A primary role for human central memory cells in tissue immunosurveillance

Key Points

• Human T_CM are tissue tropic, have impressive effector functions, and are found in noninflamed human tissues.

• T_CM can act alone to induce inflammation in human skin–grafted mice; results suggest a role for human T_CM in primary immunosurveillance.

Abstract

Central memory T cells (T_CM) patrol lymph nodes, providing central immunosurveillance against known pathogens, but have not been described as conducting primary tissue immunosurveillance. We analyzed the expression of tissue-homing addressins in human T_CM vs effector memory T cells (T_EM) from the same donors. In humans, the majority of human T_CM were tropic for either skin or gut, and the overall tissue tropism of T_CM was comparable to that of T_EM. T_CM were present in healthy, noninflamed human skin, lung, colon, and cervix, suggesting a role for T_CM in the primary immunosurveillance of peripheral tissues. T_CM also had potent effector functions; 80% of CD8⁺ T_CM produced TC1/TC2/TC17/TC22 cytokines. T_CM injected into human skin–grafted mice migrated into skin and induced inflammatory eruptions comparable to T_EM-injected mice. In summary, human T_CM express peripheral tissue-homing receptors at levels similar to their effector memory counterparts, are found in healthy human tissues, have impressive effector functions, and can act alone to induce skin inflammation in human engrafted mice. Our studies support a novel role for human T_CM in primary immunosurveillance of peripheral tissues and highlight the important role of this long-lived cell type in tissue-based immune responses.

Visual Abstract

Introduction

Central memory T cells (T_CM) coexpress L-selectin and CCR7 and provide central immunosurveillance by patrolling the lymph nodes draining peripheral tissue sites.^1,2 In mice and nonhuman primates, T_CM provide effective long-term protection because they persist long term in the circulation, have a high proliferative potential, and can give rise to both effector and effector memory T cells (T_EM) after antigen reencounter.^3-6 Although they can be drawn into inflamed tissues, T_CM have not been identified in animals as providing primary tissue-based immunosurveillance.⁷ We report that human T_CM express tissue-homing receptors, are found in noninflamed human tissues, and have potent effector functions, supporting a role for these cells in primary tissue immunosurveillance.

Methods

Samples

All studies were performed in accordance with the Declaration of Helsinki. Approval of the Partners Institutional Review Board committee was obtained for all studies. Deidentified blood, skin, foreskin, lung, and colon were obtained from Brigham and Women’s Hospital, and cervix was obtained from Johns Hopkins. Lung and colon were obtained from distant, uninvolved tissue obtained from patients undergoing resection of small isolated tumors. Normal cervix was obtained from patients undergoing hysterectomy for nonmalignant disorders. T cells were isolated by collagenase digestion or short-term explant culture.^8,9

Flow cytometry and CyTOF

A list of antibodies used to immunostain cells is included in the supplemental Methods. Flow cytometry samples were run on a Becton Dickinson FACSCanto instrument, and CyTOF samples were analyzed on a Fluidigm CyTOF 2 mass cytometer. Data were analyzed with FCS Express 5.0 or FACSDiva 8.0. Cells were sorted on a FACSAria cell sorter.

Human engrafted mouse model

Human engrafted mice were prepared as described previously.⁹ Briefly, neonatal foreskins were grafted onto the backs of 6- to 8-week-old nonobese diabetic/severe combined immunodeficiency/interleukin-2 (IL-2) receptor γ chain^null mice (Jackson Laboratories). One week later, 3 × 10⁶ flow-sorted T_CM, T_EM, or naive T cells from an unrelated adult human blood donor were injected IV. The gating strategy and flow cytometry profiles of infused cells are included in supplemental Figure 1. Skin grafts were harvested after 3 weeks for analysis.

Statistical analyses

Primary methods of data analysis included descriptive statistics (means, medians, and standard deviation). Differences between 2 sample groups were detected using the 2-tailed Wilcoxon-Mann-Whitney test, α = 0.05. For comparisons of multiple groups, a Kruskal-Wallis 1-way analysis of variance with a Bonferroni-Dunn’s posttest for multiple means test was used, α = 0.05.

Results

We compared the expression of tissue-homing addressins in T_CM vs T_EM from healthy human blood donors by CyTOF (Figure 1). T_EM are generated in response to tissue-based infections and should all be tropic for peripheral tissues.^10,11 Skin (cutaneous lymphocyte antigen, CLA)¹² and gut (α₄β₇ integrin)^13,14 homing receptors have been identified, but receptors that direct T cells to lung, brain, and other peripheral tissues remain uncharacterized. We therefore measured the expression of skin and gut-homing addressins in T_CM vs T_EM from healthy human donors. Subsets of T_CM and T_EM expressed either gut-homing or skin-homing addressins, with very few cells expressing both (Figure 1A). Both CD8⁺ and CD4⁺ T_CM contained skin-tropic and gut-tropic populations (Figure 1A-B,G). The expression of skin and gut-homing receptors was comparable in T_CM and T_EM, except that significantly more CD4⁺ T_EM expressed gut-homing receptors and significantly more CD8⁺ T_CM expressed skin-homing receptors (Figure 1C-D). When the total number of T cells expressing skin or gut-homing receptors were compared, T_CM and T_EM expressed tissue-homing addressins at similar levels (Figure 1E-F).

