Peripheral Tolerance Can Be Modified by Altering KLF2-Regulated Treg Migration

Peripheral tolerance can be modified by altering KLF2-regulated Treg migration

Sudheer K. Pabbisettya, Whitney Rabacala, Emmanuel J. Volanakisb, Vrajesh V. Parekha, Danyvid Olivares-Villagómeza, Delphine Cendrona, Kelli L. Boyda, Luc Van Kaera, and Eric Sebzdaa,1

aDepartment of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232; and bDepartment of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232

Edited by Rafi Ahmed, Emory University, Atlanta, GA, and approved June 20, 2016 (received for review April 12, 2016) Tregs are essential for maintaining peripheral tolerance, and thus diseases. Together, these results suggest that KLF2 is a therapeutic targeting these cells may aid in the treatment of autoimmunity and target for fine-tuning self-tolerance in a temporal fashion by altering cancer by enhancing or reducing suppressive functions, respectively. Treg migration patterns. Before these cells can be harnessed for therapeutic purposes, it is necessary to understand how they maintain tolerance under phys- Results iologically relevant conditions. We now report that transcription KLF2 Acts as a Rheostat to Control Expression of Treg Homing Receptors. factor Kruppel-like factor 2 (KLF2) controls naive Treg migration In contrast to conventional T cells that recirculate between SLOs patterns via regulation of homeostatic and inflammatory homing via blood and lymph, Tregs exhibit an expanded migratory pattern receptors, and that in its absence KLF2-deficient Tregs are unable that includes tertiary tissues (14). A previous report demonstrated to migrate efficiently to secondary lymphoid organs (SLOs). Diminished that KLF2 maintains naive T cells in a homeostatic migration Treg trafficking to SLOs is sufficient to initiate autoimmunity, pattern and, much like activated lymphocytes that quickly degrade indicating that SLOs are a primary site for maintaining peripheral this transcription factor, Klf2 excision causes quiescent T cells to tolerance under homeostatic conditions. Disease severity correlates vacate SLOs and enter peripheral tissues (15). Interestingly, Tregs + − with impaired Treg recruitment to SLOs and, conversely, promotion express half the amount of KLF2 as conventional CD4 CD25 of Tregs into these tissues can ameliorate autoimmunity. Moreover, T cells (Fig. 1A), possibly explaining the hybrid migration pattern stabilizing KLF2 expression within the Treg compartment enhances exhibited by this regulatory lineage. In addition, we identified a set peripheral tolerance by diverting these suppressive cells from tertiary of inhibitory compounds that elevated KLF2 levels within the Treg tissues into SLOs. Taken together, these results demonstrate that compartment (16), raising the possibility that we could manipulate peripheral tolerance is enhanced or diminished through modulation Treg migration pharmaceutically by targeting KLF2. To test this of Treg trafficking to SLOs, a process that can be controlled by exciting prospect, we initially identified an array of homing recep- adjusting KLF2 protein levels. + tors that were differentially expressed in Tregs relative to CD4 T cells both at the mRNA (Fig. 1B) and protein level (Fig. 1C). KLF2 | regulatory T cell | Treg migration | autoimmunity | Focusing on these receptors, we next confirmed that KLF2- peripheral tolerance fl/fl deficient Tregs harvested from Foxp3-cre; Klf2 mice displayed an altered transcription pattern that was consistent with KLF2- regs are necessary to maintain peripheral tolerance, and in their promoting (e.g., CCR7 and S1P1) or -suppressing (e.g., CCR4, Tabsence autoimmunity is 100% penetrant in both humans and CCR8, and CCR9) homeostatic and inflammatory receptors, re- mice (1, 2). This discovery highlights a key feature of self-tolerance, spectively (Fig. 1D). Where available, commercial antibodies namely that autoreactive lymphocytes are present in all healthy were used to verify RT-PCR results; alternatively, ex vivo migration individuals and that these cells must be continuously suppressed by Tregs to avoid disease. Conversely, reports indicate that tumor cells Significance co-opt the Treg compartment to suppress tumor surveillance (3–10), thereby allowing cancer progression toward malignancy. Modifying Treg activity offers a potential means of treating these Tregs are necessary to prevent autoimmunity; however, these diseases, either by promoting (autoimmunity) or diminishing (tumor same cells suppress tumor-specific immune responses and con- surveillance) self-tolerance. As such, targeting Treg migration is an tribute to malignancy. Before Treg-based therapies are devised to appealing technique that could increase or decrease self-tolerance treat these diseases, it is important to understand how Tregs in a spatially and temporally limited manner. Studies of in vivo Treg function under physiological conditions. We now report that Tregs activity are further complicated by the finding that naive Tregs carry out their immune-suppressive functions in secondary lym- traffic freely between secondary lymphoid organs (SLOs) and ter- phoid organs (e.g. spleen, lymph nodes) and factors that impair or tiary tissues (11–13), raising the question as to where Treg-mediated enhanceTreghomingtothesesitesdiminishorincreaseself- tolerance is carried out. We now report that intermediate levels of tolerance, respectively. Importantly, Treg migration patterns are regulated by Kruppel-like factor 2 (KLF2), and increasing expression Kruppel-like factor 2 (KLF2) are responsible for a hybrid expres- of this transcription factor within the Treg compartment promotes sion of homeostatic and inflammatory homing receptors displayed self-tolerance. The present study demonstrates that Treg traffick- on the surface of Tregs that underpin regulatory circulation pat- ing to lymphoid tissues underpins peripheral tolerance, which can terns. Excision of Klf2 within the Treg compartment shifts the be modified by targeting KLF2 with therapeutic drugs. balance of Treg migration away from SLOs and toward peripheral

