CD4+ T cells persist for years in the human small intestine and display a TH1 cytokine profile

Bartolomé-Casado, Raquel; Landsverk, Ole J. B.; Chauhan, Sudhir Kumar; Sætre, Frank; Hagen, Kjersti Thorvaldsen; Yaqub, Sheraz; Øyen, Ole; Horneland, Rune; Aandahl, Einar Martin; Aabakken, Lars; Bækkevold, Espen S.; Jahnsen, Frode L.

doi:10.1038/s41385-020-0315-5

Article
Published: 22 June 2020

CD4⁺ T cells persist for years in the human small intestine and display a T_H1 cytokine profile

Raquel Bartolomé-Casado ORCID: orcid.org/0000-0002-8966-9047¹,
Ole J. B. Landsverk¹,
Sudhir Kumar Chauhan^1,2,
Frank Sætre¹,
Kjersti Thorvaldsen Hagen¹,
Sheraz Yaqub³,
Ole Øyen⁴,
Rune Horneland⁴,
Einar Martin Aandahl^2,4,
Lars Aabakken⁵,
Espen S. Bækkevold¹ &
…
Frode L. Jahnsen¹

Mucosal Immunology volume 14, pages 402–410 (2021)Cite this article

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Abstract

Studies in mice and humans have shown that CD8⁺ T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4⁺ T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4⁺ T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4⁺ T cells were still donor-derived one year after transplantation. In contrast to memory CD4⁺ T cells in peripheral blood, intestinal CD4⁺ T_RM cells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4⁺ T_RM cells were very potent cytokine producers; the vast majority being polyfunctional T_H1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4⁺ T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4⁺ T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.

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Introduction

Studies in mouse models of infection have shown that CD8⁺ T cells remain in peripheral tissues long after pathogen clearance.¹ These long-lived CD8⁺ T cells have limited potential to recirculate and have been termed resident memory T (T_RM) cells.^2,3,4 Moreover, CD8⁺ T_RM cells show an extraordinary ability to mount rapid and potent in situ responses after infectious re-exposure.^5,6,7 The currently most established markers to identify CD8⁺ T_RM cells in barrier tissues are CD69 and CD103.^8,9,10 CD69 is rapidly upregulated after arrival into the tissue,¹¹ and plays a key role preventing tissue egress by antagonizing sphingosine 1-phosphate receptor (S1PR1).¹² CD103 (also known as α_E integrin) is highly expressed on intraepithelial lymphocytes (IELs) and the heterodimer α_Eβ₇ binds E-cadherin on the surface of epithelial cells,^13,14 promoting the accumulation of IELs in the epithelium.

Although CD4⁺ T cells are more abundant than CD8⁺ T cells in most peripheral tissues,¹⁵ studies to understand T_RM cell biology have mainly focused on CD8⁺ T cells. Over the last decade, CD4⁺ T_RM cells have been identified in the lung,^16,17 skin^18,19 and reproductive tract.²⁰ However, the CD4⁺ T_RM population seems to be more heterogeneous and functionally plastic compared to CD8⁺ T_RM cells,^21,22 and whether CD4⁺ T cells in peripheral tissues are truly resident, non-circulatory cells is still a matter of debate.²³

CD103 as well as CD69 are induced by TGF-β, which is constitutively produced by gut epithelial cells.²⁴ At human mucosal sites, most CD4⁺ T cells express CD69, but few express CD103 compared to CD8⁺ T_RM cells,¹⁵ and both CD103^- and CD103⁺ CD4⁺ T subsets have been described in different tissues, such as lung^9,25 and skin.¹⁸ Intestinal CD4⁺ T_RM cells have shown to play a critical role in protection against different pathogens, including C. rodentium²⁶ and Listeria²⁷ in mouse models. Although our knowledge about the role of intestinal CD4⁺ T-cell effector subsets in the pathogenesis of inflammatory bowel disease (IBD)^28,29,30 and coeliac disease^31,32 have substantially progressed over the last decade, our current understanding of CD4⁺ T-cell immunosurveillance and long-term persistence in the human intestine remains incomplete.

