Here, we provide novel insights in constitutive and activation-dependent mRNA and protein dynamics of CD74 in CD4+ T cells. Our analyses reveal CD74 upregulation, post-translational modification with CS and MHC II-independent translocation to the cell surface upon T-cell activation. Surface CD74 forms heterocomplexes with the classical chemokine receptor CXCR4 and is mechanistically involved in MIF-elicited T-cell chemotaxis. Dysregulated CD74 expression in severe COVID-19 disease patients demonstrates the translational relevance of our findings.
Most likely due to its classical and well-established MHC II-related functions, CD74 was initially overwhelmingly studied in antigen-presenting cells, most notably monocytes/macrophages and B cells [1]. The discovery of CD74 as the cognate MIF receptor has partially changed this picture. In the course of these studies, MIF/CD74 pathways were not only examined in monocytes and macrophages, but it turned out that CD74 can be abundantly expressed in several types of cancer cells and may be upregulated in certain other cell types such as endothelial cells or cardiomyocytes upon inflammatory stimulation or stress [21, 26–28]. However, MHC class II-negative T cells have mostly been neglected in this regard. Only a handful of descriptive reports on CD74 expression in human T cells exist, mainly in context of disease, and without scrutinizing any mechanisms. Yang et al. investigated CD74 surface expression in PBMCs after stroke and amongst other cell types found a significant increase in the number of CD74-expressing CD4+ T cells but not CD8+ T cells [26]. Fagone et al. showed an upregulation of CD74 gene expression in CD4+ T cells upon activation, that was unchanged in T cells from healthy donors vs. patients with multiple sclerosis [27]. In contrast, in the chronic inflammatory context of rheumatoid arthritis, Sánchez-Zuno et al. observed the percentage of CD74 expressing T cells to be below 1% [72]. To our knowledge, Gaber et al. provided the only functional evidence of CD74 in human CD4+ T cells reporting on an inhibition of MIF-induced T-cell proliferation using a neutralizing CD74 antibody [21]. However, the relevance of this observation has remained unclear, as no isotype control immunoglobulin was used in that study. In contrast to CD74, regulation of CXCR4 in T cells has been studied comprehensively, also as it plays an important role in the docking-process of the human immunodeficiency virus and mediates CXCL12-driven co-stimulatory and migratory T cell responses [73–76].
Our MIF receptor profiling of freshly isolated primary human CD4+ T cells revealed the expected abundant expression of CXCR4, whereas no substantial surface expression of CD74, CXCR2 and ACKR3 could be detected. This identifies non-activated human CD4+ T cells as a suitable cell type to study the MIF/CXCR4 axis. In previous reports, CXCR4 expression was shown to be downregulated in the context of T-cell activation, which is confirmed by our study [73, 75]. Nevertheless, CXCR4 remained abundantly expressed also in activated T cells.
An unanticipated effect was the observation of a significant upregulation of CD74 surface expression upon T-cell activation. Of note, this upregulation was independent of HLA-DR pointing towards an MHC II-independent role of CD74 in CD4+ T cells. Interestingly, CD74 surface expression correlated with donor age, indicating a potentially more pronounced CD74 upregulation in memory and effector T cells compared to naive T cells, due to physiologically increased abundance of these phenotypes upon enhanced antigen encounters during aging [77–79].
Our MIF receptor profiling of resting and activated CD4+ T cells as well as re-analysis of CD4+ T-cell proteome data from Wolf et al. revealed no expression of CXCR2 in T cells, which is in line with multiple literature reports, but stands in contrast to the recent finding of CXCR2/CD74 co-expression in T cells as reported by Westmeier et al. [80]. Expression of ACKR3 in T cells still remains controversial [81, 82].
