Spontaneous and inducible CD8 T cell lesions in the brain and spinal cord of HLA-DR15-positive MS PBMC humanized mice

Reviewer #1 (Public Review):

Papazian et al. demonstrate that human peripheral blood mononuclear cells (PBMCs) can be successfully and stably grafted into immunodeficient mice. They demonstrate that the adoptive transfer of PBMCs from multiple sclerosis (MS) patients is capable of inducing damage to the central nervous system (CNS). Furthermore, they demonstrate that the CNS inflammatory properties of these transferred cells are more dependent on HLA restriction rather than the disease status of the donor. Specifically, T cells restricted by HLA-DR15 (from both an MS patient and a healthy control) showed a greater propensity to induce neuroinflammation than T cells transferred from an MS patient that are restricted by a different MHC haplotype. This observation suggests that the differences in the peptide repertoire presented by this MHC haplotype biases adaptive immune responses toward encephalitogenic T cell generation. The conclusions of this paper are partially supported by their data, but the lack of important considerations and various controls limits the overall impact of this study. Major weaknesses of this study include:

1. The extent to which various immune cell quantification is performed. Two of the reasons the authors cite for the use of this model rather than a traditional EAE model are: i) the lack of involvement of CD8 T cells in the pathogenesis of EAE and ii) the marginal importance of B cells in EAE pathogenesis. However, throughout their paper, the authors never quantify the difference in CD4 vs CD8 T cell infiltration into the CNS. While repeatedly claiming that there are fewer CD4 T cells present than CD8 T cells within the CNS, this data is not included. Further, spinal cord numbers of CD4 and CD8 are not provided in lieu of CD3 T cell characterization. Given that there are far more hCD4 T cells in the periphery in these mice than CD8 T cells as well as the fact that the lack of B2m expression in this mouse model biases cells towards a CD4 fate, the omission of these data is concerning. Additionally, B cells don't make up any significant component of the cells transferred from HLA-DR15 donors. While the cells transferred from the HLA-DR13 donor are composed of a considerable number of B cells, the mice that received these cells didn't develop any signs of neurologic disease.

2. Incomplete exploration of potential experimental autoimmune encephalomyelitis (EAE) modeling. The authors justify the use of an extremely high amount of myelin peptide when immunizing their mice by citing that another humanized mouse model had such a requirement to induce clinical EAE. However, a demonstration of this technical requirement in their own model is not provided. Rather, they show that C57BL/6 mice get milder disease when such large doses of peptides are administered, leading to speculation that this is due to a tolerizing immune response that occurs at such high doses. Comparison of the susceptibility of B2m-NOG mice to EAE dependent on various peptide doses would be highly informative. Given that the number of hCD45+ in the periphery of NOG mice decreases following this immunization it would be prudent for the authors to determine if such a high peptide dose is truly ideal for EAE development in this mouse model.

3. The degree of myelin injury is not presented. The statement is repeatedly made that "demyelination was not observed in the brain or spinal cord" but no quantification of myelin staining is shown. A central feature of multiple sclerosis and related diseases is demyelination of the CNS. Hence, while compartmentalized inflammatory responses are detailed in this report, the utility of the humanized model for the exploration of human CNS demyelinating diseases remains unclear and in doubt.

Minor points:

- Method of quantification (e.g. cells per brain slice in figures 2E; 4E) is not very quantitative and should be justified or more appropriately updated to be more rigorous in methodology.

- Fig. 4 data should be shown from un-immunized DR15 MS and DR15 HI mice.

The premise of this work carries great potential. Namely, developing a humanized mouse system in which features of adaptive immunity that contribute to inflammatory demyelination can be interrogated will allow for traction into therapeutics currently unavailable to the field. Immediate questions stemming from the current study include the potential effect of ex vivo activation of PBMCs (or individual T and B cells) in vitro prior to transfer as well as the TCR and BCR repertoire of CNS vs peripheral lymphocytes before and after immunization. This group has been thoughtful and clever about their approach (e.g. use of subjects treated with natalizumab), which gives hope that fundamental aspects of pathogenesis will be uncovered by this form of modeling MS disease. Overall, while the current study makes several unique observations, the data collection is incomplete and the impact of this study could be greatly improved by addressing the limitations noted.

Reviewer #2 (Public Review):

Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system where immune cells play an important role in disease pathobiology. Increased incidence of disease in individuals carrying certain HLA class-II genes plus studies in animal models suggests that HLA-DRB1*15 restricted CD4 T cells might be responsible for disease initiation, and other immune cells such as B cells, CD8 T cells, monocytes/macrophages, and dendritic cells (DC) also contribute to disease pathology. However, a direct role of human immune cells in disease is lacking to a lag between immune activation and the first sign of clinical disease. Therefore, there is an emphasis on understanding whether immune cells from HLA-DR15+ MS patients differ from HLA-DR15+ healthy controls in their phenotype and pro-inflammatory capacity. To overcome this, authors have used severely immunodeficient B2m-NOG mice that lack B, T cells, and NK cells and have defective innate immune responses and engrafted PBMCs from 3 human donors (HLA-DR15+ MS and HI donors, HLA-DR13+ MS donor) in these B2m-NOG mice to determine whether they can induce CNS inflammation and demyelination like MS.
The study's strength is the use of PBMCs from HLADRB1-typed MS subjects and healthy control, the use of NOG mice, the characterization of immune subsets (revealing some interesting observations), CNS pathology etc. The major weaknesses are i) lack of sufficient sample size (n=1 in each group) to make any conclusion, ii) lack of phenotype in mice, iii) no disease phenotype even in humanized mice immunized for disease using standard disease induction protocol employed in an animal model of MS, and iv) mechanistic data on why CD8 T cells are more enriched than CD4+ T cells. The last point is very important as postmortem human MS patients' brain tissue had been shown to have more CD8+ T cells than CD4+ T cells.

Thus, this work is an important step in the right direction as previous humanized studies have not used HLA-DRB1 typed PBMCs however the weaknesses as highlighted above make the findings incremental to the field.