tissues, whereas increasing KLF2 expression encourages Treg re- Author contributions: S.K.P. and E.S. designed research; S.K.P., W.R., E.J.V., V.V.P., D.O.-V., D.C., cruitment into SLOs. With regard to peripheral tolerance, hindering and K.L.B. performed research; E.J.V. and L.V.K. contributed new reagents/analytic tools; S.K.P., Treg entry into SLOs is by itself sufficient to induce autoimmunity, W.R., L.V.K., and E.S. analyzed data; and S.K.P. and E.S. wrote the paper. the severity of which correlates with defective Treg trafficking into The authors declare no conflict of interest. these tissues. Conversely, enhancing Treg migration toward SLOs, This article is a PNAS Direct Submission. either by augmenting KLF2 levels or increasing the expression of 1To whom correspondence should be addressed. Email: [email protected]. SLO-centric homing receptors governed by this transcription factor, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. reestablished peripheral tolerance and ameliorated T-cell–mediated 1073/pnas.1605849113/-/DCSupplemental.

E4662–E4670 | PNAS | Published online July 26, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1605849113 Downloaded by guest on September 24, 2021 PNAS PLUS A KLF2 B 0.003 tubulin 1.0 1.0 <0.0001 0.002 + + 0.75 0.75 KLF2 CD4 T cell KLF2+ Treg 0.5 0.5

0.25 0.25 (RT-PCR) 0.04 0.01 0.140.14 0.030.03 (relative to tubulin)

KLF2 protein levels 0 0 Relative mRNA levels Relative mRNA Treg Treg 1 Klf2 S1P naive T cell CCR4 CCR6 CCR7 CCR8 CCR9 activated T cell memory T cell Treg+ Tregrapamycin + LY294002 conventional T cellTreg + simvastatin D 0.008 0.0005 + C 100 100 0.02 KLF2 Treg 80 80 KLF2-/- Treg

60 60 0.008

40 40 (RT-PCR) 0.001 0.04 <0.0001 20 20

0 0 Relative mRNA levels Relative mRNA 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 1 CCR4 CCR6 CCR7 Klf2 CCR4 CCR6 CCR7 CCR8 CCR9 S1P

100 100 100

80 80 80 F 100 100 60 60 60 80 80 Relative cell number 40 40 40 60 60

20 20 20

40 40 0 0 0 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 20 20

CCR8 CD44 CD62L 0 0 2 3 4 5 0102 10 3 10 4 10 5 010 10 10 10 P=0.001 CCR4 CCR8 100 100 100 E 50 50 100 100 80 80 80 40

80 80 60 60 60 30

Relative cell number 60 60 40 40 40 25 20

40 40 20 20 20 10 to CCL25

0 20 20 0 0 2 3 4 5 0 2 3 4 5 010 10 10 10 2 3 4 5 010 10 10 10 010 10 10 10 0 WT cKO % Treg migration % Treg fl/fl Foxp3-cre; 0 0 Klf2 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 CCR4 CCR6 CCR7 Klf2fl/fl 100 100 100 CD44 CD62L

15 P=0.01 80 15 80 80 KLF2+ Treg

Relative cell nubmer 60 60 60 10 KLF2+ Treg + simvastatin 40 40 40 7.5 -/- 20 20 20 5 KLF2 Treg + simvastatin to S1P

0 0 0 2 3 4 5 010 10 10 10 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 INFLAMMATION 0 P=0.002 WT cKO IMMUNOLOGY AND

% Treg migration % Treg 0 P<0.0001 CCR8 CD44 CD62L Klf2fl/fl Foxp3-cre; 50 1000 1000 fl/fl Klf2 800

600 25 500 G Klf2fl/fl mice Foxp3-cre; Klf2fl/fl mice 400 to S1P

200 to CCL19 migration % migration 0.03 0 25 0 untreated simvastatin 25 media 0 20 0.72 20 simvastatin 0.21 0.5 0.62 15 15 0.12 untreated untreated 0.02 10 10 + simvastatinP<0.0001 + simvastatin 0.7 50 50 Treg numbers Treg 5 numbers Treg 5 40

0 0 25 30 4 ) 4 ) 5 ) 4 ) 4 ) 5 ) 20 0 3 /ml) 0 0 3 /ml) 10 to CCL25 % migration

0 0 control KO Ax LN (x1 Ms LN (x10 Spleen (x10 Ms LN (x1Ax LN (x1Spleen (x10 Blood (x10 Blood (x10

