HHS Public Access Author manuscript Author Manuscript
J Immunol. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: J Immunol. 2015 November 1; 195(9): 4154–4161. doi:10.4049/jimmunol.1500945.
Requirement for CD28 in Effector Treg Differentiation, CCR6 induction, and Skin Homing Ruan Zhang1, Christopher M. Borges1,2, Martin Y. Fan1,2, John E. Harris3, and Laurence A. Turka1,2 1Center
for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA
Author Manuscript
2Division
of Medical Sciences, Harvard Medical School, Boston, MA
3Division
of Dermatology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA
Abstract
Author Manuscript
The skin, like most non-lymphoid tissues, contains substantial numbers of T cells. Among these, memory T cells serve a sentinel role to protect against pathogens, and regulatory T cells terminate immune responses as a check against unrestrained inflammation. Previously, we created conditional knockout mice with Treg-specific deletion of CD28. Although these mice have normal numbers of Tregs, these cells have lower levels of CTLA-4, PD-1 and CCR6, and the animals develop systemic autoimmunity characterized by prominent skin inflammation. Here, we have performed a detailed analysis of the skin disease in these mice. Our data show that Treg-expressed CD28 is required for optimal maturation of CD44loCD62Lhi central Tregs (cTreg) into CD44hiCD62Llo effector Tregs (eTregs), and induction of CCR6 among the cells that do become eTregs. While CD28-deficient Tregs are able to regulate inflammation normally when injected directly into the skin, they fail to home properly to inflamed skin. Collectively, these results suggest a key role for CD28 costimulation in promoting a cTreg to eTreg transition with appropriate upregulation of appropriate chemokine receptors such as CCR6 that are required for tissue homing.
Introduction Author Manuscript
The skin is the major contact point between “self” and the environment, and continually encounters foreign antigens and inflammatory substances. While the stratum corneum and other layers of the epidermis serve a primary barrier function, inevitably there are numerous breaches. Consistent with this, the skin is an immune cell-rich organ, with large numbers of T lymphocytes that serve multiple functions. Among other cell populations, the skin contains large numbers of memory T cells (Tmem), which like other tissue resident Tmem, are able to respond rapidly in situ to antigen challenge.
Correspondence: Laurence A. Turka, Center for Transplantation Sciences, MGH-East, Bldg. 149-9019, 13th Street, Boston, MA 02129, Phone: 617 724-7740; Fax: 617-726-6925, [email protected].
Zhang et al.
Page 2
Author Manuscript
Prior work has shown that the skin also contains significant numbers of CD4+Foxp3+ regulatory T cells (Tregs)(1). As with naïve T cells and Tmem, they gain access to the skin via complex regulation of chemokine receptors, including upregulation of CCR4 and CCR6, and downregulation of CCR7. Importantly, almost all cutaneous Tregs bear a memory phenotype, and are thought to exist to counterbalance and terminate locally induced inflammation(2, 3).
Author Manuscript
Like non-regulatory CD4 T cells, Tregs constitutively express the CD28 costimulatory receptor. Previous studies have shown that CD28 is required for thymic Treg development(4, 5). Recently, our own group created a mouse with conditional targeting of CD28. Using Foxp3-Cre to delete CD28 in Tregs, we uncovered an obligate cell intrinsic role for CD28 in Treg function and Treg survival(6). Specifically, mice lacking CD28 in Foxp3+ cells (termed CD28-ArialΔTreg mice) developed lymphadenopathy and splenomegaly characterized by the accumulation of activated effector T cells (Teff) and Tmem. In addition, tissue-specific inflammation was seen in the lung, and more prominently, the skin. Consistent with this, the most notable phenotypic alterations in CD28ΔTregs were decreased expression of PD-1, CTLA-4, and CCR6 (of particular interest, given the role of CCR6 in Treg migration to the skin).
