Nick D. Jones

T-cell activation, proliferation, and memory after cardiac transplantation in vivo.

OBJECTIVEnTo study the response of alloantigen (H2Kb)-specific T cells to a H2b+ cardiac allograft in vivo.nnnSUMMARY BACKGROUND DATAnThe response of T cells to alloantigen has been well characterized in vitro but has proved more difficult to assess in vivo. The aim of these experiments was to develop a model of T-cell-mediated rejection where the response of T cells after transplantation of a cardiac allograft could be followed in vivo.nnnMETHODSnPurified CD8+ T cells from H2Kb-specific TCR transgenic mice (BM3; H2k) were adoptively transferred into thymectomized, T-cell-depleted CBA/Ca (H2k) mice. These mice were then transplanted with a H2Kb+ cardiac allograft. Using four-color flow cytometry, the proliferative response, modulation of activation markers, and potential cytokine production of the H2Kb-specific T cells was assessed after transplantation.nnnRESULTSnConsistent rejection of H2Kb+ cardiac allografts required the transfer of at least 6 x 10(6) CD8+ H2Kb-specific T cells. Short-term analyses revealed that the transgenic-TCR+/ CD8+ T cells proliferated and became activated after transplantation of an H2Kb+ cardiac allograft. Fifty days after transplantation, the transgenic-TCR+/CD8+ T cells remained readily detectable, bore a predominantly memory phenotype (CD44hi), and rapidly produced interleukin 2 and interferon-gamma on in vitro restimulation.nnnCONCLUSIONSnThese data show that the activation of alloantigen-specific T cells can be followed in vivo in short-term and long-term experiments, thereby providing a unique opportunity to study the mechanisms by which T cells respond to allografts in vivo.

Visualization of the in vivo generation of donor antigen-specific effector CD8+ T cells during mouse cardiac allograft rejection: in vivo effector CD8+ T cell generation during allograft rejection.

BACKGROUNDnAn adoptive transfer system was used to study the fate of alloreactive CD8+ H-2Kb-specific TCR transgenic (DES+) T cells in vivo after transplantation.nnnMETHODSnA trace population of 2.0x10(6) CD8+DES+ T cells were adoptively transferred into syngeneic CBA.Ca (H-2k) mice 24 hr before transplantation of an H-2Kb+ or H-2Kb- cardiac allograft.nnnRESULTSnH-2Kb specific T cells proliferated and produced interleukin-2 and interferon-gamma in response to H-2Kb+, but not H-2Kb- cardiac allografts. CD8+DES+ T cells that infiltrated the H-2Kb+ cardiac allografts developed a distinct cell surface and cytokine phenotype compared with the CD8+DES+ T cells that remained in the periphery. H-2Kb-specific graft infiltrating T cells (a) underwent a larger number of cell divisions (> =3), (b) increased in size, (c) up-regulated CD69, and (d) down-regulated CD62L.nnnCONCLUSIONSnThese results demonstrate that alloantigen-specific T cells can be monitored in vivo during the immune response to an allograft and that the fate of CD8+ T cells specific for the allogeneic class I molecules expressed by the graft is different between cells in the periphery and those that infiltrate the graft.

Memory T cells: How might they disrupt the induction of tolerance?

Clinical and experimental evidences suggest that alloreactive memory T cells may be part of the normal T-cell repertoire and that such cells are detrimental to the survival of foreign organ allografts induced by the administration of conventional immunosuppression or experimental tolerance-inducing therapies. The potential mechanisms by which alloreactive memory T cell may form a barrier to the induction of tolerance will be discussed.

A Common Biomarker Signature for Tolerated Allografts and Self Tissues

For obvious reasons, there is an urgent need to define reliable biomarkers of transplantation tolerance that will allow better tailoring of immunosuppression and a safer withdrawal of immunosuppressive drugs. Simultaneously, this will make clinical trials of tolerance induction easier to perform and promote. However, to date there is no reliable means to discriminate between the absence of rejection due to the effects of conventional immunosuppression and the absence of rejection due to the successful induction of an active process of tolerance. Consequently, most clinicians administer a classical immunosuppressive protocol to their patients or prefer to include patients in clinical trials driven by pharmaceutical companies. n nThe problem is not easy. We already know from basic research performed with peripheral blood mononuclear cells (PBMCs) from the rare patients that are “by chance” tolerant to a kidney allograft or the more frequent tolerant liver transplant recipients, that putative biomarkers of operational tolerance (that remain to be validated in large prospective studies) are distinct for each organ (Martinez-Llordella et al., 2008; Sagoo et al., 2010; Sanchez-Fueyo and Strom, 2011). This suggests that the underlying mechanisms are distinct too. In experimental transplantation models (small and large animals), the number of cells and the mechanisms involved in tolerance are numerous and are increasing (regulatory T cells, mast cells, myeloid derived suppressor cells, immune privilege, enzyme activity such as iNOS, arginase, heme oxygenase-1, NO, etc…; Waldmann, 2010). If the success of experimental protocols of tolerance induction or its maintenance depends on those mechanisms, as demonstrated by specific models, they do not represent a biomarker as such. This is well illustrated by the example that regulatory T cells have been found to be present in tolerated but also rejected allografts. Beyond the scope of alloreactivity, the mechanisms implicated in tolerance to self antigens are still incompletely understood. Therefore, it looks tricky to define a promising strategy for identifying universal biomarkers of tolerance. Besides, the potential effects of immunosuppressive drugs on those tolerogenic mechanisms further complicate the problem. n nIn the present issue, Cobbold et al. (2011)chose an elegant and sophisticated experimental alternative to overwhelm these difficulties (ref). They compared gene profile expression in draining lymph nodes, spleen, and transplanted tissue of three apparently different immunological situations: skin allograft tolerance, skin allograft rejection, and syngeneic transplants. To ensure that observations were robust and unbiased, the allogeneic situation included transplantation across multiple minor antigen disparities, minor plus major (full mismatch) or in the presence of graft-reactive, monospecific TCR transgenic T cells. This confirmed that multiple innate and adaptive mechanisms are recruited in reliable forms of tolerance and operate within the graft, not systematically, strongly suggesting that fishing for surrogate markers of tolerance in patients should be performed with grafted tissue (or perhaps by-products) rather than PBMCs. n nAt least, two other important messages are contained in this manuscript. First, foxp3 mRNA is definitely not a biomarker of tolerance as such, and the fate of allograft depends on the balance between “regulatory-associated” genes and effector genes. The second important message is that tolerated allografts behave like syngeneic transplants. So far, this gold standard syngeneic control is of course missing in clinical transplantation research although this might provide some keys in the understanding of mechanisms involved in transplantation tolerance. Perhaps, further clinical research addressing specific biomarkers of tolerance should include experimental equivalent of the syngeneic transplants reported here as suggested by this nice study.