We have recently reported that the majority of CD8⁺ T cells persists for years in human small intestine,⁸ however, it is still unknown whether CD4⁺ T cells share these features with their CD8⁺ counterparts. Here, we present a comprehensive study of the longevity and phenotype of intestinal CD4⁺ T cells in humans. In a unique transplantation setting we followed the persistence of donor-derived CD4⁺ T cells in grafted duodenum over time and found that the majority of donor CD4⁺ T cells are maintained for at least one year in the graft. Furthermore, both CD103^- and CD103⁺ CD4⁺ T cell populations presented very similar turnover rates, suggesting that both constitute T_RM populations. Finally, we showed that the vast majority of both CD103^- and CD103⁺ CD4⁺ T_RM cells were polyfunctional T_H1 cells and a fraction produced cytotoxic granules after activation.

Results

Human intestinal CD4⁺ T cells are phenotypically distinct from their circulating counterparts

To identify CD4⁺ T cells with a T_RM-phenotype in the human small intestine (SI) we first studied the CD4⁺ T-cell compartment under steady state conditions. For this purpose we collected SI specimens from proximal duodenum-jejunum resections of patients undergoing pancreatic cancer surgery (Whipple procedure, n = 35; mean age 63 yr; 16 female), and from donors and recipients during pancreatic-duodenal Tx (baseline samples, donors: n = 52; mean age 31 yr; 24 female; patients: n = 36; mean age 41 yr; 14 female). All tissue samples were evaluated by experienced pathologists and only histologically normal SI was included. Single-cell suspensions from epithelium (intraepithelial, IE) and enzyme-digested lamina propria (LP) were obtained and analyzed by flow cytometry together with peripheral blood mononuclear cells (PBMCs) from the patients. To characterize the phenotypic profile of SI CD4⁺ T cells, we performed flow-cytometry analysis using a panel of antibodies that we recently implemented to study SI CD8⁺ T_RM cells.⁸ CD4⁺ T cells comprised almost 60% of LP T cells (with a CD4⁺: CD8⁺ ratio similar to PB), but constituted only 10% of T cells in the epithelium (Supplementary Fig. S1A-B). The relative distribution of T cell subsets was conserved in mucosal biopsies sampled up to 35 cm apart from the same intestinal resection (Supplementary Fig. S1C).

Applying a dimensionality reduction technique (UMAP, Uniform Manifold Approximation and Projection) on the compiled flow cytometry data we found that all SI CD4⁺ T cells clustered separate from PB CD4⁺ T cells (Fig. 1a, left). The vast majority of the SI CD4⁺ T cells presented a CD45RA⁻ CD45RO⁺ L-Sel⁻ CCR7⁻ effector memory (T_EM) phenotype (Fig. 1a–c). In contrast, PB CD4⁺ T cells contained a substantial fraction of naïve (T_N, CD45RO⁻ CD45RA⁺ CCR7⁺ L-Sel⁺) and central memory (T_CM, CD45RO⁺ CD45RA⁻ CCR7⁺ L-Sel⁺) CD4⁺ T cells (Fig. 1a–c). Virtually all SI CD4⁺ T cells expressed the T_RM marker CD69 whereas all PB CD4⁺ T cells were CD69-negative (Fig. 1a, c). Intestinal CD4⁺ T cells (LP and IE) separated into three clusters. The lower cluster was enriched in CD103⁺KLRG1^- cells, whereas the middle and upper clusters contained mainly CD103⁻KLRG1⁻ and CD103⁻KLRG1⁺ cells, respectively (Fig. 1a). Looking at the expression of each individual marker in a larger number of intestinal samples, the population expressing CD103 comprised on average 18% of the LP and 66% of IE CD4⁺ T cells (Fig. 1c, right), whereas KLRG1 was expressed by 26 and 5% of LP and IE CD4⁺ T cells, respectively (Fig. 1d). PB CD4⁺ T cells were completely negative for CD103, however a fraction (mean 19%) of PB CD4⁺ T_EM cells expressed KLRG1 (Fig. 1a, d). PB CD4⁺ T_EM and SI CD4⁺ T cells showed similar expression of PD1, CD127 (IL-7 receptor- α) and NKG2D. In contrast, CD28 was significantly higher expressed on PB CD4⁺ T_EM cells, whereas CD161 was expressed at higher levels on SI CD4⁺ T cells (Fig. 1a, d). In addition, the immunomodulatory receptor CD244 (2B4) was expressed higher on the IE subset. In line with other reports,^28,33 we also found that CD49a, CXCR6, and the negative regulator CD101 were highly expressed by the SI CD4⁺ T cells (Supplementary Fig. S1d). Given that one of the SI CD4⁺ T-cell clusters was enriched in cells expressing the T_RM marker CD103 (Fig. 1a), we examined the differential phenotypic profile of CD103⁺ and CD103⁻ CD4⁺ T cells in LP and in the epithelium. CD103⁻ CD4⁺ T cells presented a higher fraction of KLRG1 positive cells in both compartments, while IE CD103⁺ CD4⁺ T cells exhibited significantly higher expression of 2B4. For the rest of the markers analyzed, only small differences were found between the CD103⁺ and CD103^- subsets (representative histograms in Fig. 1e and compiled data in Supplementary Fig. S1E).

