Lymphoma-Like T Cell Infiltration in Liver Is Associated with Increased Copy Number of Dominant Negative Form of TGFβ Receptor II

Hepatosplenic T cell lymphoma (HSTCL) is a distinct and lethal subtype of peripheral T cell lymphoma with an aggressive course and poor outcome despite multiagent chemotherapy. Contradictory literature, an unknown etiology, and poor response to treatment highlight the need to define the malignant process and identify molecular targets with potential for successful therapeutic interventions. Herein, we report that mice homozygously expressing a dominant negative TGFβRII (dnTGFβRII) under the control of the CD4 promoter spontaneously develop lymphoma-like T cell infiltration involving both spleen and liver. Splenomegaly, hepatomegaly and liver dysfunction were observed in homozygous dnTGFβRII mice between 10 weeks and 10 months of age associated with a predominant infiltration of CD4−CD8−TCRβ+NK1.1+ or CD8+TCRβ+NK1.1− T cell subsets. Notch 1 and c-Myc expression at the mRNA levels were significantly increased and positively correlated with the cell number of lymphoid infiltrates in the liver of dnTGFβRII homozygous compared to hemizygous mice. Further, 2×104 isolated lymphoma-like cells transplant disease by adoptive cell transfers. Collectively, our data demonstrate that increased copy number of dnTGFβRII is critical for development of lymphoma-like T cell infiltration.


Introduction
Transforming growth factor beta (TGFb) is a multifunctional protein that acts as an important regulator of cell growth, proliferation, differentiation, morphogenesis and inflammation. TGFb exerts biological effects by ligation of its cognate cell surface TGFb receptors with activation of downstream effectors including the TGFbR Smad family [1,2,3]. Alterations of specific components involved along the TGFb signaling pathway results in loss of TGFb receptor function and disruption of the intracellular TGFb signaling cascade. Such loss of TGFbR function is implicated in the pathogenesis of aortic pathology, various cancers and fibrotic and inflammatory disease [1,2,4]. There is a reduction of TGFbRII expression in Burkitt's lymphoma [5] and advanced cutaneous T cell lymphomas [6,7]. Similarly, loss of surface TGFbRII or mutations in the TGFbRII gene have been reported in human T cell malignancies [8] and colorectal cancer [2,9,10], suggesting that abnormal expression of TGFbRII is associated with malignant progression. Thus, abnormalities in the TGFb signaling pathway may be involved in the molecular pathogenesis of lymphoid malignancy. In the study herein, we generated homozygous dominant negative TGFbRII mice in which a 2-fold increase in expression of dnTGFbRII transgene was detected under control of the CD4 promoter. We demonstrated that mice homozygous for dnTGFbRII spontaneously developed lymphoma-like T cell infiltration involving both the spleen and liver with a significantly elevated pro-oncogene expression of Notch 1 and c-Myc. Further, we demonstrated that 2610 4 lymphoma-like cells were able to transplant disease by adoptive cell transfer.
For intracellular cytokine analysis, mononuclear cells isolated from splenic tissues were cultured in media containing Leukocyte Activation Cocktail, with BD GolgiPlug TM (BD Pharmingen, San Diego, CA) for 4 hours. Cells were stained for cell surface markers with PerCP anti-CD8a (clone 53-6.7, Biolegend), APC-conjugated anti-TCR-b (clone H57-597, eBiosciences), and APC-eFluorH 780-conjugated anti-NK1.1 (clone PK136, eBiosciences). After staining, the cells were fixed with BD Cytofix/Cytoperm TM solution and permeabilized with BD Perm/Wash TM buffer (BD Pharmingen, San Diego, CA). Aliquots of these cells were stained with FITC-or PEconjugated antibodies against IFN-c, IL-2 as well as their respective isotype control antibodies. Stained cells were analyzed using a FACScan flow cytometer (BD Bioscience) that was upgraded by Cytec Development (Fremont, CA), which allows for five-color analysis. Data were analyzed utilizing CELL-QUEST software (BD Bioscience). Appropriate known positive and negative controls were used throughout.

