CXCR5+ T follicular helper (Tfh) cells are associated with aberrant autoantibody production in patients with antibody-mediated autoimmune diseases including lupus. Follicular regulatory T (Tfr) cells expressing CXCR5 and Bcl6 have been recently identified as a specialized subset of Foxp3+ regulatory T (Treg) cells that control germinal center reactions. In this study, we show that retroviral transduction of
Effective B cell responses are essential for host defense against pathogens, particularly in case of a viral infection, via the generation of neutralizing or opsonizing antibodies; however, excessive B cell responses can also be detrimental in case of autoimmunity. For instance, elevated autoantibodies in circulation have been associated with various systemic autoimmune diseases such as systemic lupus erythematosus, Sjögren’s syndrome and rheumatoid arthritis (Wahren-Herlenius and Dorner, 2013; Suurmond and Diamond, 2015). The existence of autoantibodies indicates insufficient immune tolerance to self-reactive B cells in the patients. Given that a large fraction of autoantibodies are IgG isotypes, it is evident that auto-reactive T helper cells are also involved in the development of autoantibodies. Among diverse helper T cell subsets, follicular helper T (Tfh) cells are tightly associated with autoantibodies in humans with systemic autoimmune diseases (Crotty, 2014; Ueno
Tfh cells play crucial roles in germinal center reactions to exogenous antigens as well as to self-antigens in autoimmunity (Crotty, 2014; Ueno
CD4+ T cells expressing Foxp3 are essential for maintaining immune tolerance to self-antigens (Lutz, 2016). Mutation in Foxp3 causes detrimental systemic autoimmune responses as shown in scurfy mice as well as IPEX syndrome in humans (Sakaguchi
Using Treg cells for the prevention and/or treatment of immune disorders has been investigated in experimental animals as well as in clinical setting in humans (Riley
Although the use of Tfr cells can be efficacious for the treatment of antibody-mediated immune disorders, obtaining sufficient number of Tfr cells is an obvious obstacle for the development of such Treg-cell therapy. The reason for that is because it only consists of ~5–10% of Treg cells which is only ~10% of CD4+ T cells (Chung
C57BL/6, Foxp3-IRES-mRFP (Foxp3RFP), B6.SJL (CD45.1+), and
For cell sorting, lymphoid cells isolated from mouse spleens or draining lymph nodes, were obtained and stained with PerCp-Cy5.5-conjugated anti-CD4 (clone GK1.5, BioLegend, San Diego, CA, USA), Alexa488-conjugated anti-CD62L (clone MEL-14, BioLegend), PE-conjugated anti-CD25 (clone PC61, BioLegend), Alexa647-conjugated anti-CD44 (clone IM7, BioLegend), APC-conjugated anti-CD45R/B220 (clone RA3-6B2, BioLegend), Alexa488 anti-GL7 (clone GL7, BD Pharmingen, San Jose, CA, USA), PE-conjugated anti-IgD (clone 11-26c.2a, BioLegend), FITC-conjugated anti-CD279 (PD-1, clone J43, eBioscience, San Diego, CA, USA), Biotinconjugated anti-CXCR5 (clone L138D7, BioLegend), and APC-conjugated Streptavidin (BioLegend).
The stained cells were analyzed by FACSAria II (BD Bioscience, San Jose, CA, USA), and the data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
CD4+ T cells and B220+ B cells were isolated by anti-CD4 and anti-CD45R microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. B220+GL7–IgD+ naïve B cells, and CD4+CD25–CD44–CD62L+ naïve T cells were isolated from pooled spleen and peripheral lymph nodes of naïve C57BL/6 mice. CD4+PD-1+CXCR5+ Tfh cells were isolated from the draining lymph nodes of mice immunized with KLH by FACSAria II. Treg cells isolated from Foxp3RFP mice using Treg isolation kit (Miltenyi Biotec) were stimulated using Treg expansion kits (Miltenyi Biotec), according to the manufacturer’s protocols with a small modification (50 U/ml of mIL-2, instead of 1000 U/ml).
Cells were cultured in RPMI 1640 medium (Lonza, Houston, TX, USA) supplemented with 10% FBS, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 units penicillin-streptomycin (all from Gibco, Carlsbad, CA, USA), and 10 μg/ml gentamicin (Sigma-Aldrich, St. Louis, MO, USA). 293T cells were cultured in DMEM medium (Lonza) supplemented with 10% FBS 4.5g/l glucose, 2 mM L-glutamine, and 100 units penicillin-streptomycin.
Cell proliferation dye eFluor670 (eBioscience, 5 μM) labeled conventional CD4+ T cells (Tconv, 1.0×105) isolated from congenic B6. SJL mice were co-cultured with indicated number of FACS-sorted GFP+RFP+ retrovirally transduced Treg cells in a round-bottomed 96-well plate in the presence of 0.5 μg/ml of anti-CD3 and irradiated (3000 cGy) T cell-depleted splenocytes (1.0×105) for 3 days. The proliferation of the Tconv cells was measured based on eFluor670 dilution by the CD4+CD45.1+ cell population by flow cytometry.
