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  1. Author Response:

    Reviewer #1 (Public Review):

    The manuscript by Liu et al investigates how MRI can be used to detect the earliest stages of CNS infections and how MRI can also be used as a surrogate readout for treatment efficacy. Authors demonstrate convincingly that microbleeds, as evidenced by unusual dark spots in the brain of mice infected with a virus that infects the brain, occurred at the earliest stages of viral infection. Authors also convincingly demonstrate that the infusion of virus-specific immune cells, when delivered at the right time and at the right dose, could reduce these microbleeds. Importantly, authors showed that the wrong dose could be detrimental.

    The authors cast this study as a method for improving research and discovery in immunotherapy context and the study is convincing in its conclusions regarding imaging microbleeds and the immunotherapy tested herein. While authors do not directly suggest so, these findings extend the significance of this work beyond research and development of immunotherapies by providing a potential early detection mechanism for viral infection in the brain. This may be feasible as the MRI methodologies for detecting these phenomena are generally translatable to clinical imaging scenarios, though the imaging resolution may not.

    Weaknesses in the report revolves around the value of and the ability to image magnetically labeled T cells in the presence of microbleeds.

    1. Authors developed a magnetic particle coated with fluorescent molecules and antibodies specific for CD8+ T cells. They labeled these T cells with particles for detection by MRI. They then wanted to follow the accumulation of these cells in the brain following infusion and viral infection by performing MRI using parameters that amplify the signal of the attached label. The rationale for these experiments was to determine if immune cell infiltration preceded vascular compromise. This suggests the expectation for active chemotactic migration or other signaled accumulation rather than leakage. When authors tested their magnetically labeled T cells for functional impairment due to the presence of attached magnetic particles, they did not test for deficits to migratory capabilities, such as in standard transwell migration assays. Others have shown (see https://doi.org/10.1038/nm.2198 for example) that T cell migration is very sensitive to the type of attached nanoparticle as well as the surface coverage. Perhaps authors should temper their claims that magnetically labeling of T cells does not alter T cell function without at least an assay of this critical function. Further, the fluorescence microscopy shown in Figure 7D is of insufficient resolution to claim that MPIOs are inside cells. Electron microscopy should be used to determine this.

    We thank this Reviewer for the comments. In this Revision, we added EM data to confirm the cellular location of MPIOs (Fig 7D and S7D). The EM experiment also added another layer of information for improving our cell isolation method. We improved our FACS experiment by narrowing down the MPIO positive gating to exclude the T cell population that labeled with high numbers of MPIO particles, which may affect T cell functions, and some crosslinked MPIO particles that formed during conjugation (Fig 7B and S7A). The yield of FACS of MPIO-labeled T cells is ~8.3%. As quantified from EM images, 91% MPIOs were localized intracellularly (Fig 7E). We agree that labeling T cells with nanoparticles might alter key T cell functions. We have improved the manuscript by putting this caution and reference. We also added T cell migration assay results (Fig 7G). Labeling CD8 T cells with MPIO did not affect T cell migration. This adds to our other in-vitro assays that T cell function is not significantly affected. There is in-vivo evidence as well that labeled T cells are functional. In Fig 8E-I, MPIO-labeled T cells were found in the brain, which showed that labeled T cells can migrate into the brain. In addition, a key phenotype of virus specific CD8 T cells in this model is the therapeutic function described in the manuscript. Labeling virus specific CD8 T cells with MPIO did not affect their therapeutic function. Quantification of bleeding in the OB and brain on day 6 and 11 verified the therapeutic effects of MPIOlabeled OT-I T cells (Fig 1E and 2C vs Fig S9C and D). We added discussion of these points in this Revision.

    1. Regarding the use of imaging the accumulation of magnetically labeled T cells, authors show evidence that magnetically labeled T cells accumulate in areas of the brain that as yet do not present with microbleeds but do have the histological hallmarks of vascular inflammation. This corroboration is intriguing but only provable with a serial imaging study in the same animal, which was not performed. Authors are also encouraged to report on the frequency in which a magnetically labeled T cell was present in a pre-vascular compromised inflammatory environment. The bulk of the results on imaging magnetically labeled T cells essentially show that the accumulation of magnetically labeled T cells enhances the ability to detect microbleeeds that otherwise were perhaps too small to detect (Sup Fig 8). Given the lack of data supporting the retained migratory capacity of magnetically labeled T cells, one wonders then, whether magnetically labeled T cells are indeed trafficking to the brain or are passively arriving in the brain, and might some vascular magnetic particle accumulate in an early inflammation or leak into the microbleed on its own and similarly enhance the ability to detect the otherwise undetectable microbleed. A series of controls would be useful to answer these questions, perhaps testing the administration of magnetic particles alone, and/or magnetically labeled non-CD8+ T cells. Authors are also encouraged to report on the frequency in which a magnetically labeled T cell was present in a pre-vascular compromised inflammatory environment versus in the microbleed, as measured by MRI and histology.