Histopathology
Immediately after sacrifice, the lung, spleen, liver and colon were harvested, fixed in 4% paraformaldehyde (PFA) at room temperature for 2 days, embedded in paraffin, and cut into 4micrometer sections. The liver sections were de-paraffinized, stained with hematoxylin and eosin (H&E), and evaluated using light microscopy.

Real time RT-PCR Analysis
To determine the relative mRNA levels of the genes including Notch-1, the Notch-1 ligands DLL1/DLL4, the Hes-1 gene which involved in Notch signaling, and for purposes of control the gene PTEN that is associated with DNA repair were quantitated in RNA extracted from liver tissues of the appropriate strains of mice. Total RNA was extracted from individual liver tissues using the QIAGEN RNeasy Mini Kit (Qiagen, Valencia, CA). For real time PCR analysis, 1 mg of total RNA was reverse transcribed and then quantified on an ABI ViiA TM 7 Real-Time PCR System (Applied Biosystems, Foster City, CA). Amplification was performed for 40 cycles in a total volume of 20 ml and products were detected using SYBR Green (Applied Biosystems, Foster City, CA). The relative level of expression of each target gene was determined by normalizing its mRNA level to an internal control gene GAPDH.

Clonality Analysis
The clonal T cell expansions were identified by CDR3-length analysis of TCRVb gene segments as described [13].

Relative Quantification of Transgene Copy Number
Relative quantification of transgene copy number was established as previously described [14]. Briefly, genomic DNA was obtained from mouse ear using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA), and then diluted to 2 ng/ml. Amplification was performed for 40 cycles in a total volume of 10 ml and products were detected using SYBR Green (Applied Biosystems, Foster City, CA) and quantified on an ABI ViiA TM 7 Real-Time PCR System. The primer sequences for dominant negative TGFbRII were as follows.
Forward: GCTGCA-CATCGTCCTGTG, Reverse: ACTTGACTG-CACCGTTGTTG. A single copy gene within the mouse genome, Survival Motor Neuron (SMN) gene [15,16] was used as a reference gene. The primer sequences for SMN were as follows. Forward: TGGGAGTCCATCCATCCTAA, Reverse: CGACTGGGTAGACTGCCTTC. Relative quantification methods (2 2DDCt methods) were used for relative quantification of transgene in mouse genome by normalizing target gene to the reference gene SMN.

Statistical Analysis
The data are presented as the mean 6 SEM. Two-sample comparisons were analyzed using the two-tailed unpaired t-test. The correlation between two parameters was analyzed using Spearman Correlation Method. A value of p,0.05 was considered statistically significant.

Lymphoma-like T Cell Infiltration in Intercrossed dnTGFbRII IL-6 2/2 Littermates
Our lab has previously documented that deletion of the IL-6 gene from the hemizygous dnTGFbRII mice significantly improved colitis as indicated by substantially reduced intestinal lymphocytic infiltration, reduced diarrhea and increased body weight, while maintaining the autoimmune cholangitis. In the follow-up study of cholangitis in this mouse model, we expanded this colony by intercrossing hemizygous TGFbRII IL-6 2/2 litters. In this process hemizygous dnTGFbRII +/2 IL-6 2/2 mice were generated along with homozygous TGFbRII +/+ IL-6 2/2 and TGFbRII 2/2 IL-6 2/2 littermates, in a ratio that follows Mendel's law of segregation. When individual animals generated by intercrossing were examined for the liver infiltrating mononuclear cells (MNCs), two distinct subsets of littermates with TGFbRII transgene were found to have dramatically different (6-to 12-fold) numbers of liver infiltrating MNC ( Figure 1A). One of the subsets, approximately one third of the TGFbRII littermates, had 152.7622.0610 6 hepatic mononuclear cells (HMNCs), while the rest of the animals only had 20.961.8610 6 HMNCs. Such massive HMNC increase