FACS-sorted GFP+RFP+ retrovirally transduced Treg cells (3.0×105) were rested at 37°C for 2 hours in complete RPMI media. Cells were placed in the upper chamber [(Corning, Corning, NY, USA), Polycarbonate, 6.5 mm diameter, 5 μm pore size] containing 100 μl of complete RPMI media. The lower chamber was filled with 600 μl complete RPMI media containing various concentrations of CXCL13 (PeproTech, Rocky Hill, NJ, USA). After 4 hours of incubation, cells from the lower chamber were collected and the cell count was determined by running samples at a fixed flow rate (60 μl/min) for 1 min by FACS Calibur (BD Bioscience, San Jose, CA, USA). Migration index was calculated as follows: ((number of migrated cells/number of input cells)*100).
RV-empty vector or RV-
Wild-type or CXCR5−/− Treg cells (CD4+CD25hi, 5.0×105) were adoptively transferred into
In some experiments, RV-empty vector or RV-Cxcr5-transduced Treg cells (5.0×105) were adoptively transferred into
FACS-sorted RV-empty vector or RV-
To measure the levels of IgG produced by B cells
Total RNA was extracted from retroviral vector transduced cells (3.0×105 cells) with TRIzol (Invitrogen) and reverse transcribed using amfiRivert reverse transcriptase (GenDEPOT, Baker, TX, USA) according to the manufacturer’s protocol. Gene expression was measured with iTaq-SYBR Green Supermix (BIORAD Laboratories, Hercules, CA, USA) and the ABI-PRISM 7900 detection system (Applied Biosystems, Foster City, CA, USA). Data were normalized to expression of the
Data were analyzed with GraphPad Prism 5 (GraphPad, La Jolla, CA, USA). Statistics was calculated with the two-tailed Student’s
We and others previously demonstrated that follicular regulatory T cells (Tfr cells) expressing Foxp3 and CXCR5 regulates germinal center reactions (Chung
One of the key features of Tfr cells is its ability to emigrate into B cell follicles via CXCR5-mediated chemotaxis (Sage and Sharpe, 2015). Therefore, we hypothesized that enforced expression of CXCR5 onto Foxp3+ Treg cells would endow the ability of preferential migration to B cell follicles in the secondary lymphoid organs in response to the CXCL13 gradient. As a first step to test this hypothesis, we first cloned mouse
We next compared the expression of Treg cell-associated genes in the transduced cells. To this end, we purely isolated CD4+RFP+GFP+ Treg cells by flow cytometry four days after RV transduction using a sorting strategy described in Fig. 3A. As a result, we obtained >95% of RFP+GFP+ Treg cells from each transduction condition. As expected, RV-
Treg cells are known to be converted into conventional T cells in response to inflammatory cytokines by downregulating Foxp3 (Zhou
We next determined if the
We finally determined whether the
In the present study, we aimed to determine if ectopic expression of CXCR5 on Treg cells can induce Tfr cell properties. We demonstrate that (i) CXCR5-deficient Treg cells were inefficient in suppressing germinal center reactions, (ii) retroviral transduction of
Due to the low frequency of Treg cells in the peripheral blood of human patients, polyclonal Treg cells were expanded from precursor cells
Several preclinical studies proved that the inhibitory function of Ag-specific Treg cells is superior to that of polyclonal Treg cells in suppressing autoimmune diseases or graft rejection (MacDonald
There still reamins an issue with stability and persistence of the engineered Tfr cells for their clinical application. The stability of Treg cells
Compared to conventional Treg cells, Tfr cells highly express several costimulatory molecules including ICOS, PD-1, CTLA-4 and GITR (Chung
It still remains to be elucidated whether Tfr cells have unique characteristics other than CXCR5 expression that are essential for the regulation of the germinal center reaction. In this regard, Bcl6, a Tfh cell defining transcription factor, would be one of the attractive candidates for Tfr engineering to assure full differentiation of Tfr cells. Unfortunately, however, since Bcl6 transduction itself was not sufficient to induce CXCR5 expression in naïve T cells, it has been demonstrated that Bcl6 transduction alone could not initiate Tfh cell programing in naïve T cells (Crotty, 2011). A recent study demonstrated that achaete-scute homologue-2 (ASCL2) facilitates early Tfh differentiation by inducing upregulation of CXCR5 (Liu
In summary, we found that forced expression of CXCR5 endowed Tfr-like features in Treg cells. These engineered Tfr-like cells efficiently migrated along with CXCL13 gradient and effectively suppressed B cell antibody production. Our study provides new insights into the development of adoptive Treg cell immunotherapy for the regulation of autoimmune disorders.
We thank Mr. Inbo Shim for editing manuscript. This work was supported by research grants SNU invitation for distinguished scholar (to YC), 2014R1A2A1A11054364 (to YC) from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST), 1R01HL118361 (to YC and RAW) from National Institutes of Health.