Tocilizumab increases regulatory T cells, reduces natural killer cells and delays graft‐versus‐host disease development in humanized mice treated with post‐transplant cyclophosphamide

Abstract

Graft-versus-host disease (GVHD) is a life-threatening complication following donor hematopoietic stem cell transplantation, where donor T cells damage host tissues. This study investigated the effect of tocilizumab (TOC) combined with post-transplant cyclophosphamide (PTCy) on immune cell engraftment and GVHD development in a humanized mouse model. NOD-scid-IL2Rc null (NSG) mice were injected intraperitoneally with 2 9 10 7 human (h) peripheral blood mononuclear cells and cyclophosphamide (33 mg kg À1 ) or saline on days 3 and 4, then TOC or control antibody (0.5 mg mouse À1 ) twice weekly for 28 days. Mice were monitored for clinical signs of GVHD for either 28 or 70 days. Spleens and livers were assessed for human leukocyte subsets, and serum cytokines and tissue histology were analyzed. In the short-term model (day 28), liver and lung damage were reduced in PTCy + TOC compared with control mice. All groups showed similar splenic hCD45 + leukocyte engraftment (55-60%); however, PTCy + TOC mice demonstrated significantly increased (1.5-2-fold) splenic regulatory T cells. Serum human interferon gamma was significantly reduced in PTCy + TOC compared with control mice. Long-term (day 70), prolonged survival was similar in PTCy + TOC (median survival time, > 70 days) and PTCy mice (median survival time, 56 days). GVHD onset was significantly delayed in PTCy + TOC, compared with TOC or control mice. Notably, natural killer cells were reduced (77.5%) in TOC and PTCy + TOC mice. Overall, combining PTCy with TOC increases regulatory T cells and reduces clinical signs of early GVHD, but does not improve long-term survival compared with PTCy alone.

Graft‐versus‐leukaemia immunity is retained following treatment with post‐transplant cyclophosphamide alone or combined with tocilizumab in humanised mice

“…with 1 × 10 7 human peripheral blood mononuclear cells (hPBMCs) from donors 1 (D1), 2 (D2) and 3 (D3) and intravenously (i.v.) with 1 × 10 6 THP‐1 cells were collected at day 42 or ethical endpoint and analysed by flow cytometry using an established gating strategy 23 . Data from mice injected with hPBMCs from the donor that was unable to elicit a GVL response (No GVL) were compared to those injected with hPBMCs from donors that did establish a GVL response (GVL).…”

“…After establishing a humanised mouse model of GVL, we next aimed to examine whether the GVL response was maintained following treatment with PTCy at days 3 and 4. PTCy has been shown to reduce GVHD in a humanised mouse model 12,23,30 but studies examining the impact of PTCy on GVL immunity and the mechanisms behind GVL responses are limited.…”

“…Survival outcomes of both hPBMC and hPBMC + PTCy mice were significantly shorter than Ctrl mice ( P < 0.001 and P = 0.003, respectively). Given the clinical data above, time to GVHD onset, defined as a clinical score ≥ 3, 23 was evaluated. Time to GVHD onset was significantly delayed in hPBMC + PTCy mice compared to hPBMC mice (30 days vs 21 days, respectively) ( P = 0.006) (Figure 4e).…”

“…In one study, the hCD4 + :hCD8 + ratio was increased at endpoint in mice injected with 2 × 10 7 hPBMCs and treated with PTCy compared to mice treated with PBS 12 . In a second study, the hCD4 + :hCD8 + ratio was not impacted in mice injected with 2 × 10 7 hPBMCs and treated with PTCy, using the same dose and route of administration, either early in disease or at endpoint 23 . Future studies should seek to inject isolated hCD4 + or hCD8 + T cells into mice to assess the impact of each T cell subset in mediating GVL responses and to assess the impact of PTCy on each of these subsets alone.…”

“…Clinically, the use of TOC as a prophylactic therapy for GVHD has had limited success, where a single 8 mg kg −1 dose on day –1 with cyclosporine/methotrexate did not significantly reduce acute GVHD or improve survival outcomes 22 . However, we have previously shown that combining PTCy with TOC delays GVHD onset, reduces weight loss and improves survival in humanised mice 23 but the impact of this combination therapy on GVL immunity is yet to be explored.…”