The Invisibility of Stem Cells

Adult stem cells (Mesenchymal Stem Cells - MSCs) are intensely studied due to their many different properties. According to a review by Pardis Saeedi et. al. in 2019 (1) MSCs are quite versatile. Most of their effects do come from being able to differentiate into a plethora of different cell types. Even so, other functions of theirs include site-homing, immunomodulation, tissue regeneration, and activation of resident stem cells already within the host (1). Additionally, MSCs do not generally express MCHII or only express very low levels of it. They do express some levels of MHCI so donor matching is necessary, but MHCII matching is probably thought to not be as important due to their low to none expression levels.

Today, I will be reviewing the 2020 paper published in Nature's Cell Death and Disease journal. The paper is from the Abu-El-Rub group and is called: Hypoxia-induced shift in the phenotype of proteasome from 26S toward immunoproteasome triggers loss of immunoprivilege of mesenchymal stem cells (2).

Note: There is a small number of technical names and other jargon. I will do my best to keep the technical jargon to a minimum, but there will be names of proteins that are necessary to know. I will have a short Glossary at the end of the post that can be referred to.

HLA-DRα is an MHCII protein that is a vital part of the immune system. It is used to present antigen between immune cells and, in certain cases, from non-immune cells to immune cells in order to induce an immune response. MSCs are supposed to have immunoprivilege status, meaning that they can practically hide from the immune system. Therefore, they probably don't need to be matched quite as much as differentiated cells would need to be matched.

Previously, the same group showed that MSCs do actually produce HLA-DRα but it gets degraded before being expressed on the surface by a proteasome called 26S proteasome (2). They wanted to further study the relationship behind 26S proteasome, HLA-DRα, and the hypoxic condition that MSCs would be in within injured and damaged environments (2).

When the MSCs were incubated in a hypoxic state, it was found that some subunits of the 26S proteasome had disappeared and other protein subunits may have replaced them. The replacement leads to the 26S proteasome becoming an immunoproteasome. Most of their data does show a decrease in proteins and enzymes specifically used in the 26S proteasome with a corresponding increase in most proteins and enzymes necessary for immunoproteasome production. Additionally, they observe a decrease in 26S enzymatic activity when using a specific substrate. This also correlates with an increase in enzymatic activity from two enzyme subunits that are a part of the immunoproteasome. Importantly though, the enzymatic activity for the immunoproteasome was measured with two different substrates each of which was specific for each of the two different enzymes. Both of these are necessary for the immunoproteasome's function, but they did not use one substrate that was specific for the whole immunoproteasome itself. I am concerned about this because their activity measurements prove that these two subunits (LMP2 and LMP7) have enzymatic activity in the hypoxic state and have no activity within the normal state, but that may not actually correlate with enzymatic activity within the immunoproteasome. LMP2 and LMP7 may have their own enzymatic activity and this may change when they are complexed with the immunoproteasome. Additionally, they may complex with other proteins to create other protein complexes that also have the ability to break down proteins. So these experiments conclude that the 26S proteasome is active in a normal state of MSC growth but switches to the active immunoproteasome when the growth conditions become hypoxic.

Since the relationship between 26S proteasome, the immunoproteasome, and the hypoxic condition is more defined now, they move on to the relationship between HLA-DRα and these two proteasomes within the normal and hypoxic state. They confirm that the protein concentration of HLA-DRα increases in the normal state when 26S proteasome is inhibited and then look at the relationship between HLA-DRα maturity, the proteasomes, and the growth conditions. HLA-DRα seems to be in an immature state within the normal conditions, but when switched to hypoxic conditions these same proteins lose their immaturity and become mature. This is done through their binding to either CD74 or HLA-DM. When HLA-DRα is bound to CD74 it is immature, but when it is bound to HLA-DM it becomes mature and presents antigen. The maturity of HLA-DRα was modified when an inhibitor for the immunoproteasome was used. Binding to CD74 is recovered within the hypoxic state when the immunoproteasome is inhibited. In this same state, there is still some binding of HLA-DRα to HLA-DM. This suggests that though the immunoproteasome is necessary to move HLA-DRα from immature to mature, the MHCII protein is still able to mature through a different mechanism that is also dependent on the hypoxic state.

