Memory CD8 T Cells Protect against Cytomegalovirus Disease by Formation of Nodular Inflammatory Foci Preventing Intra-Tissue Virus Spread

Abstract

Cytomegaloviruses (CMVs) are controlled by innate and adaptive immune responses in an immunocompetent host while causing multiple organ diseases in an immunocompromised host. A risk group of high clinical relevance comprises transiently immunocompromised recipients of hematopoietic cell transplantation (HCT) in the "window of risk" between eradicative therapy of hematopoietic malignancies and complete reconstitution of the immune system. Cellular immunotherapy by adoptive transfer of CMV-specific CD8 T cells is an option to prevent CMV disease by controlling a primary or reactivated infection. While experimental models have revealed a viral epitope-specific antiviral function of cognate CD8 T cells, the site at which control is exerted remained unidentified. The observation that remarkably few transferred cells protect all organs may indicate an early blockade of virus dissemination from a primary site of productive infection to various target organs. Alternatively, it could indicate clonal expansion of a few transferred CD8 T cells for preventing intra-tissue virus spread after successful initial organ colonization. Our data in the mouse model of murine CMV infection provide evidence in support of the second hypothesis. We show that transferred cells vigorously proliferate to prevent virus spread, and thus viral histopathology, by confining and eventually resolving tissue infection within nodular inflammatory foci.

Keywords: adoptive cell transfer; antiviral protection; cytomegalovirus (CMV); growth kinetics; histopathology; immunotherapy; liver infection; memory CD8 T cells; nodular inflammatory focus (NIF); virus spread.

Figure 1

Characterization of the donor CD8 T-cell population used for CD8-AT. (A) Sketch of the experimental protocol. Polyclonal CD8 T cells were isolated from the spleen of donor BALB/c mice primed 10 months earlier by infection with mCMV. The cells were transferred into immunocompromised recipient BALB/c mice. (Flash symbol) γ-irradiation was applied to both recipients of CD8-AT and no-transfer control mice. (B) Cytofluorometric analysis documenting the enrichment of CD8 T cells by positive immunomagnetic cell sorting; (SSC) sideward scatter. (C) Frequencies of memory CD8 T cells specific for the viral epitopes indicated. Bars represent the frequencies of cells stimulated by viral antigenic peptides to secrete IFNγ in an ELISpot assay. Error bars represent the 95% confidence intervals.

Figure 2

CD8-AT inhibits exponential virus growth reflected by a prolonged viral doubling time (vDT) of infectious virus in the spleen (A) and lungs (B). AT was performed with 10⁵ CD8 T cells, of which ~1.6% were memory cells specific for mCMV epitopes (recall Figure 1C). Symbols represent data from individual mice. Median values are marked. Red-rimmed empty circles—no AT; Red-filled circles—AT. Left panels—raw data. Right panels—calculated log-linear regression lines and the associated 95% confidence regions. The calculation was based on all data from individual mice at the indicated times. For clarity, symbols show only the median values. The dashed lines represent the detection limit of the virus plaque assay. vDT values represent the most probable values. The respective 95% confidence intervals for vDT are shown in parentheses.

Figure 3

CD8-AT inhibits exponential virus spread in liver tissue reflected by a prolonged viral doubling time (vDT) of the number of infected liver cells, corresponding to enhanced tissue infiltration by CD8 T cells indicated by a shorter cellular doubling time (cDT). Data refer to the same experiment as shown in Figure 2. 2C-IHC was performed to quantitate infected liver cells (mostly hepatocytes) by red staining of the intranuclear viral protein IE1 (A) and tissue-infiltrating CD8 T cells by black staining of the CD8a molecule (B). Red- or black-rimmed empty circles—no AT. Red or black filled circles—AT. For further details, see the legend of Figure 2. The dashed lines in (A) represent the detection limit of the IE1-specific IHC. For comparisons of most interest, significance levels were determined. For the comparison of groups that include data below the detection limit, as is the case in (A), the distribution-free Wilcoxon–Mann–Whitney test was applied. Otherwise, the Student’s t-test with Welch’s correction of unequal variance was performed for log-transformed data in (B). Significant with p < 0.01 (**) or p < 0.001 (***).

Figure 4

2C-IHC images showing the time course of NIF formation after CD8-AT. Infected liver cells, which are primarily hepatocytes, are identified by red staining of the intranuclear viral protein IE1. Tissue-infiltrating, NIF-forming CD8 T cells are identified by black staining of the CD8a molecule. Light hematoxylin counterstaining reveals the context of liver tissue. In the absence of CD8-AT, NIF are not formed, and the virus spreads unhindered. The bar marker represents 50 µm and applies to all images.

Figure 5

2C-IHC images showing proliferating CD8 T cells accumulated in a perivascular nodular inflammatory focus (NIF). Liver tissue-infiltrating CD8 T cells are identified by black staining of the CD8a molecule. Proliferating CD8 T cells are identified by red nuclear staining of PCNA and black cytoplasmic as well as membrane staining of CD8a. Infected liver cells, which are primarily hepatocytes, also express PCNA in their nuclei. (Left image) lower-magnification overview. (Right image) the framed area in the overview image is resolved to greater detail. Light hematoxylin counterstaining reveals the context of liver tissue. CV—central vein; iHc—infected hepatocyte; PCNA⁺ T—proliferating PCNA⁺ CD8 T cell. Bar markers, 50 µm.

Figure 6

Extensive expansion of memory CD8 T cells in transfer recipients. (Left panel) for the indicated times after CD8-AT, infected IE1⁺ cells (filled red circles) and tissue-infiltrating CD8 T cells (filled black circles) were counted in representative 2C-IHC-stained liver tissue sections, and the absolute numbers of infected cells and CD8 T cells present in the whole liver were calculated by extrapolation. Symbols represent data of individual AT recipients. Median values are marked. (Right panel) calculated numbers of CD8 T-cell divisions based on initially transferred ~1600 viral epitope-specific cells. Symbols represent values for individual AT recipients. Median values are marked. As not all transferred cells home to the liver, the number of cell divisions represents a minimum estimate. Significant with p < 0.05 (*), calculated for log-transformed data. Significant with p < 0.01 (**), calculated for linear data. Student’s t-test with Welch’s correction of unequal variance.