Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation.

Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were implicated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting properties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic alloresponses refractory to current immunosuppression.

Immunology in the Clinic Review Series; focus on host responses: invariant natural killer T cell activation following transplantation.

Invariant natural killer T (iNKT) cells have been shown to play a key role in the regulation of immunity in health and disease. However, iNKT cell responses have also been found to influence both rejection and the induction of tolerance following transplantation of allogeneic cells or organs. Although a number of mechanisms have been identified that lead to iNKT cell activation, how iNKT cells are activated following transplantation remains unknown. This review will attempt to identify potential mechanisms of iNKT cell activation in the context of transplantation by applying knowledge garnered from other disease situations. Furthermore, we put forward a novel mechanism of iNKT cell activation which we believe may be the dominant mechanism responsible for iNKT activation in this setting, i.e. bystander activation by interleukin-2 secreted by recently activated conventional T cells.

Bystander activation of iNKT cells occurs during conventional T-cell alloresponses.

It is well established that iNKT cells can be activated by both exogenous and a limited number of endogenous glycolipids. However, although iNKT cells have been implicated in the immune response to transplanted organs, the mechanisms by which iNKT cells are activated in this context remain unknown. Here we demonstrate that iNKT cells are not activated by allogeneic cells per se, but expand, both in vitro and in vivo, in the presence of a concomitant conventional T-cell response to alloantigen. This form of iNKT activation was found to occur independently of TCR-glycolipid/CD1d interactions but rather was a result of sequestration of IL-2 produced by conventional alloreactive T cells. These results show for the first time that IL-2, produced by activated conventional T cells, can activate iNKT cells independently of glycolipid/CD1d recognition. Therefore, we propose that the well-documented involvement of iNKT cells in autoimmunity, the control of cancer as well as following transplantation need not involve recognition of endogenous or exogenous glycolipids but alternatively may be a consequence of specific adaptive immune responses.

Lack of complex I is associated with oncocytic thyroid tumours.

Oncocytic tumours are characterised by hyperproliferation of mitochondria. We immunohistochemically analysed all enzymes of the oxidative phosphorylation system in 19 oncocytic thyroid tumours. A specific lack of complex I was detected, which was expressed at <5% of the level determined in surrounding non-cancerous tissue.

Alloantigen-induced specific immunological unresponsiveness.

When the immune system encounters alloantigen it can respond in any one of a number of different ways. The choice that is made will take into account factors such as where, when and how the contact with the alloantigen takes place, as well as the environmental conditions that prevail at the time the alloantigen is encountered. Alloantigen administration before transplantation either alone or in combination with therapeutic agents that modulate the functional activity of the responding leucocytes can be a powerful way of inducing specific unresponsiveness to alloantigens in vivo. The molecular mechanisms that influence the way the outcome of the immune response to alloantigen develops, either activation or unresponsiveness to the triggering antigen, hold the key to our ability to manipulate the immune system effectively by exposing it to donor antigen for therapeutic purposes. This review will focus on alloantigen-induced immunological unresponsiveness and how insights into the mechanisms of unresponsiveness have driven the development of novel tolerance-induction strategies that show promise for translation into the clinic in the future.

Differential susceptibility of heart, skin, and islet allografts to T cell-mediated rejection.

Although it is widely accepted that there is a hierarchy in the susceptibility of different allografts to rejection, the mechanisms responsible are unknown. We show that the increased susceptibility of H-2K(b+) skin and islet allografts to rejection is not based on their ability to activate more H-2K(b)-specific T cells in vivo; heart allografts stimulate the activation and proliferation of many more H-2K(b)-specific T cells than either skin or islet allografts. Rejection of all three types of graft generate memory cells by 25 days posttransplant. These data provide evidence that neither tissue-specific Ags nor, surprisingly, the number of APCs carried in the graft dictate their susceptibility to T cell-mediated rejection and suggest that the graft microenvironment and size may play a more important role in determining the susceptibility of an allograft to rejection and resistance to tolerance induction.

Cell science and protein crystal growth research for the International Space Station.

