Mechanism of action of ECP in GvHD

Graft-versus-host disease (GvHD) is a major complication of allogeneic hematopoietic stem cell transplantation, arising when donor-derived immune cells recognize recipient tissues as foreign and mount an immune response against them.^1,2 Acute GvHD (aGvHD) typically develops within the first 100 days after transplantation, although onset can occur later, and most commonly affects the skin, liver, and gastrointestinal tract.¹ Chronic GvHD (cGvHD) generally develops later and can affect multiple organs, with clinical manifestations that may resemble autoimmune and fibrotic disorders.² Conventional treatment relies largely on systemic immunosuppression, which can increase susceptibility to infection and other long-term toxicities.^3,4 Management therefore requires a balance between suppression of harmful alloreactive immune responses while preserving the beneficial effects of donor-derived immune cells.⁵ This has driven increasing interest in immunomodulatory approaches that promote immune regulation, rather than broad immunosuppression.

Extracorporeal photopheresis (ECP) is an immunomodulatory leukapheresis-based procedure used in the treatment of aGvHD and cGvHD.^4,6 A summary of the clinical evidence supporting the use of ECP in GvHD, alongside the latest guidelines and recommendations, has been published previously on the GvHD Hub. In this article, we explore the proposed immunomodulatory mechanisms underlying the therapeutic effects of ECP in GvHD.

Principles of ECP

ECP involves collection of peripheral blood and separation of leukocytes from other blood components through centrifugation, while red blood cells and plasma are returned to the patient. The collected leukocytes are then exposed to 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light. Photoactivation of 8-MOP induces DNA crosslinking and other cellular changes that promote apoptosis in leukocytes. The treated cellular product is then reinfused into the patient (Figure 1).^4,6

ECP can be performed using either an open or closed system. In open systems, separate devices are used for cell separation, photoactivation, and reinfusion, whereas closed systems integrate these steps within a single device.^7,8

Immunomodulatory mechanisms of ECP

The precise mechanisms underlying the clinical effects of ECP in GvHD remain incompletely understood. Current evidence suggests that its effects arise from several immunomodulatory processes, including induction of leukocyte apoptosis, modulation of monocyte and dendritic-cell (DC) function, changes in cytokine profiles, and alterations in regulatory and effector immune-cell populations (Figure 2).^6,9

Induction of leukocyte apoptosis

Exposure of leukocytes to 8-MOP/UV-A induces apoptosis within 36–48 hours post-ECP through DNA crosslinking and subsequent cellular damage, with evidence suggesting that this may occur through upregulation of Fas and a resultant increase in Fas-initiated apoptotic signaling.^6,10,11 Following reinfusion, these apoptotic leukocytes are recognized and phagocytosed by antigen-presenting cells (APCs).¹¹ The resulting apoptotic-cell derived material, once phagocytosed, is thought to actively modulate the immune system, although the precise mechanisms remain incompletely understood.¹² As fewer than 10% of circulating leukocytes are directly exposed to 8-MOP/UV-A during an individual treatment, direct induction of leukocyte apoptosis alone is unlikely to fully explain the broader immunomodulatory effects of ECP.^6,13

Monocyte-to-DC differentiation

One proposed downstream effect of ECP is the differentiation of circulating monocytes towards a DC phenotype. Monocytes have a central role in processing apoptotic lymphocyte antigens and presenting them to the patient’s immune system.¹⁴ During passage through the ECP device, plasma fibrinogen can coat the inner surface of the treatment chamber. Platelets adhere to immobilized fibrinogen via their αIIbβ3 integrin and become activated, resulting in surface expression of P-selectin. Activated platelets can then interact with circulating monocytes through P-selectin and P-selectin glycoprotein ligand-1, promoting monocyte activation and entry into the DC differentiation pathway.^14–17 Evidence from experimental models has further demonstrated transcriptional changes in ECP-exposed monocytes consistent with differentiation towards specialized DC populations.^11,18

