This work was supported by grants from the Foundation Blanceflor Boncompagni Ludovisi nee Bildt (http://blanceflor.se/), the Foundation Swedish Physicians against AIDS (http://www.aidsfond.se/, ref. FOa2014-0006), and Fondazione Andrea e Libi Lorini (http://fondazionelorini.it/) to Andrea Introini.&#160;

The protocol for collection of human tissues may require ethical approval by the local competent authorities. In case of indicated surgeries such as tonsillectomy and hysterectomy, the specimens are not collected specifically for research purpose and the study is not considered human subjects research (National Institutes of Health. Research on Human Subjects. https://humansubjects.nih.gov/walkthrough-investigator#tabpanel11). However, obtaining tissue donors&#39; personal and medical data (e.g., sex, age, current drug use, history of infections, etc.) that may help interpret experimental results, poses concerns about informed consent and privacy that must be addressed in the protocol for tissue collection.
Caution: The entire procedure should be carried out in a biological safety cabinet. All human specimens, including those from &#39;healthy&#39; individuals, may harbor infectious agents. The operator should receive training to work with blood-borne pathogens to safely handle tissue specimens, even if experiments do not involve the use of HIV. A risk assessment of the procedure of tissue dissection and infection with HIV should be carried out by trained staff to ensure the safety of the operator and coworkers. The use of infectious HIV requires dedicated biosafety level 2 or 3 environments depending on the country and institute-specific regulations on hazardous biologicals and blood-borne pathogens.
1. Preparation of Gelatin Sponges
NOTE: Although it is not strictly necessary to prepare gelatin sponges before tissue dissection, it is more convenient to follow the order outlined here, especially when handling a large amount of tissue and/or from many donors.
<ol>
	<li>Prepare culture medium (CM) and supplement it with antibiotic solution (Tircacillin-clavulanate mix) before use to make CMT (Table of Materials). Discard any leftover antibiotic solution. 
	NOTE: Ticarcillin and clavulanate in CMT are poorly stable. If needed, re-supplement CMT with antibiotic solution after about 24 h from preparation. It is not required to add antibiotic solution to explant medium every 24 h during culture.</li>
	<li>Fill a 100 mm x 20 mm Petri dish with about 20&#8211;30 mL of CMT. 
	NOTE: This setting is optimal to prepare enough gelatin sponges for two 6-well plates or one 12-well plate at the time. If many plates are required, use a wider Petri dish and larger CMT volume to speed up the preparation.</li>
	<li>Transfer the gelatin sponge(s) into the Petri dish using sterile forceps. Wet the sponges on both sides and allow the sponges to soak up medium for 1 min. Press the sponges against the bottom of the Petri dish for about 2 min using a sterile spatula. 
	NOTE: This step is of crucial importance because the presence of air bubbles in the sponge will block the capillaries through which culture medium nutrients reach the tissue. Gelatin sponges are extremely brittle when dehydrated. Gently push down on the sponge with the spatula, especially at the beginning, to avoid breaking the sponge.</li>
	<li>Use sterile scissors to cut the rehydrated gelatin sponges into pieces of equal size of about 1 cm2. Transfer 1 sponge piece using forceps into each well of a culture plate. Add 3&#8211;4 mL of CMT into each well of a 6-well plate (tonsils) and 1 mL per well into a 12-well plate (cervix). Place the plates in the incubator, set at 37 &#176;C, 5% CO2, &#8805; 90% humidity, until the tissue explants are ready to be loaded on the sponges. 
	Note: The volume of CMT to add into the plate should be adjusted to the thickness of the sponge to keep the explants at the liquid-air interface.</li>
</ol>
2. Dissection of Human Tonsillar Tissue
NOTE: Tonsils should be processed as soon as possible after surgery, ideally the same day of surgery. Alternatively, specimens can be left overnight submerged in CMT at 4 &#176;C.
<ol>
	<li>Allow the CM enough time to reach room temperature (RT) or put it in a water bath pre-warmed at 37 &#176;C. Supplement the CM with antibiotic solution (CMT) before use. Discard any antibiotic solution left over. Pour 70% ethanol solution into a clean container to soak forceps and scalpel whenever needed during the dissection procedure. Clean the tools and change the scalpel blade between dissections of specimens from different donors.</li>
	<li>Transfer the tonsils from the transportation container into a 100 mm-Petri dish containing 10-20 mL of CMT using sterile forceps. 
	NOTE: Specimens with widespread areas of cauterized, bloody, or necrotic tissue should be discarded.</li>
	<li>Use the lid of the Petri dish as a cutting surface to dissect tissue. Holding the tissue gently with forceps, cut each tonsil into several big pieces using sterile scalpel and blade. 