suggested a lymphoma-like disease. Therefore we examined the mRNA levels of the lymphomarelated proto-oncogenes involved in the Notch-1 signal pathway, including Notch-1, DLL1/4, Hes-1, PTEN and c-Myc in the liver tissues of individual animals. Among these genes, the relative mRNA levels of Notch-1 and c-Myc were the most pronounced and significantly increased in the littermates with massive HMNCs when compared to those with fewer HMNC infiltration (c-Myc: 9.261.7 vs. 1.760.6, n = 6, p = 0.0016) ( Figure 1B), or compared with the hemizygous dnTGFbRII mice (c-Myc: 9.261.7 vs. 2.460.6, n = 6, p = 0.0032) ( Figure 1B). A significant positive correlation was detected between the number of HMNCs and Notch-1 (r = 0.8502, p = 0.0005, Spearsman correlation), and between HMNCs and c-Myc in interbred mice (r = 0.9347, p,0.0001, Figure 1C). These results indicate that lymphoma-like T cell infiltration occurred in the interbred dnTGFbRII + IL-6 2/2 mice. Since the lymphoma-like HMNCs are seen in approximately one third of the dnTGFbRII + IL-6 2/2 mice generated by intercross breeding that resulted in a mixture of littermates with hemizygous (+/2) and homozygous (+/+) dnTGFbRII gene, but not seen in pure hemizygous dnTGFbRII +/2 IL-6 2/2 litters generated without intercrossing, the results also suggest that the lymphoma-like T cell infiltration occurred in mice with the homozygous dnTGFbRII gene.

Characterization of the Lymphoma-like HMNCs
Flow cytometric analysis was performed to determine the phenotype of the lymphoma-like HMNCs. As shown in Fig. 2A, in mice with greatly elevated HMNC infiltration (HMNC high), or hepatic lymphoma, the HMNCs were comprised of two major phenotypes. Seven out of 12 lymphoma-like mice were characterized by the predominance of CD4 2 CD8 2 TCRb + NK1.1 + cells (termed NK1.1), whereas the other 5 lymphoma-like mice had a predominant CD8 + TCRb + NK1.1 2 T cell subset (termed NK1.1 2 ) in the liver tissues. The percentage of HMNCs with these phenotypes ranged from 70-98%, indicating that these mice developed lymphoma-like T cell infiltration. Mice with HMNCs in these two phenotypes had a significant increase in splenic weight ( Figure 2B) and hepatic MNC count ( Figure 2C) compared to mice without such predominant HMNC phenotypes. Histological examinations were performed on lymphoid ( Figure 2D, a-h) and non-lymphoid ( Figure 2D, i-s) organs. Massive atypical lymphoid infiltration was observed in the grossly enlarged spleen and liver, but not in the small intestine or colon, of the mice with these predominant HMNC phenotypes ( Figure 2D). Marked lymphoid aggregation was only found in the lung of 1/7 mouse with predominant CD4 + TCRb + NK1.1 + infiltrates. In liver sections, a diffuse infiltration with atypical lymphocytes was observed in mice with CD4 2 CD8 2 TCRb + NK1.1 + HMNC phenotype, whereas the CD8 + TCRb + NK1.1 2 phenotype was associated with focal lymphoid aggregates ( Figure 2D, d:6200, t:640). These histological changes were not present in the inbred dnTGFbRII IL-6 2/2 littermates without massive HMNC infiltration. NK1.1 + lymphoma-like T cells isolated from the liver had a markedly reduced ability to produce IFN-c and IL-2 compared to NK1.1 2 lymphoma-like cells and HMNCs of non-lymphoma dnTGFbRII IL-6 2/2 mice ( Figure 3A), while such difference was not seen in the spleen ( Figure 3B). We next determined the clonality of the T cells in HMNC by examining T cell receptor Vb repertoire (Vb1-20) CDR3 length and joining beta (Jb1.1-1.6 and Jb 2.1-2.7). The results demonstrated that both lymphoma-like and non-lymphoma dnTGFbRII IL-6 2/2 mice displayed restricted Vb repertoires. However, Vb2/cp Jb2.7, Vb4 Jb 2.3, Vb7 Jb1.5 and Vb9 Jb2.3 were highly restricted in lymphoma-like mice compared to the non-lymphoma littermates, indicating that expanded lymphoid populations are clonally heterogeneous ( Figure 4).