untreated + simvastatin

Fig. 1. KLF2 levels determine homeostatic and inflammatory homing receptor expression patterns on Tregs. (A) Immunoblot and densitometry reading of KLF2 + − + + and tubulin using total cell lysate from conventional T cells (CD4 CD25 ), activated T cells (αCD3/CD28), and Tregs (CD4 CD25 ) cultured for 12 h in medium + − + − supplemented with the indicated inhibitors. Alternatively, KLF2 and tubulin were measured using cell lysate from naive T cells (CD4 CD25 CD62L CD44 ), Tregs (CD4+CD25+), and memory T cells (CD4+CD25−CD62L−CD44+). This experiment was repeated twice. (B) Transcriptional levels of Klf2 and select homing receptors expressed on KLF2-sufficient CD4+ T cells versus KLF2-sufficient Tregs as determined by RT-PCR. This experiment was performed three times in triplicate. Error bars + − indicate SD. When significant, P values (Student’s t test) are shown. (C) Cell-surface expression of select homing receptors on KLF2-sufficient CD4 CD25 Tcells + + + (gray histogram) versus KLF2-sufficient CD4 CD25 FoxP3 Tregs (open histogram) as determined by flow cytometry. n = 4experiments.(D) Transcriptional levels of Klf2 and select homing receptors expressed on Tregs harvested from Klf2fl/fl (black bars) or from Foxp3-cre; Klf2fl/fl (gray bars) littermates as determined by RT-PCR. n = 3 experiments performed in triplicate. P values (Student’s t test) are shown; error bars indicate SD. (E) Flow cytometric analysis of homing receptors expressed on the surface of Tregs harvested from Klf2fl/fl (gray histogram) or Foxp3-cre; Klf2fl/fl (open histogram) animals. n = 4 experiments. Alternatively, ex vivo

Treg migration was measured using CCL25 or S1P as chemotactic ligands for CCR9 and S1P1, respectively. n = 2experimentsperformedintriplicate.P values (Student’s t test) are shown; error bars indicate SD. (F) Histogram overlays of homing receptors expressed on the surface of KLF2-sufficient Tregs treated with simvastatin or medium alone or KLF2-deficient Tregs treated with simvastatin. n = 3 experiments. Alternatively, KLF2-sufficient Tregs treated with simvastatin or

medium alone were used to measure ex vivo migration toward CCL19 (CCR7 ligand), S1P (S1P1 ligand), or CCL25 (CCR9 ligand). n = 2 experiments performed in triplicate, except CCL19 which was performed three times. P values (Student’s t test) are shown; error bars indicate SD. (G) Tissue-specific Treg numbers in Klf2fl/fl (Left)versusFoxp3-cre; Klf2fl/fl (Right) mice following 72-h treatment with simvastatin (20 mg/kg, open bars) or vehicle control (black bars). n = 2experiments performed in triplicate. P values (Student’s t test) are shown; error bars indicate SD.

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Fig. 2. Inefficient Treg trafficking to SLOs is sufficient to cause autoimmunity. (A)Kaplan–Meier curve documenting disease incidence in Lck-cre; Klf2fl/fl (black line, n = 30 mice) versus Foxp3-cre; Klf2fl/fl (red line, n = 30 mice) animals. Visible signs of disease included cachexia, hair loss and irritated skin, inflamed eyelids, damaged ears or tail, and hind limb paralysis. (B) Histopathology of tissues harvested from Foxp3-cre; Klf2fl/fl and littermate control Klf2fl/fl mice at 4 mo of age. Foxp3-cre; Klf2fl/fl mice were visibly ill when they were killed. n = 2 mice per group. (Magnification: 20× for skin; 40× for remaining tissues.) (C) Suppression of T-cell proliferation using Tregs harvested from Klf2fl/fl and from visibly sick Foxp3-cre; Klf2fl/fl mice. This experiment was performed twice. Error bars indicate SD. No significant differences were + + found, as determined by ANOVA. (D) Recovery of CD4 T cells cotransferred from Klf2fl/fl (black bar) and Lck-cre; Klf2fl/fl (gray bar) mice into CD90.1 recipient animals. + + n = 4 recipients. (Left) Ratio of KLF2-sufficient versus KLF2-deficient CD4 T cells recovered in various tissues. (Right) Frequency of Tregs relative to CD4 T cells found in specified tissues. Ax LN, axillary lymph nodes; Ms LN, mesenteric lymph nodes. *P < 0.005; **P < 0.001. n = 2 experiments; error bars indicate SD. (E)AsinD,except cotransferred CD4+ T cells were derived from Klf2fl/fl (black bar) and Foxp3-cre; Klf2fl/fl (gray bar) mice. (Left) P > 0.05 for each tissue. (Right) P values are shown; error bars indicate SD. (F) A mouse model of colitis using KLF2-sufficient effector T cells transferred into lymphopenic recipients. Effector T cells from Klf2fl/fl mice were transferred into scid animals alone (black lines) or in combination with Tregs from Klf2fl/fl (blue lines) or from Foxp3-cre; Klf2fl/fl (red lines) mice. Average weight of each cohort at 3 mo is shown along with statistical differences between groups. This experiment was repeated three times; n = 4 recipients per cohort. (G) Frequency of recovered Tregs following the cotransfer of wild-type effector T cells + wild-type Tregs (black bars) or KLF2-deficient Tregs (gray bars) into scid recipients. Animals were killed 3 d (Left)or3mo(Right) posttransfer. P values (Student’s t test) are shown; error bars indicate SD. This experiment was performed twice, with four recipients per cohort. (H)AsinF, except effector T cells were derived from Lck-cre; Klf2fl/fl animals. Scid mice received effector T cells alone (black lines) or in combination with Tregs harvested from Lck-cre; Klf2fl/fl (red lines) or Klf2fl/fl (blue lines) animals. This experiment was repeated three times; n = 3 mice per cohort.