    Distinguishing bleeding from T cells is a key challenge for doing a serial MRI study in the same animal. In the new Fig 8I and Fig S8, we did a study using time-lapse MRI on the same mouse from 20 to 24 hr-post infection. We observed the appearance of hypointensities at the center of the bulb at 22 hr which is prior to bleeding in this area. Bleeds were observed at the GL, but not at the center of the bulb by IHC. Thus, we were able to time the entrance of T cells in this area of the brain. We were not able to find migration tracks of T cells from the outer GL layer into the center of the bulb. This is consistent with the idea that T cells infiltrate directly into areas with virus prior to vessel breakdown and microbleeds. We didn’t observe a very significant change in the location of T cells from 22 to 24 hr on the distance scale of MRI. There are two possibilities to explain our inability to detect T cell movement over a 2 hr time interval: 1.) the T cells under investigation may have been attached to blood vessels and required more time to extravasate. surface due to inflammation, and it might take some time for extravasation, or 2.) although T cell velocities in the CNS have been clocked at ~10 µm/min (Herz et al., 2015), their paths are often tortuous and influenced by antigen presenting cells displaying cognate peptide MHC as well as local chemokine gradients. Thus, upon entering a site of viral infection, the labeled T cells may not have traveled far enough in 2 hrs for us to detect their movement by MRI. We did not image mice beyond 24 hrs post-infection due to the possibility of bleeding. We added this discussion. Quantification of the frequency in which a MPIO labeled T cell was present in a region where no bleeding was detected versus in a region with a microbleed was added in Fig 8H. In the ONL/GL, 85% of MPIO-labeled T cells were in the region with microbleeds and 15% were in a region where no tissue bleeding was detected. In the MCL/GCL areas, no evidence for bleeding was detected. Magnetic labeling of CD8 T cells doesn’t reduce their migratory capacity in an in-vitro migration assay (Fig 7G). This adds to other in-vitro assays that the labeled T cells are functioning. Labeled T cells had therapeutic efficacy like unlabeled T cells and labeled T cells were found at the center of the bulb (Fig 8F-I) with no bleeds as well as in other brain regions. Based on these observations, we think that MPIO-labeled T cells are functioning and trafficking in the brain. A previous study showed that non-CD8 T cells, such as monocytes/macrophages, CD4 T cells, and neutrophiles also migrate into the OB and are involved in the immune responses in this model [(Moseman et al., 2020), Fig 2E]

    Reviewer #2 (Public Review):

    [...]

    Weaknesses:

    • Individuals with systemic infections or other underlying condition may have microbleeds due to inflammation or hypertension. The etiology of microbleeds is thus not necessarily tied to CNS infections. Investigation of potential cerebrovascular microbleeds following systemic or respiratory infections not affecting the CNS may shed light on this possibility which may also provide alternative interpretation of neurological symptoms associated with on CNS invasive infections.

    This is an important issue. Prior work has shown that virus in this model is cleared quickly (2 to 3 days) from the periphery (Ramsburg et al., 2005; Roberts et al., 1999). This is likely due to the fact the virus is inoculated through the nose. It is clear in this model that virus infects the brain, that bleeding corresponds to sites of high viral load, and bleeding can be modulated by blocking immune infiltration into the brain. However, the quantitative role of peripheral influences such as high blood pressure could be important and will be checked as this work proceeds.

    • Representative colocalization of virus infected endothelial cells with red blood cells (RBCs) is shown in Fig 4. However, a more quantitative assessment indicating how many areas or hypointensities were evaluated for virus-localization with RBCs, and how many of these revealed colocalization versus virus or RBC only would strengthen interpretation.

    Fig 4 shows that VSV can infect vascular endothelial cells and cause bleeding. Hypointensities were not measured in this Figure. We quantified the numbers of VSV infected vessels, colocalizing and not colocalizing with bleeds. Fig 4D was added with this new data.

