B cells and regulatory T cells in Graft versus Host Disease: a clinicopathological study in humanized mice

Morbidity and mortality are major concerns in graft-versus-host disease (GvHD), a response often seen after hematologic cell transplantation. Treatment of GvHD remains difficult and more knowledge is necessary. In this thesis we tried to contribute a small piece to the puzzle of GvHD by analyzing the possible relation between B cells and regulatory T cells (Tregs), from a primarily histological perspective. Humanized mice may serve as promising models in studies to GvHD. Designing the right model for the topic of interest is however subject to many choices. Based on our thorough review of the pearls and pitfalls from different mouse strains, transfer routes and sources of material, we decided that RAG2-/-γc-/- mice with intravenous transfer of huPBMC’s would be a sensible choice for our experiments. A thorough analysis was performed to evaluate the basic kinetics and pathology of our model. The induced GvH response in our model appeared to show many similarities with the sclerotic variant of human GvHD. Our analysis continued with an evaluation of our model with additional modifications: macrophage depletion and early B cell depletion. Macrophage depletion accelerated the induction and morbidity of the clinical GvH response and histologically increased the number of B-cells in the infiltrating cell signature, with only limited changes to the amount of Tregs. In early B cell depletion however, a clear improvement on survival was seen. Despite clinical improvement though, our histology only showed a significant effect on liver fibrosis and human TGF-β production within the liver. Other affected organs did not show signs of improvement or alterations. Our next exploration analyzed the effect of B cell activation on Tregs, using a combination of multiple in vitro studies with an in vivo study. Although based on our in vitro studies additional Tregs should temper the GvH reaction (even after B-cell activation), our in vivo study with myeloma was suprising. Clinical symptoms and survival of our mice did improve with additional Tregs, but no significant improvement was shown on a histological level. This contradiction between clinical symptoms and histology is known from human GvHD, as we continued to show with our last evaluation in this thesis, regarding human cutaneous GvHD. In this last analysis however, we’ve found that nowadays neither clinical grading nor histological grading seems to accurately predict a patients survival. With regard to the aim of these thesis, three major conclusions/considerations come forward. First, B cells and Tregs in GvHD have a complex relation, which is subject to the activation of B cells. Second, the lack of correlation between histology and clinical symptoms in GvHD might be explained by cytokines. Last but not least, we need to consider that Rituximab might serve best as a treatment for GvHD when used as an early B cell depletion therapy. However, since there might be a huge contrast between clinical symptoms and histology, we need to decide on our goal of therapy first: do we treat the patient or the disease? The answer to this, will ultimately decide whether our therapy really works

B cells and regulatory T cells in Graft versus Host Disease: a clinicopathological study in humanized mice

Morbidity and mortality are major concerns in graft-versus-host disease (GvHD), a response often seen after hematologic cell transplantation. Treatment of GvHD remains difficult and more knowledge is necessary. In this thesis we tried to contribute a small piece to the puzzle of GvHD by analyzing the possible relation between B cells and regulatory T cells (Tregs), from a primarily histological perspective. Humanized mice may serve as promising models in studies to GvHD. Designing the right model for the topic of interest is however subject to many choices. Based on our thorough review of the pearls and pitfalls from different mouse strains, transfer routes and sources of material, we decided that RAG2-/-γc-/- mice with intravenous transfer of huPBMC’s would be a sensible choice for our experiments. A thorough analysis was performed to evaluate the basic kinetics and pathology of our model. The induced GvH response in our model appeared to show many similarities with the sclerotic variant of human GvHD. Our analysis continued with an evaluation of our model with additional modifications: macrophage depletion and early B cell depletion. Macrophage depletion accelerated the induction and morbidity of the clinical GvH response and histologically increased the number of B-cells in the infiltrating cell signature, with only limited changes to the amount of Tregs. In early B cell depletion however, a clear improvement on survival was seen. Despite clinical improvement though, our histology only showed a significant effect on liver fibrosis and human TGF-β production within the liver. Other affected organs did not show signs of improvement or alterations. Our next exploration analyzed the effect of B cell activation on Tregs, using a combination of multiple in vitro studies with an in vivo study. Although based on our in vitro studies additional Tregs should temper the GvH reaction (even after B-cell activation), our in vivo study with myeloma was suprising. Clinical symptoms and survival of our mice did improve with additional Tregs, but no significant improvement was shown on a histological level. This contradiction between clinical symptoms and histology is known from human GvHD, as we continued to show with our last evaluation in this thesis, regarding human cutaneous GvHD. In this last analysis however, we’ve found that nowadays neither clinical grading nor histological grading seems to accurately predict a patients survival. With regard to the aim of these thesis, three major conclusions/considerations come forward. First, B cells and Tregs in GvHD have a complex relation, which is subject to the activation of B cells. Second, the lack of correlation between histology and clinical symptoms in GvHD might be explained by cytokines. Last but not least, we need to consider that Rituximab might serve best as a treatment for GvHD when used as an early B cell depletion therapy. However, since there might be a huge contrast between clinical symptoms and histology, we need to decide on our goal of therapy first: do we treat the patient or the disease? The answer to this, will ultimately decide whether our therapy really works

