Therapeutic regulatory T-cell adoptive transfer ameliorates established murine chronic GVHD in a CXCR5-dependent manner

Chronic graft-versus-host disease (cGVHD) is the primary cause of long-term morbidity and mortality after allogeneic hematopoietic stem cell transplantation.¹  The germinal center (GC) reaction between T-follicular helper cells (Tfh) and GC B cells plays a critical role in cGVHD pathogenesis, and inhibition of this reaction significantly reduces cGHVD in mouse models.^2,3  A specialized subset of CD4+Foxp3+ regulatory cells (Tregs), T-follicular regulatory cells (Tfr), migrates to lymphoid follicles where they help quell GC reactions.⁴  However, Treg frequency is reduced in cGVHD patients,⁵  and this may contribute to cGVHD pathogenesis.^2,6  Low-dose interleukin-2 (IL-2) therapy increases Tregs in some cGVHD patients, but does not always reverse all symptoms, and long-term dosing is required to maintain efficacy.^7,8 

IL-2 complexed with the JES6-1 clone of anti-IL-2 antibody (IL-2/monoclonal antibody [mAb] complexes) has a longer in vivo half-life compared with IL-2 alone.⁹  These complexes preferentially bind to CD25^hi cells, which results in Treg expansion in a variety of disease models.^9-11  As a result, IL-2/mAb complexes may be superior to IL-2 for Treg expansion in cGVHD. Treg infusions also increase Treg numbers, and, unlike IL-2-based therapies, only a single dose may be required.^12,13  Prophylactic Treg infusions appear to reduce acute GVHD (aGVHD),¹⁴  but the efficacy of therapeutic Treg infusions in cGVHD has not yet been fully assessed.¹⁵  In this study, we analyzed the therapeutic efficacy of IL-2/mAb complexes and Treg infusions for preventing and treating cGVHD.

C57BL/6 (B6) (Charles River), B10.BR, and B6-CXCR5^−/− (Jackson Laboratory) mice were housed in a pathogen-free facility and used with Institutional Animal Care Committee approval. B6→B10.BR (cGVHD) and B6→BALB/c (aGVHD) models, including disease severity assessments, were used as described.^16-18  For cGVHD, cyclophosphamide-treated (120 mg/kg/d, day −3, −2), irradiated (8.3Gy, day −1) recipients received B6 bone marrow (BM) ± 0.75 × 10⁵ conventional T cells (Tcon) on day 0, ±0.5 × 10⁶ Tregs on day 0 or day 28. For aGVHD, irradiated (7Gy, day −1) BALB/c recipients were given B6 BM ± 2 × 10⁶ Tcon ± 1 × 10⁶ Tregs on day 0. Tcon and Tregs were purified as described.¹⁹  IL-2 (0.5 µg)/JES6-1 anti-IL-2 mAb (25 µg) complexes were injected intraperitoneally days 0-3 (aGVHD) or days 28-56 (cGVHD).

Flow cytometry for Tfh, Tfr, and GC B cells, immunofluorescence, and histopathology scoring were performed as described.^16,20  Pulmonary function tests assessing cGVHD-associated bronchiolitis obliterans syndrome (BOS) were performed as described.¹⁶ 

Consistent with patient data,^5,21  cGVHD mice have significantly fewer Tregs and Tfr and more Tfh compared with no cGVHD (BM only) recipients (Figure 1A-C). Daily therapeutic dosing of IL-2/mAb complexes (days 28-56) increased Treg and Tfr levels (Figure 1A-B), reduced Tfh (Figure 1C) and tissue pathology scores (Figure 1D-E), and ameliorated cGVHD-associated BOS lung dysfunction¹⁶  (Figure 1F-H). Survival range was 90% to 100% (90% IL-2/mAb complex group), and neither survival nor weights differed among groups (not shown). These data suggest that therapeutic injections of IL-2/mAb complexes can expand Tregs, including Tfr, and reverse established cGVHD.

As some cGVHD patients can have aGVHD overlap syndrome,²²  we next assessed the efficacy of IL-2/mAb complexes in aGVHD. A 4-day course (days 0-3) of IL-2/mAb complexes accelerated mortality when given alone and when given in combination with otherwise highly effective Treg infusions (Figure 1I). IL-2/mAb complexes also increased severity of disease, as indicated by rapid recipient weight loss and increases in clinical GVHD scores from days 3-7 in mice given IL-2/mAb complexes alone, and days 13-17 when given with Tregs (Figure 1J-K). This exacerbation of aGVHD severity was due to expansion of CD25^hiCD4+ and CD8+Tcon (Figure 1L-M). Therefore, although IL-2/mAb complexes may be effective in some cGVHD patients, they may not be ideal for cGVHD patients with aGVHD overlap, as IL-2/mAb complexes may expand CD25^hi Tcon and overwhelm Treg expansion and GVHD suppression.