assays were conducted to confirm changes in homing receptor Fig. 1F, KLF2-sufficient Tregs treated with simvastatin, a com- surface expression (Fig. 1E). Of note, FACS analysis of KLF2- monly prescribed cholesterol-lowering drug, decreased the ex- sufficient versus KLF2-deficient Tregs failed to establish an inverse pression of receptors associated with tertiary tissues (CCR4, relationship between KLF2 and CCR6 surface expression, and thus CCR8, CCR9, and CD44) while simultaneously increasing the we eliminated this homing receptor from further studies. Having expression of molecules used to circulate throughout SLOs identified a series of candidate molecules, we then tested our hy- (CCR7, CD62L, and S1P1). In contrast, simvastatin had no effect pothesis that migratory receptors could be altered via drugs that on KLF2-deficient Tregs. To extend these findings to an in vivo increase KLF2 levels within the Treg compartment. As shown in setting, Treg compartmentalization was examined in wild-type

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D INFLAMMATION IMMUNOLOGY AND E

+ Fig. 3. KLF2 regulates Treg entry and placement within SLOs. (A) Frequency of Tregs relative to CD4 T cells in Klf2fl/fl, Lck-cre; Klf2fl/fl,andFoxp3-cre; Klf2fl/fl mice. n = 8 mice per group. No significant (P > 0.05) differences were detected between cohorts (ANOVA). (B) Immunohistochemistry of spleen (Top and Middle)and mesenteric lymph nodes (Bottom) from a recipient wild-type mouse following the cotransfer (1:1) of KLF2-sufficient (red) or KLF2-deficient (green) Tregs derived from Klf2fl/fl and Foxp3-cre; Klf2fl/fl mice, respectively. Treg localization is shown relative to MOMA1+ metallophilic macrophages that envelop the splenic white pulp and/or CD3+ T-cell–rich and B220+ B-cell–rich areas of SLOs. Arrowheads and asterisks highlight KLF2-deficient and KLF2-sufficient Tregs, respectively. n = 3ex- periments. (C) Number and frequency of KLF2-sufficient versus KLF2-deficient Tregs in defined SLO compartments following adoptive transfer, as described in B. Transferred Tregs in spleen, white pulp, splenic T-cell area, and mesenteric lymph nodes (Ms LN) and the associated T-cell area were quantified from 25, 20, 15, 16, and 16 individual field of views (magnification: 10×), respectively. Significance was determined using paired Student’s t test. (D) Immunohistochemistry of trans- + + ferred Tregs (as in B) relative to CD11c cells in the spleen. (Right) Average number of Treg–CD11c interactions observed per field of view at 20× magnification + + (25 fields). (E) Flow cytometric analysis of Treg–CD11c conjugate formation 16 h after cotransfer of KLF2 (Klf2fl/fl) and KLF2-deficient (Foxp3-cre; Klf2fl/fl)Tregs. (Left) CD11c expression associated with Tregs before transfer (solid histogram) and with transferred Tregs (open histograms). (Right) Frequency of CD11c+ cells associated with transferred Tregs. n = 2 experiments performed in triplicate. Significance was determined using paired Student’s t test.

mice following 3-d treatment with simvastatin. Consistent with a we conclude that KLF2 acts as a rheostat that balances the ex- role for KLF2-mediated Treg recruitment to SLOs, mice treated pression of homeostatic and inflammatory homing receptors within with simvastatin had an increased frequency of Tregs in SLOs the Treg compartment and that drugs that increase KLF2 levels relative to untreated littermates (Fig. 1G). Importantly, this shift this balance toward lymph nodes. altered migration pattern was not present in simvastatin-treated fl/fl Foxp3-cre; Klf2 mice, indicating that drug-induced Treg re- Alteration of KLF2-Regulated Treg Migration Is Sufficient to Cause cruitment to SLOs was KLF2 dependent and was not caused by Autoimmunity. While generating animals to conduct homing re- fl/fl increased generation of peripheral Tregs (16). From these results ceptor studies, we discovered that Foxp3-cre; Klf2 mice, but not