The Development of Lymphoma-like T Cell Infiltration was Associated with Homozygosity of dnTGFbRII Gene
It is critical to exclude the possibility that the observed severe HMNC infiltration in dnTGFbRII IL-6 2/2 littermates actually reflects severe autoimmune liver inflammation rather than hepatic lymphoma. We have previously demonstrated that the autoimmune cholangitis in dnTGFbRII mice is mediated by Th1 cells, and that abrogation of the Th1 pathway by depleting IL-12 p40 gene completely protect dnTGFbRII mice from both cholangitis and colitis [12]. In order to further confirm the relationship between dnTGFbRII homozygosity and lymphoma-like T cell infiltration and to differentiate lymphoma from severe autoimmune cholangitis, we intercrossed our previously reported hemizygous dnTGFbRII p40 2/2 mice, which were cholangitisfree, to generate a mixture of hemizygous and homozygous dnTGFbRII p40 2/2 littermates with a ratio that follows Mendel's law of segregation. As expected, substantially increased HMNCs (9.460.12610 7 ) were observed in the liver tissues of two out of seven, similar to the expected one-third frequency, intercrossed dnTGFbRII p40 2/2 mice at the age of 10 weeks. Liver pathology examination demonstrated massive atypical lymphoid hepatic infiltration in these mice, indicating that introduction of a homozygous dnTGFbRII gene into the cholangitis-free dnTGFbRII p40 2/2 mice resulted in development of lymphomalike T cell infiltration ( Figure 5A).
Finally we generated intercrossed dnTGFbRII mice to determine if homozygous dnTGFbRII gene alone is sufficient to cause lymphoma-like T cell infiltration. Severe IBD in female hemizygous dnTGFbRII gene mice reduced the rate of fertility. Therefore only 3 interbred littermates were obtained. In one out of these 3 littermates a predominant CD4 2 CD8 2 TCRb + NK1.1 + cell subset was found in the liver, which also demonstrated massive atypical lymphoid hepatic infiltration at 12 weeks ( Figure 5B). Taken together, the findings in the intercrossed dnTGFbRII IL-6 2/2 , dnTGFbRII p40 2/2 and dnTGFbRII mice indicate that dnTGFbRII homozygosity is associated with occurrence of lymphoma-like T cell infiltration.

Increased Copy Number of dnTGFbRII is Associated with the Development of Lymphoma-like T Cell Infiltration
To directly confirm that lymphoma-like disease occurs with a higher copy number of dnTGFbRII gene in homozygous dnTGFbRII mice, we determined the relative dnTGFbRII transgene copy number in intercrossed dnTGFbRII littermates by quantitative real-time PCR. Given the fact that the parental generations are hemizygous, Mendel's law of segregation predicts that the entire litter would be comprised of 50% of hemizygous (dnTGFbRII +/2 ), 25% homozygous (dnTGFbRII +/+ ) and 25 percent negative (dnTGFbRII 2/2 ) in the dnTGFbRII transgene. We determined the relative levels of the transgene in genomic DNA in comparison to a reference single copy gene SMN in 42 mice derived from 7 litters generated by intercrossing mice that  carried hemizygous dnTGFbRII gene. These included 5 litters of intercrossed dnTGFbRII IL-6 2/2 mice, 1 litter of dnTGFbRII p40 2/2 mice and 1 litter of dnTGFbRII mice. In 22 mice (52%), the relative transgene level was 1.2960.03; in 8 mice (19%) the relative transgene levels was 2.8360.11, while in 12 mice (29%) the transgene was not detected ( Figure 5C). Among the 8 mice with the higher level of dnTGFbRII transgene, 4 mice died around 12 weeks, two mice had splenomegaly, hepatomegaly and jaundice with an 18-fold increase of total HMNCs at age 12 weeks compared to the lower transgene level controls; the last two mice had an approximate 3-fold higher number of HMNCs at age of 15 weeks. Importantly, all mice with lymphomatous lesions, including dnTGFbRII and dnTGFbRII p40 2/2 mice shown in Figure 5A and 5B, had higher levels of TGFbRII transgene than those without lymphomatous lesions (2.4160.22, n = 8 vs. 1.2760.03, n = 15, p,0.0001). There was a highly significant positive correlation between hepatic cellular infiltrates and the copy number of dnTGFbRII transgene (r = 0.9342, p,0.0001), indicating that abrogation of TGFb signaling by higher copy number of dnTGFbRII contributes to the emergence of T cell lymphomalike T cell infiltration.