**Fig. 1: Human intestinal CD4⁺ T cells are phenotypically distinct from their circulating counterparts.**

Taken together, these results show that SI CD4⁺ T cells were clearly different from their blood counterparts, showing a T_RM-like surface phenotype (CD69⁺ CD103⁺/⁻ CD49a⁺ CXCR6⁺ CD101⁺ CD161⁺ CD28^low).

CD4⁺ T_RM cells persist for >1 yr in the transplanted SI

To directly examine the longevity of CD4⁺ T_RM cells in human SI, we assessed the long-term persistence of donor CD4⁺ T cells in endoscopic biopsies obtained from grafted duodenum at 3, 6 and 52 weeks after pancreatic-duodenal transplantation (Tx) of type I diabetic patients.³⁴ Only patients without histological or clinical signs of rejection were included (n = 32). Most donors and recipients expressed different human leukocyte antigen (HLA) type I molecules rendering it possible to distinguish donor cells from incoming recipient cells in the graft by flow cytometry (Fig. 2a, b). Since CD103 is frequently used as a marker to infer tissue residency, the replacement kinetics of CD103^- and CD103⁺ CD4⁺ T cells was analyzed independently. At 3 and 6 weeks, LP and IE CD4⁺ T cells exhibited very low replacement (median >85% donor cells), with no significant differences between the CD103⁻ and CD103⁺ CD4⁺ T subsets (Fig. 2b). Importantly, also 1-yr after Tx the majority of SI CD4⁺ T cells in both the LP and IE compartments were donor-derived, the fraction being slightly higher for LP CD103⁺ compared to CD103⁻ CD4⁺ T cells (medians 77 and 60%, respectively). The fact that the majority of CD103⁻ CD4⁺ T cells were still of donor origin at 1 yr post-Tx suggests that CD103 expression is not absolutely necessary for the persistence of CD4⁺ T_RM cells in human SI. In line with this, the turnover of both IE and LP CD103⁺ and CD103^- cells was highly correlated at 1 yr post-Tx (Fig. 2c). Moreover, at 1 yr post-Tx the CD103^- and CD103⁺ CD4⁺ T-cell subsets contained a similar (or higher) proportion of donor cells compared to donor CD8⁺ T cell subsets (Fig. 2d).⁸ To confirm the persistence of donor CD4⁺ T cells we performed immunostaining with anti-CD3 and anti-CD4 antibodies combined with fluorescent in situ hybridization probes specific for X/Y-chromosomes on tissue sections where recipients and donors were of different gender and consistently observed donor-derived CD4⁺ T cells in the graft 1-yr after Tx (Fig. 2e).

These results showed that the majority of donor-derived SI CD4⁺ T cells persisted at least 1 yr (possibly years) in the tissue. However, to exclude effects of the surgical trauma, immunosuppressive treatment and leukocyte chimerism on the SI CD4⁺ T-cell population, we examined the absolute T-cell counts in SI over time. Serial tissue sections were stained for CD3 and CD8, scanned and counted. The density of CD4⁺ T cells was determined by subtracting the number of CD8⁺ cells from the total CD3⁺ cell count. We found that the overall density of both CD4⁺ and CD8⁺ T cells in Tx duodenum was stable throughout the 1-yr follow-up period (Supplementary Fig. S2A, B). Intracellular staining of single cell suspensions from Tx biopsies with the proliferation marker Ki67 showed few Ki67-positive cells among the donor CD4⁺ T cells (Supplementary Fig. S2C, D). The percentage of Ki67⁺ CD4⁺ T cells was similar to that seen in the native duodenum in Tx patients and in steady state controls (Supplementary Fig. S2D), indicating that proliferation did not contribute substantially to the large number of persisting donor CD4⁺ T cells in transplanted SI. Finally, we confirmed that the CD4⁺ T cells in the native (recipient) duodenum were exclusively recipient-derived (Supplementary Fig. S3), demonstrating that migration of donor cells out of the graft was not occurring.