Figure 1.

Human T_CMexpress tissue-homing addressins and are found in healthy human peripheral tissues. (A-B) Comparison of the expression of skin-homing (CLA) and gut-homing (α4β7) addressins in T_CM and T_EM by CyTOF analyses from a representative human donor. The gating strategy used to identify T_CM and T_EM among human peripheral blood CD45RO⁺ CD45RA⁻ FOXP3⁻ memory T cells is shown. (C-D) Aggregate data of skin and gut addressin expression by (C) CD4⁺ and (D) CD8⁺ T_CM (white bars) and T_EM (black bars). T cells that expressed neither CLA nor α4β7 were designated as unassigned. The mean and standard error of the mean (SEM) of 4 donors are shown. (E-G) T_CM and T_EM have comparable expression of skin- and gut-homing addressins. The mean and SEM of the total assignable tissue-tropic populations (total tropism, the sum of skin and gut-tropic populations) for (E) CD4⁺ and (F) CD8⁺ T cells are shown. The mean and SEM of 6 donors are shown. (H) T_CM are present in noninflamed human peripheral tissues. T cells were isolated from noninflamed human tissues and analyzed by flow cytometry. T_CM as a percentage of the total T-cell population are shown for (H) individual representative samples and (I) pooled data. The mean and SEM of 5 skin, 2 lung, 3 colon, and 6 cervix samples are shown.

These results suggest human T_CM have intrinsic tissue tropism and may play a role in primary immunosurveillance of peripheral tissues. To evaluate this possibility, we isolated T cells from noninflamed human skin, lung, colon, and cervix. We observed populations of T_CM in each of these healthy peripheral tissues, confirming that T_CM do gain access to noninflamed human tissues (Figure 1H-I).

T_CM are known for their ability to persist in the circulation, to proliferate, and to give rise to effector T cells in animal models, but their production of cytokines that can directly combat infection has not been characterized in humans. We studied cytokine production of T_CM and T_EM and found that both CD4 and CD8 T_CM had impressive effector functions (Figure 2A-J). Interferon-γ (IFN-γ) was the most highly produced signature cytokine in T_CM, as it was in T_EM. CD8⁺ T_CM were particularly cytokine rich, with a mean 70% and 97% of CD8⁺ T_CM producing signature cytokines among the skin-tropic and gut-tropic populations, respectively. The proportion of CD8⁺ T_CM producing signature cytokines was not significantly different from T_EM from the same donors (Figure 2G). Gut-tropic CD8⁺ T_CM produced significantly more IL-2 than CD8⁺ T_EM, but levels produced by all other subsets were comparable (Figure 2C,H). Between 40% and 55% of CD4⁺ T_CM also produced signature cytokines, and IFN-γ was the most frequently produced cytokine in both CD4⁺ and CD8⁺ T_CM (Figure 2A-B,F). Tumor necrosis factor-α (TNF-α) was produced by the majority of T cells tested, and IL-10 was produced at low levels in all subsets (Figure 2D-E,I-J). These results demonstrate that all T_CM, and CD8⁺ T_CM in particular, have considerable effector functions that endow them with the capacity to contribute to frontline antipathogen responses in peripheral tissues.

Figure 2.

Human T_CMhave potent effector functions, home to skin, and induce dermatitis in human engrafted mice. (A,F) Both CD4⁺ and CD8⁺ T_CM produced T-cell signature inflammatory cytokines. Cytokine production was assayed by CyTOF following stimulation with phorbol 12-myristate 13-acetate/ionomycin. Production of T_H1/T_C1 (IFN-γ), T_H2/T_C2 (IL-4, IL-13), T_H17/T_C17 (IL-17A), T_H22/T_C22 (IL-22) signature cytokines by T_CM and T_EM is shown. Figures represent the mean of 6 donors. (B,G) Total production of signature cytokines by T_CM (white bars) compared with T_EM from the same donors (black bars) for (B) CD4⁺ and (G) CD8⁺ T cells. (C-E,H-J) Production of IL-2, TNF-α, and IL-10 by (C-E) CD4⁺ and (H-J) CD8⁺ T cells is shown. The mean and SEM of 6 donors are shown. (K-W) Human T_CM home to skin and induce an inflammatory dermatitis comparable to T_EM in human engrafted mice. (K) The human engrafted mouse experimental model. (L-P) Hematoxylin and eosin evaluation of human skin grafts 3 weeks after injection of (L-M) saline, (N) T_EM, (O) T_CM, or (P) naive T cells. T_CM homed to human skin grafts and induced T-cell–mediated inflammatory dermatitis. (P) Injection of naive T cells led to minimal inflammation. (Q-S) The inflammatory patterns induced by purified T_CM included (Q) interface dermatitis, (R) spongiotic dermatitis, and (S) epidermal necrosis. Results shown are representative of those obtained with 6 different human blood cell donors. (T) T-cell migration into the skin as assessed by NanoString CD3/CD4/CD8 gene expression analysis was comparable in T_CM- and T_EM-injected mice. (U-V) The production of inflammatory T-cell cytokines in skin (TNF-α, IFN-γ, IL-17A, and IL-22) and cytotoxic effector molecules (PRF1, perforin; GZMA, granzyme A; GZMB, granzyme B) was comparable in T_CM- and T_EM-injected mice. (W) Production of inflammatory chemokines in skin was comparable in T_CM- and T_EM-injected mice. For panels T-W, the mean and SEM of messenger RNA copies detected by NanoString analyses from 6 different human T_CM/T_EM donors are shown. Scale bars, 100 μm. GVHD, graft-versus-host disease; NS, normal skin; ns, not significant.