Adoptive Cell Transfer to Rag1 2/2 Mice
To evaluate whether the lymphoma-like T cell infiltration in dnTGFbRII mice are transferable, we carried out standard adoptive transfer studies. We reported previously that transferring CD8 + T cells from hemizygous dnTGFbRII mice induced changes consistent with autoimmune cholangitis in Rag1 2/2 mice only when the number of transferred cells reached one million [17]. We thus isolated HMNCs from hemizygous and homozygous dnTGFbRII mice and transferred 5-to 50-fold fewer cells (2610 4 or 2610 5 ) than the previous transfer studies [18,19] into Rag1 2/2 mice. The frequency of T cells expressing NK1.1 phenotype was approximately 95% in the liver of homozygous TGFbRII donors, 95% of TCRb + NK1.1 + cells were CD4 and CD8 double negative ( Figure 6A). Six weeks after intravenous injection, pathological and phenotypic changes identical to that seen in the donor homozygous mice were observed in spleen and liver in 8/8 recipient mice ( Figure 6B and 6D). Histopathological studies revealed massive atypical lymphoid cell infiltrates and hepatocellular damage in recipient mice, even in mice that received as few as 2610 4 HMNCs from TGFbRII homozygous mice ( Figure 6D). Consistent with the loss of cytokine function noted in the donor lymphoma-like cells, T cells from the recipient mice showed reduced cytokine production ( Figure 6C). In contrast, no obvious lymphoid cell infiltrates were found in Rag1 2/2 recipients of donor cells from hemizygous TGFbRII mouse ( Figure 6D

Adoptive Transfer of Hepatic CD8ab Cells from TGFbRII Hemizygous Mice Resulted in Lymphoma-like Infiltration
Since two distinct phenotypic T cell expansions were found in the TGFbRII homozygous mice, we addressed whether they were derived from the same precursors and transited through a TCRb + NK1.1 2 stage before they became NK1.1 + T cells during the terminal stage [20]. Flow cytometry sorted populations of hepatic CD8ab cells from TGFbRII hemizygous mice were adoptively transferred to Rag1 2/2 mice (10 6 sorted CD8 T cells per mouse, n = 3). The purity of the sorted CD8ab cells was .95% as assessed by flow cytometric analysis ( Figure 7A). Recipients were euthanized six weeks after adoptive transfer. Lung, heart, kidney, intestine, colon, spleen and liver were removed and examined histologically. The wet weight of spleen and liver were determined as shown in Figure 7C. Two of the three recipients had increased liver mass and cell count of mononuclear cells in both spleen and liver. Only 53% of MNCs from recipient 2 retained the TCRb + NK1.1 2 phenotype, while 45% of donor cells became CD4 CD8 double negative TCRb + NK1.1 + in liver ( Figure 7B). The frequency of TCRb + NK1.1 + was higher in liver compared to that in spleen ( Figure 7B). Histology revealed that bridging necrosis with severe to massive atypical lymphoid infiltration and hepatocellular damage were found in recipient 1 and recipient 3. On the other hand, only mild focal necrosis with mild lymphoid infiltration was observed in recipient 2. Our data collectively suggest that CD8 T cells from dnTGFbRII mice possess a capability to develop an NK1.1 + phenotype (Figure 7) with high pathogenic potential. In contrast, no NK1.1 positive T cells turned into NK1.1 negative T cells in immunodeficient Rag1 2/2 mice ( Figure 6).

