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H. Schmid et al.
third-party donors also inhibited alloreactive T-cell activa- tion and proliferation (Figure 4E).
Discussion
Allogeneic HCT is an established therapeutic option for the treatment of advanced and high-risk hematologic malignancies. Efforts to optimize donor selection, tailored preparative conditioning regimes and advanced support- ive care have significantly contributed to improved out- comes and enabled long-term survival even in aged and comorbid patient populations. Nevertheless, GvHD and relapse still represent the most important reasons for sig- nificant morbidity and mortality after allogeneic HCT.1,2 Various strategies have been applied to prevent or treat GvHD such as immunosuppressive medications and in vivo or ex vivo donor T-cell depletion. However, these approaches are suboptimal since they also inhibit immune reconstitution, pathogen control and beneficial GvL effects, leading to higher relapse rates.23,34 Therefore, strategies that prevent GvHD while preserving the capac- ity of the graft to promote GvL effects are urgently need- ed.
A convincing body of evidence has demonstrated the potential of iNKT cells as a promising alternative for the prevention of GvHD in both mice and humans. Early murine studies demonstrated that the reinfusion of NK1.1+ T cells after transplantation resulted in GvHD sup- pression.25 In particular, low doses of CD4+ iNKT cells pre- vented GvHD lethality in mice by promoting the expan- sion of Tregs while maintaining GvL effects.14 We also showed previously that third-party iNKT cells are equipo- tent due to the highly conserved invariant TCR of iNKT cells.15,26 Based on these findings, we used iNKT cells from third-party donors in our present study. In humans, sever- al groups have shown that high numbers of iNKT cells were associated with a decreased incidence of GvHD.17,18,27 Malard et al. also showed in a study of 80 patients that high iNKT-cell numbers in the graft correlat- ed with an increased GvHD-free, relapse-free survival; however, the frequency of Tregs did not seem to correlate with iNKT-cell numbers.19 Moreover, Cheng et al. ana- lyzed Treg expansion after infusion of a-galactosylce- ramide (a-GalCer), a potent iNKT-cell stimulator, but expansion of Tregs could only be observed in a subset of patients.28 Thus, the role of Treg expansion as mediator of therapeutically used iNKT cells is not well established in humans and suggests further mechanisms that contribute to the immunoregulatory properties of iNKT cells.
GvHD can be characterized as a response of donor T cells to host antigens presented by MHC molecules through APC1: first, host APC become activated and pres- ent allo-antigens to donor T cells, which are stimulated and expand. Consequently, cellular effectors promote cell damage and apoptosis.1,8,29 In this context, several studies have emphasized the role of DC as potent APC in the pathogenesis of GvHD and therefore, they represent an interesting target for prophylactic and therapeutic strate- gies against GvHD.30,31 In the present study, we therefore focused on the cellular and humoral interplay of human culture-expanded iNKT cells with DC. Thereby, we could show that iNKT cells induce DC apoptosis and conse- quently, impair alloreactive T-cell activation and prolifera- tion. Liu and Coman reported similar findings previously
hypothesizing a relevant mechanism for the modulation of immune responses and GvHD suppression.32,33 We add significant knowledge by showing that preferential apop- tosis induction of cDC leads to a relative expansion of beneficial pDC. In contrast, we did not find significant functional differences regarding distinct iNKT-cell subsets.
iNKT cells are activated upon recognition of glycolipids presented by the MHC-I-like molecule CD1d, which is highly expressed on DC.11,34 Hence, T-cell receptor-CD1d engagement induces cytokine release by iNKT cells, which confers immunoregulatory properties and the abil- ity to orchestrate immune responses of several cell types. For instance, the release of cytokines such as IFN-g, tumor necrosis factor-a (TNF-a), interleukin-2 (IL-2), IL-4, IL-17 and IL-21 has been noted.13,22 Beyond immunoregulatory properties, iNKT cells exert potent direct cytotoxic effects using different pathways.13,20 In this context, stimulation via CD95 (Fas)35,36 and TRAIL pathways36,37 has been demonstrated, resulting in a classical lymphocytotoxic response against tumor cells. Moreover, several studies have shown that perforin/granzyme B is involved in iNKT-cell tumor cytotoxicity.38-40 Using different blocking reagents and specific antibodies, we could demonstrate that DC apoptosis induced by iNKT cells relies on degran- ulation of perforin, granzyme B and granulysin and par- tially on the interaction of the invariant T-cell receptor with CD1d .
DC originate from either myeloid or lymphoid hematopoietic stem cell progenitors in the bone mar- row.41,42 They constitute a heterogeneous cell group of dif- ferent subsets playing distinct roles in regulating immune responses.43 DC have been categorized in cDC, pDC and mo-DC, considering their lineage and expression of tran- scription factors such as IFN regulatory factors 8 and 4.44 In humans, cDC are potent producers of IL-12 and harbor excellent cross-priming properties. In the context of GvHD, cDC turned out to be important stimulators of alloreactive T-cell responses.45 Also, Markley et al. demon- strated that donor cDC are critical for allo-antigen presen- tation and consequently potentiate GvHD.46 Besides, cDC are most likely responsible for the replenishment of tissue- specific DC such as migratory Langerhans cells of the skin after inflammation and therefore might contribute to the occurrence and perpetuation of skin GvHD.47,48 In con- trast, the functional hallmark of pDC is the release of high quantities of type I and type III interferon (IFN) in response to viral antigen recognition.49,50 Interestingly, pre- cursor and fully differentiated pDC are associated with an improved outcome after allogeneic HCT due to a decreased incidence of GvHD and optimized GvL effects.51,52 Thus, the modulation of cDC and pDC by iNKT cells could represent a useful approach to reduce the incidence of GvHD.
In this study, we focused on human blood DC, which are mainly composed of cDC and pDC and can be easily obtained from healthy volunteers and GvHD patients. Our results suggest an additional mechanism of how human culture-expanded iNKT cells prevent GvHD: pref- erential apoptosis of cDC leads to a relative expansion of beneficial pDC. This bias results in decreased activation and proliferation of alloreactive T cells from healthy vol- unteers and GvHD patients. However, we could also observe a minor direct impact of iNKT cells on T- cell acti- vation and proliferation when higher numbers of iNKT cells were used. Given the high plasticity and functional
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haematologica | 2022; 107(2)