Distinctive Leukocyte Subpopulations According to Organ Type in Cynomolgus Macaques

Abstract

Cynomolgus macaques (CYNO; Macaca fascicularis) are a well-established NHP model used for studies in immunology. To provide reference values on the baseline cell distributions in the hematopoietic and lymphoid organs (HLO) of these animals, we used flow cytometry to analyze the peripheral blood, bone marrow, mesenteric lymph nodes, spleen, and thymus of a cohort of male, adult, research-naïve, Mauritian CYNO. Our findings demonstrate that several cell distribution patterns differ between CYNO and humans. First, the CD4(+):CD8(+) T-cell ratio is lower in CYNO compared with humans. Second, the peripheral blood of CYNO contains a population of CD4(+)CD8(+) T cells. Third, the CD31 level was elevated in all organs studied, suggesting that CD31 may not be an accurate marker of recent thymic emigrants within the CD4(+) T cells of CYNO. Finally the B-cell population is lower in CYNO compared with humans. In summary, although the majority of immune cell populations are similar between cynomolgus macaques and humans, several important differences should be considered when using CYNO in immunologic studies. Our current findings provide valuable information to not only researchers but also veterinarians working with CYNO at research centers, in zoos, or in the wild.

Figure 1.

Size and granularity of cells in each of the HLO. (A) Peripheral blood (PB), (B) spinal bone marrow (SBM), (C) ilial bone marrow (IBM), and (D) spleen all have large populations of granular cells. These populations were not seen in the (E) mesenteric lymph nodes or (F) thymus, which contain only populations with less granularity. (G) We assessed the percentage (mean ± SEM) of high side-scatter (granular) cells in PB, SBM, IBM, and spleen.

Figure 2.

Analysis of CD3⁺, CD4⁺, and CD8⁺ T cells in thymus. When gating on thymocytes, the CD3 marker emerges as a smear that appears to comprise high and low/negative populations. When further analyzed, the CD3^low/negative population is shown to contain CD4⁺ and CD8⁺ single- and double-positive cells.

Figure 3.

Distributions of T-cell populations within the hematopoietic and lymphoid organs of cynomolgus macaques. (A) Comparison of the percentage of cells in each organ that are CD3⁺. (B) CD3⁺ cells from PB are subdivided into 4 populations (from top left, clockwise: CD4^–CD8⁺, CD4⁺CD8⁺, CD4⁺CD8^–, and CD4^–CD8^–). Between the studied organs, we compared the percentages of (C) CD4⁺CD8^– and (D) CD4^–CD8⁺. CD4⁺ and CD8⁺ single-positive cells can be directly compared by calculating (E) the CD4⁺CD8^–: CD4^–CD8⁺ T cell ratio. We examined the distribution of (F) CD4⁺CD8⁺and (G) CD4^–CD8^– cells in each organ. Data are given as mean ± SEM.

Figure 4.

Distribution of CD31 in T-cell populations within the hematopoietic and lymphoid organs of cynomolgus macaques. We examined the expression of CD31 in (A) CD4⁺ single-positive T cells, (B) CD8⁺ single-positive T cells, (C) CD4⁺CD8⁺ T cells, and (D) double-negative T cells. Data are given as mean ± SEM.

Figure 5.

Distribution of CD45RA in T-cell populations within the hematopoietic and lymphoid organs of cynomolgus macaques. Expression of CD45RA was determined among (A) CD4⁺ single-positive T cells, (B) CD8⁺ single-positive T cells, (C) CD4⁺CD8⁺ T cells, and (D) double-negative T cells. Data are given as mean ± SEM.

Figure 6.

Distribution of regulatory T cells within the hematopoietic and lymphoid organs of cynomolgus macaques. All flow data are representative plots derived from the mesenteric lymph nodes. Regulatory T-cell populations are enriched in the mesenteric lymph nodes. Regulatory T cells were assessed in multiple ways. (A) Initially the percentage of CD4⁺ T cells expressing FoxP3 was determined in each organ. (B) When assessing nonpermeabilized CD4⁺ T cells, the expression of high levels of CD25 can be used as a proxy for FoxP3. (C) Costaining of CD25 and FoxP3 is a more reliable method for the identification of regulatory T cells. Data are given as mean ± SEM.

Figure 7.

Distribution of NK and CD56 across the hematopoietic and lymphoid organs of cynomolgus macaques. (A) Initially we defined NK cells as CD3^–CD56⁺ cells. In each organ, we determined the presence of (B) NK cells and (C) CD3⁺CD56⁺ cells. We also examined (D) CD56 expression within CD3⁺ T cells. (E) Within T cells, we assessed the relationship between CD8 and CD56 expression. We examined both (F) coexpression of CD8 and CD56 and (G) expression of CD56 in the absence of CD8. (H) A population of CD3^–CD8⁺ cells that has been suggested to have NK-like function in NHP was (I) observed and quantified in each tissue and (J) further examined for CD56 expression. (K) CD56⁺ expression within the CD3^–CD8^– population was evaluated as well. Data are given as mean ± SEM.

Figure 8.

Distribution of nonT-cell lymphocytes across the hematopoietic and lymphoid organs of Mauritian cynomolgus macaques. Cells shown in (E) are from Iliac Bone Marrow. (A) Using peripheral blood, we defined B cells as CD3-CD20+ cells and (B) determined their distribution. (C) The T:B cell ratio was calculated for each tissue. (D) Monocytes were defined as less granular cells that were CD3^–CD11b⁺. (E) Their distribution was determined in each tissue. (F) We identified CD34⁺ hematopoietic stem cells and determined (G) the mean percentage of CD34⁺ hematopoietic stem cells acquired by harvesting bone marrow from either the spinal vertebrae or the iliac crest of Mauritian cynomolgus macaques; we compared that value with the proportion of CD34⁺ cells circulating in the peripheral blood. Gates were drawn on the basis of an isotype control and on cell size. Data are given as mean + SEM.