Amanda L. Williams

Cognate antigen directs CD8+ T cell migration to vascularized transplants

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Graft-infiltrating host dendritic cells play a key role in organ transplant rejection

Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection.

Memory T Cells Migrate to and Reject Vascularized Cardiac Allografts Independent of the Chemokine Receptor CXCR3

Background. Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here, we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T-cell migration to vascularized allografts. Methods. CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3−/− and wildtype B6 mice and cotransferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hr later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues. Results. We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype versus CXCR3−/− CD4 and CD8 memory T cells to allografts could be detected in wildtype or aly/aly hosts. In the latter, wildtype and CXCR3−/− memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a chemokine receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3−/− memory T cells. Conclusions. CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell-homing to transplanted organs may be distinct from those required for their migration to sites of infection.

Allograft Outcomes in Outbred Mice

Inbreeding depression and lack of genetic diversity in inbred mice could mask unappreciated causes of graft failure or remove barriers to tolerance induction. To test these possibilities, we performed heart transplantation between outbred or inbred mice. Unlike untreated inbred mice in which all allografts were rejected acutely (6–16 days posttransplantation), untreated outbred mice had heterogeneous outcomes, with grafts failing early (<4 days posttransplantation), acutely (6–24 days) or undergoing chronic rejection (>75 days). Blocking T cell costimulation induced long‐term graft acceptance in both inbred and outbred mice, but did not prevent the early graft failure observed in the latter. Further investigation of this early phenotype established that it is dependent on the donor, and not the recipient, being outbred and that it is characterized by hemorrhagic necrosis and neutrophilic vasculitis in the graft without preformed, high titer antidonor antibodies in the recipient. Complement or neutrophil depletion prevented early failure of outbred grafts, whereas transplanting CD73‐deficient inbred hearts, which are highly susceptible to ischemia‐reperfusion injury, recapitulated the early phenotype. Therefore, outbred mice could provide broader insight into donor and recipient determinants of allograft outcomes but their hybrid vigor and genetic diversity do not constitute a uniform barrier to tolerance induction.

Innate immunity alone is not sufficient for chronic rejection but predisposes healed allografts to T cell-mediated pathology.

BACKGROUND Acute allograft rejection is dependent on adaptive immunity, but it is unclear whether the same is true for chronic rejection. Here we asked whether innate immunity alone is sufficient for causing chronic rejection of mouse cardiac allografts. METHODS We transplanted primarily vascularized cardiac grafts to recombinase activating gene-knockout (RAG(-/-)) mice that lack T and B cells but have an intact innate immune system. Recipients were left unmanipulated, received adjuvants that stimulate innate immunity, or were reconstituted with B-1 lymphocytes to generate natural IgM antibodies. In a second model, we transplanted cardiac allografts to mice that lack secondary lymphoid tissues (splenectomized aly/aly recipients) and studied the effect of NK cell inactivation on T cell-mediated chronic rejection. RESULTS Acute cardiac allograft rejection was not observed in any of the recipients. Histological analysis of allografts harvested 50 to 90 days after transplantation to RAG(-/-) mice failed to identify chronic vascular or parenchymal changes beyond those observed in control syngeneic grafts. Chronic rejection of cardiac allografts parked in splenectomized aly/aly mice was observed only after the transfer of exogenously activated T cells. NK inactivation throughout the experiment, or during the parking period alone, reduced the severity of T cell-dependent chronic rejection. CONCLUSIONS The innate immune system alone is not sufficient for causing chronic rejection. NK cells predispose healed allografts to T cell-dependent chronic rejection and may contribute to chronic allograft pathology.

Type I Interferons Are Not Critical for Skin Allograft Rejection or the Generation of Donor‐Specific CD8+ Memory T Cells

Type I interferons (IFN‐I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN‐I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor‐specific CD8+ T‐cell responses in mice that lack the IFN‐I receptor (IFN‐IR−/−). We found that IFN‐IR−/− mice reject fully allogeneic wild‐type skin grafts at the same rate as wild‐type recipients. Similarly, allograft rejection was not delayed if IFN‐IR−/− male skin was transplanted to syngeneic IFN‐IR−/− female mice. Quantitation of the male (H‐Y)‐specific CD8+ T‐cell response in these mice revealed normal generation of donor‐specific CD8+ effector T cells but fourfold reduction in CD8+ memory T cells. Memory CD8+ T cells generated in the absence of IFN‐IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN‐IR−/− mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN‐1. Our findings indicate that IFN‐I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor‐specific CD8+ T cells into long‐lived, functional memory T cells.

Renal dendritic cells sample blood-borne antigen and guide T-cell migration to the kidney by means of intravascular processes

Bony fish are among the first vertebrates to possess an innate and adaptive immune system. In these species, the kidney has a dual function: filtering solutes similar to mammals and acting as a lymphoid organ responsible for hematopoiesis and antigen processing. Recent studies have shown that the mammalian kidney has an extensive network of mononuclear phagocytes, whose function is not fully understood. Here, we employed two-photon intravital microscopy of fluorescent reporter mice to demonstrate that renal dendritic cells encase the microvasculature in the cortex, extend dendrites into the peritubular capillaries, and sample the blood for antigen. We utilized a mouse model of systemic bacterial infection as well as immune complexes to demonstrate antigen uptake by renal dendritic cells. As a consequence, renal dendritic cells mediated T-cell migration into the kidney in an antigen-dependent manner in the setting of bacterial infection. Thus, renal dendritic cells may be uniquely positioned to play an important role not only in surveillance of systemic infection but also in local infection and autoimmunity.

Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts

Detection of nonself SIRPα by CD47 triggers an innate immune response to allografts in mice that lack T, B, and NK cells. Looking beyond MHCs in transplant rejection Mice engineered to lack T, B, and NK cells generate mature dendritic cells in response to allogeneic transplants. Precisely how these mice recognize allografts to be “nonself” has remained a mystery. Using an elegant positional cloning approach, Dai et al. have identified polymorphisms in the mouse gene encoding signal regulatory protein α (SIRPα) to be key in this innate self-nonself recognition. They show that SIRPα receptor CD47 binds SIRPα variants with distinct affinities and propose this affinity sensing to be the mechanism that triggers dendritic cell maturation, the first step in the initiation of the alloimmune response. Given that the SIRPα gene is also polymorphic in humans, it remains to be seen whether human SIRPα variations influence transplantation success. Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2−/−γc−/− mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein α (SIRPα) is a key modulator of the recipient’s innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRPα to recipient CD47 and was modulated by the strength of the SIRPα-CD47 interaction. Therefore, sensing SIRPα polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.

CD8+ Effector T Cell Migration to Pancreatic Islet Grafts Is Dependent on Cognate Antigen Presentation by Donor Graft Cells

Pancreatic islet transplantation is a promising therapy for diabetes, but acute rejection of the islets by host effector T cells has hindered clinical application. In this study, we addressed the mechanisms of CD8+ effector T cell migration to islet grafts because interrupting this step is key to preventing rejection. We found that effector T cell migration to revascularized islet transplants in mice is dependent on non-self Ag recognition rather than signaling via Gαi-coupled chemokine receptors. Presentation of non-self Ag by donor cells was necessary for migration, whereas Ag presentation by recipient cells was dispensable. We also observed that deficiency of SKAP1, an immune cell adaptor downstream of the TCR and important for integrin activation, prolongs allograft survival but does not reduce effector T cell migration to the graft. Therefore, effector T cell migration to transplanted islets is Ag driven, not chemokine driven, but SKAP1 does not play a critical role in this process.

MEMORY T CELLS MIGRATE TO AND REJECT VASCULARIZED CARDIAC ALLOGRAFTS INDEPENDENT OF CXCR3 AND CCR5: 1763

Introduction: Unlike their naïve counterparts, memory T cells migrate to allografts and effect rejection without the need for priming in secondary lymphoid tissues. The mechanisms by which memory T cells migrate to transplanted organs, however, are not known. Here we tested whether CXCR3 and CCR5, known to direct the migration of effector T cells to sites of inflammation, are also required for the migration of memory T cells to a vascularized graft. Methods: Alloreactive memory T cells were generated in WT or CXCR3-/B6 mice by i.p. immunization with BALB/c splenocytes. CD4+ and CD8+ CD44hi T cells were high-speed sorted >8 wks after immunization. To study migration, WT (Thy1.1) and CXCR3-/(Thy1.2) memory T cells were CFSE-labeled and co-transferred to B6 WT (CD45.1) or splenectomized B6 aly/aly (CD45.1) mice 2 days after transplanting BALB/c hearts (n = 6/grp). Graft infiltration with transferred cells was quantitated by flow cytometry 20 hrs and 3 days later. To study rejection, WT and CXCR3-/memory T cells were transferred separately into splenectomized aly/aly recipients of BALB/c hearts (n = 4/grp). These mice do not reject allografts unless effector or memory T cells are transferred, thus providing an in vivo model to study memory function. Results: WT CD4+ and CD8+ memory T cells expressed higher levels of CXCR3 and CCR5 than naïve T cells. CXCR3-/did not express CXCR3 but had similar CCR5 expression and produced similar IFNγ levels in response to allostimulation as WT memory T cells. These findings were confirmed in tetramer+ H60-specific CD8+ memory T cells. WT and CXCR3-/CD4+ and CD8+ memory T cells were recovered in equal numbers from cardiac allografts at 20 hrs and 3 days after transfer to either WT or splenectomized aly/aly recipients. On day 3, both WT and CXCR3-/cells had begun proliferating in the graft. Likewise, WT and CXCR3/memory T cells precipitated equal rejection of heart allografts in splenectomized aly/aly recipients (MST=18 vs 20d). Treatment of mice adoptively transferred with CXCR3-/memory T cells with anti-CCR5 mAb (100 μg x 14 days) did not delay allograft rejection (MST=10d). Conclusion: The data demonstrate that CXCR3 and CCR5 are not critical for memory T cell migration to and rejection of vascularized cardiac allografts. This suggests that the requirements for memory T cell migration to sites of inflammation differ from those proposed for effector T cells, possibly due to inherent differences between the two cell types.