In conclusion, these results show that the CD4⁺ T_RM cell population includes both CD103⁻ and CD103⁺ cells, and that CD4⁺ T_RM cells are at least as persistent as CD103⁺ CD8⁺ T_RM cells⁸ in the transplanted SI.

Incoming recipient CD4⁺ T cells undergo gradual phenotypic changes over time in transplanted duodenum

Transplanted SI gives us a unique opportunity to study the differentiation of recruited incoming CD4⁺ T cells and whether they acquire a T_RM phenotype in SI mucosa. To this end, we compared the expression of T_RM associated markers on donor- and recipient- derived LP CD4⁺ T cells from biopsies of transplanted duodenum over time. Already at 3 wk post-Tx, virtually all recipient LP CD4⁺ T cells expressed CD69 (Fig. 3a). More than half of recipient CD4⁺ T cells expressed CD161 at 6 weeks and that was further increased at 1-yr post Tx to similar levels as donor CD4⁺ T cells (Fig. 3b). CD103 was expressed on a minor subset of recipient-derived CD4⁺ T cells at both 6 and 52 weeks; slightly lower than that on donor CD4⁺ T cells (Fig. 3c, e). In contrast, the fraction of KLRG1-positive cells within donor and recipient-derived CD4⁺ T cells remained almost unchanged (Fig. 3d, e). Similarly to the steady state conditions (Fig. 1a), the majority of the LP CD4⁺ T cells were CD103⁻ KLRG1⁻ at all the time points regardless of their origin (Fig. 3e). Furthermore, the turnover of donor LP CD103⁻ KLRG1⁻ and CD103⁻ KLRG1⁺ CD4⁺ T cells was very similar, evidenced by the high correlation of donor-derived cells within both subsets over time (Fig. 3f).

Together, we find that recipient CD4⁺ T cells recruited to the transplanted duodenum rapidly acquire phenotypic features similar to persistent donor CD4⁺ T cells, suggesting that they gradually differentiate into T_RM in situ.

The majority of SI CD4⁺ T cells exhibits a polyfunctional T_H1 profile

To examine the functional properties of SI CD4⁺ T cells we studied their cytokine expression profile and ability to produce cytotoxic granules. First, LP CD4⁺ T cells isolated from histologically normal SI were short-term stimulated with PMA and ionomycin and intracellular staining was performed with antibodies targeting specific cytokines (Supplementary Table S1). By flow-cytometric analysis we found that the majority of the LP CD4⁺ T cells, both CD103^- and CD103⁺, produced IFN-γ, IL-2 and TNF-α (Fig. 4a). Almost half of the cells produced all these three cytokines simultaneously (Fig. 4b, c), and we did not find significant differences between CD103⁻ KLRG1⁺ and KLRG1⁻ cells (Supplementary Fig. S4A). In contrast, triple-producing cells constituted only 4% of the memory CD4⁺ T cells in PB (Fig. 4b, c). Comparing the LP CD103⁻ and CD103⁺ subsets, we found significantly higher fraction of IL-17 and MIP1-β-producing cells within the CD103⁺ subset compared to CD103⁻ CD4⁺ T cell subset (Fig. 4a). Furthermore, CD103⁺ CD4⁺ T cells contained a higher fraction of IFN-γ⁺ IL-17⁺ double producing cells (Fig. 4d). In contrast, CD103⁻ CD4⁺ T cells presented higher numbers of IL-13-producing cells than their CD103⁺ counterparts, whereas comparable expression of IL-10 and IL-22 was found in the two subsets (Fig. 4a).

Murine CD4⁺ T_RM cells exhibit upregulation of granzyme-B upon reactivation with their cognate antigen.³⁵ We therefore analyzed the capacity of SI CD4⁺ T cells to produce granzyme-B and perforin at the steady state or after stimulation with anti-CD3/CD28 beads. In the absence of stimulation, very few cells expressed these cytolytic proteins (Fig. 5a), however, both LP CD103⁻ and CD103⁺ subsets increased their expression of granzyme-B and perforin after activation (Fig. 5b, c). We found a significantly higher proportion of granzyme-B producing cells within the LP CD103⁺ subset as compared to the CD103⁻ CD4⁺ T cell subset (Fig. 5c). On the other hand, no significant differences were found in the activation-induced production of perforin between either subset (Fig. 5c). Comparing the KLRG1⁺ and KLRG1⁻ cells in the LP CD103⁻ compartment, we found higher basal levels of granzyme-B among the KLRG1⁺ cells (Supplementary Fig. S4B), but similar levels of granzyme-B and perforin after stimulation (Supplementary Fig. S4B, C).