Modifying Graft-versus-Host Disease in a Humanized Mouse Model by Targeting Macrophages or B-Cells

Humanized mouse models can well be modified to study specific aspects of Graft-versus-Host Disease (GvHD). This paper shows the results of both macrophage depletion and (early) B-cell depletion in a humanized mouse model using RAG2-/-γc-/- mice injected with HuPBMCs. Macrophage depletion showed a significant decrease in survival and also lead to a change in the histomorphology of the xenogeneic reaction. Higher levels of infiltrating B-cells were observed in various organs of mice depleted for macrophages. With (early) B-cell depletion using Rituximab, a clear improvement on clinical symptoms was observed, even when probably only inactivated B-cells were deleted. However, the histological examinations only showed a significant morphological effect on liver fibrosis. This may be related to a difference in the mRNA levels of TGF-β. Also, lower mRNA levels of Tregs in some organs were observed after Rituximab treatment, which contradicts that a higher number of Tregs would always be related to less severe GvHD. Our data show that both macrophage depletion and (early) B-cell depletion in a xenogeneic mouse model can influence the clinical, histological, and cytokine production of a GvHD response

The influence of genetic factors on the osteoinductive potential of calcium phosphate ceramics in mice.

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration

A new xenograft model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2-/- gammac-/- double-mutant mice

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-/- gammac-/-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 XGVHD 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-/- gammac-/- mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this mode

Interobserver Variation in the Assessment of Immunohistochemistry Expression Levels in HER2-Negative Breast Cancer: Can We Improve the Identification of Low Levels of HER2 Expression by Adjusting the Criteria? An International Interobserver Study

The classification of human epidermal growth factor receptor 2 (HER2) expression is optimized to detect HER2-amplified breast cancer (BC). However, novel HER2-targeting agents are also effective for BCs with low levels of HER2. This raises the question whether the current guidelines for HER2 testing are sufficiently reproducible to identify HER2-low BC. The aim of this multicenter international study was to assess the interobserver agreement of specific HER2 immunohistochemistry scores in cases with negative HER2 results (0, 1+, or 2+/in situ hybridization negative) according to the current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Furthermore, we evaluated whether the agreement improved by redefining immunohistochemistry (IHC) scoring criteria or by adding fluorescent in situ hybridization (FISH). We conducted a 2-round study of 105 nonamplified BCs. During the first assessment, 16 pathologists used the latest version of the ASCO/CAP guidelines. After a consensus meeting, the same pathologists scored the same digital slides using modified IHC scoring criteria based on the 2007 ASCO/CAP guidelines, and an extra "ultralow" category was added. Overall, the interobserver agreement was limited (4.7% of cases with 100% agreement) in the first round, but this was improved by clustering IHC categories. In the second round, the highest reproducibility was observed when comparing IHC 0 with the ultralow/1+/2+ grouped cluster (74.3% of cases with 100% agreement). The FISH results were not statistically different between HER2-0 and HER2-low cases, regardless of the IHC criteria used. In conclusion, our study suggests that the modified 2007 ASCO/CAP criteria were more reproducible in distinguishing HER2-0 from HER2-low cases than the 2018 ASCO/CAP criteria. However, the reproducibility was still moderate, which was not improved by adding FISH. This could lead to a suboptimal selection of patients eligible for novel HER2-targeting agents. If the threshold between HER2 IHC 0 and 1+ is to be clinically actionable, there is a need for clearer, more reproducible IHC definitions, training, and/or development of more accurate methods to detect this subtle difference in protein expression levels

Better survival of renal cell carcinoma in patients with inflammatory bowel disease

Background: Immunosuppressive therapy may impact cancer risk in inflammatory bowel disease (IBD). Cancer specific data regarding risk and outcome are scarce and data for renal cell carcinoma (RCC) are lacking. We aimed(1) to identify risk factors for RCC development in IBD patients (2) to compare RCC characteristics, outcome and survival between IBD patients and the general population. Methods: A PALGA (Dutch Pathology Registry) search was performed to establish a case group consisting of all IBD patients with incident RCC in The Netherlands (1991-2013). Cases were compared with two separate control groups: (A) with a population-based IBD cohort for identification of risk factors (B) with a RCC cohort from the general population to compare RCC characteristics and outcomes. Results: 180 IBD patients with RCC were identified. Pancolitis (OR 1.8-2.5), penetrating Crohn's disease (OR 2.8), IBD related surgery (OR 3.7-4.5), male gender (OR 3.2-5.0) and older age at IBD onset (OR 1.0-1.1) were identified as independent risk factors for RCC development. IBD patients had a significantly lower age at RCC diagnosis (p < 0.001), lower N-stage (p = 0.025), lower M-stage (p = 0.020) and underwent more frequently surgical treatment for RCC (p < 0.001) compared to the general population. This translated into a better survival (p = 0.026; HR 0.7) independent of immunosuppression. Conclusions: IBD patients with a complex phenotype are at increased risk to develop RCC. They are diagnosed with RCC at a younger age and at an earlier disease stage compared to the general population. This translates into a better survival independent of immunosuppressive or anti-TNFa therapy