Often, clinicians and scientists know the mechanism of how a drug or intervention works by the time it makes it into patients in the form of a clinical trial. Yet sometimes, an intervention is shown to have an effect in a clinical trial before the ‘how’ is uncovered. This type of study can prove to be an interesting path to understanding a biological process, as the investigators have to unweave the mystery of how or why something works. This was the case for the recently published article in the Journal of Clinical Investigation by Dr. Ping Zhang and Dr. Hill in the Translational Science and Therapeutics Division. Dr. Hill’s research group, whilst at the QIMR Berghofer Medical Research Institute in Australia, had completed a phase I/II clinical trial and Dr Tey there noted that blockade of the immune protein (cytokine) interleukin (IL)-6 was associated with reduced CMV reactivation in hematopoietic stem cell transplantation (HSCT)/bone marrow transplantation (BMT) patients. Noting this observation, “[a] collaboration subsequently started among three research labs (Tey, Degli-Esposti and Hill) to validate this finding and investigate potential mechanisms, using pre-clinical models and analyses of patient samples,” commented Dr. Hill.
Reactivation of a latent CMV infection is a common complication of clinical BMT and can result in significant disease and in severe cases, death. It has been long recognized that the presence of CMV-specific T cells after BMT is associated with decreased risk for reactivation. Furthermore, the development of graft-versus-host-disease (GVHD) has been associated with impaired cellular immune reconstitution and increased risk for CMV reactivation. The research team had previously shown that IL-6 is a dominant and dysregulated cytokine following BMT that can contribute to GVHD. The findings from the initial clinical trial, combined with another recent discovery from Drs. Hill and Degli-Esposti’s groups demonstrating the protective effect of CMV-specific antibody on CMV reactivation (Martins et al., Science 2019), led them to assess how IL-6 may be acting in this context.
To first address how IL-6 may be contributing to CMV reactivation, the researchers knocked out the IL-6 receptor (IL-6R) on T cells (referred to as Il6r-/- mice from now on) and transplanted cells from these donors into recipient mice that were latently infected with murine CMV (MCMV). As might be predicted from the clinical study, reactivation of MCMV was significantly reduced in mice lacking IL-6R on T cells. Looking to gain further mechanistic insights, they assessed T cell responses in their model but found no overt differences in MCMV-specific T cell numbers or function. Interestingly, they did observe improved reconstitution of donor B cells and plasma cells from donors in their Il6r-/- mice, but by using B cell deficient donors, they found that these donor-derived B cells did not contribute to protection from MCMV reactivation. One of the next observations the team made was that there were significantly higher levels of MCMV-specific Immunoglobulin G (IgG), a type of protective antibody, in recipients of Il6r-/- cells and noted that these antibodies were recipient-derived. This suggested that persistence of recipient humoral (antibody-based) responses was a dominant immune pathway impacted by IL-6 signaling after BMT. They next confirmed that this effect was not a result of a persistence of recipient B cells or plasma cells (which make the antibody). They thus assessed the kinetics of IgG in the circulation following BMT with or without IL-6 signaling. By administering exogenous IgG, they noted that it was more rapidly cleared in mice who received T cell replete BMT compared to those who did not, but in their Il6r-/- mice, the IgG had a longer half-life than mice with intact IL-6 signaling. These findings were exciting for the team, as Dr. Zhang explained “It was very unexpected that IL-6 inhibition could preserve CMV-specific IgG.”
To start unraveling the mystery of how IgG degradation was enhanced in the presence of IL-6 signaling, they looked for potential explanations that were independent of IgG production. The team knew that IgG homeostasis was regulated by the neonatal Fc receptor (FcRn), which binds IgG that has been internalized during normal cellular uptake, a process which recycles it back to the surface of the cell. If IgG is not bound to FcRn, it is targeted for degradation. In their model, they found that FcRn was most abundantly expressed on endothelial cells (ECs) and to a lesser extent, macrophages, and they also showed that these cells had high capacity for IgG uptake. When they performed BMT in Fcrn-/- recipient mice, meaning recipient mice lacked FcRn, including in nonhematopoietic cells (i.e. ECs), IgG half-life was markedly shortened. In that experiment, as well as mice where FcRn was temporarily inhibited, they observed increased reactivation of MCMV. Intriguingly, BMT was associated with decreased EC viability and FcRn activity, which was partially restored if donor T cells lacked IL-6R signaling, highlighting to the researchers that EC injury was resulting in decreased FcRn-mediated IgG recycling. They next investigated the link between IL-6 signaling and EC injury, finding that loss of IL-6R in T cells was associated with a decrease in the long term expression of the T cell derived cytokine IFNg. When they performed BMT with Ifny-/- T cells, they found that these mice had decreased EC injury, and increased FcRn and IgG levels. Impressively, moving their data back into patients, they had also completed a Phase III clinical trial assessing IL-6 blockade with tocilizumab, , an antibody therapeutic that binds to and blocks the IL-6R and is already used to treat arthritis, to study effects on GVHD. They found that for patients who were at risk of CMV reactivation, those receiving tocilizumab had lower rates of reactivation and higher levels of CMV-specific IgG, consistent with the preclinical data.
Overall, this study showcases an impressive body of work that encompasses clinical observations, translational mechanistic studies, and subsequent clinical validation. Dr. Hill explained that performing clinical studies can be a challenging endeavor, “It can be highly rewarding but requires considerable effort and patience.” But their hard efforts resulted in a new understanding of the connection between cytokines produced after BMT and how they mediate antibody degradation and subsequent reactivation of CMV. Additionally, and something many of us strive for with our work, they have uncovered clinically targetable components, as Dr. Zhang emphasized that “cytokine inhibition (IL-6 or IFNg, in this case) is a rapidly translatable approach to prevent endothelial injury, antibody loss and viral reactivation after HSCT.” The research team is interested in whether this pathway is involved in CMV reactivation after CAR-T cell therapy, where cytokine release syndrome is common and dominated by IL-6 and IFNg production, highlighting how this study has generated new questions that may be broadly applicable to other forms of immunotherapy.
Zhang P, Fleming P, Andoniou CE, Waltner OG, Bhise SS, Martins JP, McEnroe BA, Voigt V, Daly S, Kuns RD, Ekwe AP, Henden AS, Saldan A, Olver S, Varelias A, Smith C, Schmidt CR, Ensbey KS, Legg SR, Sekiguchi T, Minnie SA, Gradwell M, Wagenaar I, Clouston AD, Koyama M, Furlan SN, Kennedy GA, Ward ES, Degli-Esposti MA, Hill GR, Tey SK. IL-6-mediated endothelial injury impairs antiviral humoral immunity after bone marrow transplantation. J Clin Invest. 2024 Apr 1;134(7):e174184. doi: 10.1172/JCI174184. PMID: 38557487; PMCID: PMC10977988.
This spotlight work was supported by the National Health and Medical Research Council (NHMRC) of Australia, QIMRB, EverGreen, and the NIH.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Scott Furlan and Geoffrey Hill contributed to this work.