The safe application of new strategies for the treatment of graft-versus-host disease (GVHD) is hampered by the lack of a clinically relevant model for preclinical testing. Current models are based on intraperitoneal transfer of human peripheral blood mononuclear cells (huPBMCs) into NOD-SCID (nonobese diabetic-severe combined immunodeficient)/SCID mice. Intravenous transfer would be preferred but this has always been ineffective. We developed a new model for xenogeneic GVHD (X-GVHD) by intravenous transfer of huPBMCs into RAG2 ؊/؊ ␥c ؊/؊ mice. Our results show a high human T-cell chimerism of more than 20% (up to 98%) in more than 90% of mice, associated with a consistent development of X-GVHD within 14 to 28 days and a total mortality rate of 85% shorter than 2 months. After murine macrophage depletion, engraftment was earlier and equally high with lower doses of huPBMCs. Human macrophages were also absent in these mice. Purified huCD3 ؉ cells showed a similar X-GVH effect with contribution of both CD4 and CD8 phenotypes. Human immunoglobulins and cytokines were produced in diseased mice. One of 30 mice developed chronic X-GVHD with skin histology similar to human GVHD. In conclusion, we present a new model for X-GVHD by intravenous transfer of huPBMCs in RAG2 ؊/؊ ␥c ؊/؊ mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this model.

Purpose:The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20 + follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. Experimental Design: We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximabmediated CDC, ADCC, and the combination. Results: We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. Conclusions:These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary.These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20 + tumors.

Purpose: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4 + CD25+ regulatoryTcells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell^dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2 administration of human peripheral blood mononuclear cells (PBMC).Experimental Design: Human PBMC, depleted of or supplemented with autologous CD25 + Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of humanTcells, and secretion of human cytokines were monitored at weekly intervals. Results: Depletion of CD25 + cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25 + cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4 + and CD8 + Tcells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-g, suggesting the de novo development of TR1cells. Conclusions:These results show, for the first time, the potent in vivo capacity of naturally occurring humanTregs to control GvHD-inducing autologousT cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring humanTregs.

Purpose: Unleashing the immune system by PD-1 and/or CTLA-4 blockade can cause severe immune-related toxicity necessitating immunosuppressive treatment. Whether immunosuppression for toxicity impacts survival is largely unknown.Experimental Design: Using data from the prospective nationwide Dutch Melanoma Treatment Registry (DMTR), we analyzed the association between severe toxicity and overall survival (OS) in 1,250 patients with advanced melanoma who were treated with immune checkpoint inhibitors (ICI) in first line between 2012 and 2017. Furthermore, we analyzed whether toxicity management affected survival in these patients.Results: A total of 1,250 patients were included, of whom 589 received anti-PD1 monotherapy, 576 ipilimumab, and 85 combination therapy. A total of 312 patients (25%) developed severe (grade ≥3) toxicity. Patients experiencing severe ICI toxicity had a significantly prolonged survival with a median OS of 23 months compared with 15 months for patients without severe toxicity [hazard ratio (HR adj ) ¼ 0.77; 95% confidence interval (CI), 0.63-0.93]. Among patients experiencing severe toxicity, survival was significantly decreased in patients who received anti-TNF AE steroids for steroid-refractory toxicity compared with patients who were managed with steroids only (HR adj ¼ 1.61; 95% CI, 1.03-2.51), with a median OS of 17 and 27 months, respectively.Conclusions: Patients experiencing severe ICI toxicity have a prolonged OS. However, this survival advantage is abrogated when anti-TNF is administered for steroid-refractory toxicity. Further prospective studies are needed to assess the effect of different immunosuppressive regimens on checkpoint inhibitor efficacy.

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