Discussion
The TGFb signaling pathway plays an important role in T cell development and proliferation, naïve T cell homeostasis, peripheral T cell tolerance and effector T cell differentiation [20,21]. To date, different strategies have been employed to generate mice with a T cell-targeted disruption of TGFb signaling. In these models, consequences of TGFb defects are limited to T cells. Mice that have a CD4-Cre-mediated deletion of a TGFbRII allele [20] develop a progressive lymphoid infiltration into multiple organs before 5 weeks of age [22]. Inserting a truncated TGFbRII under a CD2 promoter/enhancer in mice results in a CD8 T cell lymphoproliferative disorder with small lymphocyte infiltration [23]. Expression of a dominant-negative form of TGFbRII (dnTGFbRII) under a CD4 promoter without a CD8 silencer leads to spontaneous activation and differentiation of both CD4 and CD8 T cells and development of autoimmune diseases at 4-6 months of age [24]; the different pathological outcomes demonstrated that the TGFb signaling pathway is not completely abrogated by the expression of the dominant negative form of TGFbRII.
We previously reported that hemizygous dnTGFbRII mice manifest autoimmune cholangitis with elevated Th1 cytokines in serum and liver [25]. In the present study, we unexpectedly found shown in the middle and right panels were gated on TCRb + NK1.1 2 or TCRb + NK1.1 + populations as indicated in the left panels. B. The spleen weight of dnTGFbRII, dnTGFbRII IL-6 2/2 and dnTGFbRII IL-6 2/2 mice with a predominant NK1.1 positive or negative phenotype at age of 24-40 weeks. C. The total HMNC counts of dnTGFbRII, dnTGFbRII IL-6 2/2 and dnTGFbRII IL-6 2/2 mice with a predominant NK1.1 positive or negative phenotype at age of 24-40 weeks. D. Representative H&E stained sections of tissue sample including liver (a-d), spleen (e-h), small intestine (i-l), colon (m-p) and lung (qs) were prepared from dnTGFbRII and dnTGFbRII IL-6 2/2 mice at age of 24-40 weeks (a-s,6200; t,640). Typical diffuse lymphomatous lesions were found in liver (c) and spleen (g) of dnTGFbRII IL-6 2/2 mice with a predominant NK1.1 + phenotype, while large focal lymphomatous lesions were found in liver (d,6200)(t,640) and spleen (h) of dnTGFbRII IL-6 2/2 mice with a predominant TCRb + NK1.1 2 phenotype. No obvious lymphomatous lesions were found in lung, small intestine and colon in dnTGFbRII and dnTGFbRII IL-6 2/2 mice. doi:10.1371/journal.pone.0049413.g002 MNCs were CD4 2 CD8 2 TCRb + NK1.1 + or CD8 + TCRb + NK1.1 + cells. Second, a significantly increased expression of the proto-oncogenes Notch-1 and c-Myc, which correlates significantly with the number of HMNCs in mice homozygous for dnTGFbRII, compared to hemizygous dnTGFbRII mice. This observation has to be taken with caution partly because Myc and Notch can be induced upon normal T cell activation [26,27]. Third, histological analysis demonstrated massive atypical lymphoid cell infiltration in the grossly enlarged IL-2-producing T cells is lower in liver of dnTGFbRII IL-6 2/2 mice with a predominant NK1.1 + phenotype than dnTGFbRII IL-6 2/2 mice with a predominant NK1.1 2 phenotype (IFN-c + T: 6.13% vs. 50.13%; IL-2 + T: 7.45% vs. 29.10%). The data are representative of three independent experiments. doi:10.1371/journal.pone.0049413.g003 spleen and liver from the homozygous dnTGFbRII, but not hemizygous dnTGFbRII mice. Fourth, adoptive transfer of very small numbers of infiltrating cells (2610 4 ) isolated from the liver of homozygous dnTGFbRII, but not from hemizygous dnTGFbRII mice, resulted in massive atypical lymphoid cell infiltration with CD4 2 CD8 2 TCRb + NK1.1 + phenotype in Rag1 2/2 recipients, which were identical to that of the donor. However, our present data here do not clearly discriminate lymphoma from inflammatory T-cell infiltration. NKT cells are most abundant in the liver. TGFb signaling is critical for the differentiation of NKT subsets [28], but how TGFb signaling involved in the regulation and differentiation of NKT in our mice model with abrogated TGFbRII needs to be further elucidated.
