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Optimizing IHC for Evaluating�Immune cells in Guinea Pig Tissues�Understanding the Immune Landscape During cCMV Infection

Lynna Ngo

University of Minnesota Medical School

T35 NIH Summer Infection and Immunity Research Grant

Bierle Lab

Aug 21, 2024

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What is Congenital Cytomegalovirus (cCMV)?

Infographic source: www.nationalCMV.org

Cytomegalovirus, or CMV, is a highly common virus. In pregnancy, if the virus is transmitted to the fetus, it can lead to congenital CMV, cCMV. This is the most common infectious cause of birth defects in the United States. It’s estimated that 1 in 3 pregnant women with primary CMV infection will pass the virus onto their offspring. Roughly 1 in 150 children are born with cCMV. Babies with cCMV may suffer from a range of health problems, including microcephaly, seizures, behavioral issues, and hearing loss. In fact, cCMV is the leading NON-genetic cause of childhood hearing loss.
Most cCMV infections are asymptomatic. Roughly 80% to 90% of newborns with cCMV will not show any immediate symptoms, although they may develop issues like hearing loss later in life.

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Currently, no vaccine exists for this virus and treatment is limited to some antiviral meds and supportive interventions such as cochlear implants and speech therapy.

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The Guinea Pig Model of cCMV infection (GPCMV)

Why guinea pigs?

Human-like placenta structure
Comparable pregnancy progression time points
Similar hormonal signaling
Mature neonates at birth
GPCMV causes cCMV while mouse CMV does not

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Placental Histology

GP placenta has 3 main regions.
placenta – top section, labyrinth that facilitates the exchange of nutrients, and gases between the mother and offspring.
A unique feature of the guinea pig placenta is the sub placenta in green, which connects the main placenta to the decidua. This structure is not found in humans thought that SB plays a role in anchoring the placenta during pregnancy.
You also see a third section in orange, the decidua, which is the maternal tissue that interacts with the developing fetus, much like in the decidua in the human placenta.

By studying these structures in the guinea pig, we can gain insights into how infections like CMV might alter placental function and contribute to fetal injury. The similarities in the labyrinth structure make the guinea pig a relevant model, but the differences such as the SB in the GP also highlight the need for careful interpretation when trying to translate these findings.

RNA scope stained placenta, highlight 3 regions within the placenta..

Subplacenta thought to be analogous to

Decidua – endometrial lining of uterous, where whole embryo originally implanted.

Human placenta image: https://www.sciencedirect.com/science/article/pii/B978012394445000014X

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Research Objective:

To optimize Immunohistochemistry (IHC) protocols for detecting immune cells (CD3, CD4, CD8) and cytokeratin in GPCMV placental tissue.

Localize, visualize, and quantify the type of immune cells at site of inflammation in response to CMV infection.

Challenges:

Limited standardized protocols for guinea pig tissues, lack of available reagents for GP research.

With new antibodies 🡪 need for refining antigen retrieval methods, antibody concentrations, and incubation times in IHC.

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IHC Workflow and Antibodies of Interest

	Purpose	Specs
CD3	T cell lineage	Rabbit monoclonal IgG
Guinea Pig CD4	T helper cell lineage	Lab – rat monoclonal IgG Flow - mouse
Guinea Pig CD8	Cytoxic T cell lineage	Lab – mouse monoclonal IgG Flow – mouse
Cytokeratin	Epithelial cells, trophoblastic cells	Mouse monoclonal Igg1/IgG2a

While IHC is well-established, the main challenge lies in optimizing each step for the target antigen, to get a strong signal with minimal background noise.
The general workflow involves several key steps: first, tissue is fixed, embedded, and sectioned onto slides. This is followed by antigen retrieval to unmask the epitopes, then using blocking reagents to reduce non-specific binding. After that, primary antibodies are added to bind to the target antigen, followed by a secondary antibody. Detection reagents such ad DAB then produce a colored precipitate, which allows us to visualize the target under the microscope. See example, cells are brown.
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In this project, we tested several key primary antibodies, particularly because we know that leukocytes are recruited to infection sites. Look at CD3, CD4, and CD8. also interested in cytokeratin as that marks epithelial cells, help us identify trophoblastic cells throughout the placenta
Optimization considerations: I varied diff parameters like antigen retrieval methods, primary antibody concentrations, and incubation times to find the optimal conditions for staining.