We used a human engrafted mouse model to measure the relative ability of T_CM and T_EM to enter skin and induce inflammation. NSG mice were grafted with neonatal human foreskin, a tissue that lacks T cells, and infused IV with allogeneic purified peripheral blood T_EM, T_CM, or naive T cells isolated from healthy adult human donors (Figure 2K; supplemental Figure 1).⁹ A robust inflammatory dermatitis was observed in both T_EM- and T_CM-injected mice (Figure 2L-S). T_CM effectively entered human skin and induced inflammatory changes, including interface dermatitis, epidermal spongiosis, and epidermal necrosis (Figure 2Q-S). One caveat is that T_CM may differentiate into effector cells within the skin graft, and we cannot rule out a contribution of newly generated effector cells to skin inflammation. In contrast, injection of naive T cells led to minimal infiltration of T cells into skin and little if any visible inflammation (Figure 2P). Very few human T cells migrated into mouse skin adjacent to the grafts, and no inflammation in mouse skin was appreciated (supplemental Figure 2). Although this is not a model of immunosurveillance per se, it does measure in vivo the ability of human T cells to enter human skin and induce inflammation.

NanoString-based gene expression profiling of the skin grafts demonstrated comparable levels of T cells in the skin of T_CM- and T_EM-injected mice (Figure 2T). Moreover, the levels of TNF-α, IFN-γ, IL-17A, IL-22, perforin, granzyme A, granzyme B, and inflammatory chemokines were similar in the skin of T_CM- and T_EM-injected mice (Figure 2U-W). These studies demonstrate and confirm the ability of T_CM to enter the skin and initiate inflammation in the absence of other T-cell subsets.

Discussion

T_CM provide effective long-term memory responses because they have the capacity to persist long term in the circulation, have a high proliferative capacity, and can replenish other memory T-cell subsets, including T_EM.^3-6,15 The role of T_CM in immunosurveillance has been assumed to be limited to patrolling the lymph nodes for evidence of pathogen exposure. The initial description of human T_CM characterized these cells as having poor effector functions and little tissue tropism.¹ However, these studies did not evaluate the expression of the gut-homing addressin α₄β₇, used a different antibody to detect CLA than the one that identifies cutaneous T cells (HECA-205 vs HECA-452¹²), and did not study the cytokine production of polyclonally stimulated T_CM. With the benefit of updated and more comprehensive approaches, it is clear that T_CM express tissue-homing addressins at levels similar to T_EM, and indeed, these cells are present in healthy human peripheral tissues. This is consistent with a prior report that CCR7 is expressed by the majority of CLA- and α₄β₇-expressing T cells in human blood.¹⁶ Human T_CM, particularly CD8⁺ T_CM, also have significant effector functions. T_CM alone were capable of entering human skin and initiating inflammation comparable to that induced by T_EM.

These findings demonstrate that T_CM express tissue-homing receptors, are found in healthy human peripheral tissues, have potent effector functions, and can migrate into and initiate tissue-based inflammation. Our findings suggest that human T_CM, much like T_EM, are imprinted with both tissue-homing addressin expression and specialized programs of cytokine production and likely participate directly in the immunosurveillance of peripheral tissues.

Tissue-tropic T_CM have not yet been described in animal models, perhaps because young mice kept in pathogen-free conditions, the animals used in most experiments, lack the large numbers of pathogen-specific recirculating T_CM that human patients have accumulated over decades of pathogen exposures. Alternatively, there may be key differences in homing of human vs mouse T_CM.

Our work demonstrates that human T_CM enter and have the capacity to provide primary immunosurveillance of peripheral tissues. Added to their known abilities to persist long term in the circulation, proliferate, and give rise to additional memory T-cell subsets, our work supports a critical role for T_CM in providing long-term protection against known pathogens.

Supplementary Material

The full-text version of this article contains a data supplement.

Authorship

Contribution: A.G., J.E.T., and T.R.M. carried out the experiments and assisted in analyzing data; V.H., C.Y., and R.W. participated in developing the human engrafted mouse model; J.T.O. helped in analyzing gene expression data; R.A.C. designed experiments, analyzed data, and drafted the manuscript and figures; C.L.T. provided human cervix; and T.S.K. provided access to human lung and colon.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Rachael A. Clark, Department of Dermatology, Brigham and Women’s Hospital, Room 501A, 221 Longwood Ave, Boston, MA 02115; e-mail: rclark1@partners.org.