Histological and immunophenotypical analysis

Aged E-NKX2-3 mice showed clinical signs of disease and exhibited shorter survival in comparison to WT animals. The median of survival for L1 mice was 17.5 months, for L2 mice was 20.4 months, and for WT mice, 25.6 months (p<0.0001) (Fig 4.1).

80

Figure 4.1: Kaplan-Meier overall survival curves for the two E-NKX2-3 transgenic lines and WT mice.

Upon necropsy, all 38 examined mice (26 from L1, 12 from L2) showed enlarged spleens, whereas 34 mice (89%) also displayed extranodal tumors: 30 in the small intestine (78%), 22 in kidneys (57%), 19 in liver (50%), 7 in salivary glands (18%) and 6 in lungs (15%) (Table 4.1).

Table 4.1: Location of lymphomas developed in E-NKX2-3 mice.

ID Line Splenomegaly Small intestine

Liver Lung Kidney Salivary gland

Age (months)

6 1 YES YES NO NO YES NO 20 39 1 YES YES YES YES NO NO 23 785 1 YES YES NO NO YES YES 19 782 1 YES YES YES YES YES YES 18 757 1 YES YES NO NO YES NO 19 761 1 YES NO NO NO NO NO 19 202 1 YES YES YES YES YES YES 20 846 1 YES NO NO NO NO NO 18 962 1 YES YES NO NO YES YES 19 964 1 YES YES NO NO YES YES 20 956 1 YES NO NO NO NO NO 17 168 1 YES YES NO NO YES NO 18 484 1 YES YES NO NO NO NO 23 498 1 YES YES YES NO YES NO 23 489 1 YES YES YES NO YES YES 16 497 1 YES YES YES YES YES YES 12 669 1 YES YES YES NO YES NO 17 627 1 YES YES NO NO NO NO 18 257 1 YES YES NO NO NO NO 19 180 1 YES YES YES NO YES NO 18 179 1 YES NO YES NO NO NO 22 687 1 YES YES YES NO NO NO 20 646 1 YES YES YES NO YES NO 18 582 1 YES NO NO NO NO NO 12 563 1 YES YES YES YES YES NO 12 580 1 YES YES NO NO YES NO 12

81

ID Line Splenomegaly Small intestine

Liver Lung Kidney Salivary gland

Age (months)

194 2 YES YES YES NO YES NO 25 833 2 YES YES YES YES YES NO 25 622 2 YES YES NO NO NO NO 16 142 2 YES YES YES NO NO NO 22 169 2 YES YES YES NO NO NO 20 831 2 YES YES YES NO YES NO 20 826 2 YES NO NO NO YES NO 20 581 2 YES YES YES NO YES NO 18 487 2 YES YES NO NO NO NO 24 833 2 YES YES YES NO YES NO 25 841 2 YES NO NO NO NO NO 25 999 2 YES NO NO NO NO NO 18

Histological analysis of transgenic spleens revealed a prominent white pulp, composed of large B-cell infiltrates and poorly developed T-cell areas, with an almost absence of the red pulp. Compartmentalization of the follicle mantle into a marginal-zone and lymphocytic corona completely disappeared. The mantle-zone was almost entirely populated by small B lymphocytes and clusters of these cells could also be seen in the red pulp, sometimes invading the sinuses (Fig.4.2a). Immunofluorescence (IF) and immunohistochemical (IHC) studies detected expression of NKX2-3 in the nuclei of tumor B220+ B cells, while NKX2-3 expression was not detected in wild-type mouse lymphocytes (Fig.4.2b). Immunophenotypic characterization showed that B lymphocytes showed high CD20 expression, lacked CD5 expression, and were marked by high surface expression of IgM and low IgD expression. Staining revealed the presence of infiltrated CD3+ T cells (Fig.4.2c).

82

Figure 4.2: (A) Hematoxilin-esosin (H&E) staining of fixed paraffin-embedded spleen tissues of

E-NKX2-3 transgenic mice. Scale represents 100 m in all cases. (B) Immunohistochemical (IHC) and immunofluorescence (IF) studies of transgenic spleens using Nkx2-3 and B220 antibody. (C) Immunohistochemical (IHC) studies of transgenic spleen biopsies using CD20, IgD, IgM, and CD3 antibodies.

Splenic tissue architecture was characterized by IF in WT and transgenic mice. At 12 months, IgM+MadCAM-1+ B cells were detectable, initially at the T:B-cell boundary and the adjacent region between the follicles and marginal zone. These transgenic B cells substantially expanded by 18 months, while

83 both the MadCAM-1+ marginal sinus and its marginal reticular cell support were destroyed (Fig.4.3). This process was accompanied by a severe disorganization of marginal zone macrophage architecture, leading to the near-complete loss of MARCO-positive marginal zone macrophages and a substantial reduction in the number of sialoadhesin-positive metallophilic macrophages. Both types of macrophages are located surrounding the marginal zone B cells and are important in the integrity and organization of marginal zone (Mebius and Kraal 2005). In addition, gradual loss of follicular-stromal architecture (evidenced through fragmentation and collapse of follicular dendritic meshwork) was detected.

84

Figure 4.3: (A) Gradual expansion of MadCAM-1 positive IgM and plasma cells and dissolution

of follicular architecture in aging Eµ-NKX2-3 mice: in WT mice (top) marginal reticular cells (arrow, green) adjacent to MAdCAM-1 sinus-lining cells (red) separate follicular (FoB) and MZ B cells (MZB, blue, IgM) mice. Scale bar= 200 µm. (B) Loss of MZ macrophages in aging Eµ- NKX2-3 mice: in WT mice (top) two concentric layers of MZ macrophages consist of an expanded rim of MARCO-positive (red) MZ macrophages encircling sialoadhesin-positive metallophilic macrophages (green) intermingled with IgM-positive MZ B cells (blue; Fo). At 12 months (middle) focal thinning and loosening of MZ MARCO-positive macrophages is visible (indicated with *), while by 18 months the MARCO-positive rim disappear and only sialoadhesin- positive metallophilic macrophages arranged in a discontinuous arc remain (arrows). Scale bar= 200 µm. (C) Dissolution of follicular stromal architecture in aging Eµ-NKX2-3 mice: in WT mice (top) CR1/2 receptors (green) mark FDC reticula (arrow) and outline several layers of marginal zone B cells (arrowhead) outside the MadCAM-1 rim of marginal sinus-lining cells (red; Fo, follicle; MZ, marginal zone). At 12 months (middle) FDC networks are convoluted and

85

discontinuous, coupled with a marked reduction of MZ B-cells outside the MadCAM-1 positive rim, while MadCAM-1 positive cells appear within the white pulp. At 18 months (bottom) the FDC reticula collapse, the substantially enlarged MadCAM-1 positive rim is dissolved. Scale bar= 200 µm.

Although there is a loss in the integrity of marginal sinus and an expansion in the follicular stromal reticulum, the segregation of white pulp vasculature and red pulp venous sinus network (lost in Nkx2-3-/- mice) were intact in E-NKX2-3 mice (Figure 4.4) (Balogh et al. 2007).

Figure 4.4: Vascular topography in Eµ-NKX2-3 mice. Gradual loss of marginal sinus integrity

86

sinus (red [arrowheads in upper and middle panels]) and expansion of MadCAM-1-positive follicular stromal reticulum (arrow in lower panel), with preserved red pulp vessels displaying IBL-9/2 marker1 (blue). Scale bar = 200 µm.

Similarly to human SMZL, bone marrow was infiltrated by tumoral B cells (Figure 4.5).

Figure 4.5: Infiltration of B220+ B-cells in bone marrow in 18 months-old transgenic mouse.

Analysis of Igh, Igk and Igl gene rearrangements by genomic PCR revealed that 18-month-old splenic B cells harboured clonal rearrangements without IGVH somatic hypermutation, but this clonality was absent in splenic B cells from 12-month-old transgenic mice. (Fig.4.4). These features are consistent with the diagnosis of human-like SMZL, which in a fraction of cases showed unmutated IGVH sequences (Traverse-Glehen et al. 2011, Baliakas et

87

Figure 4.4: Detection of Ig gene rearrangements by PCR on genomic DNA from CD19+ cells isolated from transgenic spleens in 12 and 18-month-old mice. VDJH rearrangements were identified by direct sequencing (marked with red arrowheads). B and T, healthy murine B (CD19+) and T (CD3+) lymphocytes, respectively.

Additionally, tumors developed in extranodal sites were composed of a heterogeneous population of small and centrocyte-like mature B cells that infiltrated the epithelium and formed lymphoepithelial lesions, therefore mimicking human MALT lymphomas (Isaacson and Du 2004, Jaffe 2009) (Fig.4.5a). Notably, few splenic and extranodal lymphomas (5 of 32 cases,

88 16%) showed areas with diffuse infiltrates of large B lymphoblasts that indicated transformation to DLBCL, which is also observed in human SMZL and MALT lymphoma (Camacho et al. 2001, Sagaert et al. 2006), and was also detected during the evolution of index case 1 with the t(10;14)(q24;q32). Mouse DLBCLs showed expression of Irf4 and Foxp1, with negativity for Bcl6, CD10 and Gcet1 staining consistent with a non-germinal center origin (Choi et al. 2009) (Fig.4.5b).

89

Figure 4.5: (A) H&E staining of extranodal lymphomas developed in kidney, lung and gut,

showing a lymphoepithelial lesion. (B) H&E staining and IHC studies with CD20, FoxP1 and Mum1 (Irf4) antibodies in spleen tissue biopsies showing transformation areas resembling diffuse large-cell lymphomas. Gcet1, Bcl10 and Bcl6, not shown.

Finally, splenic lymphomas could be propagated in immunodeficient Rag2-/-IL2γc-/- mice following tumor cell injection in the tail vein (Figure 4.5). Overall, these data show that E-NKX2-3 mice generated tumors mirroring the spectrum of human NKX2-3-expressing B-cell lymphomas.

90

Figure 4.5: Kaplan-Meier overall survival curves for immunodeficient Rag2-/-IL2γc-/- mice

intravenously injected with 2.5x106 splenic NKX2-3-expressing lymphoma cells from 18-month- old mice (P0). Survival curves for secondary (P1) and tertiary (P2) recipients are also shown.