Recirculation of innate lymphocyte subsets in the skin

The trafficking of innate-like lymphocytes, such as γδ T cells and B-1 B cells, has garnered comparatively little attention from the immunological community relative to conventional T and B cells. However, recent studies have shown that innate-like cell subsets are critical for immune regulation and host defense. In this study, we use a classic ovine lymph cannulation model to describe the phenotype and function of γδ T cells migrating through the skin. We find that γδ T cells traveling in the skin-draining afferent lymph are IFN-γ- and/or IL-17-producing effector cells that express high levels of the skin- and inflammation-seeking molecule E-selectin ligand. Notably, they also lack expression of CCR7, indicating that they use alternative receptors for egress. Next, we analyze B cell subset composition, repertoire, and trafficking in the skin of sheep in the lymphatic cannulation model. We find a heterogeneous population of B cells in the skin and skin-draining lymph increases in inflammation that contains a subset of B-1-like B cells coexpressing IgM and CD11b. Furthermore, we show that skin accumulation of B cells and antibody-secreting cells during inflammation increases local antibody titers, which may augment host defense and autoimmunity. We then extend our findings of cutaneous B-1 B cells to the mouse, analyzing both uninflamed skin and skin with chronic inflammation from complete Freund\u27s adjuvant, well as human tissue. We find that B-1 B cells, unlike conventional follicular B-2 B cells, efficiently enter into the inflamed skin and differentially express the trafficking molecule α4β1 integrin (VLA-4), which facilitates their entry. Furthermore, innate B cells are a contributing source of cutaneous IL-10 in both IL-10-reporter mice and normal human skin. These findings, initiated in the sheep model then followed up and supported by experiments in mice and human tissues, demonstrate the evolutionary similarity between mammalian species. They also validate the utilization of multiple models to allow for experimental setups not possible in all species. More importantly, the further characterization of γδ T cells and the new description of skin B and B-1 cells uncovers additional targets for regulating the cellular composition of a cutaneous immune response. In summary, the data support a model in which innate-like lymphocytes are poised to migrate into barrier sites, including the skin, where they rapidly provide requisite effector functions, such as cytokine and/or antibody production, and fulfill an emerging role in skin immunity

Recirculation of innate lymphocyte subsets in the skin

The trafficking of innate-like lymphocytes, such as γδ T cells and B-1 B cells, has garnered comparatively little attention from the immunological community relative to conventional T and B cells. However, recent studies have shown that innate-like cell subsets are critical for immune regulation and host defense. In this study, we use a classic ovine lymph cannulation model to describe the phenotype and function of γδ T cells migrating through the skin. We find that γδ T cells traveling in the skin-draining afferent lymph are IFN-γ- and/or IL-17-producing effector cells that express high levels of the skin- and inflammation-seeking molecule E-selectin ligand. Notably, they also lack expression of CCR7, indicating that they use alternative receptors for egress. Next, we analyze B cell subset composition, repertoire, and trafficking in the skin of sheep in the lymphatic cannulation model. We find a heterogeneous population of B cells in the skin and skin-draining lymph increases in inflammation that contains a subset of B-1-like B cells coexpressing IgM and CD11b. Furthermore, we show that skin accumulation of B cells and antibody-secreting cells during inflammation increases local antibody titers, which may augment host defense and autoimmunity. We then extend our findings of cutaneous B-1 B cells to the mouse, analyzing both uninflamed skin and skin with chronic inflammation from complete Freund\u27s adjuvant, well as human tissue. We find that B-1 B cells, unlike conventional follicular B-2 B cells, efficiently enter into the inflamed skin and differentially express the trafficking molecule α4β1 integrin (VLA-4), which facilitates their entry. Furthermore, innate B cells are a contributing source of cutaneous IL-10 in both IL-10-reporter mice and normal human skin. These findings, initiated in the sheep model then followed up and supported by experiments in mice and human tissues, demonstrate the evolutionary similarity between mammalian species. They also validate the utilization of multiple models to allow for experimental setups not possible in all species. More importantly, the further characterization of γδ T cells and the new description of skin B and B-1 cells uncovers additional targets for regulating the cellular composition of a cutaneous immune response. In summary, the data support a model in which innate-like lymphocytes are poised to migrate into barrier sites, including the skin, where they rapidly provide requisite effector functions, such as cytokine and/or antibody production, and fulfill an emerging role in skin immunity

CXCR4 is dispensable for T cell egress from chronically inflamed skin via the afferent lymph.

