Variants in human CD48 lead to impaired T cell immunity and increased inflammation
Samantha Milanesi, Tiziana Lorenzini, Tommaso Marchetti, Diana Tintor, Raquel Planas, Ola Sabet, Lars Malmström, Sudip Acharya, Carson D. Williams, Zoe E. Manning, Jack H. Roser, Angelica C. Ehler, Michael Huber, Seraina Prader, Stefano Vavassori, Cullen M. Dutmer, Jordan K. Abbott, Jana Pachlopnik Schmid
Samantha Milanesi, Tiziana Lorenzini, Tommaso Marchetti, Diana Tintor, Raquel Planas, Ola Sabet, Lars Malmström, Sudip Acharya, Carson D. Williams, Zoe E. Manning, Jack H. Roser, Angelica C. Ehler, Michael Huber, Seraina Prader, Stefano Vavassori, Cullen M. Dutmer, Jordan K. Abbott, Jana Pachlopnik Schmid
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Research Article Immunology Inflammation

Variants in human CD48 lead to impaired T cell immunity and increased inflammation

Abstract

CD48 is a surface molecule with immunoregulatory functions. Following our initial report of a patient with a de novo heterozygous variant at amino acid S220 in the CD48 gene, we describe a second, unrelated patient with similar features of immune dysregulation and a missense change affecting the same residue. To further elucidate the specific pathogenic mechanisms of the identified variants, we reviewed patient records, analyzed patient-derived cells, and employed complementary in vitro and in vivo model systems, including transfected cell lines and CD48-deficient mice. We demonstrate that the variants are associated with altered distribution of CD48, characterized by diminished CD48 surface expression, intracellular retention, and activation of ER stress signaling. Patient T cells displayed increased susceptibility to apoptosis, reduced antiviral responses, and enhanced inflammation. Both patients exhibited T cell lymphopenia, a restricted T cell receptor repertoire diversity, and oligoclonal expansions consistent with antigen-driven selection. In parallel, virally infected CD48-deficient mice recapitulate key aspects of the human phenotype, including delayed antiviral immune responses, impaired viral clearance, and pronounced inflammation. We conclude that identified variants compromise CD48 cell surface localization, impair T cell survival and function, and predispose to inflammation, thereby highlighting the role of CD48 in immune regulation and the prevention of excessive inflammation.

Authors

Samantha Milanesi, Tiziana Lorenzini, Tommaso Marchetti, Diana Tintor, Raquel Planas, Ola Sabet, Lars Malmström, Sudip Acharya, Carson D. Williams, Zoe E. Manning, Jack H. Roser, Angelica C. Ehler, Michael Huber, Seraina Prader, Stefano Vavassori, Cullen M. Dutmer, Jordan K. Abbott, Jana Pachlopnik Schmid

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Figure 2

Human S220 CD48 variants lead to altered CD48 cellular distribution.

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Human S220 CD48 variants lead to altered CD48 cellular distribution. (A)...
(A) Microscopy analysis and quantification of the CD48 fluorescence intensity in HEK293 cells transfected with a plasmid encoding CD48 WT (light blue), S220F (purple), or S220Y (orange). Scale bars: 10 μm. (B) Box-and-whisker plots showing the median and quartiles of ATF4 reporter activity in HEK293 cells cotransfected with ATF4-mScarlet and either WT, S220F, or S220Y CD48 expression constructs. mScarlet fluorescence was measured by flow cytometry 24 hours after transfection. Expression levels are reported as the gMFI of the mScarlet signal. P values were determined using 1-way ANOVA followed by Dunnett’s post hoc test, with the WT used as reference. (C and D) Flow cytometry quantification of surface CD48 expression in HEK293 cells transfected with an empty vector (red), WT (blue), S220F (purple), or S220Y (orange) CD48 expression constructs, and either left untreated (–) or treated with PI-PLC (+). A representative experiment (C) and the relative quantification of the CD48 gMFI (D) are shown. The experiment was conducted twice. P values were determined by 2-tailed t tests. (E) Scatterplot of estimated binding free energies of the interaction between CD48 WT (light blue), CD48 S220F (purple), and CD48 S220Y (orange) with 2B4 and CD2. For each sample, the median of 3 measurements is shown. For each ligand, free energies were compared using Friedman’s test followed by Dunn’s multiple-comparison test. No significant differences were detected. (F) Flow cytometry quantification of surface CD48 expression in HEK293 cells cotransfected with equal quantities of CD48 WT and either an empty vector, or S220F, S220Y, or WT expression vectors, at 24 hours (left panel) and 48 hours (right panel) after transfection. Statistical comparisons between each experimental group and the control group (WT + empty vector) were performed using separate 2-tailed t tests. P values were not adjusted for multiple comparisons. The box-and-whisker plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median.