Pabbisetty et al. PNAS | Published online July 26, 2016 | E4665 Downloaded by guest on September 24, 2021 fl/fl fl/fl Klf2 or Lck-cre; Klf2 cohorts, spontaneously developed overt between dynamic versus homeostatic Treg localization (24), which signs of autoimmunity including cachexia, inflamed and damaged suggested that Treg trafficking defects were being underestimated ears, oily fur, flaky skin, and hair loss. Although severity and symp- during steady-state conditions. Mechanistically, decreased expres- fl/fl toms varied among animals, ∼70% of Foxp3-cre; Klf2 mice sion of CCR7 and CD62L would delay the entry of KLF2-deficient developed visible signs of autoimmunity with a median disease Treg into SLOs; however, additional defects, including decreased A onset of 15 wk (Fig. 2 ). As well, histochemistry confirmed that S1P1 expression leading to impaired SLO exit, could obscure these tertiary tissues from afflicted mice contained elevated lymphocyte observations by “averaging out” aberrant homing patterns over infiltrates (Fig. 2B). Disease severity was reduced relative to time. To verify that KLF2-deficient Tregs were impaired in their Foxp3-deficient animals [100% penetrant, lethality within 2 mo (17– ability to access tissues associated with peripheral tolerance efficiently, 20)], suggesting that the autoimmunity associated with Foxp3-cre; fl/fl dynamic localization was assessed by immunohistochemistry fol- Klf2 cohorts was not caused by a global loss of Treg function. Klf2fl/fl Foxp3-cre; Klf2fl/fl fl/fl lowing the cotransfer of Tregs from or Indeed, direct analysis of Tregs harvested from sick Foxp3-cre; Klf2 mice into wild-type recipients (Fig. 3B). In agreement with prior + mice demonstrated that these cells retained their immunosup- adoptive transfer experiments, KLF2 Tregs entered lymph nodes pressive capabilities ex vivo, despite their inability to maintain self- and splenic white pulp to a greater extent than KLF2-deficient tolerance in vivo (Fig. 2C). Instead we speculated that aberrant Tregs (Fig. 3 B and C). Although Treg frequencies were comparable Treg migration was responsible for the breakdown in peripheral within T-cell zones, we observed fewer KLF2-deficient Treg–dendritic tolerance. Therefore, we examined KLF2-deficient versus KLF2- cell conjugates relative to control Tregs (Fig. 3D). This latter re- sufficient Treg trafficking under dynamic conditions. Consistent E + lationship was confirmed by flow cytometry (Fig. 3 ), indicating with previously published work (15), coinjection of CD4 T cells that KLF2 controls Treg migration toward and within SLOs; how- Lck-cre; Klf2fl/fl from and littermate control mice resulted in al- ever, this regulation was evident only under dynamic conditions. tered homing patterns: KLF2-sufficient T cells remained in SLOs, whereas KLF2-deficient T cells had a propensity to leave SLOs The Degree of Impaired Treg Migration to SLOs Correlates with D and enter tertiary tissues such as the liver (Fig. 2 ). However, Autoimmune Severity. The extent of autoimmunity exhibited by fl/fl Treg frequencies remained the same in all tissues examined, in- individual Foxp3-cre; Klf2 mice varied both in severity and dicating that KLF2-deficient Tregs trafficked similarly to KLF2- + + tissues affected, leading us to speculate that peripheral tolerance deficient conventional CD4 T cells. As expected, CD4 Tcellsfrom was not a binary state but instead was a spectrum that could be Foxp3-cre; Klf2fl/fl animals, which were 90% KLF2 sufficient, comi- diminished by obstructing Treg access to SLOs. To test this hy- grated with control T cells (Fig. 2E). In contrast, KLF2-deficient Klf2 + pothesis, we crossed gene-targeted animals onto a CCR7- Tregs no longer aped the trafficking patterns of conventional CD4 deficient background to impair further Treg entry/positioning Foxp3-cre; Klf2fl/fl Tcellsfrom animals. These results suggested that within SLOs (23, 25, 26). Although single-gene–targeted animals desynchronized Treg–conventional T-cell migration underpinned the −/− fl/fl fl/fl were viable for the course of the study, all CCR7 ; Lck-cre; Klf2 autoimmunity in Foxp3-cre; Klf2 mice while sparing Lck-cre; CCR7−/−;Foxp3-cre;Klf2fl/fl Klf2fl/fl and mice were moribund within 24 wk, animals. To test this possibility, we used a mouse model of with a median lifespan of 8 and 6 wk, respectively (Fig. 4A). inflammatory bowel disease (IBD) (21) that allowed us to control With regard to peripheral tolerance, both conventional T cells separately for effector T-cell versus Treg migration patterns. In our and Tregs use CCR7 to enter SLOs; however, increased disease −/− fl/fl fl/fl initial set of experiments, wild-type effector T cells were transferred severity in CCR7 ; Lck-cre; Klf2 mice relative to Lck-cre; Klf2 into lymphopenic recipients, either alone or in combination with littermates indicated that loss of CCR7 had a greater functional wild-type or KLF2-deficient Tregs. As expected, animals that solely impact on the Treg compartment relative to potential target cells received wild-type effector T cells exhibited significant weight loss (i.e., autoreactive, CCR7-deficient T cells). Based on this obser- (Fig. 2F), which was prevented by the cotransfer of wild-type Tregs. fl/fl vation, we posit that a minimum number of Tregs must enter In contrast, Tregs from Foxp3-cre; Klf2 mice were unable to SLOs to ward off autoimmunity. Above this threshold, peripheral prevent colitis. Analysis of Treg migration to IBD-associated SLOs tolerance is subject to the relative efficiency of Treg versus target revealed an early defect in KLF2-deficient Treg homing that was cell entry into SLOs; below this threshold, a limited number of offset at later time points (Fig. 2G). Alternatively, some of the autoreactive T cells are allowed to go unchecked, ultimately transferred effector T cells differentiated into peripheral-derived leading to severe disease. Consistent with a systemic breakdown Tregs that failed to prevent colitis. The development of IBD in the + − + in self-tolerance, CD4 (Foxp3 )andCD8 T cells harvested from presence of KLF2-deficient Tregs suggested that regulatory and −/− fl/fl CCR7 ; Lck-cre; Klf2 mice expressed the activation marker effector T cells must traffic in a similar manner to prevent auto- B immunity. Consistent with this hypothesis, KLF2-deficient Tregs CD25 (Fig. 4 ). [Note: surface expression of CD69 and CD44 were were able to prevent colitis induced by KLF2-deficient effector not evaluated, because both of these activation markers are known fl/fl TcellsharvestedfromLck-cre; Klf2 mice (Fig. 2H). Surprisingly, to be elevated on KLF2-deficient T cells (15, 27).] Despite these wild-type Tregs were also able to suppress disease induction by autoreactive phenotypes, Treg frequencies were relatively normal C KLF2-deficient effector T cells despite the dichotomy in trafficking when tissues were examined by flow cytometry (Fig. 4 ), sup- patterns. Therefore, comigration of Tregs and effector T cells was porting our contention that transitory defects in Treg migration not necessary to maintain viability; instead, these results suggested have a significant impact on peripheral tolerance. To demonstrate that Treg migration to specific anatomical locations dictated the that inefficient recruitment of Tregs to SLOs underpinned these balance between tolerance and autoimmunity. various autoimmune phenotypes, we repeated IBD experiments using KLF2-deficient Tregs that were stably transduced with KLF2 Controls Dynamic Treg Circulation Patterns Within SLOs. Naive CCR7. Retroviral transduction with CCR7 shifted KLF2-deficient T-cell responses are initiated in SLOs and, consistent with this, Treg trafficking from tertiary tissues toward SLOs (Fig. 4D). As a reports indicate that peripheral tolerance is enforced in these lo- result, cotransfer of CCR7 transduced KLF2-deficient Tregs into cations (22, 23). For these reasons we were surprised to discover scid mice suppressed wild-type effector T cells in vivo and pre- that Treg frequencies within SLOs and tertiary tissues were similar vented colitis (Fig. 4E). From these experiments, we conclude that fl/fl fl/fl in control, Lck-cre; Klf2 ,andFoxp3-cre; Klf2 mice (Fig. 3A). KLF2 regulates a series of homing receptors on Tregs that are Moreover, Treg localization within T-cell–rich areas of SLOs oc- necessary to preserve peripheral tolerance. Normally the degree of curred independently of KLF2 expression under homeostatic peripheral tolerance is subject to Treg versus conventional T-cell conditions (Fig. S1). A previous study that focused on CCR4- entry into SLOs; however, a minimum Treg threshold appears to dependent Treg migration patterns similarly noted a dichotomy exist, below which tolerance cannot be maintained in SLOs.