As CD74 is known to be expressed only in small percentages on cell surfaces and is mainly stored in intracellular deposits, we next evaluated CD74 protein expression after membrane permeabilization via flow cytometry. Unexpectedly and to date unknown, we detected an abundant intracellular expression of CD74 in freshly isolated T cells, which was further enhanced by T-cell activation. WB experiments confirmed enhanced CD74 expression with detection of protein bands corresponding to the known p33 and p41 isoforms in humans [1, 52, 83, 84]. However, due to the small difference in size a clear differentiation between short and long isoforms of the protein regarding p33 vs. p35 and p41 vs. p43 isoforms was not possible. Interestingly, we observed an additional pronounced protein band at approximately 55 kDa, which appeared only after 24 h of T-cell activation and further increased in abundance during activation, even exceeding the most abundant p33 protein band. Previous reports identified a specific CD74 isoform, CD74–CS that is being reported to run at a similar molecular weight and is product of a post-translational modification with the glycosaminoglycan CS at Ser 201. The modification was shown to enable the translocation of CD74 molecules towards the cell surface, while due to following rapid endocytosis only a small proportion can be transiently detected on the cell surface [54–62]. In fact, when we treated our T-cell samples with chondroitinase, an enzyme that specifically cleaves CS, we noticed the signal intensity of the observed p55 isoform to be significantly decreased in comparison to untreated controls. Nevertheless, we acknowledge that treatment with chondroitinase did not lead to a complete disappearance of the observed band, which could be explained by sub-optimal buffer conditions due to the strong pH-dependency of the enzyme or non-sufficient incubation time. Furthermore, several other post-translational modifications, such as O- and N-silylation, palmitoylation and phosphorylation, have been reported for CD74 that were not studied in this work [85–87]. Despite these limitations, we speculate that post-translational modification of CD74 with CS might be the underlying mechanism of CD74 translocation to the cell surface during the process of T-cell activation. Immunofluorescent co-staining of CD74 with ER and lysosomal markers verified the typical localization of CD74 in the ER and suggested a functional trafficking of CD74 within the endolysosomal compartment. Re-analysis of two independent RNAseq data sets from the Cano-Gamez et al. and Szabo et al. studies and two proteomic data sets from the Cano-Gamez et al. and Wolf et al. publications comparing resting and activated T-cell states, complemented our data and provided substantial corroborating evidence that CD74 is constitutively expressed in resting T cells and becomes rapidly upregulated upon T-cell activation in a sustained manner [28, 35, 39]. The proteome data suggested a maximum CD74 protein abundance after 72 h and again identified a counter-regulation of CD74 and CXCR4 in the early activation phase. After the initial downregulation, CXCR4 expression was then found to be reconstituted after approximately 3 to 4 d. Of note, CD74 upregulation occurred after upregulation of the early activation marker CD69, but before the intermediate activation marker CD25 [65, 66]. Cytokine polarization to T-cell effector phenotypes had no additional effects on CD74 protein abundance. In contrast, CXCR4 protein expression was upregulated after 5 d of Treg and Th17 polarization, possibly linked to an already described TGF-β-induced CXCR4 expression mechanism [88].
The study by Cano-Gamez et al. caught our attention as CD74 incidentally appeared as a strong marker protein of natural Tregs and effector memory T cells re-expressing CD45RA (TEMRA) in their presented data, possibly linking CD74 protein expression to T-cell effectorness [28]. Since observations of CD74 expression have often been made under inflammatory conditions, as for instance IFN-γ-rich environments, or in a disease context, we compared DEGs of regularly activated T cells (Th0) with activated T cells that were additionally differentiated towards specific Th0, Th1, Th2, iTreg and Th17 phenotypes through established cytokine polarization protocols [89]. Notably, except for the observed reduction of CD74 in Th17 conditions, cytokine conditions did not trigger significant changes. Therefore, T-cell activation represents the main stimulus for CD74 upregulation independent of the surrounding inflammatory cytokine milieu. Interestingly, Th17-polarized cells were also the only phenotype with significantly upregulated MIF expression compared to non-polarized CD4+ T cells, fitting to previous data indicating a role of MIF in Th17 T-cell differentiation [18, 20, 24]. Re-analysis of proteomic data further identified CD74 to be rapidly renewed in resting memory CD4+ T cells, potentially pointing towards a role of CD74 in memory T-cell homeostasis.
We also aimed to identify potential MHC II-independent CD74 transcriptional gene regulation. Combining a database analysis of the GTRD, PathwayNet and STRING network databases enabled us to narrow down relevant and potential MHC II-independent transcription factors within a 500 bp distance from the CD74 gene locus. However, we like to emphasize that the here provided database research approach mainly relies on the quality of the included pathway/protein interaction prediction tools and can only be interpreted as a first approximation to the subject. The list of eight CIITA-independent transcription factors with high confidence predictions included ETS1, a crucial transcription factor for T-cell survival and activation [63, 64]. In this context, Wolf et al. identified ETS1 as the most rapidly renewed transcription factor in T cells reflecting preparedness towards activating stimuli [39]. Accordingly, by performing an ATAC assay, Wolf et al. found that the ETS1 transcription factor binding motif can be detected in most accessible promoter regions of the resting naive CD4+ T-cell genome. About half of these binding sites were located in promoter regions, suggesting ETS1 as a transcriptional regulator of the promoter-associated genes. Interestingly, supplementary data of Wolf et al. shows that the CD74 gene is located in accessible chromatin regions in naive CD4+ T cells. Based on a ChIP analysis, showing actual ETS1 binding in the CD74 promoter region, transcriptional regulation of CD74 by ETS1, a transcription factor associated with T-cell preparedness for rapid activation, seems conceivable. Binding of ETS1 to other MHC II-associated genes was not observed, which may be either related to insufficient accessibility of the MHC II-related genes in resting naive CD4+ T cells or differential ETS1 gene binding.