**Fig. 5: LP CD4⁺ T cells produce cytotoxic granules after stimulation.**

These data show that the majority of the SI CD4⁺ T_RM cells are polyfunctional T_H1 cells, with a large fraction co-producing IFN-γ, IL-2 and TNF-α. A subset of CD4⁺ T_RM cells also produces the cytotoxic proteins granzyme-B and perforin after stimulation.

Discussion

Over the last years it has been demonstrated that immunosurveillance by memory CD8⁺ T cell in barrier tissues is largely mediated by durable, resident cell populations. However, whether memory CD4⁺ T cells use similar surveillance strategies is less clear.^9,18,23,36 Here, we show that the majority of CD4⁺ T cells are persistent for at least 1 yr in the human SI mucosa, where they exhibit a polyfunctional T_H1 cytokine profile.

There is conflicting evidence with regards to the long-term residency of memory CD4⁺ T cells in barrier tissues. Studies of CD4⁺ T_RM cells using parabiotic mice have suggested that CD4⁺ T-cell surveillance in the skin was dependent on continuous recirculation rather than permanent residency.^37,38 However, evidence of CD4⁺ T_RM cells persistence has been reported in other peripheral tissues, such as the reproductive mucosa and lung.^16,20 Similarly, Beura et al. recently demonstrated that residency is the dominant mechanism of memory CD4⁺ T-cell immunosurveillance in non-lymphoid tissues, but they did not evaluate the longevity.³⁵ Moreover, in a recent study Klicznik and colleagues discovered a population of skin CD103⁺ CD69⁺ CD4⁺ T cells that were able to downregulate CD69 expression and enter the circulation, indicating that some CD4⁺ T_RM cells may retain migratory potential.³⁹

In mouse models of infection, the number of antigen-specific memory CD4⁺ T cells in lymphoid and non-lymphoid tissues seem to decline faster than CD8⁺ T cells,^36,40 suggesting that memory CD4⁺ T cells are less durable. In line with these results, donor CD4⁺ T cells in lung transplanted patients were more rapidly lost than CD8⁺ T cells.⁹ Here, we found that donor CD4⁺ T cells were maintained in duodenal grafts at equal or even higher numbers than CD103⁺ CD8⁺ T cells. At 6 weeks post-transplantation more than 80% of CD4⁺ T cells were of donor origin and at 1 yr the average of donor CD4⁺ T cells was still over 60%, but with high variation between grafts. This variation was not likely caused by an increased influx of recipient CD4⁺ T cells outnumbering persisting donor cells in the graft, as the overall density of CD4⁺ T cells was unchanged at 1 yr post-transplantation (Figure S2).

Several reports on organ transplantation, including intestinal transplantation,^9,41,42 have shown that rejection episodes dramatically increase the replacement kinetics of immune cells. Although we only included patients without histological and/or clinical signs of cellular and antibody-mediated rejection, the patients were not routinely examined by a clinician between 6 and 52 weeks. Therefore, a likely explanation for the large variation in persisting donor CD4⁺ T cells after 1 yr is that some patients have had undiagnosed intermittent rejection episodes (or other clinical problems) between 6 and 52 weeks after transplantation. Consequently, the grafts with low replacement might more closely represent the steady-state situation in healthy, non-transplanted gut. Together, these results indicate that most CD4⁺ T cells in human SI under normal conditions are non-circulating, resident cells that most likely perpetuate for years.

Similar to intestinal CD8⁺ T_RM cells,⁸ we found that virtually all the SI CD4⁺ T cells expressed CD69 and CD161. However, unlike CD8⁺ T cells, only a minor fraction of LP CD4⁺ T cells expressed the α_E integrin, CD103. While CD103⁻ CD8⁺ T cells very rapidly turned over in transplanted duodenum (Fig. 2d and ref. ⁸), both CD103⁻ and CD103⁺ donor CD4⁺ T cells were maintained at high numbers one year after transplantation, with CD103⁺ cells displaying slightly higher persistence. These results suggest that both CD103⁻ and CD103⁺ CD4⁺ T cells constitute resident populations and that retention mechanisms independent of CD103 exist, in line with previous reports of intestinal CD4⁺ T cells in mice models.²⁷