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100 100 100 100 100 Ms LN 10 Ax LN 8 Ms LN 10 Ax LN 8 80 80 80 80 CCR7-/- 5 6 5 6 60 60 60 60 -/- fl/fl 28 49 25 40 CCR7 ; Foxp3-cre; Klf2 40 40 40 40

-/- fl/fl 20 CCR7 ; Lck-cre; Klf2 20 20 20

0 0 0 0 2 3 4 5 2 3 4 5 50 0102 103 104 105 0102 103 104 105 010 10 10 10 010 10 10 10

100 100 100 100 Liver Colon Liver 34 Colon 8 28 13 % (Max)

% (Max) 80 80 80 80 39 10 41 10 60 60 60 60

Percent survival Percent 71 70 56 60 40 40 40 40

0 20 20 20 20

01020 0 0 0 0 2 3 4 5 2 3 4 5 Weeks 0102 103 104 105 0102 103 104 105 010 10 10 10 010 10 10 10 CD25 CD25 CCR7-/- Lck-cre; Klf2fl/fl CCR7-/-; Lck-cre; Klf2fl/fl C Spleen Ms LN Ax LN Sm. Intestine D P=0.003 P=0.008

) 10 fl/fl 12 7 6 7 2 Tregs from Foxp3-cre; Klf2 8 transfected with: C57Bl/6 6 MigR1 4 MigR1-CCR7 17 24 27 32 2 numbers (x10 Transferred Treg CCR7-/- 0 Liver Ms LN E 60

Foxp3 61418 14 x=44.9±12.6 CCR7-/-; Lck-cre; 40 P=0.06 Klf2fl/fl x=27.5±8.8 9 9 30 11 20 P=0.001 -/-

CCR7 ; % weight change Foxp3-cre; x=5.1±5 Klf2fl/fl 0

0 2 4 6 8 10 12 14 INFLAMMATION Weeks post-transfer IMMUNOLOGY AND CD25 KLF2+ T cells + KLF2+ Tregs KLF2+ T cells + KLF2-/- Tregs KLF2+ T cells + CCR7-transduced KLF2-/- Tregs

Fig. 4. The degree of peripheral tolerance correlates with Treg migration to SLOs. (A) Kaplan–Meier survival curves of CCR7−/− (black line, n = 10), CCR7−/−; Foxp3-cre; Klf2fl/fl (red line, n = 10), and CCR7−/−; Lck-cre; Klf2fl/fl (blue line, n = 7) mice. (B) Histogram overlays of CD25 surface expression on CD4+FoxP3− + − − − − T cells (Left) and CD8 T cells (Right) harvested from CCR7 / (gray line), Lck-cre; Klf2fl/fl (black line), or CCR7 / ; Lck-cre; Klf2fl/fl (red line) mice. Corresponding + + + mean fluorescent intensity is displayed in each quadrant. n = 3 experiments. (C) Percentage of Tregs (CD25 FoxP3 ) relative to CD4 T cells recovered from − − − − − − wild-type (C57BL/6), CCR7 / , CCR7 / ; Lck-cre; Klf2fl/fl, and CCR7 / ; Foxp3-cre; Klf2fl/fl mice. Contour plots are representative of four mice per cohort. (D) Quantification of adoptively transferred KLF2-deficient Tregs in wild-type recipients. Tregs from Foxp3-cre; Klf2fl/fl animals were transduced with MigR1-CCR7 (or control vector), and their tissue localization was analyzed 16 h posttransfer. This experiment was performed once in quadruplicate. (E) Colitis mouse model using KLF2-sufficient effector T cells transferred into lymphopenic recipients. Effector T cells from Klf2fl/fl animals were cotransferred into scid mice along with Tregs from Foxp3-cre; Klf2fl/fl (red line) or Klf2fl/fl (blue line) animals. Alternatively, Tregs from Foxp3-cre; Klf2fl/fl mice were transduced with a CCR7 expression vector before cotransfer with KLF2-sufficient effector T cells (black line). Average change in weight is charted over time. This experiment was performed twice; n = 5 mice per cohort. P values are shown; error bars indicate SD.

