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.