C-kit-derived CD11b+ cells are critical for cardiac allograft prolongation by autologous C-kit+ progenitor cells.

Autologous C-kit+ cells robustly prolong cardiac allografts. As C-kit+ cells can transdifferentiate to hematopoietic cells as well as non-hematopoietic cells, we aimed to clarify the class(es) of C-kit-derived cell(s) required for cardiac allograft prolongation. Autologous C-kit+ cells were administered post-cardiac transplantation and allografts were evaluated for C-kit+ inoculum-derived cells. Results suggested that alloimmunity was a major signal for trafficking of C-kit-derived cells to the allograft and demonstrated that C-kit+ inoculum-derived cells expressed CD11b early after transfer. Allograft survival studies with CD11b-DTR C-kit+ cells demonstrated a requirement for C-kit+-derived CD11b+ cells. Co-therapy studies demonstrated near complete abrogation of acute rejection with concomitant CTLA4-Ig therapy and no loss of prolongation in combination with Cyclosporine A. These results strongly implicate a C-kit-derived myeloid population as critical for allograft preservation and demonstrate the potential therapeutic application of autologous C-kit+ progenitor cells as calcineurin inhibitor-sparing agents and possibly as co-therapeutics for durable graft survival.

Disruption of Transplant Tolerance by an "Incognito" Form of CD8 T Cell-Dependent Memory.

Several approaches successfully achieve allograft tolerance in preclinical models but are challenging to translate into clinical practice. Many clinically relevant factors can attenuate allograft tolerance induction, including intrinsic genetic resistance, peritransplant infection, inflammation, and preexisting antidonor immunity. The prevailing view for immune memory as a tolerance barrier is that the host harbors memory cells that spontaneously cross-react to donor MHC antigens. Such preexisting "heterologous" memory cells have direct reactivity to donor cells and resist most tolerance regimens. In this study, we developed a model system to determine if an alternative form of immune memory could also block tolerance. We posited that host memory T cells could potentially respond to donor-derived non-MHC antigens, such as latent viral antigens or autoantigens, to which the host is immune. Results show that immunity to a model nonself antigen, ovalbumin (OVA), can dramatically disrupt tolerance despite undetectable initial reactivity to donor MHC antigens. Importantly, this blockade of tolerance was CD8+ T cell-dependent and required linked antigen presentation of alloantigens with the test OVA antigen. As such, this pathway represents an unapparent, or "incognito," form of immunity that is sufficient to prevent tolerance and that can be an unforeseen additional immune barrier to clinical transplant tolerance.

What's Hot, What's New From the 2016 American Transplant Congress.

From June 11-15, 2016 the American Transplant Congress, the joint meeting of the American Society of Transplantation and the American Society of Transplant Surgeons, held its annual meeting in Boston, MA. The meeting, attended by 5200 registrants, included pre-meeting conferences, focused topic sessions, and hundreds of high-quality presentations from the transplant field. This meeting report highlights key findings from specific basic science, translational, and clinical research presentations deemed to have notable impact in thematic areas. In particular, there were a number of transformative studies indicating important advances in the understanding of alloimmunity, chronic rejection, tolerance, and organ-specific outcomes. Many of these results are discussed in the context of the published literature to showcase rapid advances in the transplant field.

Interferon Gamma and Contact-dependent Cytotoxicity Are Each Rate Limiting for Natural Killer Cell-Mediated Antibody-dependent Chronic Rejection.

Natural killer (NK) cells are key components of the innate immune system. In murine cardiac transplant models, donor-specific antibodies (DSA), in concert with NK cells, are sufficient to inflict chronic allograft vasculopathy independently of T and B cells. In this study, we aimed to determine the effector mechanism(s) required by NK cells to trigger chronic allograft vasculopathy during antibody-mediated rejection. Specifically, we tested the relative contribution of the proinflammatory cytokine interferon gamma (IFN-γ) versus the contact-dependent cytotoxic mediators of perforin and the CD95/CD95L (Fas/Fas ligand [FasL]) pathway for triggering these lesions. C3H/HeJ cardiac allografts were transplanted into immune-deficient C57BL/6 rag-/- γc-/- recipients, who also received monoclonal anti-major histocompatibility complex (MHC) class I DSA. The combination of DSA and wild-type NK cell transfer triggered aggressive chronic allograft vasculopathy. However, transfer of IFN-γ-deficient NK cells or host IFN-γ neutralization led to amelioration of these lesions. Use of either perforin-deficient NK cells or CD95 (Fas)-deficient donors alone did not alter development of vasculopathy, but simultaneous disruption of NK cell-derived perforin and allograft Fas expression resulted in prevention of these abnormalities. Therefore, both NK cell IFN-γ production and contact-dependent cytotoxic activity are rate-limiting effector pathways that contribute to this form of antibody-induced chronic allograft vasculopathy.