Author Manuscript
Recently, based on the expression of CD62L and CD44, Treg cells were divided into two subsets with distinct characteristics: CD44loCD62Lhi central Tregs (cTreg), mainly recirculating in secondary lymphoid tissues, and CD44hiCD62Llo effector Tregs in nonlymphoid tissues (7). Quiescent cTregs (which express CCR7 to enable lymphoid homing and retention) can differentiate into eTregs under inflammatory signals and migrate to nonlymphoid tissues, but the mechanism of how eTregs are activated and function in lymphoid and nonlymphoid organs remains unknown. Here we have performed a detailed characterization of the skin disease seen in CD28-ΔTreg mice. We find that loss of CD28 in Tregs leads to failure to induce appropriate maturation of cTregs into eTregs as well as defective induction of CCR6 among the cells that do become eTregs. While CD28-deficient Tregs are able to regulate inflammation normally when injected directly into the skin, they fail to home properly to inflamed skin. Together, these results suggest a key role for CD28 costimulation in promoting a cTreg to eTreg transition with appropriate upregulation of appropriate chemokine receptors required for tissue homing.
Materials and methods Author Manuscript
Mice Mice with conditional targeting of CD28 in Foxp3+ cells (CD28fl/fl x Foxp3YFP-Cre, termed CD28-ΔTreg mice) have been previously described (6). CD28-ΔTreg mice, and CD45.1 or CD45.1/2 congenic B6 mice were bred in our facility and maintained under specific pathogen free conditions. All experiments described were approved by the Institutional Animal Care and Use Committee at the Massachusetts General Hospital.
J Immunol. Author manuscript; available in PMC 2016 November 01.
Zhang et al.
Page 3
Media, reagents, antibodies, and flow cytometry
Author Manuscript
Cell culture media was RPMI 1640 (Mediatech Inc.) supplemented with 10% heatinactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 50 mM 2-mercaptoethanol (Sigma-Aldrich). Fluorescent anti-CD4, anti-CD8α, anti-CD8β, anti-CD25, anti-CD44, anti-CD45, anti-CD45.1, anti-CD45.2, anti-CD62L, anti-CD103, anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR-9, anti-TCRβ and anti-TCRγδ antibodies were purchased from Biolegend. Anti-CTLA-4, anti-PD-1, anti-GITR, anti-CD127, anti-IFNγ, anti-CXCR3 and anti-CXCR4 were purchased from BD Biosciences-Pharmingen. AntiMHCII, anti-CD207, anti-Gr-1, anti-CD11b, and anti-Foxp3 staining kit were purchased from eBioscience. Murine recombinant IL-2, IL-6 and E-selection-FC were purchased from R&D Systems. BD Cytofix/Perm buffer was used for intracellular staining. Cells were run on a BD LSR II flow cytometer or a Beckman Coulter Gallios flow cytometer and analyzed by Flowjo (Flowjo LLC).
Author Manuscript
T cell isolation Spleen and lymph nodes were harvested from 4 to 8 week old CD28-ΔTreg mice and agematched Foxp3-YFP-Cre littermates. Total cells were enriched for CD4+ cells by negative selection using a mouse CD4+ enrichment kit (Stemcell Technologies, Canada). For in vitro Treg activation assays, cells were stained with CD44-Percpcy5.5, CD62L-PE and CD4A700. CD4+YFP+ CD44hiCD62Llo (eTregs) and CD4+YFP+CD44loCD62Lhi (cTregs) were sorted by flow cytometry on a FACSAria Cell Sorter (BD Biosciences). Cell purity was routinely greater than 90%. Tissue digestion and cell isolation
Author Manuscript
As previously reported(8), ears or skin grafts were minced into small pieces and put into a gentle MACS C tube (Miltenyi) with 3 to 5 ml DMEM digestion solution containing 2% FCS, 10mM Hepes, 0.1mg/ml Liberase TM (Sigma), 0.1 mg/ml DNAse I (Sigma) and 0.5 mg/ml hyaluronidase (Sigma). After 1.5 hour shaking at 37°C, ear tissues were further broken down in a gentle MACS dissociator (Miltenyi) and debris were filtered. Cells were washed in FACS buffer and stained for flow cytometry. To separate the epidermis and dermis, ears were split into dorsal and ventral halves and incubated with 20 mM EDTA in PBS at 37°C for 2 hr. Then the separated epidermis and dermis were digested as above. Adoptive transfer of T cells and in vivo migration Enriched CD4 T cells were adoptively transferred to congenic B6 mice by intraperitoneal injection.