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Each of these markers is crucial for understanding the immune response at the maternal-fetal interface, especially in the context of congenital CMV (cCMV) infection, which can disrupt normal placental function.

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CD4 and CD8 IHC Results in GP Placenta

1:100 lab CD8

nonspecific binding

1:6000 lab CD8

Sniper Blocker

1:6000 lab CD8

Sniper + 5% NFDM

CD4

CD8

1:100 flow antibodies, validated in frozen but not fixed tissue

Did not work

Tested different blocking conditions:

Nonfat dairy milk – rich casein protein coat binds to nonspecific sites in tissue, reducing chance for antibody to bind, reducing background noise.

Starting with results from CD4 and CD8, our lab had flow antibodies on GP tissue, antibody was validated for IHC on frozen tissue, want to see if work for fixed tissue, unfortunately we got no brown signal in fixed tissue even at a high concentration with 1:100 dilution.
Also tested two other CD4 and CD8 lab made antibodies, high dilution of 1:100, significant background noise, and non specific binding, which presents a challenge that we are working to overcome.
Tried to test diff blocking conditions to reduce background noise, reagent called sniper, adding 5% NFDM, and lowering antibody conc would help.. see that NFDM helped reduce background noise, even if we didn’t get specific CD4 and CD8 biding. Starting using sniper + NFDM as blocking reagent for future tests.

Nonfat milk blocks nonspecific sites on the tissue that could otherwise bind to antibodies, reducing unwanted background staining and improving the specificity of the IHC signal.

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CD3 IHC Results

Human Tonsil

1:1600 dilution

No secondary antibody on

GP placenta

Isotype control

on GP placenta

Placental Labyrinth

Placenta

Syncytium

RBC

Best results:

1:1600 dilution

(tested 1:100-1:3200)

Sniper block + 5% NFDM
5 minute DAB incubation

For CD3, here, I tested various dilutions from 1:100-1:3200, found best to be 1:1600.
You can see the three controls I have on the left. Together, these controls demonstrate the specificity and accuracy of our IHC protocol. The positive control confirms that our staining process works, the no secondary antibody control shows that the detection is antibody-dependent, and the isotype control rules out non-specific primary antibody binding. While some background noise is present in the isotype control, higher concentration than antibody, waiting from manufacturer for concentration.
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On the right, distinct brown staining in some cells in the placental labyrinth and syncytium (placenta edge), indicating successful CD3+ T cell detection. Suspect these cells are likely maternal t cells circulating within the maternal blood space. Zoomed up image of the cells more clearly

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Cytokeratin IHC Results

Decidua

Placenta

Best results:

1:3200 dilution (tested starting from 1:100)
Sniper block + 5% NFDM
4 minute DAB incubation

Subplacenta

Decidua

Placenta

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Conclusions

CD3 and Cytokeratin:

Distinct brown staining observed in placental tissues
Successfully found optimal antibody concentrations and blocking conditions (longer sniper incubation with NFDM) in GP tissue.

CD4 and CD8:

Faced challenges due to nonspecific binding, requiring further optimization or tests with other antibodies

Implications of Findings:

Successful CD3 and Cytokeratin staining provides insight into T cell localization at the maternal-fetal interface.
Challenges with CD4 and CD8 suggest the need for further protocol refinement.

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Future Directions

Quantification of CD3+ Cells:

Developing methods for systematic random imaging and cell counting to better characterize abundance and location of immune response during cCMV infection 🡪 comparing infected vs non-infected models.

Continued Refinement:

Further optimize CD4 and CD8 staining protocols.
Explore additional markers like CD56 for NK cells.

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Acknowledgements and Q&A

Acknowledgements:

NIH T35 Medical Student Summer Research Program in Infection and Immunity.
Dr. Craig Bierle’s Lab, Jason Hatfield, Colleen Forster, and UMN BLS Histology and IHC Laboratory.

Thank You & Questions:

Open the floor for questions and discussion.