73% nucleotide similarity, respectively, with humans. Both cytokines are expressed in lymphocytes, with IL-9 expression also evident in CD4+ T cells. Recombinantly expressed rhesus IL-9 and IL-33 were each biologically active in vitro, including enhancing the proliferation of a rhesus B cell line.

Conclusions: The recombinant rhesus IL-9 and IL-33 constructs produce biologically active cytokines that can act upon rhesus B cells.

Keywords: B cell; IL-33; IL-9; proliferation; rhesus.

Figure 1:. Expression of IL-9 and IL-33 in rhesus macaque.

(A) RT-PCR amplification of IL-9 and IL-33 from rhesus PBMC and CD4+ T cells. (B) Expression of IL-9 by CD4+ T cells. PBMC from two rhesus macaques were stimulated with PMA and ionomycin for four hours, permeabilized, stained with anti-human IL-9 antibody, and analyzed by flow cytometry. Plots are gated on live, CD19-CD20-CD8-CD3+CD4+ T cells. Nucleotide alignment of rhesus observed (Rmobsv), predicted (Rmpre), mouse, and human (Hum) IL-9 (C) and IL-33 (D). The boxes indicate codon differences between the rhesus observed and human.

Figure 2:. Amino acid sequences of rhesus macaque IL-9 and IL-33.

Amino acid alignments of mouse, human, predicted, and synthesized (syn) IL-9 (A) and IL-33 (B) proteins.

Figure 3:. Determination of recombinant IL-9 and IL-33 protein expression.

HEK293T cells were transfected with cloned rhesus IL-9 and IL-33 and supernatant was collected to measure the production of IL-9 (A) and IL-33 (B) protein by ELISA. Decreasing titrations of recombinant human cytokine included as positive control, supernatant from empty vector control transfected cells (pUNO1) included as negative control. Bars represent mean ± SEM of triplicate samples.

Figure 4:. Cell proliferation induced by recombinant IL-9 and IL-33.

(A) Human MO7e cells were cultured with rhesus macaque IL-9 (RmIL-9), in the absence or presence of IL-9 neutralizing antibody for 3 days. (B) Mouse D10.G4.T1 cells were cultured with rhesus macaque IL-33 (RmIL-33) in the absence or presence of IL-33 neutralizing antibody for 3 days. Recombinant human IL-9 and IL-33 were used as positive control and empty vector pUNO1 as negative control. Bars represent mean ± SEM of triplicate samples. * indicates p<0.05 compared to pUNO1.

Figure 5:. BLCL-C162 cell proliferation induced by recombinant IL-9 and IL-33.

Rhesus BLCL-C162 cells were cultured for 3 days with rhesus macaque IL-9 or IL-33 in the presence or absence of neutralizing antibody. * indicates p<0.05 compared to pUNO1.