DCs take up and process antigens from apoptotic leukocytes before migrating to lymphoid organs to prime naïve T cells becoming tolerogenic or immunogenic antigen-specific effector cells. Accordingly, DC induced by ECP are considered important mediators of antigen-specific T-cell responses, contributing to the immunomodulatory effects of ECP through regulation of immune activation and tolerance.^6,19

Modulation of T-cell populations and cytokine profiles

Tolerogenic DCs, generated following the phagocytosis of apoptotic cells, are thought to promote the expansion and activation of regulatory T cells (Tregs).¹⁴ Tregs are a subset of CD4+ T cells that play a central role in maintaining peripheral immune tolerance and immune homeostasis. They can suppress alloreactive immune responses through multiple mechanisms, including modulation of APC co-stimulatory activity, cytolytic mechanisms, metabolic disruption, and secretion of anti-inflammatory cytokines, including interleukin (IL)-10, transforming growth factor beta (TGF-β), and IL-35.^6,20,21 ECP has also been associated with changes in cytokine profiles, including reductions in pro-inflammatory cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-2.^6,21 These findings are consistent with a shift towards a more regulatory immune environment.

In addition to promoting regulatory responses, ECP may influence effector T-cell populations involved in GvHD pathogenesis.⁶ CD4+ effector T cell lineages can be classified into T helper (Th)1, Th2, Th17, and Tfh cells, according to their transcriptional profiles, cytokine production, and chemokine receptor expression.^6,22 Th1 and Th17 cells predominantly produce pro-inflammatory cytokines and can contribute to tissue inflammation, whereas Th2-associated responses are generally considered more anti-inflammatory.^23,24 Long-term ECP therapy may modulate these effector T-cell populations, contributing to a more balanced Th-cell profile.^6,25

Together, these processes may promote immune tolerance by enhancing regulatory pathways and reducing pathogenic alloreactive responses, contributing to a more tolerogenic immune environment in patients with GvHD.^{4,6,11,14,25,26}

Potential effects on B cells and natural killer (NK) cells

ECP may also modulate immune-cell populations beyond DCs and T cells, although the contribution of these changes to its therapeutic effects remains under investigation. In patients with steroid-refractory/resistant GvHD, ECP has been associated with a reduction in CD19^hiCD20^hi B cells, a population implicated in cGvHD pathophysiology, alongside an increase in regulatory B cells, which have established immunoregulatory properties.²⁷ Similarly, a study in patients with steroid-refractory/resistant aGvHD found that ECP was associated with changes in the NK-cell compartment, including a reduction in CD56^briCD16⁻ NK cells with a shift towards a more regulatory phenotype, alongside maturation of CD56^dim cells.²⁸ Further studies are needed to determine whether these changes directly contribute to the immunomodulatory effects of ECP.

Remaining knowledge gaps

Despite increasing understanding of the immunomodulatory effects of ECP, important knowledge gaps remain. Further studies are needed to further elucidate the immune pathways underlying its clinical efficacy, alongside the identification and validation of predictive biomarkers that could help identify patients most likely to respond to treatment.^6,11 Circulating micro RNAs have emerged as potential biomarkers of GvHD and response to ECP, but further research is needed to determine their predictive value and clinical applicability.^29,30 It is also important to determine the extent to which observed immunological changes can be attributed to ECP itself, as changes in immune cell composition may also reflect fluctuations in GvHD activity or the effects of steroid withdrawal.⁶

Conclusion

ECP represents a distinct immunomodulatory therapeutic approach in GvHD, with proposed effects spanning leukocyte apoptosis, modulation of monocyte and DC function, changes in cytokine profiles, and alterations in regulatory and effector immune-cell populations. Although the precise mechanisms underlying its clinical efficacy remain incompletely understood, the available evidence supports a model in which these interconnected processes contribute to modulation of the alloreactive immune responses that drive GvHD, while promoting a more regulatory immune environment. Further elucidation of these pathways and identification of predictive biomarkers may enable more personalized treatment approaches and optimization of ECP use in GvHD.

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