T cell recirculation through extralymphoid tissues is essential to immune surveillance, host defense and inflammation. In this process, T cells enter the tissue from the blood and subsequently leave via the afferent lymph. In the absence of inflammation, T cells require CCR7 expression to egress from the skin or lung, which is consistent with the constitutive expression of the CCR7 ligand CCL21 on lymphatic endothelium. However, during chronic inflammation alternative chemoattractants come into play, allowing Ccr7-deficient (Ccr7-/-) T cells to egress efficiently from affected skin. As T cell egress from inflamed sites is a potential control point of the inflammatory response, we aimed to determine alternative T cell exit receptors using a mouse and a sheep model. We show that CCR7+ and CCR7- T cells exiting from the chronically inflamed skin were highly responsive to the CXCR4 ligand CXCL12, which was induced in the lymphatics in the inflamed site. Based on these findings, we hypothesized that CXCR4 mediates T cell egress from inflamed skin. However, pharmacological inhibition of CXCR4 did not affect the tissue egress of wildtype or Ccr7-/- CD4 and CD8 T cells after adoptive transfer into chronically inflamed skin. Similarly, adoptively transferred Cxcr4-/- Ccr7-/- and Ccr7-/- T cells egressed from the inflamed skin equally well. Based on these data, we conclude that, while CXCR4 might play an essential role for other cell types that enter the afferent lymphatics, it is dispensable for T cell egress from the chronically inflamed skin

Cxcr4 deficiency does not reduce the tissue exit of Ccr7^−/− T cells from chronically inflamed skin.

<p>(<b>A–C</b>) Adoptive transfer exit experiments with fetal liver chimeric mice as a source of donor splenocytes. As depicted in the experimental scheme (<b>A</b>), fetal liver chimeras were generated by reconstituting lethally irradiated CD45.1⁺ wildtype recipient mice with fetal liver cells from (CD45.2⁺) <i>Cxcr4</i>^−/−<i>Ccr</i>7^−/− or (CD45.2⁺) <i>Ccr7</i>^−/− or (CD45.1⁺) wildtype donors. Splenocytes from the fetal liver chimeras were labeled with eFlour670 and CFSE, so that the combination of congenic maker and fluorescent label uniquely marked cells of each genotype. Labeled splenocytes of all three genotypes were mixed and co-injected into chronically inflamed footpads. (<b>B</b>) Analysis of migrated lymphocyte subsets recovered in the draining popliteal lymph nodes 12 h post cell transfer. Data is presented as the ratio of migrated to input cells for <i>Ccr7</i>^−/− or <i>Cxcr4</i>^−/−<i>Ccr</i>7^−/− relative to wildtype cells of each subset. Connected lines represent individually analyzed recipient mice. Horizontal, non-connecting lines indicate the mean of each group. (<b>C</b>) Flow cytometric analysis of memory (CD45RB^l°) versus naïve (CD45RB^hi) phenotype CD4 and CD8 T cells in injected and migrated populations for each genotype. Data combined from 2 experiments analyzing 5–10 recipient mice per experiment (<b>B</b>) or the cell phenotype of one out two experiment with similar results (<b>C</b>) is shown. WT, wildtype.</p

Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7^−/− T cells.

<p>(<b>A–B</b>) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 (<b>A</b>) and CCL21 (<b>B</b>) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. (<b>C–E</b>) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled <i>Ccr7</i>^−/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype (<b>C</b>) and <i>Ccr7</i>^−/− (<b>D</b>) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results (<b>C</b> and <b>D</b>) or both experiments combined (<b>E</b>) are shown. WT, wildtype.</p

Ovine T cells that exit the inflamed and uninflamed skin efficiently migrate to CXCL12.

<p>Lymphocytes traveling in afferent lymph draining uninflamed or chronically inflamed skin (>d15 post induction of inflammation with CFA) were collected from sheep, and chemotaxis was tested toward mouse CCL21 and human CXCL12 in a Transwell chemotaxis assay. Migration of CD4, CD8 and γδ T cells is expressed as the percentage of each T cell subset that migrated to the lower chamber. Data points represent the mean migration of T cells from individual animals based on triplicate wells for each concentration, and the mean ± SEM for all animals analyzed is shown. N = 5–7 animals per condition and T cell subset.</p

Afferent lymph vessels in chronically inflamed skin express CXCL12.

<p>Immunofluorescence staining of frozen sections of chronically inflamed skin (21 days after inflammation elicited by subcutaneous injection of CFA) for CXC12 and CCL21 by LYVE-1⁺ lymph vessels using DAPI counterstaining. One representative image of lymph vessels analyzed in 5 mice is shown. Scale bars, 10 µm.</p