	Caution: Handling sharp tools to dissect human specimens increases the risk of injury and contamination. The operator should consider wearing metal, mesh, cut-resistant gloves as additional protection.</li>
	<li>Remove cauterized, bloody, and fibroid tissue, and any parts containing tonsilloliths (calcified material) and/or with green-brownish color using forceps and scalpel. 
	NOTE: While working on one piece of tissue, it is important to keep the rest of the tissue submerged in medium to avoid tissue desiccation.</li>
	<li>Cut each tissue piece into slices of about 2 mm in thickness. Remove any unwanted part as in the previous step. Cut the tissue slices into 2 mm-thick strips. Cut the tissue strips into 2 mm-thick blocks. This should result in tissue explants of roughly 8 mm3.</li>
	<li>Transfer the explants into a new 100 mm Petri dish containing 10&#8211;20 mL of CMT to avoid tissue desiccation while proceeding with the dissection. At the end of dissection, swirl the plate to randomize the explant distribution. Place 9 tissue explants on top of each gelatin sponge in a 6-well plate using forceps, allowing space between them. Return the plates to the incubator. 
	NOTE: Typically, explants are cultured overnight and inoculation of cultures with HIV-1 is performed the next day. This is to make sure that no bacterial or fungal contamination develops in the culture. In addition, cells tend to egress tonsillar tissue explants after dissection. This process is largely completed within the first 24 h of culture1.</li>
	<li>Dispose of all biological waste in apposite containers according to the institute&#39;s regulations on handling hazardous biologicals and the specific risk assessment.</li>
</ol>
3. Infection of Tonsillar Tissue Explants with HIV-1
NOTE: To reduce result variability between independent experiments, the same virus preparation should be used for an entire study. Virus can be propagated in exogenously activated peripheral blood mononuclear cells and then the culture supernatant can be aliquoted for storage at -80 &#176;C to avoid repeated freezing and thawing (Table of Materials).
<ol>
	<li>Put the CM in a water bath pre-warmed at 37 &#176;C. Supplement the CM with antibiotic solution (CMT) before use. Discard any leftover antibiotic solution.</li>
	<li>Aspirate the medium in the 6-well plate(s) with a pipette and discard it in a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom, and aspirate and discard it. Add 3-4 mL of CMT to each well.</li>
	<li>Thaw the HIV-1 virus preparation in the water bath at 37 &#176;C. If necessary, dilute the virus stock with CM (Table 1). 
	NOTE: The virus stock should be diluted so that the selected inoculum is contained in a volume of 5&#8211;8 &#181;L (Table 1). For an efficient infection, it is critical to use the minimum time required between thawing the HIV-1 preparation and infecting tissue explants.</li>
	<li>Pipette the virus inoculum directly on top of each of the 9 explants on a gelatin sponge. Return the plate(s) to the incubator as soon as the infection is completed. Discard any residual virus preparation. Continue to Section 5. 
	NOTE: To accurately deliver the virus inoculum the operator should consider using a reverse pipetting technique and changing the tip for every well. This involves pushing the pipette piston down to the purge position (the second stop) to draw the virus inoculum up, and pushing to the first stop to deliver the inoculum, which helps minimizing bubble formation and the error associated with repeated sampling of small volumes, i.e., 5&#8211;8 &#181;L.</li>
</ol>
4. Dissection and Infection of Uterine Cervical Mucosa
NOTE: For optimal results, explants of cervical mucosae should be processed and infected as soon as possible after surgery, ideally the same day of surgery. Alternatively, specimens can be stored submerged in CMT at 4 &#176;C overnight, and infected immediately after dissection.
<ol>
	<li>Allow the CM enough time to reach RT or put it in a water bath pre-warmed at 37 &#176;C. Supplement the CM with antibiotic solution (CMT) before use. Discard any leftover antibiotic solution. Pour 70% ethanol solution into a clean container to soak the forceps and scalpel whenever needed during the dissection procedure. Clean the tools and change the scalpel blade between dissections of specimens from multiple donors.</li>
	<li>Using sterile forceps, transfer the sample from the transportation container into a 100 mm Petri dish containing 10&#8211;20 mL of CMT.</li>
	<li>Use the lid of the Petri dish as a cutting surface to dissect the tissue. Holding the tissue gently with forceps, separate the mucosa of the ectocervix and/or endocervix from the underneath stromal and muscular tissue (submucosa) with a scalpel and blade to obtain strips of mucosa. 
	NOTE: While working on one piece of tissue, it is important to keep the rest of the tissue submerged in medium to avoid tissue desiccation.</li>