Increased KLF2 Expression Within the Treg Compartment Augments KLF2 expression within the Treg lineage improved disease out- Peripheral Tolerance. Based on results demonstrating that KLF2 come as reflected by significantly less weight loss and decreased regulates Treg trafficking to SLOs (Fig. 2E), the active re- tissue damage in the colon and small intestine relative to controls cruitment of which prevents IBD (Fig. 4E), we speculated that (Figs. 5 B and C). Having demonstrated that augmenting KLF2 enhancing KLF2 expression within the Treg compartment could levels within the Treg compartment is sufficient to promote pe- be used to promote peripheral tolerance. To test this premise, we ripheral tolerance, we next tested if this event could be replicated established an acute model of graft-versus-host disease (aGVHD) by simvastatin. Of note, statins have an immunosuppressive effect using irradiated major histocompatibility complex (MHC) on endothelial cells, including decreased MHC class II expression, haplotype H2d animals that received allogeneic T cells (MHC decreased expression of P-selectin and CD40, increased surface haplotype H2b) in concert with CD62Llow Tregs; alternatively, expression of complement inhibitory molecules, and increased CD62Llow Tregs were transduced with a nondegradative form of production of nitric oxide (31–41). Because the endothelial com- KLF2 (16, 28) before cotransfer. Consistent with previous reports partment contributes to symptoms of aGVHD (42–45), these (29, 30), CD62Llow Tregs were unable to prevent aGVHD (Fig. 5A) pleiotropic effects precluded the systemic use of simvastatin to because of inefficient entry into SLOs. In contrast, increased test Treg-mediated tolerance. Instead, CD62Llow Tregs were treated

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Fig. 5. Peripheral tolerance is reestablished by enhancing KLF2 expression within the Treg compartment. (A–C) aGVHD in H2d mice that received H2b T cells + transduced H2b CD62Llow Tregs. This experiment was performed twice; n = 5 mice per cohort. (A) Average changes in weight over time were graphed for mice that received Tregs transduced with empty vector (black line) or a nondegradative form of KLF2 [KLF2(ND)] (red line). P value was determined by Mann– Whitney test; error bars indicate SD. (B) Histology (H&E staining) of colon (Upper) and small intestine (Lower) from mice that received Tregs transduced with empty vector or a nondegradative form of KLF2. (Magnification: 20×.) (C) Pathologic scores of colon (Left) and small intestine (Right) from mice receiving control Tregs or KLF2(ND)-transduced Tregs. P values (Student’s t test) are displayed. (D–G) aGVHD in H2d mice that received H2b T cells ± cultured CD62Llow Tregs. This experiment was performed twice; n = 5 mice per cohort. (D) Average changes in weight over time were graphed for mice that received no Tregs (gray line) or Tregs that were treated with simvastatin (red line) or vehicle control (black line). P values (Student’s t test) at termination are shown; error bars indicate SD. (E) Histology of colon from mice that received Tregs that were treated with simvastatin or vehicle control. (Magnification: 20×.) (F) Pathologic scores of colon from mice that received effector T cells ± Tregs treated with simvastatin or vehicle control. P values (Student’s t test) are shown. (G) Histogram overlays of CD25 (Left) and CD44 (Right) surface expression on CD4+Foxp3− T cells harvested from the spleen and mesenteric lymph nodes of mice that received effector T cells alone (gray) or in combination with Tregs that were treated with simvastatin (red line) or vehicle control (black line). Corresponding average mean fluorescent intensity (± SD) and P values (ANOVA) are shown below the histograms.

