Immunology Services Case Studies

IMMUNOLOGY SERVICES CASE STUDY #1

Client: Dr. Dixon Kaufman, MD, PhD, Professor and Chair, Division of Transplantation, Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health

Problem or goal:  In solid organ transplant research, it is important to monitor neutrophil maturation in the bone marrow. However, the current methodology (Wright-Giemsa staining and microscopy) is time-consuming and fails to provide information about the functional capabilities of neutrophil progenitors.

Solution: Immunology Services developed a flow cytometry-based method to differentiate subsets of neutrophil progenitors in humans and rhesus macaques (a model system for transplant research).

How did Immunology Services work with the Kaufman team:

  1. During an initial consultation Dr. Kaufman’s research team described the specific aims of the study that included monitoring neutrophil leukocyte differentiation in the bone marrow of rhesus macaques following kidney transplants.
  2. We determined that a flow cytometry-based method yielding high-resolution immunophenotype data would be advantageous.
  3. Immunology Services tested a wide array of reagents, optimized a 14-color staining method, and validated the rhesus model by analyzing corresponding samples in human and rhesus subjects Fig. 1
  4. We have established a standard operating procedure (SOP), and perform this assay routinely for the Kaufman laboratory and other interested collaborators.
  5. Data files are sent to clients using the UW-Madison Box Service within 48 hours of receiving the samples.

Flow cytometry data for stained bone marrow cells from human (a) and rhesus macaque (b)

Figure 1. Neutrophil progenitor immunophenotype markers in the bone marrow of (A) humans and (B) rhesus macaques (Weisgrau et al, J Leukoc Biol. 2019).

How has Immunology Services contributed to the progress of Dr. Kaufman’s research: Unit Head Dr. Eva Rakasz’s extensive immunology background and flow cytometry expertise (more than 25 years) and the multi-year immune assay development experience of other members of the unit made it possible to design multicolor flow cytometry staining panels to evaluate conditions for kidney transplant tolerance in rhesus macaques.

Client comments: Dr. Dixon Kaufman says “I have relied on the services of Immunology Services since 2013. Their services are beneficial to laboratories on the UW campus and elsewhere interested in using state-of-the-art immunological methods to generate highly reproducible data with all the necessary controls. Importantly, Dr. Rakasz provides critical support in high-dimensional data visualization and interpretation.”

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IMMUNOLOGY SERVICES CASE STUDY #2

Client: Dr. James A. Thomson, V.M.D., PhD, Diplomate A.C.V.P., Director of Regenerative Biology, Morgridge Institute for Research; Professor, Department of Cell and Regenerative Biology; University of Wisconsin-Madison School of Medicine and Public Health

Problem or goal: Dr. Thomson wanted to determine whether cellular immune responses mounted against major histocompatibility complex (MHC)-mismatched arterial transplants could be detected in the peripheral blood of recipient rhesus macaques.

Solution: Immunology Services developed a flow cytometry-based method to quantify proliferating NK cells, CD4 and CD8 T cells, and activated memory T cells of the CD8+ and CD4+ subsets.

How did Immunology Services work with the Thomson team:

  1. Although Dr. Thomson’s laboratory had access to a flow cytometer, they needed an immunologist’s insight to design an experimental approach.
  2. Dr. Rakasz suggested monitoring of Ki-67 expression (a cell proliferation marker) in NK and T cell subsets.
  3. A similar assay had previously been optimized by Immunology Services using 100 microliter whole blood samples (Pomplun et al. Cytometry 2015).
  4. Since Dr. Thomson’s laboratory was not familiar with the procedure, they hired Immunology Services to (a) modify the method according to their project’s specific aims, and (b) perform the assay from staining and flow data acquisition to data analysis and interpretation (Fig. 2).
  5. Researchers in Dr. Thomson’s laboratory received the analyzed and raw data files within 96 hours of the assay.
  6. Statistical analysis and data interpretation were provided by Dr. Rakasz (Immunology Services Unit Head) at the end of the study.

Flow cytometry analysis of proliferating NK cells before and after arterial transplant in matched and unmatched rhesus macaques.

Figure 2. Proliferating NK cell frequency is a good indicator for monitoring cellular immune responses against mismatched allotransplants. Ki-67+ (proliferating) NK cells A: before, and B: after arterial transplant. C: Comparison of integrated NK cell responses between rhesus macaques that received 100% matched, 50% matched and 0% matched arterial transplants. AUC: Area Under the Curve (Maufort et al, unpublished data).

How has Immunology Services contributed to the progress of Dr. Thomson’s research: The Thomson lab monitors responses of antigen-presenting cells, NK cells, and T cells after experimental arterial transplantation. Immunology Services detects these responses by measuring the frequency of Ki-67-positive cells expression in immune cell subsets. Accurate quantitation of this marker is crucial to decreasing inter-assay variability and providing the best possible resolution to detect immune perturbation.

Client comments: Dr. James Thomson says “I have used the expertise of Immunology Services to support my arterial transplant study for almost two years. Their contributions have been absolutely essential to our success.”

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IMMUNOLOGY SERVICES CASE STUDY #3

Client: Dr. David Evans, PhD, Professor, Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison School of Medicine and Public Health; Associate Director, Wisconsin National Primate Research Center

Problem or goal: Dr. Evans wanted to determine the effect of simian immunodeficiency virus (SIVmac239) infection on the functional capability of different natural killer (NK) cell subsets in rhesus macaques of Indian origin.

Solution: Immunology Services developed an assay, which included an 18-hour NK cell-specific stimulation in vitro followed by a 13-color flow cytometry analysis, to follow the function of five KIR3D- and CD16-defined NK cell populations.

How did Immunology Services work with the Evans team:

  1. Dr. Evans’ laboratory had access to a flow cytometer, but they needed an expert flow cytometrist to develop a multicolor flow cytometry panel, perform the necessary standardization steps, and provide the final quality controls.
  2. Dr. Evans and Dr. Rakasz selected the desired immunophenotype and functional markers of NK cell subsets.
  3. Immunology Services optimized the assay and the reagents (Fig. 3).
  4. Immunology Services established a standard operating protocol (SOP), and trained a member of Dr. Evans’ laboratory to perform the assay routinely.
  5. Dr. Evans published their results: Ries et al. PLoS Pathogens 2017.

Quantification of functional subpopulations ) Functional subpopulations, as defined by expression of IFN‐γ+, TNF‐α+, CD107a + Granzyme B_high, and CD107a+ Granzyme B_low

Figure 3. Frequency of NK cell subsets with different functional capabilities in the peripheral blood of rhesus macaques infected with simian immunodeficiency virus (SIVmac239; Weisgrau et al. Cytometry 2016).

How has Immunology Services contributed to the progress of Dr. Evans’ research: Development of a multi-parameter flow cytometry method to study changes in the phenotypic and functional properties of NK cells in KIR- and MHC class I-defined rhesus macaques infected with simian immunodeficiency virus (SIV) provided the means to discover broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection.

Client comments: According to Dr. David Evans “With Dr. Rakasz’s help, we are able to develop technically sophisticated flow cytometry assays with minimal commitment of my lab staff’s time.”

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