	<li>Cut the mucosa into 2 mm-wide strips, remove and discard as much submucosa as possible, leaving only a 2 mm-thick layer of tissue. Cut the strips into 2 mm-thick blocks. This should result in tissue explants of roughly 8 mm3. 
	Caution: Handling sharp tools to dissect human specimens increases the risk of injury and contamination. The operator should consider wearing metal, mesh, cut-resistant gloves as additional protection.</li>
	<li>Transfer tissue explants into a new 100 mm Petri dish containing 10&#8211;20 mL of CMT to avoid tissue desiccation while proceeding with the dissection. At the end of dissection, swirl the plate to randomize the explant distribution.</li>
	<li>With sterile forceps, transfer the explants into sterile 1.5-mL conical tube(s) (maximum 16 explants per tube). Remove any medium in the tube using a pipette.</li>
	<li>Thaw the HIV-1 preparation in a water bath at 37 &#176;C. If necessary, dilute the virus stock with CM. Transfer the virus into the tube(s) containing the explants. Discard any residual virus preparation. 
	NOTE: The virus stock should be diluted so that the selected inoculum is contained in a volume of minimum 0.5 mL (Table 1). To reduce result variability between independent experiments, the same virus preparation should be used for an entire study (Table of Materials).</li>
	<li>Transfer the tubes into a thermo-shaker and incubate for 2 h at 37 &#176;C rocking at 300 rpm. Gently invert the tubes a few times every 30&#8211;60 min. 
	Note: For an efficient infection, it is critical to use the minimum time required between thawing the HIV-1 preparation and transferring the tubes to 37 &#176;C.</li>
	<li>Fill a 6-well plate with 3&#8211;4 mL per well of sterile phosphate buffer saline (PBS). At the of the incubation with HIV-1, discard all virus preparation in the tube(s) into a container with disinfectant solution using a pipette. Transfer the explants into the 6-well plate containing PBS using forceps.</li>
	<li>Allow the explants to rest in PBS for 1 min at RT, and then wash them by gently pipetting up-and-down the PBS into the well 2-3 times. Discard the PBS into a container with disinfectant solution using a pipette. Add 3&#8211;4 mL of new PBS to each well. Repeat two more times for a total of three washes. Discard the PBS just before transferring the explants to the sponges to avoid tissue desiccation. 
	NOTE: Explants of cervical mucosa, and in particular endocervix, release great amounts of mucus during infection. It is critical to wash and remove as much mucus as possible because unspecific retention on explants and subsequent release of virus into culture supernatant can mask virus replication.</li>
	<li>Using forceps, transfer 5-8 explants on top of each gelatin sponge in a 12-well plate.Return the plate to the incubator.</li>
	<li>Dispose of all biological waste in apposite containers according to the institute&#39;s regulations on handling hazardous biologicals and the specific risk assessment.</li>
</ol>
5. Histoculture
<ol>
	<li>Change the CM every 3 days starting from the time of infection until day 12&#8211;15 post-infection. The CM and/or explants can be sampled at regular intervals throughout the culture time to assess HIV-1 replication, among other parameters.</li>
	<li>Put the CM in a water bath pre-warmed at 37 &#176;C. 
	NOTE: It is not required to supplement the CM with antibiotic solution at this stage.</li>
	<li>Collect a sample of CM with a pipette and transfer it into sterile 1.5&#160;or 2 mL screw cap tube(s) for storage at -80 &#176;C. 
	NOTE: The CM from replicate wells are pooled at collection.</li>
	<li>Harvest the tissue explants with sterile forceps, remove the excess medium on explants by placing them on a piece of paper (optional), and transfer explants into sterile 1.5&#160;or 2 mL screw cap tube(s). Process or store the tissue samples as required by the experiment. 
	NOTE: For flow cytometry, explants are immediately digested with an enzymatic cocktail to obtain a single-cell suspension (for more details see references 1, 9, 16, and 17). For RNA extraction, explants are kept in a RNA stabilization solution at 4 &#176;C overnight before storing them at -80 &#176;C. For DNA extraction or in situ staining (cervix), explants can be snap-frozen in liquid nitrogen or dry ice/ethanol slurry and stored at -80 &#176;C. Alternatively, tonsillar explants can be fixed by submersion in a solution of PBS and 4% paraformaldehyde for in situ staining.</li>
	<li>Aspirate the residual CM in the well with a pipette and discard it into a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom, and then aspirate and discard it.</li>
	<li>Add 3&#8211;4 mL of CM to each well. Using sterile forceps, return any tissue explants that dropped into the medium to the gelatin sponge. Return the plate to the incubator.</li>
	<li>At the end of culture, dispose of all biological waste in apposite containers according to the institute&#39;s regulations on handling hazardous biologicals and the specific risk assessment.</li>
</ol>