Finally, MSCs are known to be immunoprivilege but, their destruction has been found in the host after some time. Due to the increase in HLA-DRα production and their maturity within the hypoxic setting, the group looked for cytotoxic activity against MSCs and MSC immunomodulatory properties. They cytotoxic assays do confirm that MSCs grown within a hypoxic state are significantly more likely to be destroyed than those grown in the normal state. Additionally, the inhibition of the immunoproteasome within the hypoxic state does significantly decrease this cytotoxicity. When looking at the immunomodulatory date, I can only confirm that there is a trend of lymphocyte proliferation being inhibited by MSCs grown in the normal state. There does seem to be more proliferation happening when the MSCs are grown in the hypoxic state, and the immunoproteasome inhibitor seems to reverse this allowance of proliferation, but every error bar is so large that it is difficult to determine any actual significance. Additionally, they specifically observed the differentiation of T-reg cells induced by MSCs grown in the two different states. Again, there is about a 2% difference found in T-reg populations between the normal and hypoxic grown MSCs but that really isn't a lot of difference, and the error bars make it difficult to determine anything more than a trend. The trend is that normal MSCs induce 2% more T-reg cell differentiation than MSCs grown in the hypoxic state. This trend also shows some recovery when hypoxic MSCs are also given the immunoproteasome inhibitor. Again though, this is just a trend for now and will need to be explored further.

To summarize:

1. HLA-DRα seems to be constitutively produced and degraded by 26S proteasome.

2. Subunits necessary for the 26S proteasome are found in the normal state while subunits necessary for the immunoproteasome are not found in this state.

3. The hypoxia state induces a switch from the 26S proteasome to the immunoproteasome through the increased production and binding of 11S and corresponding proteolytic proteins LMP2, LMP7, and slight production of MECL-1. There is also a decrease in the production of 19S and corresponding proteolytic proteins PMSB6, PMSB7, and PMSB5.

4. The hypoxia state also increases the maturation of HLA-DRα by increasing the binding of HLA-DM instead of CD74. HLA-DRα binding of CD74 is recovered by inhibiting the immunoproteasome activity, but it does not drastically suppress HLA-DRα binding to HLA-DM. There seems to be another mechanism that controls this binding and is also affected by the hypoxia state.

5. The hypoxia state does increase lymphocyte cytotoxicity towards MSCs, though MSCs suppression of lymphocyte proliferation and increased differentiation of T-reg cells are not fully confirmed in this study.

Future Explorations:

1. The main thing that needs to be further explored is the immunomodulatory activity of MSCs and how it may differ between cells grown in the normal state and those grown in the hypoxic state.

2. We do see a recovery of HLA-DRα immaturity when the immunoproteasome is suppressed, but there is still some HLA-DRα maturity occurring. The mechanism for this maturity should be analyzed so that a fuller picture of how the hypoxic state changes MSCs maintenance and processes.

3. The relationship and function of immunoproteasomes within the MHCII maturation and antigen presentation pathway also needs to be further analyzed.

Therefore an interesting paper, but much more work needs to be done regarding the relationship between MSCs and immunogenicity within a hypoxic state.

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Remember to take breaks for inspiration and step back from the books and your research to look at the awe and beauty of life sciences and their functions. See beauty everywhere!

#science #lifescience #lifesciences #research #biology #mesenchymalstemcells #MSCs #MSC #stemcelltherapy #immunogenicity #lymphocytes

References:

1. Saeedi P, Halabian R, Imani Fooladi AA. A revealing review of mesenchymal stem cells therapy, clinical perspectives and Modification strategies. Stem Cell Investig. 2019;6:34. Published 2019 Sep 25. doi:10.21037/sci.2019.08.11

2. Abu-El-Rub, E., Sareen, N., Yan, W. et al. Hypoxia-induced shift in the phenotype of proteasome from 26S toward immunoproteasome triggers loss of immunoprivilege of mesenchymal stem cells. Cell Death Dis11, 419 (2020). https://doi.org/10.1038/s41419-020-2634-6

Glossary

11S: the complex that replaces the 19S complex of the 26S proteasome. This is part of the switch from 26S proteasome to immunoproteasome.

19S: the complex that houses the 26S targeting abilities for proteins that need to be broken down.

20S: the complex of proteins that houses the 26S proteasome's and immunoproteasome's enzymatic activity.

26S Proteasome: A protein that has both the ability to target and breakdown certain proteins within the cell.

Enzymatic Activity: The act of breaking down a protein. When this is blocked, the enzyme is still present within the cell, it is just not able to do its job.

HLA-DRα: MHCII protein not usually expressed on MSCs.

Immunomodularity: When the immune system is modified by something.

Immunoproteasome: A particular proteasome that is necessary for MHCI antigen presentation. Not much is known about how it associates with MHCII antigen presentation.

LMP2&7, and MECL-1: proteolytic proteins that replace the PSMB5-7 proteins within the 20S complex. This is part of the switch from 26S proteasome to immunoproteasome.

MSCs: mesenchymal stem cells - adult-derived stem cells.

PSMB5-7: proteins found in the 20S complex that actually breaks proteins down within the 26S proteasome.

Substrate: A compound molecule or chemical or protein that is broken-down, modified, or changed in some way through enzymatic activity.