Because some, but not all, cGVHD patients may respond well to IL-2/mAb complexes, we tested Treg infusions in cGVHD. Treg infusions were given either therapeutically (day 28), or prophylactically (day 0). A single infusion with wild-type (WT) Tregs on day 28 or day 0 significantly reduced cGVHD-associated BOS (Figure 2A-C) and increased Treg frequency, including Tfr (Figure 2D). Tfh and GC B-cell frequency, GC size, and tissue pathology scores were also significantly reduced in mice given WT Tregs (Figure 2E-J). Survival range was 85% to 100%, and neither survival nor weights differed among groups (not shown).

Given the importance of the GC reaction in cGVHD pathogenesis,²³  the finding that both therapeutic and prophylactic Treg infusions increased Tfr and reduced Tfh, GC B cells, and GC size suggests that Tregs may ameliorate or prevent cGVHD through GC reaction inhibition. The chemokine receptor CXCR5 is required for Tregs to home to GCs and inhibit B-cell responses.²⁴  To determine whether homing to the GC was required for therapeutic or prophylactic Treg efficacy, we compared CXCR5 knockout (CXCR5KO) with WT Tregs. We hypothesized that CXCR5KO Tregs may be less effective than WT. However, like WT, CXCR5KO Tregs given prophylactically were able to prevent cGVHD-associated lung dysfunction (Figure 2A-C), increase Tfr, and reduce Tfh, GC B cells, GC size, and tissue pathology scores (Figure 2D-J). In contrast, CXCR5KO Tregs given therapeutically had no effect on lung function, Tfh, or GC B cells, and minimal effect on GC size or tissue pathology scores (Figure 2A-C,E-J). This lack of efficacy correlated with an absence of Tfr niche restoration, as Tfr levels were similar to cGVHD mice (Figure 2D). These data indicate that Tregs can suppress alloreactive T-cell responses required for cGVHD generation prior to the establishment of GC reactions, but once GCs are formed, Tregs must home to the GC to interrupt the pathogenic process of producing antibody-secreting cells.

In conclusion, our data indicate that both therapeutic Treg infusions and IL-2/mAb complexes can increase Tregs and treat established cGVHD. The therapeutic efficacy of Treg infusions and IL-2/mAb complexes may be driven by Treg-mediated inhibition of the GC reaction. Future Treg-modifying therapies in cGVHD may be augmented by targeting Treg homing to the GC, especially in patients with confirmed auto- or alloantibodies.²⁵  Importantly, whereas Treg infusions may useful for all cGVHD patients, including those with aGVHD overlap syndrome, IL-2/mAb complexes may need to be used with more caution. Even though IL-2/mAb complexes have the capacity to expand Tregs in cGVHD, our data suggest that they also have the potential to expand activated CD25^hi Tcon, and thus, may best be reserved for cGVHD patients with no aGVHD overlap. Alternatively, lower doses or different dosing schedules of IL-2/mAb complexes could result in less Tcon expansion, which could increase the therapeutic index for cGVHD patients, including those with aGVHD overlap.

Regardless, Treg infusions could prove to be advantageous over IL-2-based therapies, because IL-2-based approaches require long-term treatment to maintain efficacy, whereas Tregs might require more limited dosing. Considering that Tregs from a single autologous Treg infusion have been shown to persist at least 1 year in type 1 diabetes patients,¹³  it is possible that a single Treg dose would be sufficient. However, the half-life of infused Tregs in cGVHD is unknown, and long-term Treg survival may be limited by concurrent use of immunosuppressive agents that minimize IL-2 bioavailability. Therefore, it may be worthwhile in future studies to explore infusions of either nonenriched or CXCR5-enriched Tregs combined with low-dose IL-2, or other approaches that deliver IL-2 to drive Treg, but not Tcon, proliferation. Alternatively, Tregs and IL-2-based treatments could be given sequentially in cGVHD patients, including those with aGVHD overlap, treating first with Tregs, to ensure dampening of any aGVHD-like components, followed by IL-2-based therapies as a novel, potentially efficacious approach.

The authors thank Colby Feser for tissue preparation.

This study was funded by the National Institutes of Health, National Cancer Institute grants P01 CA142106-08A1 (B.R.B.) and R01 CA183560 (J.R.); National Institute of Allergy and Infectious Diseases grants P01 AI056299-13 (B.R.B.) and T32 AI007313 (C.M.-H. and R.F.); National Heart, Lung, and Blood Institute F30 HL121873 (C.M.-H.); and Leukemia and Lymphoma Society Translational Research grants 6458-15 (B.R.B.) and 6462-15 (I.M. and B.R.B.).

Contribution: C.M.-H. and R.F. designed and performed experiments, discussed results, and wrote the paper; A.P.-M. and N.P. performed histologic analyses and edited the paper; K.P.A.M., G.R.H., L.L., J.S.S., W.J.M., I.M., D.H.M., L.A.T., J.K., C.S.C., J.H.A., R.J.S., and J.R. discussed results and edited the paper; B.R.B. contributed to experiments, discussed results, and edited the paper.

Conflict-of-interest statement: The authors declare no competing financial interests.

Correspondence: Bruce R. Blazar, MMC 109, University of Minnesota, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: blaza001@umn.edu.