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The authors have nothing to disclose.

The use of human tissue explants for the study of HIV-1 infection provides advantages over traditional bi-dimensional and monotypic experimental systems, such as primary cells or cell lines, due their superior ability to reproduce intercellular interactions and cellular functions (e.g., cytokine production) as they are in vivo. Nevertheless, tonsillar and cervical tissue differ in many aspects including the number, and phenotypic and functional characteristics of HIV target cells1</sup...

Monotypic bi-dimensional cell cultures, here referred to as conventional, do not account for the spatial and functional communication between the large variety of cell types that compose tissues and organs. This aspect is of paramount importance for experimental models of disease, as interference with homeostatic intercellular interactions is the driving factor of all pathologies. Tissue explants offer major advantages for modeling health and disease in humans because they retain the cytoarchitecture and many important functional aspects of organs as they are in vivo, although for a limited amount of time1. For example, upon ex vivo challenge with recall antigens, such as diphtheria toxoid or tetanus toxoid, tonsillar tissue responds with a vigorous production of antigen-specific antibodies2. Like any other ex vivo model, histoculture has its own limitations: inter-donor variability, tissue polarization, limited tissue survival, and difficulty in monitoring cells beyond the depth of confocal microscopy1. Nevertheless, human tissue explants remain a model of choice to study homeostatic and pathogenic immunological processes in humans, including host-pathogen interactions and potential therapeutic interventions3.
The critical events of HIV-1 pathogenesis take place in tissues. Infection of the genital mucosa accounts for the majority of all HIV-1 transmission events worldwide4. Lymphoid tissue is the major site of virus replication during acute infection and harbors a significant pool of latently-infected cells5, and their persistence represents the main obstacle to achieve a cure6. The culture and ex vivo challenge of human lymphoid and mucosal tissues provide some advantages over conventional systems based on isolated cells for the study of HIV-1. For instance, tissue-resident cells can support HIV-1 infection in the absence of exogenous activation, as opposed to peripheral blood mononuclear cells1. The use of lymphoid tissue explants has allowed a better understanding of some key mechanisms underlying CD4 T cell depletion, i.e., the bystander effect7, which is the hallmark of acute infection. The preservation of structures like B cell follicles in lymphoid tissue8 and the epithelium in female genital mucosa explants9 offers the unique opportunity to integrate spatial and functional features of HIV-1 infection at these sites. Finally, histocultures were successfully used to model and study HIV-1 and herpesvirus co-infection10,11, as well as for preclinical testing of antiretrovirals and multitarget microbicides12,13,14,15.
Here, we describe a detailed protocol for the culture of human tissue explants obtained from tonsils and mucosal tissue from the lower female genital tract (cervix), covering tissue dissection to explant challenge with HIV-1 in a non-polarized way. The culture of tissue explants at the liquid-air interface, using gelatin sponges as a support, maximizes exposure to air oxygen while providing access to culture medium nutrients through the sponge capillaries, thus delaying their natural decay. The most immediate way of evaluating HIV-1 replication in our system is to measure the amount of virus released in the explant culture medium over time by immunoassay or qPCR. HIV-1 infection and pathogenesis (e.g., CD4 T cell depletion) can also be evaluated in tissue explants by bulk RNA/DNA extraction and qPCR, in situ staining, or single cell-analysis upon tissue digestion by flow cytometry.

