Aaron McBride and Dr. Gregory F. Burton, Chemistry and Biochemistry

Current Human Immunodeficiency Virus (HIV) treatment is greatly hindered due to viral reservoirs throughout the body prolonging and perpetuating infection.i Large stores of HIV exist in the follicular dendritic cell (FDC) microenvironment, located primarily in the lymph nodes and spleen. Free-floating virus has a half-life of less than two hours; however, FDC-trapped virus has a half-life of over eight weeks and can be rescued in its infectious state longer than nine months after the initial infection,ii showing the role of FDCs as an HIV reservoir.iii Studies in the Gregory Burton laboratory focus on the interactions between HIV, FDCs, and CD4+ T lymphocytes to understand and ultimately disable the HIV-harboring functions of the FDCs. Our research helps provide possible targets for treatment of HIV infection—an epidemic that infects more than thirty million individuals throughout the world and eventually leads to AIDS, a pandemic that has claimed well over 25 million lives since its discovery in 1981.iv

A promising approach to interfering with the FDC reservoir of HIV is blocking the activation of NF-κB, a major transcription factor in immune cells. Infected CD4+ T lymphocytes near FDCs in the spleen or lymph nodes upregulate of NF-κB, greatly increasing viral production in infected lymphocytes.5 On the other hand, AAT, a potent serpin isolated from the liver, prevents the destruction of the NF-κB inhibitor nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), leading to complete inactivation of NF-κB even in the presence of FDCs.v Although this function of AAT in human CD4+ T lymphocytes is known,5 scientists do not know the specifics of how AAT enters the cell nor its route once inside. Our objective is to conclusively show how AAT traffics intracellularly via organelles in CD4+ T lymphocytes. Once we can show the intracellular trafficking of AAT en route to its binding of IκBα, we aretter understand the mechanism by which AAT crosses the cell membrane and where it transported. Since both CD4+ T lymphocytes and FDCs use NF-κB, the inhibition of NF-κB may also be possible in FDCs. We hope to selectively inhibit NF-κB in FDCs to neutralize the HIV-harboring abilities of FDCs, thereby possibly removing this major obstacle in the effective treatment of AIDS.1

Methods

Isolation of CD4+ T lymphocyte and incubation with AAT: We draw 12–18 ml blood from donors following our established protocol approved by the Institutional Review Board at Brigham Young University (BYU). From the blood, we will isolate CD4+ T lymphocytes by using density gradient separation. The T cells are enriched via negative selection using a CD4+ T cell isolation kit,5 activated by incubation with phytohemagglutinin (PHA) in supplemented medium for three days, and then incubated with AAT. The cells are separated into sample groups and incubated for 0, 6, and 22 hours with AAT to allow us to track the intracellular movement of AAT. Two million cells per sample group are fixed at the appropriate time point by a mixture of 4% paraformaldehyde and 1% glutaraldehyde.

Embedding and sectioning cells in agar: The two million fixed cells of each sample group are embedded in 7.5% noble agar by centrifugation. The agar are sectioned in 50 μm sections on a vibratome. The serial sections are placed on microscope slides in a humidified chamber, and the cell membranes are permeabilized with Cytofix/Cytoperm to allow labeling of the cells’ internal components. The intracellular AAT are conjugated in situ with anti-AAT immunogold particles and an isotype control antibody to track AAT with TEM. The lysosomes are labeled with rat anti-human lysosome- associated membrane protein 1 (LAMP1) antibodies to provide intracellular reference points. A rat isotype control antibody are added to confirm antibody specificity. The sections are incubated with the two primary antibodies for one hour. To minimize nonspecific signals, the sections are incubated with Bloxall to block endogenous peroxidase activity in the cells. Incubation with a horse radish peroxidase (HRP) anti-rat secondary antibody will precede development with 3, 3′-diaminobenzidine (DAB) The antibody-HRP-substrate complex will provide a dark, electron-dense labeling of the lysosomes when seen with TEM and a brown hue in low-magnification microscopy (Figure 1).

Preparation for TEM: The sections are stained with 1% OsO4 and with 4% uranyl acetate to enhance contrast of the cellular structure. vi The sections are dehydrated by a series of solutions of increasing acetone concentration. The sections are embedded in Spurr’s Resin and hardened overnight in an oven. The resin block are sectioned on a microtome in 70 nm sections, and the sections are placed on a carbon-coated copper grid for TEM imaging.

Results/Discussion/Conclusion

Under Dr. Burton’s mentoring, I have spent six months optimizing the protocol for embedding T cells in noble agar with subsequent sectioning and staining. However, after analysis under TEM, I was not able to see a sufficient number of cells per sample. I went back to the literature and consulted with Dr. Burton and Michael Standing, the electron microscopy technician at BYU. We decided upon a more efficient cell pelleting method to get them more cells per sample in the final TEM sections. In this method, we are forgoing the vibratome sectioning and the use of noble agar. Instead we are using 1nstead we are using 1% low melt agar to pellet and resuspend the cells in, producing a small block of low melt agar with many cells. We then treat this as the sample for TEM processing per aforementioned protocol. We have also decided to label the cells before fixation to avoid the Cytofix/Cytoperm step that could interfere with TEM preparation later. I am now paying special attention to ensuring all reagents are prepared fresh and at the correct pH. We are currently collecting enough T cell samples to finish optimization and continue visualization of cells. After determining the intracellular location and route of AAT at the aforementioned time points, I plan to submit the results for publication in the Journal of Immunology by the end of July of 2014 when I leave to medical school.

References

1. Smith, B.A.; Gartner, S.; et al. Persistence of Infectious HIV on Follicular Dendritic Cells. J. Immunol. 2001, 166, 690–696.
2. Heath, S.L.; Tew, J.G.; et al. Follicular Dendritic Cells and Human Immunodeficiency Virus Infectivity. Nature 1995, 377, 740–744.
3. Keele, B. F.; Tazi, L.; et al. Characterization of the Follicular Dendritic Cell Reservoir of Human Immunodeficiency Virus Type 1. J. Virol. 2008, 82, 5548–5561.
4. Kallings, L.O. The First Postmodern Pandemic: 25 Years of HIV/AIDS. J. Intern. Med. 2008, 263, 218–243.
5. Zhou, X.; Shapiro, L.; et al. HIV Replication in CD4+ T Lymphocytes in the Presence and Absence of Follicular Dendritic Cells: Inhibition of Replication Mediated by α-1-Antitrypsin through Altered IκBα Ubiquitination. J. Immunol. 2011, 186, 3148–3155.
6. Szakal, A.; Kosco, M.; et al. A Novel In Vivo Follicular Dendritic Cell-Dependent Icosome-Mediated Mechanism for Deilvery of Antigen to Antigen-Processing Cells. J. Immuno. 1988, 140, 341–353.