Prolongation of cardiac allograft survival by a novel population of autologous CD117+ bone marrow-derived progenitor cells.

Autologous CD117(+) progenitor cells (PC) have been successfully utilized in myocardial infarction and ischemic injury, potentially through the replacement/repair of damaged vascular endothelium. To date, such cells have not been used to enhance solid organ transplant outcome. In this study, we determined whether autologous bone marrow-derived CD117(+) PC could benefit cardiac allograft survival, possibly by replacing donor vascular cells. Autologous, positively selected CD117(+) PC were administered posttransplantation and allografts were assessed for acute rejection. Although significant generation of recipient vascular cell chimerism was not observed, transferred PC disseminated both to the allograft and to peripheral lymphoid tissues and facilitated a significant, dose-dependent prolongation of allograft survival. While CD117(+) PC dramatically inhibited alloreactive T cell proliferation in vitro, this property did not differ from nonprotective CD117(-) bone marrow populations. In vivo, CD117(+) PC did not significantly inhibit T cell alloreactivity or increase peripheral regulatory T cell numbers. Thus, rather than inhibiting adaptive immunity to the allograft, CD117(+) PC may play a cytoprotective role in prolonging graft survival. Importantly, autologous CD117(+) PC appear to be distinct from bone marrow-derived mesenchymal stem cells (MSC) previously used to prolong allograft survival. As such, autologous CD117(+) PC represent a novel cellular therapy for promoting allograft survival.

"Indirect" acute islet allograft destruction in nonobese diabetic mice is independent of donor major histocompatibility complex and requires host B lymphocytes.

Islet allografts are destroyed rapidly in spontaneously diabetic nonobese diabetic (NOD) mice. However, whether this process is more similar to conventional allograft immunity, islet-specific autoimmune pathogenesis, or both remains controversial. In particular, we sought to determine whether C57BI/6 donor islet major histocompatibility complex (MHC) class I or class II expression was required for islet allograft destruction in autoimmune prone NOD mice versus non-autoimmune-prone BALB/c mice. Results show that islet allografts deficient in both MHC I and II are uniformly accepted in BALB/c mice. In sharp contrast, such MHC-deficient allografts were destroyed acutely in spontaneously diabetic NOD mice. Such donor MHC-independent rejection implicates "indirect" (host MHC-restricted) immunity as a pathway responsible for islet injury. To determine whether host NOD B lymphocytes could contribute to indirect graft recognition, wild-type and MHC I/II-deficient allografts were grafted into B-lymphocyte-deficient (microMT) NOD mice. Whereas wild-type NOD mice could reject MHC-I/II-deficient islet allografts, such grafts were all accepted in B-lymphocyte-deficient NOD mice. Taken together, these results indicate that NOD mice are capable of vigorous donor MHC-independent islet allograft rejection not found in non-autoimmune-prone recipients. Importantly, B lymphocytes may play a key role as antigen-presenting cells in this exuberant host 'indirect' response found in NOD mice.

Both innate and adaptive major histocompatibility complex class I-dependent immunity impair long-term islet xenograft survival.

Natural killer (NK) cells have long been appreciated for their rapid, proinflammatory contribution to host defense. However, more recent studies show an unexpected regulatory role for host major histocompatibility complex (MHC) class I-dependent immunity and NK cells in promoting tolerance induction to islet allografts. It is unclear whether the potential tolerance induction to islet xenografts follows similar requirements to those found in allograft tolerance. In this study, we determined whether induced islet xenograft prolongation also showed a reliance on MHC class I-dependent immune pathways. In particular, we tested whether NK1.1+ cells and/or CD8 T cells were required for the long-term islet xenograft survival in a rat-to-mouse transplant model. Short-term host treatment with combined anti-CD154 plus anti-LFA-1 (CD11a) resulted in prolonged, but not indefinite, survival of WF rat islet xenografts in C57BI/6 mouse recipients. In stark contrast with similar islet allograft studies, adjunct treatment with anti-NK1.1 therapy combined wither anti-CD154/anti-LFA-1 treatment led to long-term (>100 days) survival of the majority of islet xenografts. In parallel studies, we determined whether CD8 T cells also contributed a barrier to xenograft survival. Similar to results found in anti-NK1.1-treated animals, CD8-deficient (knockout) recipients also demonstrated augmented xenograft prolongation after combined anti-CD154/anti-LFA-1 therapy. Taken together, NK1.1+ cells (NK/NKT cells) and CD8 T cells constitute differing MHC class I-dependent immune pathways forming a significant barrier to xenograft prolongation.

CD4 T cell-mediated cardiac allograft rejection requires donor but not host MHC class II.

Numerous studies indicate that CD4 T cells are required for acute cardiac allograft rejection. However, the precise role for CD4 T cells in this response has remained ambiguous owing to the multipotential properties of this T-cell subpopulation. In the current study, we demonstrate the capacity of CD4 T cells to serve as direct effector cells of cardiac allograft rejection. We show that CD4 T cells are both necessary and sufficient for acute graft rejection, as indicated by adoptive transfer experiments in immune-deficient SCID and rag1(-/-) recipients. We have analyzed the contribution of direct (donor MHC class II restricted) and indirect (host MHC class II restricted) antigen recognition in CD4-mediated rejection. Acute CD4 T cell-mediated rejection required MHC class II expression by the allograft, indicating the importance of direct graft recognition. In contrast, reciprocal experiments indicate that CD4 T cells can acutely reject allogeneic cardiac allografts established in rag1(-/-) hosts that were also MHC class II deficient. This latter result indicates that indirect presentation of donor antigens by host MHC class II is not required for acute CD4-mediated rejection. Taken together, these results indicate that CD4 T cells can serve as effector cells for primary acute cardiac allograft rejection, predominantly via direct donor antigen recognition and independent of indirect reactivity.

T-cell-T-cell collaboration in allograft responses.

T-cell-T-cell collaboration in allogeneic responses traditionally has been viewed as the requirement for CD4+ T helper cells in the activation of CD8+ cytotoxic T cells. In this regard, the role of the CD4+ T cell is primarily to provide growth factors, such as interleukin-2, on which the CD8+ T cell is dependent. However, expanding information concerning the function of T-cell subsets, and the roles of antigen-presenting cells and cytokines in regulating immune responses, requires that the basic tenets of T-cell interactions be re-evaluated.

Initial results of screening of nondiabetic organ donors for expression of islet autoantibodies.

CONTEXT: Type 1A diabetes is characterized by a long prodromal phase during which autoantibodies to islet antigens are present. Nevertheless, we lack data on the pancreatic pathology of subjects who are positive for islet autoantibodies (to islet autoantigens GAD65, insulin, and ICA512). OBJECTIVE: In this manuscript, we describe a novel strategy in obtaining pancreata and pancreatic lymph nodes from islet autoantibody-positive organ donors that involves careful coordination among the laboratory and the organ donor provider organization. DESIGN: We developed a rapid screening protocol for islet autoantibodies measurement of organ donors to allow identification of positive subjects before organ harvesting. In this way we were able to obtain pancreata and pancreatic lymph nodes from subjects with and without islet autoimmunity. SETTING: The organ donors used in this study were obtained from the general community. SUBJECTS: The population studied consisted of 112 organ donors (age range 1 month to 86 yr, mean age 39 yr). MAIN OUTCOME MEASURE: The main outcome measure of this study consisted of evaluating the pancreatic histology and identify T cells autoreactive for islet antigens in the pancreatic lymph nodes. RESULTS: To date we have identified three positive subjects and obtained the pancreas for histological evaluation from one of the autoantibody-positive donors who expressed ICA512 autoantibodies. Although this subject did not exhibit insulitis, lymphocytes derived from pancreatic lymph nodes reacted to the islet antigen phogrin. CONCLUSION: In summary, these results indicate that it is possible to screen organ donors in real time for antiislet antibodies, characterize pancreatic histology, and obtain viable T cells for immunological studies.

Autoimmune diabetes in NOD mouse is L3T4 T-lymphocyte dependent.