Like murine CD4⁺ T_RM cells,^20,27 the vast majority of LP CD4⁺ T_RM cells exhibited a polyfunctional T_H1 profile, producing high amounts of IFN-γ, IL-2 and TNF-α. The fraction of polyfunctional T_H1 cells among SI CD4⁺ T cells was much higher than among memory CD4⁺ T cells in blood. Furthermore, >40% of the CD4⁺ T_RM cells expressed granzyme-B after stimulation. These results show that SI CD4⁺ T_RM cells, like CD8⁺ T_RM cells, undergo tissue-specific changes that make them poised to provide robust T_H1 immunity in response to reinfections.³⁵ In addition to protection against pathogens,²⁷ long-lived CD4⁺ T cell responses to commensal bacteria have been found during acute gastrointestinal infection with T. gondii.⁴³ Moreover, microbiota-specific CD4⁺ T cells have been identified in blood and intestinal biopsies from healthy humans,⁴⁴ indicating that CD4⁺ T_RM cells may actively contribute to intestinal homeostasis through interactions with the microbiota.

We found that a fraction of CD4⁺ T_RM cells produced IL-17. T_H17 cells play an important role in intestinal inflammatory disorders,^28,29,45 however IL-17 is also critical for maintaining mucosal barrier integrity.^45,46 Recently it was reported that, in contrast to inflammatory T_H17 cells elicited by pathogens, gut commensal bacteria elicited tissue-resident homeostatic T_H17 cells, which showed limited capacity to produce inflammatory cytokines.⁴⁷ In our study only a very small percentage of T_H17 cells co-produced the inflammatory cytokine IFN-γ, suggesting that the majority of SI T_H17 cells during homeostasis are non-inflammatory cells that support barrier integrity. However, further studies are needed to understand the role of SI T_H17 cells under homeostatic and inflammatory conditions.

Finally, we found, although marginally, that CD103⁺ T_RM cells contained higher fractions of IL-17 single- and IL-17/IFN-γ double-producing cells than their CD103⁻ counterparts. Moreover, CD103^- and CD103⁺ CD4⁺ T cells also showed subtle phenotypic differences regarding their expression of KLRG1, CD28 and 2B4. However, to what extent the CD103⁺ and CD103^- subsets represent distinct functional compartments needs further investigation.

In conclusion, here we provide evidence that the majority of memory CD4⁺ T cells in the human SI are resident and may persist in the tissue for >1 year. This indicates that tissue residency represents a major mechanism for CD4⁺ memory T cell immunosurveillance in the human SI and has several important clinical implications. Effective vaccines depend on durable adaptive immune responses. We have previously shown that plasma cells⁴⁸ and CD8⁺ T cells⁸ are long-lived in the SI. The finding that the intestinal immune system fosters also persisting CD4⁺ memory T cells is important knowledge in the search for strategies to develop long-lasting oral vaccines. Persisting CD4⁺ T cells may also play an important role in the chronicity of CD4⁺ T-cell mediated inflammatory diseases, such as celiac disease³¹ and inflammatory bowel disease.²⁸ Furthermore, we have recently shown that intestinal CD4⁺ T cells survive conditioning regimens in allogeneic stem cell transplantation and are present in intestinal lesions of graft versus host disease (GVHD) (Divito et al. J Clin Invest, in press), suggesting that persistent host CD4⁺ T cells may play a role in GVHD pathology. Longevity of intestinal CD4⁺ T cells must therefore be taken into consideration in therapeutic interventions targeting pathogenic CD4⁺ T cells to treat immune-mediated disorders.

Methods

Patient samples

Small intestinal samples were either obtained during pancreatic cancer surgery (Whipple procedure, n = 35; mean age 63 yr; range 40–81 yr; 16 female), or from donors and/or patients during pancreas-duodenum transplantation (donors: n = 52; mean age 31 yr; range 5–55 yr; 24 female; patients: n = 36; mean age 41 yr; range 25–60 yr; 14 female) as described previously.⁸ Cancer patients receiving neoadjuvant chemotherapy were excluded from the study. Endoscopic biopsies from donor and patient duodenum were collected at 3, 6 and 52 weeks after transplantation. All tissue specimens were evaluated blindly by experienced pathologists, and only material with normal histology was included.⁴⁹ All transplanted patients received a standard immunosuppressive regimen,³⁴ and patients showing clinical or histological signs of rejection or other complications, as well as patients presenting pre-transplant or de novo donor specific antibodies (DSA) were excluded from the study. Blood samples were collected at the time of the surgery and buffy coats obtained from healthy donors (Oslo University Hospital). All participants gave their written informed consent. The study was approved by the Regional Committee for Medical Research Ethics in Southeast Norway and complies with the Declaration of Helsinki.

