Comprehensive phenotypic and clonality analysis of intraepithelial lymphocytes of intestinal T-cell lymphoma and chronic enteropathy in dogs

Canine intestinal T-cell lymphoma (ITCL) may arise from intraepithelial lymphocyte (IEL) subsets expanded in chronic enteropathy (CE). To investigate this potential cell of origin, we performed flow cytometry, immunohistochemistry, and RNA <i>in situ</i> hybridization on IELs using biopsy samples from 62 dogs, including 6 large-cell lymphomas (LCLs), 9 small-cell lymphomas (SCLs), 31 CEs with increased IELs (IEL⁺CE), and 16 CEs without increased IELs (IEL^-CE). IELs in ITCLs were predominantly CD4^-CD8α^- (LCL, 5/6; SCL, 4/9), and 4/31 IEL⁺CEs had a higher proportion of CD4^-CD8α^- IELs than the other CE cases. Forward scatter values of CD4^-CD8α^- IELs were higher in IEL⁺CEs compared with IEL^-CEs, correlating positively with the Ki-67 index, indicating proliferation of this population. An increase in IELs lacking T-cell receptor expression was observed in 1/4 LCL, 2/5 SCL, and 1/13 IEL⁺CE cases. Lack of surface CD3 was observed in 2/9 SCL cases. One of 5 LCL cases partially expressed NKp46, whereas the proportion of NKp46⁺ IELs was low (< 4%) in 32/33 CE cases examined. In addition, 3/6 LCL cases expressed <i>NKp46</i> mRNA without detectable protein expression. Clonality analysis of isolated IELs yielded at least 1 clonal peak in 1/2 LCLs, 4/7 SCLs, 12/23 IEL⁺CEs, and 4/13 IEL^-CEs, underscoring the limited diagnostic utility on distinguishing ITCL, particularly SCL, from IEL⁺CE. These findings suggest that most canine ITCLs have a CD4^-CD8α^- immunophenotype and that unconventional IELs in IEL⁺CE may be the cells of origin. Moreover, the distinction between ITCL and CE remains challenging, emphasizing the importance of integrated diagnostic approaches.