Oligoclonal expansion of T cells was detected in the liver of homozygous and hemizygous dnTGFbRII mice by clonality analysis, indicating that the expanded T cells are heterogeneous. Of note, clonality is not equivalent to malignancy, since benign and inflammatory conditions may show monoclonal rearrangement [29,30]. Our finding of heterogeneous clonal expansion of lymphoma-like T cell in homozygous dnTGFbRII mice is consistent with previously documented heterogeneous clonal restrictions of T cell populations in patients with angioimmunoblastic T-cell [31] and cutaneous T-cell lymphoma [32]. Moreover, our clonality results support a previous study in AKR/J mice demonstrating that restricted TCR Vb repertoire and lack of CDR3 conservation displayed thymic lymphomas [33]. In addition, lymphoma-like changes develop in dnTGFbRII mice with a skewed but polyclonal TCR repertoire, which is in agreement with a recently published study in which TCR-diversity suppressed development of mature T-cell lymphoma [34]. The clonal competition hypothesis might be a possible explanation for outgrowth of atypical lymphocytes in such a skewed TCR repertoire situation. Another possible explanation for local atypical infiltrates in our experimental system could be outgrowth of activated T cells accumulated in liver and mutations in certain oncogenes such as p53 [35]. The liver is a ''graveyard'' that actively sequesters activated and eventually apoptotic T cells [36]. However, activated T cells without the regulation of TGFb signaling might undergo cell divisions rather than apoptosis due to mutations in oncogenes, resulting in outgrowth of atypical massive T cells. Further studies will be required to characterize the outgrowth of atypical lymphocytes in detail, including selections of malignant clones, deep sequencing and potential gene mutations to elucidate the underlying mechanism.
Patients with various autoimmune diseases have demonstrated an increased risk of developing non-Hodgkin lymphoma and multiple myeloma. Previous studies have also shown that loss of response to TGFb is associated with the progression of different types of malignancies including T-cell lymphomas [7,8]. Since it has been previously implicated that the expression of a single copy of dnTGFbRII transgene does not completely block TGFb signaling in T cells, we reasoned that a second copy of dnTGFbRII transgene in homozygous mice further suppresses the downstream signaling, resulting in the development of T cell lymphoma. The pathological and immunological presentation in these lymphoma mice resembles the main clinical features in patients with HSTCL, although to date no cd + T-cell lymphomas have been found in homozygous dnTGFbRII mice. Strikingly, although elevated circulating IL-6 was reported to correlate with adverse clinical features and survival in non-Hodgkin lymphoma [37,38], while clinical trials have showed anticancer effects of IL-12 on cutaneous T cell lymphoma [39], genetic depletion of IL-6 or IL-12p40 did not rescue outgrowth of lymphoma-like T cell in homozygous dnTGFbRII mice indicating that dnTGFbRII homozygosity is critical for the outgrowth of lymphoma-like T cells.