<table><tbody><tr><td>Gelfoam 12-7 mm Absorbable gelatin sponge&amp;nbsp;</td><td>Pfizer</td><td>NDC:&amp;nbsp; 0009-0315-08</td><td>Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.</td></tr><tr><td>SPONGOSTAN Standard 70 &amp;times; 50 &amp;times; 10 mm Absorbable haemostatic gelatin sponge</td><td>Ferrosan Medical Devices</td><td>MS0002</td><td>Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.</td></tr><tr><td>RPMI 1640 with phenol red and glutamine</td><td>ThermoFisher Scientific</td><td>21875034</td><td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td></tr><tr><td>Modified Eagle&#039;s medium (MEM) non-essential aminoacids (100x)</td><td>ThermoFisher Scientific</td><td>11140035</td><td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td></tr><tr><td>Sodium pyruvate, 100 mM (100x)&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11360070</td><td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td></tr><tr><td>Gentamicin 50 mg/mL (1000x)&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15750037</td><td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td></tr><tr><td>Amphotericin B 250 &amp;mu;g/mL (100x)&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15290018</td><td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td></td><td></td><td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td></tr><tr><td>Ticarcillin disodium salt</td><td>Sigma-Aldrich</td><td>T5639-1G</td><td>Resuspend in sterile cell culture grade water tircacillin disodium (3 g) and potassium clavulanate (100 mg) and mix to obtain 100 mL of antibiotic solution (100x). Aliquote and store at -20 &amp;deg;C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT.</td></tr><tr><td>Potassium clavulanate</td><td>Sigma-Aldrich</td><td>33454-100MG</td><td>Resuspend in sterile cell culture grade water tircacillin disodium (3 g) and potassium clavulanate (100 mg) and mix to obtain 100 mL of antibiotic solution (100x). Aliquote and store at -20 &amp;deg;C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT.</td></tr><tr><td>Sterile phosphate buffer saline (PBS) pH 7.4 w/o calcium, magnesium and phenol red</td><td></td><td></td><td>To use for storage and transportation of surgical specimens. PBS can be replaced with another isotonic solution with physiologic pH. Culture medium is best for overnight storage.&amp;nbsp;</td></tr><tr><td>Sterile cell culture grade water</td><td></td><td></td><td></td></tr><tr><td>Sterile transportation container</td><td></td><td></td><td></td></tr><tr><td>70% ethanol solution</td><td></td><td></td><td></td></tr><tr><td>Disinfectant solution for biological waste disposal</td><td></td><td></td><td></td></tr><tr><td>Sterile metal forceps or tweezers</td><td></td><td></td><td></td></tr><tr><td>Sterile metal scissors</td><td></td><td></td><td></td></tr><tr><td>Sterile flat weighing metal spatula</td><td></td><td></td><td></td></tr><tr><td>Sterile scalpels and blades</td><td></td><td></td><td></td></tr><tr><td>6-well cell culture plates</td><td></td><td></td><td></td></tr><tr><td>12-well cell culture plates</td><td></td><td></td><td></td></tr><tr><td>Sterile Petri dish, 100 mm &amp;times; 20 mm</td><td></td><td></td><td></td></tr><tr><td>Cell-free HIV-1 viral preparation HIV-1&lt;sub&gt;BaL&lt;/sub&gt;</td><td>NIH AIDS Reagent Program</td><td>510</td><td>The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 &amp;deg;C. Avoid repeated freezing and thawing.</td></tr><tr><td>Cell-free HIV-1 viral preparation HIV-1&lt;sub&gt;LAI.04&lt;/sub&gt;</td><td>NIH AIDS Reagent Program</td><td>2522</td><td>The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 &amp;deg;C. Avoid repeated freezing and thawing.</td></tr><tr><td>Lamivudine (3TC)</td><td>NIH AIDS Reagent Program</td><td>8146</td><td>Resuspend in DMSO at 10 mg/mL, aliquote and store at -20 &amp;deg;C. Avoid repeated freezing and thawing.</td></tr><tr><td>Biological safety cabinet</td><td></td><td></td><td></td></tr><tr><td>Water-jacketed CO&lt;sub&gt;2&lt;/sub&gt; incubator, set at 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, &amp;ge; 90% humidity</td><td></td><td></td><td></td></tr><tr><td>Water bath set at 37 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>Sterile 1.5 and 2mL screw cap tubes</td><td></td><td></td><td></td></tr><tr><td>Dry bath shaker with heating block for 1.5 mL tubes, set at 37 &amp;deg;C, 300 rpm</td><td></td><td></td><td></td></tr></tbody></table>

ex vivo infection of human lymphoid tissue and female genital mucosa with human immunodeficiency virus 1 and histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Several techniques can be used to assess HIV-1 replication in tissue explants. Our standard read-out is to measure the amount of HIV-1 p24gag released in CM over time with an immunoassay18.
Tissue explants from different donors, infected with the same inoculum of the same HIV-1 stock, may yield different amounts of virus (Table 1). This is due to several factors, such as the nu...

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Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

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Immunology and Infection

9.1K Views.  Karolinska Institutet. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het HIV-gebied, zoals de identificatie van de determinanten van virusoverdracht en pathogenese binnen twee van de meest uitgevoerde organen door de infectie. In feite vindt de belangrijkste geavanceerde progressie van de ziekte plaats in weefsels. De kenmerken en complexiteit van infectie worden slecht weerspiegeld door cellen in het verkeer of geïsoleerd en geïnfecteerd in vitro.Het belangrijkste voordeel van de ...

Video: Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Medicine

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

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