OMIP-052: An 18-Color Panel for Measuring Th1, Th2, Th17, and Tfh Responses in Rhesus Macaques.

Abstract

This 18-color panel was designed to measure vaccine-induced T cell responses in rhesus macaques by intracellular cytokine staining (ICS). It detects seven cytokines, IFN-γ, IL-2, IL-4, IL-5, IL-13, IL-17, and IL-21, two memory markers, CD45RA and CCR7, and four follicular helper T cell (Tfh) markers, CXCR3, CXCR5, ICOS, and PD-1 (Figure 1). While this panel was optimized for use on cryopreserved rhesus macaque peripheral blood mononuclear cells (PBMC) (Table 1), it can also be used on rhesus macaque tissue samples and human PBMC and tissue samples. Experimental vaccines are often tested in nonhuman primates (NHP) for immunogenicity and protection in preclinical trials before proceeding to clinical trials in humans. Historically, vaccine-induced T cell responses have been measured by IFN-γ ELISpot or intracellular cytokine staining (ICS) for IFN-γ, IL-2, and TNF. However, antibody levels, rather than Th1 responses, correlate with protection for many vaccines including the HIV vaccine used in the RV144 clinical trial 1. As such, measuring only Th1 responses may not be adequate for determining T cell correlates of protection or risk in vaccine studies. Therefore, we expanded our 11-color intracellular cytokine staining (ICS) panel, described in OMIP-005, to 18 colors in order to assess a fuller spectrum of T cell responses (Table 2). IL-17 To decrease the variability of the assay due to age-related differences in T cell differentiation, cytokine measurements can be reported as a frequency of memory T cells using the markers CD45RA and CCR7 2. Similarly, IL-21 can be reported as a frequency of Tfh using a combination of the markers CXCR3, CXCR5, ICOS, and PD-1. This combination of markers allows for the delineation of Tfh cells by all currently accepted methods 3-5. This 18-color rhesus macaque panel is an extension of the 11-color panel described in OMIP-005. The cytokines IL-4, IL-5, IL-13, IL-17, and IL-21 were added as well as the Tfh subset markers CXCR3, CXCR5, ICOS, and PD-1. To accommodate these additional markers, CD28 and TNF were removed. It is similar to the following human panels also used to measure antigen-specific responses by intracellular staining: OMIP-001, OMIP-008, OMIP-009, OMIP-14, OMIP-16, OMIP-22, OMIP-25. However, this panel is designed for staining rhesus macaque PBMC (using NHP cross-reactive antibody clones) and includes additional Tfh markers, as well as IL-5 and IL-13. It is also the only panel with this combination of seven cytokines. We thank Mario Roederer and Pratip K. Chattopadhyay for advice and help in developing and optimizing this panel. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.