Background
Regulatory T cells (Tregs) are a heterogeneous population essential for maintaining homeostasis and controlling immune responses. Tregs can also suppress tumor immunity. Their plasticity enables them to effectively regulate immune responses based on mechanisms optimized by tissue development, thereby adapting to their microenvironment. However, Treg adaptation to external signals can also cause Treg instability, which is associated with a shift from suppressive to proinflammatory functions and can lead to the development of autoimmunity or enhance anti-tumor responses. Therefore, comprehensive characterization of Treg signatures associated with individual tissues or pathologies should facilitate the design of optimized immunotherapies.
On April 17, 2024, Elisa Bonnin et al. published an article titled "CD74 supports accumulation and function of regulatory T cells in tumors" in Nature Communications. The researchers found that human tumor-infiltrating Tregs selectively overexpressed the MHC class II invariant chain CD74. CD74 has previously been described as a regulator of antigen-presenting cell biology, but its function in Tregs remains unknown . To understand the role of CD74 in Tregs, the researchers generated CRISPR-Cas9 from primary human Tregs. CD74 knockout (CD74KO) cells were found to be completely dysfunctional in tumor tissues. Under steady-state conditions, CD74KO-Treg phenotype, survival, and suppressive capacity were unaffected both in vitro and in vivo. Therefore, CD74 is a specific regulator of tumor-infiltrating Tregs and a target for interfering with Treg anti-tumor activity.
Tumor-associated Tregs overexpress CD74
By analyzing single-cell RNA sequencing (scRNAseq) datasets of CD4+ conventional T (Tconv) and Treg cells from patients with non-small cell lung cancer (NSCLC), researchers found that genes associated with activated tumor Tregs, such as CD25, HLA-DR, CTLA-4, and CCR8, as well as CD74, were significantly differentially expressed in tumor Tregs compared with tumor Tconvs. Tumor Tregs overexpressed CD74 compared with blood Tregs and both blood and tumor Tconvs. Flow cytometry analysis of CD74 surface expression levels in Tregs and Tconvs from blood and tumor samples of NSCLC patients revealed that few blood and tumor Tconvs, as well as blood Tregs, expressed CD74 on their surfaces, while 6% to 22% of tumors expressed Tregs.
CD74 deficiency in vitro does not affect the survival, phenotype, and suppressive capacity of Tregs
Using CRISPR-Cas9 technology to knock out the CD74 gene in Tregs, flow cytometry was used to assess CD74 expression after electroporation, revealing that 70-90% of Tregs lost CD74 protein expression four days after electroporation. CD74 deletion had no effect on Treg proliferation, activation, or suppressive function in vitro.
Tregs lacking CD74 retain suppressive function
The requirement for CD74 for Treg suppressive activity in vivo was assessed using a xenogeneic graft-versus-host disease (GvHD) model. In this model, GvHD was induced by injecting human PBMCs into non-glandular stem (NSG) immunodeficient mice, and co-injection of Tregs limited effector T cell activation, thereby attenuating clinical symptoms of the disease. The dynamics of injected expTregs in the blood were followed, revealing that both WT and CD74KO Tregs were detected in the blood from day 4 to day 8 post-injection, with similar dynamics. Mice that received PBMCs alone developed severe GvHD (over 20% weight loss) starting around day 20, whereas co-injection of WT or CD74KO expTregs delayed the onset of clinical symptoms. Tregs lacking CD74 controlled xenogeneic GvHD to a similar extent as WT Tregs, indicating that CD74 is dispensable for Treg suppressive activity in vivo in this GvHD model.
CD74 stabilizes the suppressive phenotype of intratumoral Tregs
The researchers transplanted MDA-MB231 triple-negative breast cancer cells into NSG mice. On day 10, when tumors were palpable, the mice were either left untreated (control group) or injected with fresh PBMCs alone or co-injected with ex vivo expanded WT or CD74KO Tregs (expTregs). In control mice, tumors grew exponentially, and injection of PBMCs led to tumor rejection. The addition of WT expTregs slightly delayed tumor elimination, but CD74KO expTregs significantly accelerated tumor rejection. This suggests that CD74 expression in Tregs is required for Tregs to maintain their suppressive function in tumors.
The loss of Foxp3 expression in Tregs is associated with an unstable Treg phenotype. The geometric mean of Foxp3 expression under different conditions was analyzed. The CD74KO spleen was expressed 6 days after transplantation. The geometric mean was slightly lower in Tregs. WT Tregs showed significantly elevated Foxp3 levels in tumors compared to the spleen, whereas tumor-infiltrating CD74KO Tregs showed a significant decrease in Foxp3 expression. These results suggest that CD74 is required to ensure Treg stability under certain inflammatory conditions. Treg activation in the presence of tuSN alone or in the presence of IL-12 induced apoptosis in CD74KO but not in WT Tregs, suggesting that CD74 helps increase Treg survival in the presence of tumor-derived soluble factors. The researchers found that incubation of tuSN with IL-12 induced the highest IFNγ production, with the greatest increase observed in CD74KO Tregs, indicating that CD74 plays a crucial role in the preservation of Treg suppressive function in the TME. CD74 plays a regulatory role in maintaining TSDR demethylation and provides a molecular basis for CD74-mediated maintenance of Foxp3 stability in Tregs.
CD74 facilitates tumor infiltration and maintains Foxp3 levels
Studies have found that CD74 can regulate the migration ability of Tregs, as well as the size and cytoskeletal contractility of Tregs. Using immunocompetent C57BL/6 mice transplanted with B16-F10 melanoma cells, the surface expression of CD74 in tumor Tregs was higher than that in peripheral Tregs, and also higher than that in tumor and peripheral CD4 + Tconvs and CD8 + T cells. CD4 + CD25 high Tregs sorted from CD45.1WT or CD45.2CD74 complete KO mice were co-injected into MCA tumor-bearing Rag-KO mice. Compared with the spleen, the KO/WT Treg ratio in the tumor was slightly lower, and was accompanied by a significant decrease in Foxp3 levels. These results suggest that CD74 has a potential role in the accumulation of Tregs in tumors.
Summarize
Tregs are a major barrier to tumor immune rejection because they induce tumor immune tolerance and promote tumor angiogenesis. Selectively depleting or inhibiting Tregs in tumors and preserving Tregs in healthy tissues (required for maintaining immune homeostasis) is a viable strategy for tumor elimination, and the effect is enhanced when used in combination with other immunotherapies. Researchers found that CD74KO Tregs exhibit defects in actin cytoskeleton organization and intracellular organelles within tumors, resulting in reduced activation, decreased Foxp3 expression, and reduced tumor accumulation. In preclinical models, CD74KO Tregs were associated with accelerated tumor rejection, highlighting the specific role of CD74 in tumor-infiltrating Tregs. Reducing CD74 surface expression can inhibit the accumulation of Tregs in tumors, and CD74 may be a potential therapeutic target for regulating tumor Tregs.