Author Manuscript
Skin Transplantation Skin transplantation was performed as described previously(9). In brief, recipient mice were anesthetized with isoflurane, and a 1 × 1 cm area of skin was removed from the lateral trunk. A full-thickness donor skin graft was sutured to the exposed s.c. tissue bed using 4.0 chromic absorbable suture, and animals were bandaged after application of antibiotic ointment to the graft.
J Immunol. Author manuscript; available in PMC 2016 November 01.
Zhang et al.
Page 4
DTH responses
Author Manuscript
Mice were sensitized on days 0 and 1 by painting 50 μl 0.5% 1-fluoro-2, 4-dinitrobenzene (DNFB, Sigma) in acetone on a shaved abdomen. On day 4, ears were painted with 20 μl of 0.2% DNFB in acetone and ear thickness was measured every 24 hours using a micrometer (Mitutoyo, Japan) for up to 7 days. Intradermal injection of LPS To induce local skin inflammation, a single dose of 10 μg LPS (sigma) in 10μl PBS was injected into the ventral side of the ear using a 31 gauge syringe. Quantitative PCR
Author Manuscript
FACS sorted CD4+YFP+CD44lo CD62Lhi cTregs and CD4+YFP+CD44hi CD62Llo eTregs were lysed in TRIzol (Invitrogen), and total RNA was extracted using the PureLink RNA Mini kit (Ambion). For CCR6 induction, FACS sorted CD4+YFP+ Tregs were stimulated with plate-bound anti-CD3 (2 μg/mL) and soluble anti-CD28 (5 μg/mL) for 24 h before RNA extraction. Genomic DNA was removed using DNase I (Invitrogen), and cDNA was generated using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed on a Stratagene Mx3005P instrument, using the QuantiTect SYBR Green PCR Kit (Qiagen) and the following amplification protocol: 10 minutes at 95°C, 40 cycles (30 seconds at 95°C, 1 minute at 55°C, 1 minute at 72°C), followed by confirmation of amplicon melting temperature. Reactions were performed in triplicate, and genomic DNA contamination was excluded by observing no amplification from non-reverse-transcribed RNA. Primer sequences used were as follows: 18S rRNA Fwd: 5′GTAACCCGTTGAACCCCATT-3′ and Rev: 5′-CCATCCAATCGGTAGTAGCG-3′; βActin Fwd: 5′-GCGAGCACAGCTTCTTTGC-3′ and Rev: 5′TCGTCATCCATGGCGAACT-3′; and CCR6 Fwd: 5′CCTCACATTCTTAGGACTGGAGC-3′ and Rev: 5′-GGCAATCAGAGCTCTCGGA-3′. Signals from the β-Actin reactions were used to normalize signals in the CCR6 PCR reactions of the same cDNA sample to determine relative CCR6 mRNA levels for each sample.
Author Manuscript
Statistical Analysis Comparison of means between groups used the two-tailed Student t-test. Differences were considered statistically significant at p 0.05.
J Immunol. Author manuscript; available in PMC 2016 November 01.
Zhang et al.
Page 14
Author Manuscript Author Manuscript Author Manuscript
Figure 3.
Author Manuscript
CD28-deficient eTregs are defective in CCR6 expression. A/B. Percentages of CD44hiCD62Llo eTregs and CD44loCD62Lhi cTregs in lymph nodes of wild type and CD28-DTreg mice (
J Immunol. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: J Immunol. 2015 November 1; 195(9): 4154–4161. doi:10.4049/jimmunol.1500945.