Cultured BALB/c islets fail to function when transplanted into diabetic nonobese diabetic (NOD) mice; such grafted tissue is rapidly destroyed by disease recurrence. The cellular requirements for this graft damage are unclear. This study was designed to investigate the role of the L3T4+ T-lymphocyte subset in disease recurrence in the NOD mouse. L3T4+ T-lymphocytes were depleted by the in vivo administration of the L3T4-specific monoclonal antibody GK1.5. This treatment reduced the level of L3T4+ T-lymphocytes from an initial 43% of the peripheral blood lymphocytes to less than 4%. L3T4 levels remained at this low level for approximately 2 wk after withdrawal of GK1.5 treatment, after which the L3T4 levels slowly began to increase in the periphery. Grafting of cultured BALB/c islet tissue into GK1.5-treated diabetic NOD mice resulted in a rapid return to normoglycemia that persisted for 2-4 wk. The gradual return to the hyperglycemic condition roughly correlated with the reappearance of L3T4+ T-lymphocytes in the peripheral circulation. From these findings we conclude that the disease process in the NOD mouse is L3T4 T-lymphocyte dependent.

Cellular immune response to phogrin in the NOD mouse: cloned T-cells cause destruction of islet transplants.

The ability of nonobese diabetic (NOD) mice to mount a cellular immune response to the secretory granule protein tyrosine phosphatase (PTP), phogrin was evaluated by immunization of 8- to 12-week-old animals with recombinant phogrin in complete Freund's adjuvant. Draining lymph nodes displayed a robust proliferative response to the protein, as did derived T-cell lines and clones. Ten clones obtained by limiting dilution were all CD4+ and of a T-helper-1-like phenotype, but showed variation in their Vbeta usage. Of the 10 clones, 3 responded to endogenous antigens in rat islets. Two of these caused the destruction of rat islets that had been transplanted under the kidney capsule of streptozotocin-treated NOD scid mice without affecting adjacent thyroid implants. The results demonstrate the feasibility of generating antigen-specific diabetes-inducing CD4+ cells by direct immunization of NOD mice and their potential use for further studies of the antigenic epitopes in the PTP family members. The conclusion, based on serological studies, that PTP members do not play a role in the pathogenesis of type 1 diabetes in rodent models needs reevaluation in light of these findings.

The basis of immunogenicity of endocrine allografts.

Two signals are required for optimal T-cell activation: the engagement of the antigen-specific receptor and the provision of a second non-antigen-specific inductive signal, or costimulator (CoS). Regarding allograft immunity, two primary pathways of donor antigen presentation can fulfill this two-signal requirement, resulting in cellular immunity to a transplant: (1) "direct" (donor MHC-restricted) presentation in which the antigen-presenting cells (APCs) resident within the transplant directly activate host T lymphocytes and (2) "indirect" (host MHC-restricted) presentation in which host-derived APCs acquire donor antigens that are then presented to host T lymphocytes. It appears that endocrine allografts, such as pancreatic islets and thyroid, are highly dependent on donor-derived APCs, or "passenger leukocytes," to trigger acute graft rejection. Tissue pretreatment aimed at selectively eliminating APCs within endocrine tissues can result in indefinite allograft survival in immune-competent recipients. Although such results implicate the "direct" pathway as the predominant route of host sensitization, the role of donor APCs in rejection appears to be more complex. Recently, we have found that indirect, CD4 T-cell-dependent reactivity can contribute to islet allograft rejection. However, such indirect recognition nevertheless requires donor-derived APCs as a source of antigen. Thus, whereas the donor-type APC is a critical limiting step for initiating islet allograft rejection, such cells can trigger both direct and indirect forms of immune responses that can result in graft rejection. That is, donor hematopoietic cells, rather than tissue parenchymal cells, probably play a major role in providing antigens that stimulate cellular immunity.

Tolerance induction to cultured islet allografts. I. Characterization of the tolerant state.

The immunogenicity of murine pancreatic islets can be reduced by culture in 95% O2 prior to transplantation. Such cultured tissue can reverse diabetes indefinitely in nonimmunosuppressed, allogeneic recipients. Although the cultured graft does not trigger a rejection response, the graft retains recognizable alloantigens in that the graft is acutely rejected when the host is immunized with donor-type antigen-presenting cells. However, over time the recipients bearing cultured islet allografts become increasingly resistant to rejecting the established graft following APC challenge. Data show that this process of graft "stabilization" is a function of time postgrafting and initial graft mass. Graft stabilization is not due to a change in the vulnerability of the graft to immune recognition--that is, stabilization cannot be accounted for by the spontaneous adaptation of the long-term graft. Rather, graft stabilization is associated with a change in host reactivity (tolerance induction). This conclusion is based on the findings that (1) recipients of long-term established grafts (> 120 days) resist rejection of both the primary and secondary donor-type grafts, and (2) donor-specific tolerance can be transferred to severe-combined-immune-deficient (scid) recipient mice.

