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, 33 (S1), S31-S39

An Optimized and Validated Method for Isolation and Characterization of Lymphocytes From HIV+ Human Gut Biopsies

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An Optimized and Validated Method for Isolation and Characterization of Lymphocytes From HIV+ Human Gut Biopsies

Martin Trapecar et al. AIDS Res Hum Retroviruses.

Abstract

The gastrointestinal (GI) tract harbors most of the body's immune cells and is also a major HIV reservoir in ART-treated patients. To achieve a cure, most HIV-infected cells must be identified and eliminated. While obtaining gut biopsies is a relatively noninvasive method of sampling relevant tissue for monitoring HIV activity, immune cell isolation from these limited tissue samples has proven to be challenging. Enzymatic tissue digestion is required for maximal immune cell isolation from gut biopsies. However, these enzymatic digestions can also be detrimental for preservation of cellular surface markers that are required for accurate identification of various subsets of leukocytes. In this study, we describe an optimized protocol for isolation of lymphocytes from human gut biopsies. We also discuss our validation results, which show that compared with several other collagenase preparations, the use of CSLPA maintains high lymphocyte recovery while preserving the integrity of most cellular surface antigens that we tested. Importantly, chemokine receptors that are used to characterize various subsets of T cells, which are notorious for being digested during a typical enzymatic tissue digestion, are highly preserved using this protocol.

Keywords: CXCR5; CyTOF; collagenase; human gut biopsies; lymphocyte isolation; surface antigens.

Conflict of interest statement

All authors declare no conflict of interest.

Figures

<b>FIG. 1.</b>
FIG. 1.
Lymphocyte isolation scheme.
<b>FIG. 2.</b>
FIG. 2.
Effect of various collagenase enzymes on CD4 T cell surface antigens. (A) Human tonsil cells collected from the same donor were digested for 30 min at 37°C with 500 U/ml of either collagenase type I, II-s, IV, or CLSPA. Representative flow plots from one of four different donors with similar results are shown. (B) Tonsil cells were first incubated at 37°C for 20 min with stripping buffer containing EDTA and DTT, or were left on ice in FACS buffer without EDTA and DTT. Cells from both conditions were incubated in digestion media containing CLSPA for 30 min, followed by analysis using flow cytometry. (C) Heat map showing fold change in MFIs of treated over untreated CD4 T cells. Tonsil cells were treated with either collagenase type I or CLSPA as in A, or were left untreated. Surface antigens were analyzed by mass cytometry. All cells were first gated on live cells and singlets, followed by gating on CD3+ CD4+ cells. Blue indicates reduction and red indicates enhancement in levels of indicated surface molecules, as compared with untreated cells. The experiments described in each panel in this figure used tonsils from different donors. FACS, fluorescence-activated cell sorting; MFI, median fluorescence intensity
<b>FIG. 3.</b>
FIG. 3.
Improved retention of surface antigens on cells isolated from CLSPA-digested human rectosigmoid biopsies. (A) Flow cytometry plots of CD4 T cells from PBMC or rectosigmoid biopsies collected from an HIV (+) participant (PID #2). Tonsil cells from an HIV (−) donor are also shown. The same number of rectosigmoid biopsies were digested with either collagenase IV or CLSPA. (B) Detection of various surface markers on CD3+ cells in rectosigmoid biopsies from (A). CD3+ cells from PBMC are also shown as an undigested control. Representative plots of CLSPA digestion of biopsies from one of six different HIV (+) participants are shown. PBMC, peripheral blood mononuclear cell.
<b>FIG. 4.</b>
FIG. 4.
Cell recovery from CLSPA-digested rectosigmoid biopsies. (A) Gating strategy for isolating CD3+ cells from PBMC and rectosigmoid biopsies of HIV (+) participants. (B) Total cells recovered per biopsy. At least 18 biopsies were used to generate total cell count per participant, and then the cell number was divided by the number of biopsies from each participant. (C) Percentages of live (Aquamine −), and (D) CD3+ in total cells obtained from CLSPA-digested rectosigmoid biopsies from each participant.

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