Requirement for CD28 in Effector Treg Differentiation, CCR6 induction, and Skin Homing Ruan Zhang1, Christopher M. Borges1,2, Martin Y. Fan1,2, John E. Harris3, and Laurence A. Turka1,2 1Center
for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA
Author Manuscript
2Division
of Medical Sciences, Harvard Medical School, Boston, MA
3Division
of Dermatology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA
Abstract
Author Manuscript
The skin, like most non-lymphoid tissues, contains substantial numbers of T cells. Among these, memory T cells serve a sentinel role to protect against pathogens, and regulatory T cells terminate immune responses as a check against unrestrained inflammation. Previously, we created conditional knockout mice with Treg-specific deletion of CD28. Although these mice have normal numbers of Tregs, these cells have lower levels of CTLA-4, PD-1 and CCR6, and the animals develop systemic autoimmunity characterized by prominent skin inflammation. Here, we have performed a detailed analysis of the skin disease in these mice. Our data show that Treg-expressed CD28 is required for optimal maturation of CD44loCD62Lhi central Tregs (cTreg) into CD44hiCD62Llo effector Tregs (eTregs), and induction of CCR6 among the cells that do become eTregs. While CD28-deficient Tregs are able to regulate inflammation normally when injected directly into the skin, they fail to home properly to inflamed skin. Collectively, these results suggest a key role for CD28 costimulation in promoting a cTreg to eTreg transition with appropriate upregulation of appropriate chemokine receptors such as CCR6 that are required for tissue homing.
Introduction Author Manuscript
The skin is the major contact point between “self” and the environment, and continually encounters foreign antigens and inflammatory substances. While the stratum corneum and other layers of the epidermis serve a primary barrier function, inevitably there are numerous breaches. Consistent with this, the skin is an immune cell-rich organ, with large numbers of T lymphocytes that serve multiple functions. Among other cell populations, the skin contains large numbers of memory T cells (Tmem), which like other tissue resident Tmem, are able to respond rapidly in situ to antigen challenge.
Correspondence: Laurence A. Turka, Center for Transplantation Sciences, MGH-East, Bldg. 149-9019, 13th Street, Boston, MA 02129, Phone: 617 724-7740; Fax: 617-726-6925, [email protected].
Zhang et al.
Page 2
Author Manuscript
Prior work has shown that the skin also contains significant numbers of CD4+Foxp3+ regulatory T cells (Tregs)(1). As with naïve T cells and Tmem, they gain access to the skin via complex regulation of chemokine receptors, including upregulation of CCR4 and CCR6, and downregulation of CCR7. Importantly, almost all cutaneous Tregs bear a memory phenotype, and are thought to exist to counterbalance and terminate locally induced inflammation(2, 3).
Author Manuscript
Like non-regulatory CD4 T cells, Tregs constitutively express the CD28 costimulatory receptor. Previous studies have shown that CD28 is required for thymic Treg development(4, 5). Recently, our own group created a mouse with conditional targeting of CD28. Using Foxp3-Cre to delete CD28 in Tregs, we uncovered an obligate cell intrinsic role for CD28 in Treg function and Treg survival(6). Specifically, mice lacking CD28 in Foxp3+ cells (termed CD28-ArialΔTreg mice) developed lymphadenopathy and splenomegaly characterized by the accumulation of activated effector T cells (Teff) and Tmem. In addition, tissue-specific inflammation was seen in the lung, and more prominently, the skin. Consistent with this, the most notable phenotypic alterations in CD28ΔTregs were decreased expression of PD-1, CTLA-4, and CCR6 (of particular interest, given the role of CCR6 in Treg migration to the skin).