Preparation of intestinal and peripheral blood single-cell suspensions

Intestinal resections were opened longitudinally and rinsed with PBS, and mucosa was dissected in strips off the submucosa. For microscopy, small mucosal pieces were fixed in 4% formalin and embedded in paraffin according to standard protocols. Intestinal mucosa was washed 3 times in PBS containing 2 mM EDTA and 1% FCS at 37 °C with shaking for 20 min and filtered through nylon 100-µm mesh to remove epithelial cells. Epithelial fractions in each washing step were pooled and filtered through 100-µm cell strainers (BD, Falcon). Epithelial cells in the EDTA fraction were depleted by incubation with anti-human epithelial antigen antibody (clone Ber-EP4, Dako) followed by anti-mouse IgG dynabeads (ThermoFisher) according to the manufacture’s protocol. De-epithelialized LP was minced and digested in complete Roswell Park Memorial Institute (RPMI) medium (supplemented with 1% Pen/Strep) containing 0.25 mg/mL Liberase TL and 20 U/mL DNase I (both from Roche), stirring at 37 °C for 1 h. Digested tissue was filtered twice through 100-µm cell strainers and washed tree times in PBS. Purity of both IE and LP fractions was checked by flow-cytometry.⁸ Intestinal biopsies from transplanted patients were processed in the same way. PBMCs were isolated by Ficoll-based density gradient centrifugation (Lymphoprep™, Axis-Shield).

Flow cytometry

Single cell suspensions of intestinal LP and IE fractions and PBMCs were stained using different multicolor combinations of directly conjugated monoclonal antibodies (Supplementary Table S1). To assess the expression of L-Selectin on digested tissue, cells were rested for 12 h at 37 °C before the immunostaining. Replacement of donor cells in duodenal biopsies of HLA mismatched transplanted patients was assessed using different HLA type I allotype-specific antibodies targeting donor- and/or recipient-derived cells, and stroma cells were used as a control of specific staining. Dead cells were excluded based on propidium iodide staining (Molecular Probes, Life Technologies). For analysis of cytokine production, LP and IE cell suspensions were stimulated for 4 h with control complete medium (RPMI supplemented with 10% FCS, 1% Pen/Strep) or phorbol-12-myristate-13-acetate PMA (1.5 ng/mL) and ionomycin (1 µg/mL; both from Sigma-Aldrich) in the presence of monensin (Golgi Stop, BD Biosciences) added after 1 h of stimulation to allow intracellular accumulation of cytokines. Cells were stained using the BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions and stained with antibodies against several cytokines (Supplementary Table S1). For detection of cytotoxic granules, LP and IE cells were activated for 21 h with anti-CD3/CD28 beads (Dynabeads, ThermoFisher) or control complete medium. For detection of intranuclear Ki67 expression the FoxP3/transcription factor staining buffer set was used according to the manufacturer’s instructions. eFluor-450 or eFluor-780 fixable viability dyes (eBioscience) were used prior any intracellular/intranuclear staining procedure. All samples were acquired on LSR Fortessa flow cytometer (BD Biosciences), using FACSDiva software (BD Biosciences). Single stained controls were prepared for compensation (UltraComp eBeads™, eBioscience), and gates were adjusted by comparison with FMO controls or matched isotype controls. Flow cytometry data were analyzed using FlowJo 10.4.2 (Tree Star). For Fig. 1a, the expression of 16 phenotypic markers was analyzed at the single cell-level and compared for CD4⁺ T cells in PB, LP and IE (n = 3) using the merge and calculation functions of Infinicyt software (Cytognos). The population within the CD4⁺ T-cell gate was down-sampled for each compartment and exported to a new file as in,⁸ and then concatenated and subjected to UAMP analysis using the plugin integrated in FlowJo 10.5.3. All experiments were performed at the Flow Cytometry Core Facility, Oslo University Hospital.

Microscopy

Analysis of chimerism was performed as described previously.⁴⁸ Briefly, formalin-fixed 4-μm sections were washed sequentially in xylene, ethanol, and PBS. Heat-induced epitope retrieval was performed by boiling sections for 20 min in Dako buffer. Sections were incubated with CEP X SpectrumOrange/Y SpectrumGreen DNA Probes (Abbott Molecular Inc.) for 12 h at 37 °C before immunostaining according to standard protocol with anti-CD3 (Polyclonal; Dako), anti-CD4 (clone 1F6, Leica Biosystems) and secondary antibodies targeting rabbit IgG or mouse IgG2b conjugated to Alexa Fluor 647 and 555, respectively. Laser scanning confocal microscopy was performed on an Olympus FV1000 (BX61WI) system. Image z stacks were acquired at 1-μm intervals and combined using the Z project max intensity function in Image J (National Institutes of Health), and all microscopy images were assembled in Photoshop and Illustrator CC (Adobe).

