CCR5 plays multiple roles in mucosal immunity

From the Prlic lab, Vaccine and Infectious Disease Division

Immune cells expressing the receptor CCR5 are recruited to areas of inflammation by chemokine ligands, making CCR5 an attractive therapeutic target for preventing cell trafficking during conditions such as cancer or graft-versus-host disease. CCR5 is also the primary receptor for HIV CD4+ T cell entry, which prompted the development of Maraviroc, a small molecule CCR5-antagonist, and its use as therapy in HIV-positive individuals. Fortunately, patients tolerate Maraviroc well and do not lose barrier immunity due to the loss of CCR5. However, this outcome is somewhat surprising considering the important role of CCR5 in mucosal immune cells. Therefore, CCR5 presents an interesting paradox wherein its expression is necessary for proper T cell migration to sites of inflammation, but it appears to be dispensable once a cell arrives in the mucosa. To explain the dichotomous roles of CCR5 in mucosal barrier tissues, Dr. Amanda Woodward-Davis from the Prlic lab (Vaccine and Infectious Disease Division) investigated the CCR5+ T cell compartment in human mucosa and published their findings this month in Science Translational Medicine.

First, the authors characterized the mucosal T cell environment. They obtained healthy human oral mucosal tissue along with matched blood from the same patients, and using flow cytometry and immunofluorescence staining, observed that a high frequency of both CD4+ and CD8+ mucosal T cells, but not blood T cells, expressed CD69, which is a marker used to identify tissue-resident T cells. CD4+ T cells are known to be important for barrier immunity, so the authors focused their analysis on both the transient (CD69-) and tissue resident memory (Trm; CD69+) populations, and found that CCR5 was expressed on both populations in the mucosa. These experiments defined expression of CCR5 on mucosal CD4 T cells during homeostasis, so the authors next examined how the CCR5+ CD4 T cell population was affected by inflammation. They obtained oral mucosa samples of varied inflammation levels and used fluorescence-activated cell sorting (FACS) to isolate transient CCR5+ CD4 T cells from the blood and mucosa along with CCR5+ CD4 Trm from the mucosa, and performed bulk RNA sequencing to determine the transcriptional profiles of each subset at the population level. Here they were surprised to find that inflammation score was not associated with differential gene expression in the tissue. However, the sequencing data revealed that mucosal CCR5+ CD4 T cells were very transcriptionally distinct from blood CCR5+ CD4 T cells, suggesting that T cell entry into the tissue drives more transcriptional changes than does inflammation itself.

To further compare the CCR5+ subsets within the mucosa, they examined differentially expressed genes between the transient and Trm CCR5+ CD4 T cell populations. They saw that the Trm population was enriched for a CD4+ T cell subset called T-helper 17 (Th17), a population known to have an important role in barrier immunity. This finding contrasted earlier work that suggested CCR5+ cells are exclusively T-helper 1-type (Th1), a different subset of CD4+ T cells. To corroborate this surprising result, the authors performed single-cell RNA-sequencing to corroborate their preliminary data and confirmed that Trm and Th17 signatures were indeed co-expressed in the same cell, suggesting that mucosal CCR5+ CD4 T cells have both Trm and Th17 potential. By activating T cells in vitro, they further demonstrated that Trm cells expressed more Th17 cytokines than did transient T cells, demonstrating the CCR5+ CD4 Trm subset to be more diverse in effector function that previously appreciated.

To relate their findings back to the clinic, the authors worked with Drs. Rhonda Brand and Ian McGowan to answer if CCR5+ CD4 Trm are stably maintained in the presence of Maraviroc, a CCR5 antagonist often prescribed to HIV patients. Their collaborators provided biopsies collected before and after either topical (rectal) or oral Maraviroc treatment. Woodward-Davis and colleagues performed immunofluorescence staining and analyzed previously collected flow cytometry data and concluded that the CCR5+ CD4 Trm population remained after both local and systemic Maraviroc treatment. This demonstrated that the existing CCR5+ CD4 Trm population in the mucosa was not dependent on ongoing CCR5-mediated T cell recruitment, partially explaining why HIV+ patients prescribed Maraviroc do not lose barrier immunity.

Mucosal barrier immunity is maintained by tissue-resident T cells in the presence of CCR5 antagonist Maraviroc.
Mucosal barrier immunity is maintained by tissue-resident T cells in the presence of CCR5 antagonist Maraviroc. Image made with Biorender and provided by Amanda Woodward-Davis.

“This study offers key insights into how barrier immunity is maintained in the presence of CCR5 antagonists, which are used to treat patients with HIV and are currently being investigated for other uses such as to fight autoimmunity and graft-versus-host disease,” Woodward-Davis explained. “However, much of what we understand about the human immune system has been gained from studies with blood. Our research emphasizes the importance of considering these drug therapies in the context of barrier tissues, which hosts a large number of unique CCR5+ CD4 T cells and is where strategic immune defenses are maintained to protect from infection, survey for cancer and support a healthy relationship with our microbiome,” she elaborated. Going forward, Woodward-Davis speculated on how their data could inform gene therapy as an HIV cure strategy and said that “our data suggest that careful consideration should be given to how this may impact important T cell subsets sequestered at sites of HIV transmission.”

This work was supported by the National Institutes of Health, the Doug and Maggie Walker Fellowship.

UW/Fred Hutch Cancer Consortium member Martin Prlic contributed to this work. 

Woodward-Davis AS, Roozen HN, Dufort MJ, DeBerg H, Delaney MA, Mair F, Erickson JR, Slichter CK, Berkson JD, Klock AM, Mack M, Lwo Y, Ko A, Brand RM, McGowan I, Linsley PS, Dixon DR, Prlic M.  2019. The human tissue-resident CCR5+ T cell compartment maintains protective and functional properties during inflammation. Science Translational Medicine. Epub. ahead of print.