Author Manuscript
Recently, based on the expression of CD62L and CD44, Treg cells were divided into two subsets with distinct characteristics: CD44loCD62Lhi central Tregs (cTreg), mainly recirculating in secondary lymphoid tissues, and CD44hiCD62Llo effector Tregs in nonlymphoid tissues (7). Quiescent cTregs (which express CCR7 to enable lymphoid homing and retention) can differentiate into eTregs under inflammatory signals and migrate to nonlymphoid tissues, but the mechanism of how eTregs are activated and function in lymphoid and nonlymphoid organs remains unknown. Here we have performed a detailed characterization of the skin disease seen in CD28-ΔTreg mice. We find that loss of CD28 in Tregs leads to failure to induce appropriate maturation of cTregs into eTregs as well as defective induction of CCR6 among the cells that do become eTregs. While CD28-deficient Tregs are able to regulate inflammation normally when injected directly into the skin, they fail to home properly to inflamed skin. Together, these results suggest a key role for CD28 costimulation in promoting a cTreg to eTreg transition with appropriate upregulation of appropriate chemokine receptors required for tissue homing.
Materials and methods Author Manuscript
Mice Mice with conditional targeting of CD28 in Foxp3+ cells (CD28fl/fl x Foxp3YFP-Cre, termed CD28-ΔTreg mice) have been previously described (6). CD28-ΔTreg mice, and CD45.1 or CD45.1/2 congenic B6 mice were bred in our facility and maintained under specific pathogen free conditions. All experiments described were approved by the Institutional Animal Care and Use Committee at the Massachusetts General Hospital.
J Immunol. Author manuscript; available in PMC 2016 November 01.
Zhang et al.
Page 3
Media, reagents, antibodies, and flow cytometry
Author Manuscript
Cell culture media was RPMI 1640 (Mediatech Inc.) supplemented with 10% heatinactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 50 mM 2-mercaptoethanol (Sigma-Aldrich). Fluorescent anti-CD4, anti-CD8α, anti-CD8β, anti-CD25, anti-CD44, anti-CD45, anti-CD45.1, anti-CD45.2, anti-CD62L, anti-CD103, anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR-9, anti-TCRβ and anti-TCRγδ antibodies were purchased from Biolegend. Anti-CTLA-4, anti-PD-1, anti-GITR, anti-CD127, anti-IFNγ, anti-CXCR3 and anti-CXCR4 were purchased from BD Biosciences-Pharmingen. AntiMHCII, anti-CD207, anti-Gr-1, anti-CD11b, and anti-Foxp3 staining kit were purchased from eBioscience. Murine recombinant IL-2, IL-6 and E-selection-FC were purchased from R&D Systems. BD Cytofix/Perm buffer was used for intracellular staining. Cells were run on a BD LSR II flow cytometer or a Beckman Coulter Gallios flow cytometer and analyzed by Flowjo (Flowjo LLC).
Author Manuscript
T cell isolation Spleen and lymph nodes were harvested from 4 to 8 week old CD28-ΔTreg mice and agematched Foxp3-YFP-Cre littermates. Total cells were enriched for CD4+ cells by negative selection using a mouse CD4+ enrichment kit (Stemcell Technologies, Canada). For in vitro Treg activation assays, cells were stained with CD44-Percpcy5.5, CD62L-PE and CD4A700. CD4+YFP+ CD44hiCD62Llo (eTregs) and CD4+YFP+CD44loCD62Lhi (cTregs) were sorted by flow cytometry on a FACSAria Cell Sorter (BD Biosciences). Cell purity was routinely greater than 90%. Tissue digestion and cell isolation
Author Manuscript
As previously reported(8), ears or skin grafts were minced into small pieces and put into a gentle MACS C tube (Miltenyi) with 3 to 5 ml DMEM digestion solution containing 2% FCS, 10mM Hepes, 0.1mg/ml Liberase TM (Sigma), 0.1 mg/ml DNAse I (Sigma) and 0.5 mg/ml hyaluronidase (Sigma). After 1.5 hour shaking at 37°C, ear tissues were further broken down in a gentle MACS dissociator (Miltenyi) and debris were filtered. Cells were washed in FACS buffer and stained for flow cytometry. To separate the epidermis and dermis, ears were split into dorsal and ventral halves and incubated with 20 mM EDTA in PBS at 37°C for 2 hr. Then the separated epidermis and dermis were digested as above. Adoptive transfer of T cells and in vivo migration Enriched CD4 T cells were adoptively transferred to congenic B6 mice by intraperitoneal injection.