The recent National Research Council report, Future Biotechnology Research on the International Space Station, evaluates NASA's plans for research in cell science and protein crystal growth to be conducted on the International Space Station. This report concludes that the NASA biotechnology programs have the potential to significantly impact relevant scientific fields and to increase understanding and insight into fundamental biological issues. In order to realize the potential impacts, NASA must focus its research programs by selecting specific questions related to gravitational forces' role in cell behavior and by using the microgravity environment as a tool to determine the structure of macromolecules with important biological implications. Given the time and volume constraints associated with space-based experiments, instrumentation to be used on the space station must be designed to maximize the productivity of researchers, and NASA's recruitment of investigators and support for space station experiments should aim to encourage and facilitate cutting-edge research.

CD40-CD40 ligand-independent activation of CD8+ T cells can trigger allograft rejection.

In experimental transplantation, blockade of CD40-CD40 ligand (CD40L) interactions has proved effective at permitting long-term graft survival and has recently been approved for clinical evaluation. We show that CD4+ T cell-mediated rejection is prevented by anti-CD40L mAb therapy but that CD8+ T cells remain fully functional. Furthermore, blocking CD40L interactions has no effect on CD8+ T cell activation, proliferation, differentiation, homing to the target allograft, or cytokine production. We conclude that CD40L is not an important costimulatory molecule for CD8+ T cell activation and that following transplantation donor APC can activate recipient CD8+ T cells directly without first being primed by CD4+ T cells.

Evidence that non-deletional mechanisms are responsible for inducing and maintaining unresponsiveness after intrathymic injection of non-professional antigen presenting cells.

INTRODUCTION: Intrathymic inoculation of donor alloantigen and concomitant immunosuppressive treatment can induce immune unresponsiveness to alloantigen. To examine the role of non-deletional mechanisms in the development of unresponsiveness, fractionated splenocytes were injected into only 1 lobe of the thymus. METHODS AND RESULTS: Untreated CBA (H2(k)) mice or controls pre-treated with anti-CD4 monoclonal antibody alone (on Day -28 and -27 relative to transplantation) acutely rejected C57BL/10 (H2(b)) cardiac allografts. Intrathymic inoculation of unfractionated splenocytes, resting B (rB) cells, or dendritic cells into both thymic lobes with the antibody resulted in indefinite survival of cardiac allografts. In contrast, when donor rB cells or dendritic cells were delivered into a single lobe of the thymus with the antibody, only rB cells induced indefinite prolongation of graft survival; unfractionated splenocytes or dendritic cells were markedly less effective. Mice that had 1 of the 2 thymic lobes removed were able to reject grafts even when treated with the antibody 27 days before transplantation. Therefore, T-cell export from 1 thymic lobe was sufficient to induce graft rejection. Finally, adoptive transfer of splenocytes from mice with long-term surviving primary grafts resulting from the intrathymic injection of rB cells significantly prolonged a graft from the same donor strain in a naive syngeneic recipient. CONCLUSION: Taken together, these data suggest that regulatory mechanisms generated by intrathymic injection of a non-professional antigen presenting cell, in this study donor rB cells, suppressed the rejection response mediated by T cells exported from the uninjected lobe.

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.

BACKGROUND: An adoptive transfer system was used to study the fate of alloreactive CD8+ H-2Kb-specific TCR transgenic (DES+) T cells in vivo after transplantation. METHODS: A 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. RESULTS: H-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. CONCLUSIONS: These 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.

The visualization of T cell responses.

Transplanting allogeneic grafts is still significantly hampered by the rejection process, despite the use of powerful immunosuppressive agents. The T cell is recognized as playing a central role in the process of rejection, and it is believed that graft tolerance will ultimately be achieved by immunological manipulation of this cell (1, 2). As immunologists strive to define the role of the T cell in the fundamental processes of immunity and tolerance, new methods are emerging that will facilitate visualization of the T cells directly involved in the rejection response (3, 4). This overview addresses the visualization of T cell responses as made possible by these technological developments.

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

OBJECTIVE: To study the response of alloantigen (H2Kb)-specific T cells to a H2b+ cardiac allograft in vivo. SUMMARY BACKGROUND DATA: The 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. METHODS: Purified 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. RESULTS: Consistent 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. CONCLUSIONS: These 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.

