CARD11 Mutation Induces Oligoclonal Expansion of T-Cells, and Accelerates ATL Development in Combination with HBZ
Authors: Kameda Takuro , Shide Kotaro , Kamiunten Ayako , Yuki Tahira , Sekine Masaaki , Akizuki Keiichi , Hidaka Tomonori , Kubuki Yoko , Sugiyama Midori , Morishita Daisuke , Shimoda Kazuya 
Published: November 2020

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma that develops in about 5% of human T-cell leukemia/lymphoma virus 1 (HTLV-1) carriers. In addition to viral oncogenes, namely tax and HTLV-1 bZIP factor (HBZ), gene mutations, highly enriched for T-cell receptor (TCR)-NF-kB signaling, should be involved in the development of ATL. Among gene mutations, mutation in CARD11, a cytoplasmic scaffolding protein required for TCR-induced NF-kB activation, was detected in 24% of ATL patients. Here we generated a mouse model for conditional expression of a human ATL-derived CARD11(E626K) gain-of-function mutant, and demonstrated CARD11 activation induced oligoclonal expansion of T-cell and infiltration to many organs. We also showed that expression of HBZ accelerated mutant CARD11-induced lymphoproliferative diseases.

We introduced a human Card11(E626K) into the mouse genome at the ROSA26 locus. After crossing with CD4-Cre Tg, CARD11(E626K)CD4-Cre mice was obtained. In CD4+ cells from CARD11(E626K)CD4-Cre, the amount of cleaved BCL-10 and NF-kBp65 increased compared with those in WT CD4+cells, confirming the activation of NF-kB. About half of CARD11(E626K)CD4-Cre mice died on or after 6 months after birth. At 6 months, leukocytosis was observed in CARD11(E626K)CD4-Cre, and accordingly the number of CD4+ cells cells was about 1.43 times greater than those in WT mice. The most affected organ in CARD11(E626K)CD4-Cre mice was lung. Alveolar septum was thickened by infiltrated cells at 6 months, and worsened subsequently. CD3+ T-cell accumulated around capillary blood vessels, and had high proliferation indices (&gt;50%), as assayed by Ki-67 staining. CARD11(E626K)CD4-Cre mice developed lymphadenopathy (4/8 mice (50%) at 6M and 4/6 mice (66.7%) at 12M). Normal lymph node (LN) architecture was barely preserved, and medullary sinus was expanded with CD3+ T-cell. Some of them were positive for FoxP3, and had moderate proliferation indices (25%-50%). Among CD4+ T-cell, the proportion of naive T-cell (Tn) decreased, and that of effector/memory T-cell (Tem) and regulatory T-cell (Treg) increased compared to WT LNs. The proportion of Treg in CD4+ T-cell from LN of 6M- and 12M-old CARD11(E626K)CD4-Cre mice was 25% and 40%, respectively, which values were much larger than the normal range as 10-15%. We next examined the clonality of CD4+ cells in spleen and swollen LNs from CARD11(E626K)CD4-Cre mice. The clonality of the TCR repertoires of 20 individual Vb gene famines from Vb1-20 was assessed by a PCR. The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 5 splenic CD4+ cells, and 1 of 2 LN CD4+ cells from CARD11(E626K)CD4-Cre mice.

To assess the effect of HBZ constant expression on CARD11(E626K)CD4-Cre mice, we generated HBZ Tg, in which HBZ cDNA was expressed under the CD4 promoter. Similar to the previous report (by Satou et al.), our HBZ Tg showed increased number of Tem, destroyed architecture of lung such as thickened alveolar septum by infiltrated cells and decreased alveolar space by edema, and the lymphadenopathy after 12M (66.7%). We then crossed CARD11(E626K)CD4-Creand HBZ Tg, and obtained the compound mice. The compound mice caused more aggressive lymphoproliferative diseases compared with CARD11(E626K)CD4-Cre mice. Most of compound mice died within 8M. At 6M, architecture of lung, kidney, spleen, and LN was destroyed. In lung, alveolar space of lung was scarcely observed caused of T-cell invasion. Alveolar septum was thickened with infiltrated cells, and CD3+ cells accumulated around capillary blood vessel. Some of them were positive for FoxP3, and indicated moderate proliferation indices (25-50%). T-cell invasion was also observed in kidney. Lymphadenopathy was detected in 6 of 9 (66.7%) with completely destroyed architecture, increment of the proportion of Fox3+ cells, and moderate proliferation indices (25-50%). The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 4 splenic CD4+ cells, and 2 of 2 LNs CD4+ cells from compound mice.

These results suggest that CARD11 mutant-induced NFkB activation and constant HBZ expression may have similar effects, such as T-cell infiltration into organs and LN adenopathy, and that the simultaneous occurrence of both may have additive effects.

Disclosures

Sugiyama: Chordia Therapeutics, Japan.: Current Employment. Morishita:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Shimoda:Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Novartis: Honoraria, Research Funding; Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria.