A study of immune dynamics in experimental arthritis using CRISPR and PET techniques

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation associated with CD4 T cell dysfunction. The disease has no cure; thus, early diagnosis and interventions are crucial. DNMT3A is one of the most somatically mutated genes in the hematopoietic system of elderly people and RA patients. However, its association with CD4 T cells, particularly CD4 T follicular helper (Tfh) cells, is poorly understood. In this thesis, I aim to explore potential interventions and diagnostics related to CD4 T cells using CRISPR and PET technologies. Additionally, I address CRISPR-related issues, such as the genetic heterogeneity caused within a CRISPR-targeted cell population, and the selective advantage to p53 mutated cells due to CRISPR-associated DNA damage.

Methods: To address the genetic heterogeneity created by CRISPR, I developed the Rapid CRISPR Competitive (RCC) assay. This discovery approach uses PCR, Sanger sequencing, and sequence deconvolution to identify how different genotypes are enriched or depleted in relation to cellular behavior or phenotype of interest (Paper I). The RCC assay was applied in vivo to understand how Dnmt3a mutations affect CD4 T cell differentiation in experimental arthritis (KRN splenocyte transfer model) (Paper II). The RCC assay and custom CRISPR screens were further used to identify a CRISPR-p53 interactome relevant to CRISPR-associated DNA damage (Paper III). In addition, to test the possibility of capturing early immune system activation in arthritis, we deployed PET imagining using a novel CD69 affibody tracer in experimental arthritis (Paper IV).

Results: The RCC assay can be applied to study gene functions in different settings, such as T cell activation and development, macrophage differentiation and function, and cancer cell fitness (Paper I). Using the RCC assay, mutated Dnmt3a was identified to limit Tfh differentiation and attenuate the arthritis score in experimental arthritis (Paper II). Moreover, the RCC assay was used to identify that CRISPR-associated DNA damage can enrich cells with mutations in p53-related genes, and that this can be abrogated by specific inhibitors (Paper III). Lastly, using PET imaging, the CD69 affibody tracer can detect immune activation prior to disease onset in an animal model for arthritis (Paper IV).

Conclusions: I established the RCC assay, leveraging CRISPR-generated genetic heterogeneity. This assay was applied to identify that (i) Dnmt3a mutations negatively affect CD4 T cell differentiation, particularly follicular T helper cells, and (ii) CRISPRassociated DNA damage can enrich cells with p53 mutations (which can be prevented using inhibitors). Utilizing PET technology, I further identified that a CD69 affibody PET tracer could be employed for the early detection of arthritis development.

List of scientific papers

I. Shen, Y., Jiang, L.*, Iyer, V.*, Raposo, B., Dubnovitsky, A., Boddul, S., Kasza, Z., & Wermeling, F. A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system. Comput Struct Biotechnol J. 2021 Sep 20;19:5360-5370. *,#Authors contributed equally.
https://doi.org/10.1016/j.csbj.2021.09.020

II. Shen, Y., Li, Z., Boddul, S., Kasza, Z., Espinosa, A., Klareskog, L., & Wermeling, F. Dnmt3a mutations limit Tfh differentiation and attenuate joint inflammation. [Manuscript]

III. Jiang, L., Ingelshed, K., Shen, Y., Boddul, S., Iyer, V. S., Kasza, Z., Sedimbi, S., Lane, D., & Wermeling, F. CRISPR/Cas9-induced DNA damage enriches for mutations in a p53-linked interactome: implications for CRISPR-based therapies. Cancer Res. 2022 Jan 1;82(1):36-45.
https://doi.org/10.1158/0008-5472.CAN-21-1692

IV. Puuvuori, E.*, Shen, Y.*, Hulsart-Billström, G., Mitran, B., Zhang, B., Cheung, P., Wegrzyniak, O., Ingvast, S., Persson, J., Ståhl, S., Korsgren, O., Löfblom, J., Wermeling, F.#, Eriksson, O.# Positron emission tomography imaging of CD69 in a murine model of rheumatoid arthritis. J Nucl Med. *,#Authors contributed equally. [Accepted]
https://doi.org/10.2967/jnumed.123.266336