Abteilung für Molekulare Innere Medizin (in der Medizinischen Klinik und Poliklinik II)
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In the initiation phase of acute graft-versus-host disease (aGvHD), CD4+ T cells are activated by hematopoietic antigen presenting cells in secondary lymphoid organs whereas in effector phase by non-hematopoietic cells in the small intestine. We hypothesized that alloreactive CD4+ T cells primarily home to the secondary lymphoid organs subsequent to allogeneic hematopoietic cell transplantation in the initiation phase of aGvHD and are activated by the non-hematopoietic lymph node stromal cells via MHC class II. To test this hypothesis, we employed CD4+ T cell-dependent major mismatch aGvHD mouse model to study this correlation.
Upon analyzing the early events following allo-HCT with bioluminescence imaging, flow cytometry and whole-mount light sheet fluorescence microscopy, we found that allogeneic T cells exclusively home to the spleen, lymph nodes and the Peyer’s patches and not to the intestinal lamina propria in the initiation phase of aGvHD. Utilizing mice devoid of partial or complete hematopoietic antigen presentation we could show allogeneic CD4+ T cells activation in the lymphoid organs of MHCIIΔCD11c and MHCIIΔ BM chimeric mice early after allo-HCT. MHCIIΔ BM chimeras failure of thymic negative selection and developing tissue wasting disease upon syn-HCT deemed them unsuitable to study non-hematopoietic antigen presentation in aGvHD. To overcome this challenge, we generated MHCIIΔVav1 mice that lack MHC class II expression on all hematopoietic cells. MHCIIΔVav1 mice were susceptible to aGvHD and LNSCs from these animals activated allogeneic CD4+ T cells in mixed lymphocyte reaction. Likewise, mesenteric lymph nodes from CD11c.DTR mice surgically transplanted into a MHCIIΔ mouse could activate CD4+ T cells in vivo, clearly demonstrating LNSCs as non-hematopoietic APCs of the lymphoid organs.
We specifically target lymph node stromal cell subsets via the Cre/loxP system, we employed single cell RNA sequencing and selected Ccl19 and VE-Cadherin to specifically target the fibroblastic reticular cells and endothelial cells of the lymph nodes respectively. In MHCIIΔCcl19 mice, alloreactive CD4+ T cells activation was discreetly reduced in the initiation phase of aGvHD whereas absence of MHCII on fibroblastic reticular cells resulted in hyper-activation of allogeneic CD4+ T cells leading to poor survival. This phenotype was modulated by the regulatory T cells that were able to rescue H2-Ab1fl mice but not the MHCIIΔCcl19 subsequent to GvHD.
Knock-out of MHCII on endothelial cells MHCIIΔVE Cadherin, resulted only in modest reduction of CD4+ T cells activation in the initiation phase of GvHD, conversely MHCIIΔVE Cadherin mice showed a protective phenotype compared against littermates H2-Ab1fl mice in long-term survival. Furthermore, to pin-point endothelial cells MHCII antigen presentation we generated MHCIIΔVE Cadherin ΔVav1 animals devoid of antigen presentation in both endothelial and hematopoietic compartments. LNSCs from MHCIIΔVE Cadherin ΔVav1 were unable to activate alloreactive CD4+ T cells in mixed lymphocyte reaction.
Altogether, we demonstrate for the first time that MHC class II on the lymph node stromal cells plays a crucial role in the modulation of allogeneic CD4+ T cells in the initiation and later in the effector phase of graft-versus-host-disease.
The TRAF-binding receptor CD40 belongs to the TNFR superfamily and is broadly expressed on healthy cells, mainly on antigen-presenting cells, but also on other immune cells and non-immune cells. CD40 is bound by its ligand CD40L, which is essential for a wide range of immunological responses by inducing or inhibiting different pathways that are essential for a variety of cellular processes, including immune activation and maturation. (1,2) Dysregulated CD40 signalling has been implicated in inflammatory diseases, such as hyper-IgM syndrome, psoriasis, and cancer. (3–6) Due to its broad expression across various tumour types, it can serve as a tumour-associated antigen and has therefore been proposed as a target for antibodies for cancer treatment. (2,7,8)
Agonistic anti-CD40 antibodies have been demonstrated to induce anti-tumoural immune responses as well as therapeutic immunity. (2) Furthermore, prolonged stimulation of CD40 in tumour cells in vitro has been shown to decrease proliferation, increase expression of cytotoxic TNFSFLs and induce apoptosis. (9,10) Their effect on anti-tumoral responses has been well studied and anti-tumoral responses by DC maturation and suppression of malignant growth of B-cells have been confirmed and were found to induce cell death in tumours in vitro. (11–14)
Many agonistic anti-CD40 antibodies specifically have been reported to require secondary crosslinking by binding to either activating or inhibitory FcγRs to be agonistic in vitro, while in vivo studies have indicated inhibitory FcƴR2B expression as critical factor. (15–17) However, FcƴR independent agonism has also been reported for anti-CD40 antibodies. (18,19) While agonistic anti-CD40 IgG1, IgG3 and IgG4 antibodies have been shown to display FcƴR dependent agonism, agonistic anti-CD40 IgG2 antibodies have shown to display FcƴR independent agonism. Conversion of anti-CD40 IgG1 antibodies into IgG2 has also been shown to convert the antibody’s agonism into FcƴR independent agonism. (20)
To overcome FcƴR dependency, bispecific antibody fusion proteins containing a scFv as anchoring domain allowing for crosslink independent of FcƴR binding have been designed before. This approach has been found to display strong agonism for other antibody fusion proteins when bound to both targets, with response levels resembling that of FcƴR bound antibodies. (21,22)
The relevance of antibody isotype and idiotype for FcƴR-dependent agonism as well as the relevance of valency and antibody oligomerization for FcƴR-independent agonism were investigated in this study on a panel of different anti-CD40 antibodies. Several clinically investigated anti-CD40 antibodies (ADC-1013(23), APX005M(24), ChiLob7.4(25) and CP-870,893(26)) and one preclinical antibody (G28.5(27,28)) were considered. Selected antibodies were then cloned onto an IgG1, IgG1(N297A), IgG2 and IgG4 backbone. The IgG1(N297A) isotype is an IgG1 antibody with a point mutation (N297A) that is known to strongly reduce binding to FcƴR1, while reducing the binding affinity to FcƴR2B to undetectable levels. (29,30) In this work it is demonstrated that the investigated anti-CD40 antibody variants across different isotypes activate both the classical and alternative NFκB pathway by stimulating U2OS cells in an FcƴR dependent manner. Stimulation in the presence of both human FcƴRs as well as murine FcƴRs resulted in CD40 stimulation. A difference in binding competition was observed for the various anti-CD40 IgG1 antibodies, but no indication of a CRD-dependent mechanism responsible for their agonistic activity was found. Moreover, this FcƴR dependency could be overcome by creation of tetravalent antibody fusion proteins.