    • A limitation clearly acknowledged by the authors is that hypointensity spots detected by MRI cannot distinguish microbeads from MPIO-labeled T cells.

    As in our response to Reviewer 1, this is a critical next step since bleeding so often occurs with immune cell infiltration in the brain. We have discussed potential approaches and have added the idea that development of more sensitive MRI contrast agents and quantitative T2* analysis especially at different magnetic field strengths may be approaches to accomplish this. It will be crucial for MRI cell tracking under the condition of bleeding, which is one common pathology associated with many diseases.

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  2. Evaluation Summary:

    The manuscript by Liu et al., aims to develop a novel MRI-based approach to monitor virus specific CD8+ T cells and their relationship to cerebrovascular pathology in living brains. Using a mouse model of VSV brain infection, they show that MRI approaches can be used to identify microbleeds in the brain, and these microbleeds occur independent of immune cell influx. Furthermore, the transfer of low numbers of virus specific CD8+ T cells can reduce cerebrovascular bleeding. The capacity to track virus specific T cells and cerebrovascular pathology in real time in living brains would be a major technological advance.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The manuscript by Liu et al investigates how MRI can be used to detect the earliest stages of CNS infections and how MRI can also be used as a surrogate readout for treatment efficacy. Authors demonstrate convincingly that microbleeds, as evidenced by unusual dark spots in the brain of mice infected with a virus that infects the brain, occurred at the earliest stages of viral infection. Authors also convincingly demonstrate that the infusion of virus-specific immune cells, when delivered at the right time and at the right dose, could reduce these microbleeds. Importantly, authors showed that the wrong dose could be detrimental.

    The authors cast this study as a method for improving research and discovery in immunotherapy context and the study is convincing in its conclusions regarding imaging microbleeds and the immunotherapy tested herein. While authors do not directly suggest so, these findings extend the significance of this work beyond research and development of immunotherapies by providing a potential early detection mechanism for viral infection in the brain. This may be feasible as the MRI methodologies for detecting these phenomena are generally translatable to clinical imaging scenarios, though the imaging resolution may not.

    Weaknesses in the report revolves around the value of and the ability to image magnetically labeled T cells in the presence of microbleeds.

    1. Authors developed a magnetic particle coated with fluorescent molecules and antibodies specific for CD8+ T cells. They labeled these T cells with particles for detection by MRI. They then wanted to follow the accumulation of these cells in the brain following infusion and viral infection by performing MRI using parameters that amplify the signal of the attached label. The rationale for these experiments was to determine if immune cell infiltration preceded vascular compromise. This suggests the expectation for active chemotactic migration or other signaled accumulation rather than leakage. When authors tested their magnetically labeled T cells for functional impairment due to the presence of attached magnetic particles, they did not test for deficits to migratory capabilities, such as in standard transwell migration assays. Others have shown (see https://doi.org/10.1038/nm.2198 for example) that T cell migration is very sensitive to the type of attached nanoparticle as well as the surface coverage. Perhaps authors should temper their claims that magnetically labeling of T cells does not alter T cell function without at least an assay of this critical function. Further, the fluorescence microscopy shown in Figure 7D is of insufficient resolution to claim that MPIOs are inside cells. Electron microscopy should be used to determine this.

    2. Regarding the use of imaging the accumulation of magnetically labeled T cells, authors show evidence that magnetically labeled T cells accumulate in areas of the brain that as yet do not present with microbleeds but do have the histological hallmarks of vascular inflammation. This corroboration is intriguing but only provable with a serial imaging study in the same animal, which was not performed. Authors are also encouraged to report on the frequency in which a magnetically labeled T cell was present in a pre-vascular compromised inflammatory environment. The bulk of the results on imaging magnetically labeled T cells essentially show that the accumulation of magnetically labeled T cells enhances the ability to detect microbleeeds that otherwise were perhaps too small to detect (Sup Fig 8). Given the lack of data supporting the retained migratory capacity of magnetically labeled T cells, one wonders then, whether magnetically labeled T cells are indeed trafficking to the brain or are passively arriving in the brain, and might some vascular magnetic particle accumulate in an early inflammation or leak into the microbleed on its own and similarly enhance the ability to detect the otherwise undetectable microbleed. A series of controls would be useful to answer these questions, perhaps testing the administration of magnetic particles alone, and/or magnetically labeled non-CD8+ T cells. Authors are also encouraged to report on the frequency in which a magnetically labeled T cell was present in a pre-vascular compromised inflammatory environment versus in the microbleed, as measured by MRI and histology.