with simvastatin or control vehicle ex vivo before cotransfer with lowing T-cell activation that degrades KLF2, these lymphocytes effector T cells into allogeneic hosts. Consistent with previous vacate SLOs and enter inflamed tertiary tissues. We now report results demonstrating that simvastatin improves Treg recruitment that intermediate levels of KLF2 within the Treg compartment to SLOs in a KLF2-dependent manner (Fig. 1G), we found that create a hybrid pattern of homeostatic and inflammatory homing mice receiving simvastatin-treated Tregs had significantly im- receptors, providing these suppressor cells access to both SLOs and proved disease outcome relative to recipient controls, both in tertiary tissues. Altering KLF2 levels changes the degree to which termsofweightloss(Fig.5D) and histopathology (Fig. 5 E and F). these two sets of homing receptors are promoted/suppressed, Moreover, T cells harvested from mice receiving simvastatin- thereby shifting the balance between inflammatory and homeostatic treated Tregs were more quiescent than those from control ani- migration. Importantly, this shift in Treg migration patterns seems mals, as indicated by lower surface expression of CD25 and CD44 to be sufficient to initiate autoimmunity. (Fig. 5G). From these findings we conclude that drugs that in- Current results suggest that there is a direct relationship between crease KLF2 expression within the Treg compartment are capable the ability of Tregs to enter SLOs and the degree of peripheral of enhancing peripheral tolerance by redirecting Tregs to SLOs. tolerance maintained under homeostatic conditions. Should too few Tregs traffic to these sites, autoimmunity is initiated despite the Discussion continued presence of other tolerizing mechanisms (e.g., central Quiescent T cells express high levels of KLF2, which is necessary tolerance, activation-induced cell death, tolerogenic antigen-presenting −/− to limit naive T-cell migration to blood, lymph, and SLOs. Fol- cells) (46). Indeed, under extreme conditions (e.g., CCR7 ;Lck-cre;

E4668 | www.pnas.org/cgi/doi/10.1073/pnas.1605849113 Pabbisetty et al. Downloaded by guest on September 24, 2021 fl/fl −/− fl/fl PNAS PLUS Klf2 and CCR7 ; Foxp3-cre; Klf2 mice)asmallnumberof Materials and Methods autoreactive T cells can no longer be restrained by a similarly limited Mice. Klf2fl/fl mice were generated as previously described (53). Lck-cre, scid, number of Tregs, suggesting that a lower threshold for Treg-medi- and Rag−/−γc−/− mice were purchased from Taconic, and Foxp3-cre and − − ated tolerance exists within SLOs. Disease severity ranges from mild, CCR7 / mice were purchased from Jackson Laboratories. All experiments fl/fl chronic forms of autoimmunity (e.g., Foxp3-cre; Klf2 mice) to were performed using 6-wk-old healthy mice unless specified otherwise. All fl/fl severe cases that quickly lead to death (e.g., CCR7; Foxp3-cre; Klf2 ). mice were housed in pathogen-free conditions in accordance with the pro- tocols of the Institutional Animal Care and Use Committee at Vanderbilt This spectrum of pathogenic phenotype suggests that peripheral University. tolerance is not a binary state but instead is a continuum that can be adjusted at the level of Treg migration. Therefore pharma- Murine Model of Colitis. Experimental colitis was performed as previously ceutical drugs that augment KLF2 expression offer an opportu- described (21). Briefly, 4 × 105 CD4+CD45RBhigh naive T cells ± 2 × 105 CD4+ nity to enhance peripheral tolerance as a means of ameliorating CD45RBlow Tregs or CD4+CD45RBlow Tregs transduced with MigR1-CCR7 – were injected intraperitoneally into 7-wk-old scid recipient mice. Treg purify T-cell mediated diseases. + + + Patients suffering from autoimmunity are not typically subject (92–94% CD4 CD25 Foxp3 ) was confirmed by flow cytometry. Recipient to acute immunological defects (e.g., lack of Tregs); instead, dis- mice were weighed weekly to document colitis-associated wasting, and pathologic scores were obtained on sections of H&E-stained colons. Percent ease is likely initiated by modest deviations in tolerance that change in weight was calculated as 100 × [(experimental weight − initial promote tissue damage over time. Based on our observations that weight)/initial weight]. aberrant Treg migration causes a variety of autoimmune phenotypes in Klf2 gene-targeted mice (e.g., psoriasis-like disease, IBD, GVHD Model. aGVHD was induced as described previously (29). In brief, ir- hind limb paralysis), we speculate that this mechanism is relevant radiated (800 cGy) BALB/c hosts received 2 × 106 T-cell–depleted bone marrow cells plus 106 CD4+CD25− T cells ± 0.5 × 106 CD4+CD25+CD62L− in a subset of human cases. Of note for clinical diagnosis, Treg + + migration defects are not readily detectable under steady-state (± 10 μM simvastatin for 15 h) or MigR1-KLF2–transduced CD4 CD25 cells. conditions and instead may require dynamic assays to identify Survival, appearance, and weight change were monitored daily. For histology, mice were killed 6–8 d after cell transfer, and tissues were collected. existing abnormalities. Fortunately, our work suggests that diseases arising from Treg migration defects are responsive to treat- Additional Materials and Methods. Information concerning Western blots, RT-PCR, ments that reestablish peripheral tolerance via recruitment flow cytometry, ex vivo and in vivo migration assays, and immunohistochemistry to SLOs. In this regard, many autoimmune patients are receptive are provided in SI Materials and Methods. to drugs that are predicted to recruit (e.g., simvastatin) or retain (FTY720) (47, 48) Tregs in SLOs (49–52). If Treg migration de- Statistical Analysis. Statistical analysis was performed using Prism (GraphPad fects are indeed proven to be a significant factor contributing to Software). Values P ≤ 0.05 were considered statistically significant. autoimmunity in the clinic, these findings would encourage the development of new therapeutics that redirect Treg trafficking to ACKNOWLEDGMENTS. We thank Jenny Schafer (Vanderbilt University Medical Center) for cell imaging assistance and Mark Boothby (Vanderbilt SLOs. Some of these drugs may include compounds that augment University Medical Center) and Avinash Bhandoola (NIH) for helpful KLF2 expression within the Treg compartment. suggestions. INFLAMMATION

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