Peripheral T-cell lymphomas (PTCL) are rare and aggressive malignancies that are distinct from the more common cutaneous T-cell lymphomas. Hepatosplenic T cell lymphoma (HSTCL) is a distinct and lethal subtype of peripheral T-cell lymphoma with an aggressive clinical course and a dismal outcome despite multiagent chemotherapy. HSTCL likely arises from cytotoxic T-cells that express the cd T-cell receptor type. However, it is important to note that an ab T-cell phenotype has been described increasingly in HSTCL [40,41,42,43]. Lymphoma cells usually have the following phenotype: CD2 + , CD3 + , CD4 2 , CD5 2 , CD7 + and CD8 2 . The World Health Organization has updated the classification of lymphomas, which has led to the application of a more stringent criteria for the diagnosis of enteropathyassociated T-cell lymphoma (EATL) [44]. However, the challenges in understanding and treating PTCLs remain, since published literature consists mostly of case reports. A wide range of pathologic subdivisions with varied clinical features also impedes systematic study on PTCL. Our findings highlight a potential role for TGFb signaling in the development of HSTCL.
Consistent with the previous work by Marie and colleagues [20], we found that in the hemizygous TGFbRII mice an expanded subset of T cells expressed the NK1.1 marker, although lymphoma was not found in these mice. However, when we adoptively transfer the dysfunctionally activated TGFbRII-CD8 NK1.1 2 T cells from these mice into the immunocompromised micro-environment in the Rag1 2/2 recipients without regulatory T and B cells, expression of NK1.1 and disease pathology were observed (Figure 7), suggesting that expression of NK1.1 marker is Figure 7. CD4 and CD8 double negative T cells were detected in Ly5.1Rag1 2/2 mice six weeks after adoptively transferred with one million hepatic CD8ab T cells from hemizygous dnTGFbRII mice (lymphomatous lesion-free). A. Flow cytometry analysis demonstrated the purity of hepatic CD8ab T cells from hemizygous dnTGFbRII mice. The numbers in the plots indicate the percentage of cells. B. Flow cytometric analysis of splenic and hepatic mononuclear cells of recipient mice at 6 weeks post-transfer. Three Ly5.1Rag1 2/2 mice were adoptively transferred with 1610 6 hepatic CD8ab T cells from hemizygous dnTGFbRII mice. TCRb staining was gated on CD45.2 + cells. The numbers in the plots indicate the percentage of cells. C. Weight and total MNC counts of spleen and liver from Ly5.1Rag1 2/2 recipients 6 weeks post-transfer. D. H&E staining sections of liver tissues from Ly5.1Rag1 2/2 recipients six weeks post-transfer. R1, Recipient 1; R2, Recipient 2; R3, Recipient 3; doi:10.1371/journal.pone.0049413.g007 associated with enhanced pathogenic potential. Although some lymphomas are phenotypically and genotypically of T cell origin, there are also lymphomas that are positive for the CD56 marker and are of NK cell origin [45]. NK cell markers are frequently expressed in HSTCL and other types of T cell lymphomas except for nasal and extranodal NK/T-cell lymphomas [46]. In comparison to CD56 2 T-cell lymphoma, the CD56 + NK-like T cell lymphomas demonstrated an aggressive clinical course [45,47] associated with a poor prognosis [48]. Our present study suggests that the acquisition of the NK1.1 cell surface marker by dnTGFbRII-CD8 T cells resulted in a highly pathogenic population that leads to development of T cell lymphoma with an aggressive clinical course. We speculate that blockage of the transition from NK1.1 2 to NK1.1 + T cells could be a potential strategy for the management of lymphoma disease.
In summary, our data demonstrate that several features of human HSTCL are manifested in homozygous dnTGFbRII mice, suggesting that selective CD4 targeted functional abrogation of TGFbRII by increased copy number of dominant negative form of TGFbRII in mice can serve as a model of HSTCL for studying the disease mechanism and therapeutic strategies.