A review of CD4+ T cell differentiation and diversity in dogs

Abstract

CD4⁺ T cells are an integral component of the adaptive immune response, carrying out many functions to combat a diverse range of pathogenic challenges. These cells exhibit remarkable plasticity, differentiating into specialized subsets such as T helper type 1 (T_H1), T_H2, T_H9, T_H17, T_H22, regulatory T cells (Tregs), and follicular T helper (T_FH) cells. Each subset is capable of addressing a distinct immunological need ranging from pathogen eradication to regulation of immune homeostasis. As the immune response subsides, CD4⁺ T cells rest down into long-lived memory phenotypes-including central memory (T_CM), effector memory (T_EM), resident memory (T_RM), and terminally differentiated effector memory cells (T_EMRA) that are localized to facilitate a swift and potent response upon antigen re-encounter. This capacity for long-term immunological memory and rapid reactivation upon secondary exposure highlights the role CD4⁺ T cells play in sustaining both adaptive defense mechanisms and maintenance. Decades of mouse, human, and to a lesser extent, pig T cell research has provided the framework for understanding the role of CD4⁺ T cells in immune responses, but these model systems do not always mimic each other. Although our understanding of pig immunology is not as extensive as mouse or human research, we have gained valuable insight by studying this model. More akin to pigs, our understanding of CD4⁺ T cells in dogs is much less complete. This disparity exists in part because canine immunologists depend on paradigms from mouse and human studies to characterize CD4⁺ T cells in dogs, with a fraction of available lineage-defining antibody markers. Despite this, every major CD4⁺ T cell subset has been described to some extent in dogs. These subsets have been studied in various contexts, including in vitro stimulation, homeostatic conditions, and across a range of disease states. Canine CD4⁺ T cells have been categorized according to lineage-defining characteristics, trafficking patterns, and what cytokines they produce upon stimulation. This review addresses our current understanding of canine CD4⁺ T cells from a comparative perspective by highlighting both the similarities and differences from mouse, human, and pig CD4⁺ T cell biology. We also discuss knowledge gaps in our current understanding of CD4⁺ T cells in dogs that could provide direction for future studies in the field.

Keywords: Adaptive immunity; CD4(+) T cells; Canine; Flow cytometry; Single-cell RNA sequencing.

Fig 1.. Canine CD4⁺ T cell lineage subsets.

Lineage-defining markers are shown as cell surface molecule expression (on top of the cell), intracellular staining of transcription factors (square boxes in the center of the cell) and cytokine production (within the cell). Markers, transcription factors, and cytokines that have not yet been tested in the dog, but have been validated in other species, are shown in red text. Proposed gene signatures for each lineage subset are combined from three published canine single-cell RNA sequencing datasets (in colored boxes adjacent to the cell). Genes from the Ammons et al. dataset (2023) are taken from Table 1 in the manuscript. Genes from the Eschke et al. dataset are taken from Figure 3A in the manuscript where genes that had an average log2 fold change (avg_log2FC) > 0 were included. Genes from the Ammons et al. dataset (2024) of intratumoral CD4⁺ T_FH cells from Figure 3b and Figure 3g of the manuscript (avg_log2FC > 1) were included.

Fig 2.. Canine CD4⁺ T cell memory subsets.

A) A representative flow plot illustrates the relative frequencies of memory subsets in a healthy canine splenocyte sample with upstream gating: lymphocytes > singlets > CD3⁺ > Dump-negative (CD14, CD21, CD11b, dead) > CD4⁺CD8⁻ T cells. B) A schematic illustrates the basics of memory CD4⁺ T cell formation where there is simultaneous generation of each memory CD4⁺ T cell subset from naïve CD4⁺ T cells. Relative gene signatures across two different scRNA-seq datasets are included. Genes from the Ammons et al. dataset (2023) come from Table 1 of the manuscript. Genes from the Eschke et al. dataset come from Figure 3A of the manuscript where genes that had an avg_log2FC > 0 were included.

Fig 3.. Co-expression of CD5 and CD3 in canine CD4⁺ T cells.

Two commonly used pan-T cell markers (CD5 and CD3) are compared by flow cytometry and scRNA-seq to demonstrate their overlap in expression. A) A representative flow cytometry plot of CD5 (clone YKIX322.3) vs. CD3 (clone CA17.2A12) protein-level expression from healthy canine PBMCs ex vivo pre-gated on lymphocytes, singlets, Dump-negative (CD21/CD14/CD11b), live cells (Zombie Green-negative cells). Percentages of CD5 vs. CD3 expression are displayed in the corners of the plot (left). The quantification of % CD5 vs. CD3 expression across 5 canine PBMC and 5 splenocyte samples (right). B) The number of cells that expressed CD3E or CD5 gene transcripts, which encode for the CD3 and CD5 molecules, respectively, are shown across two publicly available scRNA-seq datasets (left). The percentage of cells expressing either the CD3E or CD5 genes (# of CD3E or CD5 cells / total # of CD4) and the %difference of cells that express CD3E, but not CD5 (%CD3E - %CD5) (right). C) In the Ammons et al. scRNA-seq dataset, expression of CD5-expressing cells (shown in gold) is overlaid on all cells that are also CD4+ (light gray) in the left-most panel. Similarly, in the middle panel, CD3E-expressing cells (shown in burgundy) are overlaid on cells that are also CD4+ (light gray). Cells that express both CD5 and CD3E transcripts are demonstrated in the right-most panel (orange) on top of cells that are solely expressing CD5 (gold) and CD3E (burgundy). In the Eschke et al. scRNA-seq dataset, CD5, CD3E, and CD5/CD3E co-expression plots are shown in same way as the Ammons et al. dataset relative to universal CD4 expression (light gray).

Fig 4.. Canine CD4+ cells express genes associated with neutrophils and monocytes.

A) A feature plot using data from Ammons et al. (2023) demonstrating the three major clusters of cells that express CD4: T cells (outlined in black), monocytes (outlined in red), and neutrophils (outlined in light blue). The dots shown in purple correspond to the relative level of CD4 gene expression. B) The proposed monocyte cluster (left) is based on high expression of monocyte-related genes (MAFB and FN1), while the proposed neutrophil cluster (right) expresses neutrophil-related genes (SGK1 and S100A12). As in (A), the dots shown in purple corresponds to the level of CD4 gene expression. This dataset provides validation that canine neutrophils and monocytes express high levels of CD4 by single-cell RNA sequencing.