Assessing the Role of Alloreactive CD28^– Memory CD8 T Cells in Allograft Injury

By Robert Fairchild, PhD

Acute allograft injury continues to contribute to the unacceptable number of renal grafts lost in patients transplanted to treat end-stage kidney disease.

Acute graft injury promotes the development of the interstitial fibrosis, tubular atrophy and occlusive vasculopathy that are the primary causes of late kidney graft loss.

An acknowledged factor contributing to the incidence of acute and chronic allograft injury is the presence of memory CD8 T cells having reactivity to graft alloantigens in patients prior to transplantation.

Higher levels of endogenous memory T cells with reactivity to allograft major histocompatibility complex (MHC) molecules in the peripheral blood of renal transplant patients result in the increased incidence of delayed graft function and acute rejection, and portend poor long-term graft outcome.

Activation and rapid infiltration

Our studies in mouse models documented that endogenous memory CD8 T cells infiltrate solid organ allografts within 8 to 12 hours after reperfusion. The infiltrating memory CD8 T cells are activated through recognition of graft-allogeneic MHC molecules expressed to proliferate within the graft tissue. This proliferation induces the graft-reactive memory CD8 T cells to produce proinflammatory mediators including interferon-gamma (IFN-γ) that amplify inflammation and increase graft tissue injury.

Unlike memory CD8 T cells in mice, terminal differentiation of effector memory CD8 T cells in humans can lead to loss of expression of the co-stimulatory molecule CD28. Increased levels of CD28– memory CD8 T cells in the peripheral blood of transplant patients are associated with increased risk of acute graft rejection, and these memory T cells are resistant to the CD28-targeted costimulatory blockade immunosuppressive drugs that are in current trials in renal transplant patients.

Paradoxically, in vitro studies have indicated that human CD28– memory CD8 T cells are poorly activated to proliferate in response to anti-T cell receptor antibodies or mitogens and to express effector functions. This poor activation in vitro makes it difficult to assess their frequency and functions in graft recipients.

Questions about risk

Our results documenting memory CD8 T cell proliferation and expression of effector functions in organ allografts in mouse models raised question regarding the risk factor constituted by a population of CD28– memory CD8 T cells in human transplant patients when their activation in response to T cell receptor-mediated stimuli in vitro was difficult to demonstrate.

We reasoned that differences in the stimulatory conditions in vitro, such as in allogeneic mixed lymphocyte cultures, versus the inflammatory conditions induced in an allograft are likely to underlie observations of the poor activation of CD28– memory CD8 T cells in vitro.

Therefore, we investigated potential factors that might be required for the activation and proliferation of human CD28– memory CD8 T cells in vitro. Since interleukin-15 (IL-15) is required for the growth and survival of memory CD8 T cells, as well as natural killer (NK) cells, we tested the effect of IL-15 on the proliferation of CD28– memory CD8 T cells in vitro. (Figure 1.)

Figure 1. Peripheral blood mononuclear cells (PBMC) were prepared from three patients and levels of alloreactivity were determined by a modification of the Panel of Reactive T Cell assay. The test PBMC were cultured with or without a mixture of three allogeneic B cell lines with or without the addition of IL-15 to detect numbers of alloreactive T cells producing interferon-gamma. Addition of IL-15 enhances the accuracy in assessing levels of alloreactivity by exposing the activation of alloreactive memory CD8 T cells that do not express CD28 (CD28– memory CD8 T cells).

Proliferation differences and insights

Our recently published results confirmed that CD28– memory CD8 T cells do not proliferate during culture with allogeneic cells whereas CD28+ memory CD8 T cells proliferate robustly in response to this stimulation.

However, addition of IL-15 to cultures with allogeneic cells does induce proliferation of the CD28– memory CD8 T cells and this activation does not occur without the alloantigen stimulation. Addition of other known T cell proliferative factors IL-2 or IL-7 did not provoke the proliferation of the CD28– memory CD8 T cells.

Importantly, the CD28– memory CD8 T cells activated in cultures with allogeneic cells plus IL-15 produce IFN-γ and express CD107a, a marker indicating granule exocytosis that is associated with cytolytic activity. Together these results indicate the ability of an inflammatory mediator that is likely to be present in allografts to activate this CD28– population of memory CD8 T cells.

On the basis of these results, we hypothesize that these properties can be used to more accurately assess the presence of alloreactive CD28– memory CD8 T cells and the frequency of alloreactive memory CD8 T cells in the peripheral blood of renal transplant patients, using a modification of the Panel of Reactive T Cell (PRT) assay.

Our initial results have indicated that addition of IL-15 to the PRT assay increases the number of alloreactive spots observed 2- to 5-fold. Current studies are directed at testing if the ability of the IL-15 to more accurately assess the level of alloreactivity provides a better indication of which renal transplant patients are more likely to have acute rejection of their graft.

Future research directions

We anticipate that these and future experiments will provide more extensive evidence regarding the function of alloreactive CD28– memory CD8 T cells, and a better assessment of their presence in transplant patients and of the impact of rejection risk.

These studies will provide novel, improved approaches for detecting these memory CD8 T cells in patients prior to transplantation and during post-transplant monitoring to better assess risk of alloimmune-mediated acute and chronic graft injury.

Dr. Fairchild is a staff member of Cleveland Clinic’s Transplantation Center and the departments of Urology and Immunology. He can be reached at fairchr@ccf.org or 216.444.3146.