The induction of transplantation tolerance by intrathymic (i.t.) delivery of alloantigen: a critical relationship between i.t. deletion, thymic export of new T cells and the timing of transplantation.

Intrathymic (i.t.) injection of donor alloantigens has proved to be an effective strategy for the induction of tolerance. However, the mechanisms by which tolerance is induced and maintained after transplantation remain unclear. In this report we show that tolerance to donor cardiac allografts can be induced across a MHC class I difference by i.t. injection of donor splenocytes and transient T cell depletion. Furthermore, using H-2K(b)-specific TCR transgenic mice (BM3), we demonstrate that prolonged deletion of donor-reactive thymocytes was essential to induce tolerance by i.t. injection and this was dependent upon donor cells persisting in the thymus. Examination of the kinetics of thymic export following i.t. injection revealed that prolonged deletion of thymocytes was required to delay export of new T cells to the periphery until the time of transplantation. Importantly, after transplantation donor cell persistence in the thymus and i.t. deletion were no longer necessary to maintain tolerance. The graft itself or cells from the graft was responsible for maintaining tolerance at this stage. These findings reveal that multiple mechanisms are responsible for the induction and maintenance phases of tolerance to alloantigens in vivo after i.t. delivery, and that a complex inter-relationship between donor cell persistence in the thymus, i.t. deletion, thymic export of T cells and the timing of transplantation is involved.

Intrathymic administration of B cells induces prolonged survival of fully allogeneic cardiac grafts without prolonged deletion of donor-specific thymocytes.

Intrathymic (IT) injection of alloantigen has been shown to induce unresponsiveness to allografts although the exact mechanisms of tolerance induction remains unclear. C57BL/10 (H2b) cardiac allografts were accepted in C3H/He (H2k) mice pretreated with IT inoculation of donor splenocytes (1 x 10(6)) in combination with a depleting anti-CD4 monoclonal antibody 27 days before cardiac transplantation. To investigate which cell types were responsible for tolerance induction by IT injection of alloantigen, resting B (rB) cells or dendritic cells were used as the thymic inoculum instead of whole splenocytes. IT injection of rB cells induced indefinite graft prolongation in all recipients while only 20% of mice that had received IT injection of dendritic cells accepted grafts for over 100 days. In contrast, IT injection of dendritic cells resulted in significant deletion of donor-specific thymocytes whereas rB cells were relatively ineffective. IT deletion is not essential for the induction of tolerance by IT injection of rB cells; nondeletional mechanisms can be involved.

Potassium tetracyanoplatinate (II) trihydrate (K2Pt(CN)4 x 3H2O) studied by high resolution solid state 13C MAS NMR.

Prudent analysis of the solid state 13C MAS NMR spectra of polycrystalline K2Pt(CN)4 x 3H2O (KTCP)- reveals that in crystals of this compound there are two types of carbon nuclei with slightly different 13C chemical shift tensors, contrary to what is found for the solution NMR spectrum and previous static powder NMR studies on this compound and the high resolution solid state NMR studies on other similar compounds. The 13C MAS spectra measured at different rotor spinning speeds are satisfactorily simulated though the use of a newly developed computer program based on a novel density matrix formulation. The present method is eminently successful even though the spectra are rather complicated because of (1) the relatively large anisotropies of the chemical shift tensors; (2) the high-order dipolar interactions between 13C and 14N nuclei because of the strong quadrupolar coupling constants of 14N nuclei; and (3) the indirect J-coupling between the 13C and 195Pt. The principal elements as well as their orientations of the two 13C chemical shift tensors are evaluated from the spectral simulations.

Deletion of alloantigen-reactive thymocytes as a mechanism of adult tolerance induction following intrathymic antigen administration.

Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2b/BL10) alloantigens can be induced in CBA/Ca (H2k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4+ T cells using a depleting anti-CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2k) expressing a CD8-independent TCR specific for H2Kb were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 x 10(7) BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2Kb-specific deletion of transgenic-TCR+ (tg-TCR) thymocytes which was dependent on the level of tg-TCR expression. IT deletion and the failure to export CD8+ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2Kb; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2Kb+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8+ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR-transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2Kb following transplantation.