Interrogating Adaptive Immunity Using LCMV

Abstract

In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: LCMV infection routes in mice Support Protocol 1: Preparation of LCMV stocks ASSAYS TO MEASURE LCMV TITERS Support Protocol 2: Plaque assay Support Protocol 3: Immunofluorescence focus assay (IFA) to measure LCMV titer MEASUREMENT OF T CELL AND B CELL RESPONSES TO LCMV INFECTION Basic Protocol 2: Triple tetramer staining for detection of LCMV-specific CD8 T cells Basic Protocol 3: Intracellular cytokine staining (ICS) for detection of LCMV-specific T cells Basic Protocol 4: Enumeration of direct ex vivo LCMV-specific antibody-secreting cells (ASC) Basic Protocol 5: Limiting dilution assay (LDA) for detection of LCMV-specific memory B cells Basic Protocol 6: ELISA for quantification of LCMV-specific IgG antibody Support Protocol 4: Preparation of splenic lymphocytes Support Protocol 5: Making BHK21-LCMV lysate Basic Protocol 7: Challenge models TRANSGENIC MODELS Basic Protocol 8: Transfer of P14 cells to interrogate the role of IFN-I on CD8 T cell responses Basic Protocol 9: Comparing the expansion of naïve versus memory CD4 T cells following chronic viral challenge.

Keywords: LCMV model; acute viral infection; chronic viral infection; immune exhaustion; immune memory.

Figure 1.. Experimental outline for evaluating immunotherapies using the conventional Cl-13 model.

In this model of chronic LCMV infection, most of the long-term persistence is limited to brain and kidney. Viremia is cleared after a month, and thus, the optimal time to perform immunotherapy (e.g. PD-1 blockade) in this model is between days 15–25 post-infection.

Figure 2.. Experimental outline for evaluating immunotherapies using the CD4-depleted Cl-13 model.

In this model of chronic LCMV infection caused by depleting CD4 T cells prior to infection, mice exhibit high-titer, lifelong multi-organ infection. The optimal time to perform immunotherapy (e.g. PD-1 blockade) in this model is between days 40–90 post-infection. After day 90, mice will exhibit deep T cell exhaustion, and suboptimal response to immunotherapy. Thus, the CD4-depleted Cl-13 model also allows one to interrogate different phases of T cell exhaustion, including early (day 90) exhaustion.

Figure 3.. LCMV plaque assay plate.

Each row represents a different sample. Each column represents a different dilution (1000-fold, 100-fold and 10-fold, from left to right, respectively). Data are from sera of mice infected intravenously with LCMV Cl-13 (day 12 post-infection). It is advisable to use the wells that have ~50 PFU to calculate viral titer.

Figure 4.. LCMV immunofluorescence images.

LCMV+ cells are represented as fluorescent foci. Monolayer of Vero e6 cells were infected with an LCMV Armstrong stock at different dilutions, and after 24 hr, cells were incubated with a primary VL4 antibody, followed by staining with a secondary antibody (Anti-Rat IgG-Alexa Fluor568).

Figure 5.. Representative FACS plots showing tetramer+ CD8 T cell responses following LCMV Armstrong infection.

A) Experimental outline. B) Representative FACS plots showing the frequencies of CD8 T cells that are LCMV-specific in PBMCs at day 28.

Figure 6.. Representative FACS plots showing LCMV-specific CD8 T cell responses by ICS following LCMV Armstrong infection.

A) Experimental outline. B) Representative FACS plots showing the frequencies of CD8 T cells that are LCMV-specific in spleen at day 28. All plots are gated from total CD8 T cells. Note that the GP61–80 peptide (known to be a CD4 epitope) also induces CD8 T cell responses.

Figure 7.. Antibody secreting cell (ASC) assay using bone marrow from LCMV Armstrong infected mice.

Data are from day 52 post-infection. Rows A-F represent six different LCMV-immune mice. Row G represents a naïve mouse. Row H received only PBS (no cells). A) 96-well plate at the end of the assay. B) Top: enlarged image of well A1 (LCMV-immune bone marrow); Bottom: enlarged image of well G1 (naïve bone marrow).

Figure 8.. ELISA plates for detection of LCMV-specific IgG.

5 μL of sera samples from LCMV-immune mice (9–13), naïve control mice (N), and one Armstrong positive control (PC) was added on the first column of wells (1:30 dilution), followed by 12 serial 3-fold dilutions across the plate. (A) After adding TMB substrate. (B) After adding stop solution. Absorbance was measured in a spectrophotometer. As shown in these images, antibody responses are detected by the development of color in the wells. It is important to sonicate the BHK21-LCMV lysate for accurate results. In this experiment, C57BL/6 Ifnar1−/− mice were immunized with 2×10⁶ PFU of a recombinant LCMV Cl-13 expressing WE glycoprotein (day 12 post-infection).

Figure 9.. Comparing memory versus naïve SMARTA cell expansion following chronic LCMV challenge.

A) Experimental outline. B) CD44 expression on naive and memory SMARTA CD4 T cells before injection to corroborate antigen experience. C) Representative FACS plots comparing the frequencies of primary and secondary SMARTA effectors (gated on total CD4 T cells). D) Ratio of primary and secondary SMARTA responses. E) Number of SMARTA cells in spleen at day 8 after lymphocytic choriomeningitis virus (LCMV) Cl-13 infection. In these experiments, 10⁵ naive and 10⁵ memory SMARTA cells (day 30 post-immunization) from spleen were co-transferred at (1:1 ratio) into congenically distinct mice, followed by LCMV Cl-13 challenge 1 day after CD4 T-cell transfer. SMARTA cells were evaluated in PBMCs in all panels unless indicated otherwise. Data are from two experiments, n = 4–5 mice/group per experiment. Error bars indicate SEM.