Functional characterization of infiltrating T lymphocytes in human hepatic allografts

Abstract

We have employed recently developed techniques in T-cell culturing to study the nature and function of infiltrating hepatic allograft T cells. Using the rationale that intragraft T cells are activated during cell mediated damage to the allograft, we were able to show that these cells would propagate and remain functionally active in the presence of the T-cell growth factor, IL-2. In several instances, phenotypic analysis of cells grown in this manner was very similar to that found within the graft. Both proliferative and cytotoxic responses could be detected from the cultured cell lines. The majority of the proliferative responses were donor-directed and immunogenetic analysis could define donor-directed HLA reactivity, to either class I or class II antigens, or both. Monoclonal anti-HLA antibodies inhibition profiles verified the apparent HLA reactivity. In a smaller percentage of cases, only IL-2 responsiveness could be detected, and no HLA reactivity could be determined. Cytotoxicity could be detected against both class I and class II antigens, however, those cells which demonstrated a greater magnitude of donor-directed cytotoxicity appeared to be directed against class I antigens. A significant correlation between donor-directed proliferation of biopsy cultured lymphocytes and cellular rejection was found. This model appears to be useful in delineating functions of the intragraft T-cell population during rejection.

FIGURE 1

In vitro propagation of lymphocytes from a liver biopsy culture. This is a 400× magnification (original magnification) taken with an inverted stage microscope 4 days after expansion in tissue culture medium supplemented with IL-2. The dark area in the left corner is the liver biopsy tissue.

FIGURE 2

PLT specificity of LB3.32A. Tritiated thymidine uptake was determined in 3-day proliferation assays. HLA antigens of the panel cells are listed, those shared with the original donor are underlined. The background count with 10% human AB serum was 2097 ± 1608 (1 SD).

FIGURE 3

PLT specificity of LB9.9A. Legend is the same as for Figure 2. The background count was 222 ± 23 (1 SD).

FIGURE 4

CML specificity of LB13.28B. Percent lympholysis is determined by the formula: $$\%\text{lympholysis}=\frac{(\text{experimental}-\text{spontaneous})\text{release}}{(\text{Triton‐X total}-\text{spontaneous})\text{release}}\times 100\%.$$ HLA antigens of the panel cells are listed; those shared with the original donor are underlined. Lymphocytes incubated with PHA-M for 4 days prior to testing are designated as PHA targets. EBV targets are splenocytes that were originally transformed with EBV.