Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Experimental autoimmune encephalomyelitis

• CD4+ T cell memory

• Multiple sclerosis

Main Findings

In this preprint, the authors wanted to understand why therapies that block T cell infiltration into the central nervous system (CNS) are efficacious for reducing the symptoms of relapsing-remitting multiple sclerosis (RRMS), but not for progressive multiple sclerosis (PMS). The authors hypothesize that the extant pool of autoreactive resident memory CD4+ T cells (CD4+ TRM) chronically present in the CNS contribute to the chronicity of PMS independently of de novo recruitment of autoreactive CD4+ T cells.

To examine the presence and function of autoreactive CD4+ TRM in the CNS, the authors used a model of experimental autoimmune encephalomyelitis (EAE) induction in mice involving immunization of MOG33-55 (myelin oligodendrocyte peptide) in complete Freund’s adjuvant (CFA). This model of EAE induction in mice results in chronic inflammation of the CNS and brain that resembles human PMS. The authors note the presence of CD4+ T cells in the brain, spinal cord (SC), and cervical lymph nodes (cLN) at the peak and chronic phases of disease (8 and 50 days following EAE induction, respectively). Importantly, the CD4+T cells present in the brain and SC at the chronic phase of disease had a surface phenotype reminiscent of TRM cells; notably, positive expression of CD69, CXCR6, and P2RX7. EAE induction in mice expressing tdTomato under the control of Hobit (a transcription factor associated with CD4+ TRMcells) confirmed the presence of CD4+ TRM cells during the chronic phase of EAE. Functionally, these cells formed large aggregates associated with areas of microglial activation and demyelination, suggesting a potential role of CD4+ TRM cells in these processes. CITE-sequencing-based characterization confirmed that CD4+ TRM present during the chronic phase of EAE display heterogeneity and have proinflammatory potential. Furthermore, PMA/Ionomycin restimulation of these cells induced their production of IFNγ and IL-17A. The authors then confirmed the presence of CD4+ TRM cells in human PMS. Indeed, the proportion and number of CD4+CD69+CD49a+ T cells from active MS brain lesions were significantly higher than those of non-associated white matter MS lesions and healthy control brain white matter.

The authors next wanted to discern the relative contribution of CD4+ TRM cells to brain inflammation during the chronic phase of EAE from that of circulating CD4+T cells. To do this, the authors induced EAE in two sets of mice, with one set receiving the S1P antagonist, FTY720 or CD4+-depleting antibody for 18 days following the disease peak (d8; before the establishment of bona fide CD4+ TRM in the CNS), and the other set receiving FTY720 or CD4+ depletion for the same duration at the chronic phase of disease (d40; when CD4+ TRM have been established). Strikingly, mice receiving FTY720 or CD4+ depletion during the early phase of disease had almost a complete remission in clinical score. In contrast, mice receiving these treatments during the chronic phase of disease had no change in the clinical scores of EAE. These findings suggest that circulating CD4+ T cells are dispensable for maintaining chronic inflammation during EAE in the presence of CD4+ TRM.

Lastly the authors sought to see if CD4+ TRM depletion can alleviate chronic EAE. To address this question, the group administered NAD to mice during the chronic phase of EAE, in the presence of CD4+ T cell depletion. NAD treatment leads to engagement of the P2RX7 receptor on CD4+ TRM cells therefore leading to NAD-induced cell death. The results of their experiment indicated that CD4+ TRM depletion can alleviate the clinical score of chronic EAE. Together, these results implicate CD4+ TRMas drivers of chronic autoimmune neuroinflammation in both mice and humans.

Limitations

• It is now being commonly appreciated that meningeal inflammation is associated with the demyelination of MS. Furthermore, recent work using mouse models of EAE stratify between the brain parenchyma, the dura mater, and the leptomeninges. The preprint does not mention whether the region they call the “brain” is the brain parenchyma, or whether it includes the leptomeninges anatomically located above the parenchyma. A further stratification of whether the CD4+ TRM cell aggregates are present in the brain, the leptomeninges, or the dura mater during chronic EAE would be helpful in strengthening the authors claims.

• The authors refer to CD4+T cells that express various combinations of surface markers such as CD69, CD49a, P2RX7, and CD44 as being “resident-memory cells”. However, residency and memory are functional characteristics, and the use of surface markers isn’t sufficient to support their claim of residency. To conclusively demonstrate that their population of CD4+ TRM  are memory, restimulation experiments of brain CD4+ TRM cells from chronic EAE mice with cognate myelin peptide and assessment of cytokine response and proliferation would be helpful. PMA/Ionomycin restimulation experiments are non-specific and will activate all effector and memory T cells. Furthermore, to claim true residency, the authors should consider parabiosis or i.v labelling experiments to demonstrate that the population of CD4+ T cells present in the brain at d50 post induction of EAE is truly resident (or i.v negative).

• The CD4+ TRM depletion experiments only seemed to have a marginal effect on the reduction of chronic EAE clinical scores that only seemed to be significant during normalization to the start of the treatment. The authors should address this modest effect size and potentially offer alternate explanations underlying the chronicity of EAE beyond the effects of CD4+ TRM cells. Furthermore, it would be helpful to see if the CD4+ TRM depletion strategy affected the histopathology of the brain as an alternate readout beyond clinical scoring. These additional verifications would make the authors’ claim stronger.

• The authors use inconsistent gating of their flow cytometry data when examining similar populations of CD4+ TRM cells across figures. For example, CD44 is used in Figures 5D, 5F, 5J, and 5L, where CD44 does not seem to be used in Figure 1D and Figure 1E.

Significance/Novelty

What is the novelty of the preprint for the specific field?

The preprint reveals the presence of CD4+ TRM cells in a chronic model of murine EAE and in human progressive MS patients. Furthermore, the authors establish a correlation between the presence of these CD4+ TRMcells and chronic autoimmune inflammation in mice. Lastly, the authors’ work offers a potential explanation as to why PMS remains refractory to traditional therapies involving the sequestering of circulating T cells away from the brain and into the lymph nodes (e.g Fingolimod).

How does the result of the preprint matter for general immunologists and/or patients?

This work offers an intriguing explanation as to why PMS remains refractory to S1P antagonists and anti-integrin therapies. The results of this preprint indicate that strategies seeking to deplete brain CD4+ TRMcells may be an alternative avenue forward when considering drug developments for PMS.

Credit

Reviewed by Boyan Tsankov as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.