CD8 and CD3 immunoenzymatic staining was performed on formalin-fixed 4-μm sections, dewaxed in xylene and rehydrated in ethanol, and prepared with Vulcan Fast red kit (Biocare Medical) following standard protocols. In brief, heat-induced antigen retrieval was performed in Tris/EDTA pH9 buffer (EnVision FLEX Dako kit, K8010), followed by staining with primary antibody (CD8 clone 4B11, Novocastra or CD3, polyclonal, Dako), secondary anti- mouse AP-conjugated antibody and incubation with substrate (Fast red chromogen, Biocare Medical). Slides were counterstained with hematoxylin and excess of dye was removed by bluing in ammoniac water. Tissue sections were scanned using Pannoramic Midi slide scanner (3DHISTECH) and counts generated with QuPath software.⁵⁰

Statistical analysis

Statistical analyses were performed in Prism 8 (GraphPad Software). To assess statistical significance among SI CD4⁺ T cell subsets, data were analyzed by one-way ANOVA (standard or repeated measures, RM-ANOVA) followed by Tukey’s multiple comparison tests. Replacement data and distribution of CD4⁺ T cell subsets at different time points were analyzed by two-way ANOVA matching across subsets followed by Tukey’s multiple comparison tests. Correlations between replacement kinetics of different CD4⁺ T cell subsets were calculated using Pearson correlation with two-tailed P-value (95% confidence interval). P-values of <0.05 were considered significant.

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Acknowledgements

We are grateful to the staff at the Endoscopy Unit and the surgical staff; Christian Naper, Institute of Immunology, for providing HLA typing; the Confocal Microscopy and Flow Cytometry Core Facilities; all at Oslo University Hospital, Rikshospitalet. This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme (project number 179573/V40) and by grant from the South Eastern Norway Regional Health Authority (project number 2015002).

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Department of Pathology, Oslo University Hospital and University of Oslo, Oslo, Norway
Raquel Bartolomé-Casado, Ole J. B. Landsverk, Sudhir Kumar Chauhan, Frank Sætre, Kjersti Thorvaldsen Hagen, Espen S. Bækkevold & Frode L. Jahnsen
Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
Sudhir Kumar Chauhan & Einar Martin Aandahl
Department of Gastrointestinal Surgery, Oslo University Hospital, Rikshospitalet, Oslo, Norway
Sheraz Yaqub
Department of Transplantation Medicine, Section for Transplant Surgery, Oslo University Hospital, Rikshospitalet, Oslo, Norway
Ole Øyen, Rune Horneland & Einar Martin Aandahl
Department of Gastroenterology, Oslo University Hospital - Rikshospitalet, Oslo, Norway
Lars Aabakken

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Raquel Bartolomé-Casado
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Ole J. B. Landsverk
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Contributions

R.B.-C., O.J.B.L., E.S.B., and F.L.J. conceived the project. R.B.-C., O.J.B.L., and S.K.C. processed samples, designed and performed experiments, and analyzed data. R.B.-C. prepared figures. F.S. and K.T.H. assisted with experiments and data analysis. S.Y. and R.H. coordinated recruitment of patients and collection of biopsies. S.Y., R.H., O.Ø., and E.M.A. performed surgery and provided samples. L.A. performed endoscopy and provided endoscopic biopsies. R.B.-C. and F.L.J. wrote the manuscript. O.J.B.L., F.S., and E.S.B. contributed to writing the manuscript. E.S.B. and F.L.J. supervised the study.

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Correspondence to Raquel Bartolomé-Casado or Frode L. Jahnsen.

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Bartolomé-Casado, R., Landsverk, O.J.B., Chauhan, S.K. et al. CD4⁺ T cells persist for years in the human small intestine and display a T_H1 cytokine profile. Mucosal Immunol 14, 402–410 (2021). https://doi.org/10.1038/s41385-020-0315-5

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Received: 17 December 2019
Revised: 30 April 2020
Accepted: 03 June 2020
Published: 22 June 2020
Issue Date: March 2021
DOI: https://doi.org/10.1038/s41385-020-0315-5

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