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  4. Reviewer #2 (Public Review):

    Imaging based methods to detect early vascular damage and to better understand the relationship to invading pathogens and immune cell infiltrates is relevant to both identify sites of pathology and develop targeted therapies to control infections while minimizing pathological events. The paper in its current form uses high resolution MRI combined with histological analysis and T cell labeling to better define the relationship between microbleeds indicative of cerebrovascular damage, viral replication and CD8 T cell accumulation. Several interrelated aims address whether the detection of microbleeds can serve as a neuroimaging marker for infection and to what extent microbleeds are associated with direct virus infection or infiltrating immune cells.
    Central nervous system (CNS) vascular bleeding has been associated with virus infections, but the underlying mechanisms are poorly defined. This paper overall demonstrates the suitability of high resolution MRI to detect both micro bleeds and preferential sites of T cell inflammation throughout the CNS in small animal models. MPIO labeling and high resolution MRI with the sensitivity to detect single cells is applied to nonphagocytic lymphocytes and should be applicable to track accumulation of other peripheral immune cells of interest in a variety of neurological disease models. The technology should be of wide interest to the field of neuroinflammation. The data support the aims and are well documented. It is also acknowledged that more research is required to distinguish causation of pathogenic signals. The ability to non invasively monitor initial insults associated with infections as well as evaluate therapies would advance both detection and treatment outcomes of brain infections, where the causative agents are often unknown. It remains to be determined if results apply uniquely to VSV or is similarities are noted in other models. It will also be of interest to pursue the role of glia activation in promoting microbleeds in the absence of peripheral leukocytes.
    Strengths:

    • This work takes advantage of high resolution MRI to detect areas of cerebrovascular breakdown following viral infection with VSV. VSV is known to enter the brain when administered intranasally and provides a good model to study entry of viruses from the nasal cavity into the olfactory bulb and other brain areas.
    • Segregating events leading to vascular breakdown has been difficult. Results from MRI and histology show that microbleeds can be directly associated with infection of vascular cells, even when entry of peripheral immune cells is blocked. On the other hand, transfer of activated CD8 T cells at onset and peak infection reduced levels of infection and coincidently microbleeds. This finding is relevant as immune cells, including anti-viral cytolytic CD8 T cells, are commonly associated with vascular damage.
    • The measurement of microbleed numbers and volume with T2* is appropriate to demonstrate affected regional sites, changes over time, and differences across treatment. Hyperintensities evident by MRI allow precise localization of affected areas for subsequent more refined histological analysis.
    • An exciting novel aspect of the paper is the development of chemically modified MPIO particles to improve internalization and labelling efficiency of T cells ex vivo for subsequent transfer and MRI detection in mice. The technique is well described and extensive data showing efficacy of labeling and no effects on select T cell functions are included. MRI combined with histological analysis revealed hypointensities were attributed to colocalization of microbleeds and labeled CD8 T cells in some areas, but labeled CD8 T cells alone in others.

    Weaknesses:
    • Individuals with systemic infections or other underlying condition may have microbleeds due to inflammation or hypertension. The etiology of microbleeds is thus not necessarily tied to CNS infections. Investigation of potential cerebrovascular microbleeds following systemic or respiratory infections not affecting the CNS may shed light on this possibility which may also provide alternative interpretation of neurological symptoms associated with on CNS invasive infections.
    • Representative colocalization of virus infected endothelial cells with red blood cells (RBCs) is shown in Fig 4. However, a more quantitative assessment indicating how many areas or hypointensities were evaluated for virus-localization with RBCs, and how many of these revealed colocalization versus virus or RBC only would strengthen interpretation.
    • A limitation clearly acknowledged by the authors is that hypointensity spots detected by MRI cannot distinguish microbeads from MPIO-labeled T cells.

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  5. Reviewer #3 (Public Review):

    The manuscript by Liu et al., aims to develop a novel MRI-based approach to monitor virus specific CD8+ T cells and their relationship to cerebrovascular pathology in living brains. Using a mouse model of VSV brain infection, they show that MRI approaches can be used to identify microbleeds in the brain, these microbleeds occur independent of immune cell influx, and that the transfer of low numbers of virus specific CD8+ T cells can reduce cerebrovascular bleeding.

    Read the original source
    Was this evaluation helpful?