Taken together, we hypothesize that ETS1-driven regulation of CD74 expression might be the underlying process of the observed rapid CD74 induction after activation, which, together with post-translational chondroitin sulfatinylation of constitutively expressed intracellular CD74, serves to rapidly establish marked CD74 surface expression. Once positioned on the cell surface, CD74, functioning as the cognate MIF receptor, can mediate downstream signaling events [90].
In the absence of an identified classical signaling-competent cytosolic domain in the short cytoplasmic tail of CD74, two alternative distinct tracks of CD74 signaling have been reported. First, CD74 signaling can be mediated by its intracytoplasmic domain (ICD), which is proteolytically cleaved by the intramembrane protease signal peptide peptidase-like (SPPL)2a and subsequently translocates into the nucleus, where it functions as a transcription factor and/or transcriptional coactivator [90–92]. Whether this process occurs in the endolysosomal compartment or on the cell surface and how it is exactly triggered by extracellular MIF has remained partly unclear. A second signaling CD74 pathway involves the association of CD74 with a co-receptor. Depending on the cellular and (patho)physiological context this can be CD44, the initially identified co-receptor of CD74, or one of the MIF chemokine receptors, i.e. CXCR2, CXCR4 or ACKR3/CXCR7 [4, 8, 11, 12]. In our study, we provide evidence for a role of CXCR4, as we obtained evidence from PLA and chemotaxis experiments for CD74/CXCR4 heterocomplex formation to facilitate MIF-elicited chemotaxis of activated T cells. We also obtained evidence for MIF-induced internalization of CD74/CXCR4 heterocomplexes from the surface of T cells.
As mentioned above, CD44 represents another potential co-receptor of CD74 in T cells that is abundantly expressed and is an established activation marker of T cells. Additional studies are necessary to evaluate the functional relevance of CD74/CD44 interactions in T cells [11, 93].
An impaired adaptive immune response linked to sustained T-cell activation and a dysregulated IFN-response is believed to be a significant determinant of COVID-19 progression [30, 32, 94, 95]. Furthermore, accumulating evidence points towards a critical role of MIF as a prognostic marker to predict disease severity and patient outcome in COVID-19 disease. Notably, a recent study by Westmeier et al. investigated MIF receptor expression in CD4+ and CD8+ T cells in COVID-19 patients with mild and severe disease and observed an increased expression of CD74 in CD4+ and CD8+ T cells compared to healthy controls [71]. Interestingly, the authors also observed an inducible expression of CXCR2 and CXCR4 upon SARS-CoV-2 infection pointing towards increased susceptibility to MIF-mediated signaling in the course of COVID-19 disease. A characterization of T-cell subpopulations in their study revealed a predominant central and effector memory phenotype of the CD74-expressing T cells that further produced higher cytotoxic molecules and expressed enhanced proliferation markers. In accordance, we observed a significant upregulation of CD74 surface expression on CD4+ and CD8+ T cells in the severe disease group, when comparing patient cohorts with mild and severe COVID-19 disease. In contrast, no significant differences between both groups regarding CXCR4 expression was observed. CD74 markedly exceeded HLA-DR expression, which showed no significant changes between both cohorts, again confirming an MHC II–independent regulation of CD74 in T cells. Of note, CXCR4 and CD74 expression was also monitored in monocyte subpopulations in the same patient cohort revealing enhanced expression of CD74 in classical monocytes again without significant changes in CXCR4 expression. We speculate that the observed upregulation of CD74 reflects increased COVID-19-induced T-cell activation states, which might enhance susceptibility towards MIF [30, 32]. However, suitability of T-cell CD74 as a potential biomarker for disease progression in COVID-19 and its relevance in other inflammatory or malignant diseases accompanied by broad T-cell activation still needs to be evaluated in future prospective trials. Furthermore, due to the small patient cohort and heterogeneity a subgroup-specific analysis based on factors such as age, gender or comorbidities was not feasible in the presented study.
In summary, our data identify CD74 as a functional MIF receptor and MHC II-independent activation marker of activated CD4+ T cells mediating MIF-driven CD4+ T-cell chemotaxis, most likely through complex formation with CXCR4. CD74 and CXCR4 expression levels behave inversely in the course of T-cell activation. Induction of CD74 occurs rapidly upon activation stimulus in naive and memory T cells leading to an activation-induced chondroitin sulfated isoform. We have thus unraveled a previously unrecognized MIF/CD74/CXCR4 signaling pathway in activated human T cells with functional relevance for T-cell motility and potentially other activities of activated T cells (Fig. 7). We confirm high CD74 surface expression in T cells under disease conditions in critically ill COVID-19 patients potentially linking dysregulated CD74 to disease severity. Thus, targeting the dysregulated MIF-CD74 axis might resemble a tractable treatment strategy to interfere with the critical role of MIF in the COVID-19 disease context. To this end, future studies will be needed to clarify whether CD74 could have implications in immunosenescence of T cells with potential relevance for the enhanced susceptibility of the aging population to infections like COVID-19 or reduced responses to vaccinations [96–98].