Author Manuscript
Skin Transplantation Skin transplantation was performed as described previously(9). In brief, recipient mice were anesthetized with isoflurane, and a 1 × 1 cm area of skin was removed from the lateral trunk. A full-thickness donor skin graft was sutured to the exposed s.c. tissue bed using 4.0 chromic absorbable suture, and animals were bandaged after application of antibiotic ointment to the graft.
J Immunol. Author manuscript; available in PMC 2016 November 01.
Zhang et al.
Page 4
DTH responses
Author Manuscript
Mice were sensitized on days 0 and 1 by painting 50 μl 0.5% 1-fluoro-2, 4-dinitrobenzene (DNFB, Sigma) in acetone on a shaved abdomen. On day 4, ears were painted with 20 μl of 0.2% DNFB in acetone and ear thickness was measured every 24 hours using a micrometer (Mitutoyo, Japan) for up to 7 days. Intradermal injection of LPS To induce local skin inflammation, a single dose of 10 μg LPS (sigma) in 10μl PBS was injected into the ventral side of the ear using a 31 gauge syringe. Quantitative PCR
Author Manuscript
FACS sorted CD4+YFP+CD44lo CD62Lhi cTregs and CD4+YFP+CD44hi CD62Llo eTregs were lysed in TRIzol (Invitrogen), and total RNA was extracted using the PureLink RNA Mini kit (Ambion). For CCR6 induction, FACS sorted CD4+YFP+ Tregs were stimulated with plate-bound anti-CD3 (2 μg/mL) and soluble anti-CD28 (5 μg/mL) for 24 h before RNA extraction. Genomic DNA was removed using DNase I (Invitrogen), and cDNA was generated using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed on a Stratagene Mx3005P instrument, using the QuantiTect SYBR Green PCR Kit (Qiagen) and the following amplification protocol: 10 minutes at 95°C, 40 cycles (30 seconds at 95°C, 1 minute at 55°C, 1 minute at 72°C), followed by confirmation of amplicon melting temperature. Reactions were performed in triplicate, and genomic DNA contamination was excluded by observing no amplification from non-reverse-transcribed RNA. Primer sequences used were as follows: 18S rRNA Fwd: 5′GTAACCCGTTGAACCCCATT-3′ and Rev: 5′-CCATCCAATCGGTAGTAGCG-3′; βActin Fwd: 5′-GCGAGCACAGCTTCTTTGC-3′ and Rev: 5′TCGTCATCCATGGCGAACT-3′; and CCR6 Fwd: 5′CCTCACATTCTTAGGACTGGAGC-3′ and Rev: 5′-GGCAATCAGAGCTCTCGGA-3′. Signals from the β-Actin reactions were used to normalize signals in the CCR6 PCR reactions of the same cDNA sample to determine relative CCR6 mRNA levels for each sample.
Author Manuscript
Statistical Analysis Comparison of means between groups used the two-tailed Student t-test. Differences were considered statistically significant at p 0.05.
J Immunol. Author manuscript; available in PMC 2016 November 01.
Zhang et al.
Page 14
Author Manuscript Author Manuscript Author Manuscript
Figure 3.
Author Manuscript
CD28-deficient eTregs are defective in CCR6 expression. A/B. Percentages of CD44hiCD62Llo eTregs and CD44loCD62Lhi cTregs in lymph nodes of wild type and CD28-DTreg mice (