Tolerance induction to cultured islet allografts. II. The status of antidonor reactivity in tolerant animals.

Murine islet tissue cultured in 95% O2 to eliminate/inactivate donor antigen-presenting cells can function indefinitely and induce a state of tolerance in nonimmunosuppressed, allogeneic recipients. Such cultured grafts represent a model of antigen presentation in which antigen (signal 1) is presented without the delivery of appropriate costimulatory activity (signal 2) necessary for T cell activation. As T cell inactivation has been proposed to result from this form of antigen presentation, we determined whether the tolerance generated in response to such cultured grafts was due to a passive (clonal deletion/inactivation) mechanism. We have shown that, although tolerant in vivo, animals bearing long-term cultured islet allografts are donor-reactive in vitro as assessed by (1) CTL precursor frequency, (2) antidonor proliferative and cytotoxic responses, and (3) lymphokine production (IL-2, IL-3, TNF, and IFN-gamma). In addition, tolerance does not appear to be tissue (islet)-specific in that primed, donor-reactive T cells from tolerant animals react to islet cells in vitro and are capable of destroying donor-type islet grafts in vivo. Thus, the notion that "signal 1" antigen presentation, as represented by cultured islet allografts, leads to the clonal deletion or inactivation (anergy) of donor-reactive T cells is not supported by these results. Since this form of tolerance does not appear to be an intrinsic property of the donor-specific lymphocyte, these results are more consistent with a model of active regulatory tolerance in vivo.

Donor antigen-presenting cell-independent rejection of islet xenografts.

Donor-derived antigen-presenting cells (APC) are thought to serve as major stimulators for triggering the rejection of tissue allografts. However, the capacity of APC to stimulate xenogeneic T cells is generally deficient relative to the corresponding response from allogeneic T cells. For this reason, the contribution of donor-type APC to xenogeneic graft rejection remains unclear. Using a concordant species combination (rat to mouse), we examined the requirement for donor-type APC in triggering islet xenograft rejection. While the depletion of donor-type APC resulted in indefinite allograft survival, similar depletion of APC from xenogeneic rat islets resulted in only modest graft prolongation. Furthermore, APC-depleted rat xenografts were rejected by a CD8+ T cell-independent mechanism, as determined by appropriate depletion of T cell subsets through monoclonal antibody therapy. This contrasts with the dependence of islet allograft rejection on both CD4+ and CD8+ T cells. Although in vitro experiments show that rat APC can directly stimulate mouse T cells, rat APC do not appear to be required for xenograft immunity in vivo. We conclude that the mechanisms of islet allograft and xenograft rejection differ both in the dependence on donor-type APC and in the role of T cell subsets in the response.

Evidence of recurrent autoimmunity in human allogeneic islet transplantation.

We transplanted 10,000 isolated, handpicked human pancreatic islets into the subfascial compartment of the forearm muscle of a type I diabetic recipient who had received a successful renal transplant one year prior. The recipient was maintained on his usual immunosuppressive therapy of cyclosporine, azathioprine, and prednisone. A biopsy performed 7 days after transplantation showed normal islets with both insulin- and glucagon-staining cells present and no lymphocytic infiltration. A second biopsy performed 14 days after transplantation showed a dense mononuclear cell infiltrate with a preferential loss of insulin-staining cells relative to glucagon-staining cells in the islets. These data are consistent with recurrent autoimmune diabetes in an isolated islet allograft in an immunosuppressed type I diabetic recipient. In addition, this forearm subfascial site may be a useful means to monitor islet rejection and autoimmune recurrence in therapeutic intraportal islet allografts.

Facilitation of specific tolerance induction in adult mice by RS-61443.

RS-61443 is an immunosuppressive agent that facilitates pancreatic islet allograft acceptance in two mouse strain combinations (BALB/c----CBA and C57Bl/6J----BALB/c). A remarkable feature of this agent is its ability to facilitate long-term graft acceptance after a short (30 days) period of treatment; following withdrawal of the agent 40-70% of islet allografts are maintained for an indefinite period. This long-term graft acceptance has been shown to result from specific tolerance induction in the recipient animal. The state of specific tolerance is an active rather than a passive form of tolerance, such as clonal deletion or anergy. Active tolerance induction is cyclosporine-sensitive, although cyclosporine enhances graft acceptance when used in combination therapy with RS-61443, this agent inhibits tolerance development under the influence of RS-61443.