Ciara N. Magee

TIM-3: A Novel Regulatory Molecule of Alloimmune Activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4+FoxP3− and CD8+ T cells in acutely rejecting graft recipients. A blocking anti–TIM-3 mAb accelerated allograft rejection only in the presence of host CD4+ T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ–, IL-6–, and IL-17–producing splenocytes, enhanced CD8+ cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4+ T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4+ T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.

Monocyte-secreted inflammatory cytokines are associated with transplant glomerulopathy in renal allograft recipients.

Background. Although there is ample evidence about the role of adaptive immunity in the development of chronic allograft dysfunction, little is known about the contribution of innate immunity to this process. Herein, we studied the relationship between inflammation, chronic biopsy scores, and anti-human leukocyte antigen (HLA) circulating alloantibodies in a cohort of 57 patients recruited at our center. Methods. Available biopsies (n=27) were graded for chronic lesion scores according to Banff criteria. The production of cytokines by peripheral blood mononuclear cells after 48 hr of culture under resting conditions was quantified by Luminex. Tumor necrosis factor (TNF)-&agr; secretion assay and depletion studies were used to identify the source of these cytokines. Results. There was a high correlation between the levels of interleukin (IL)-1&bgr;, IL-6, and TNF-&agr; (r>0.8, P<0.001 for all correlations). The levels of these cytokines were associated with transplant glomerulopathy (IL-1&bgr;, P=0.019; IL-6, P=0.015; and TNF-&agr;, P=0.006) but not with other chronic lesions or anti-HLA circulating alloantibodies. TNF-&agr; was predominantly secreted by monocytes (percent of TNF-&agr; secreting cells: 20.4±4.8 vs. 1.2±0.5 vs. 1.4±0.6 vs. 1.7±0.5 for CD14+, CD4+, CD8+, and CD19+ cells, respectively; all P<0.01 vs. CD14+). The levels of all three proinflammatory cytokines were significantly reduced after monocyte depletion. Intriguingly, cytokine levels increased after ex vivo depletion of regulatory T cells (all P<0.001). Conclusions. Taken together, these data suggest that in vivo–activated monocytes in peripheral blood spontaneously secrete proinflammatory cytokines in renal allograft recipients with transplant glomerulopathy and seem to be under the regulation of functional regulatory T cells in this setting.

Derivation and Validation of a Cytokine-Based Assay to Screen for Acute Rejection in Renal Transplant Recipients

BACKGROUND AND OBJECTIVES Acute rejection remains a problem in renal transplantation. This study sought to determine the utility of a noninvasive cytokine assay in screening of acute rejection. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In this observational cross-sectional study, 64 patients from two centers were recruited upon admission for allograft biopsy to investigate acute graft dysfunction. Blood was collected before biopsy and assayed for a panel of 21 cytokines secreted by PBMCs. Patients were classified as acute rejectors or nonrejectors according to a classification rule derived from an initial set of 32 patients (training cohort) and subsequently validated in the remaining patients (validation cohort). RESULTS Although six cytokines (IL-1β, IL-6, TNF-α, IL-4, GM-CSF, and monocyte chemoattractant protein-1) distinguished acute rejectors in the training cohort, logistic regression modeling identified a single cytokine, IL-6, as the best predictor. In the validation cohort, IL-6 was consistently the most accurate cytokine (area under the receiver-operating characteristic curve, 0.85; P=0.006), whereas the application of a prespecified cutoff level, as determined from the training cohort, resulted in a sensitivity and specificity of 92% and 63%, respectively. Secondary analyses revealed a strong association between IL-6 levels and acute rejection after multivariate adjustment for clinical characteristics (P<0.001). CONCLUSIONS In this pilot study, the measurement of a single cytokine can exclude acute rejection with a sensitivity of 92% in renal transplant recipients presenting with acute graft dysfunction. Prospective studies are needed to determine the utility of this simple assay, particularly for low-risk or remote patients.

Ex Vivo Expansion of Human Tregs by Rabbit ATG Is Dependent on Intact STAT3‐Signaling in CD4+ T Cells and Requires the Presence of Monocytes

The addition of low, nondepleting doses of rabbit antithymocyte globulin (ATG) to human peripheral blood mononuclear cells has been shown to expand functional CD4+CD25+FoxP3+ regulatory T cells (Tregs) in vitro. This report is the first to elucidate the exact cellular mechanisms of ATG‐mediated Treg expansion. CD4+ T cells require monocytes, but not other antigen presenting cell subsets, to be present in coculture to expand Tregs. However, T cells do not require direct cell–cell contact with monocytes, suggesting the importance of soluble factors. Moreover, ATG initially “reprograms” CD4+ T cells, but not monocytes, and induces STAT3 and STAT5 signaling in CD4+ cells. These reprogrammed CD4+ T cells subsequently secrete GM‐CSF and IL‐10 only in case of intact STAT3 signaling, which in turn promote the generation of tolerogenic CD14+CD11c+ dendritic cells characterized by enhanced IL‐10 and decreased IL‐12 production. Treg expansion following ATG treatment is accompanied by enhanced gene expression of both GM‐CSF and Bcl‐2, but not TGF‐β, in peripheral blood mononuclear cells. These results demonstrate that ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD4+ T cells in a STAT3‐dependent but TGF‐β‐independent manner, leading to the generation of monocyte‐derived dendritic cells with a tolerogenic cytokine profile.

Prolonged, low-dose anti-thymocyte globulin, combined with CTLA4-Ig, promotes engraftment in a stringent transplant model.

Background Despite significant nephrotoxicity, calcineurin inhibitors (CNIs) remain the cornerstone of immunosuppression in solid organ transplantation. We, along with others, have reported tolerogenic properties of anti-thymocyte globulin (ATG, Thymoglobulin®), evinced by its ability both to spare Tregs from depletion in vivo and, when administered at low, non-depleting doses, to expand Tregs ex vivo. Clinical trials investigating B7/CD28 blockade (LEA29Y, Belatacept) in kidney transplant recipients have proven that the replacement of toxic CNI use is feasible in selected populations. Methods Rabbit polyclonal anti-murine thymocyte globulin (mATG) was administered as induction and/or prolonged, low-dose therapy, in combination with CTLA4-Ig, in a stringent, fully MHC-mismatched murine skin transplant model to assess graft survival and mechanisms of action. Results Prolonged, low-dose mATG, combined with CTLA4-Ig, effectively promotes engraftment in a stringent transplant model. Our data demonstrate that mATG achieves graft acceptance primarily by promoting Tregs, while CTLA4-Ig enhances mATG function by limiting activation of the effector T cell pool in the early stages of treatment, and by inhibiting production of anti-rabbit antibodies in the maintenance phase, thereby promoting regulation of alloreactivity. Conclusion These data provide the rationale for development of novel, CNI-free clinical protocols in human transplant recipients.

Interruption of Dendritic Cell–Mediated TIM-4 Signaling Induces Regulatory T Cells and Promotes Skin Allograft Survival

Dendritic cells (DCs) are the central architects of the immune response, inducing inflammatory or tolerogenic immunity, dependent on their activation status. As such, DCs are highly attractive therapeutic targets and may hold the potential to control detrimental immune responses. TIM-4, expressed on APCs, has complex functions in vivo, acting both as a costimulatory molecule and a phosphatidylserine receptor. The effect of TIM-4 costimulation on T cell activation remains unclear. In this study, we demonstrate that Ab blockade of DC-expressed TIM-4 leads to increased induction of induced regulatory T cells (iTregs) from naive CD4+ T cells, both in vitro and in vivo. iTreg induction occurs through suppression of IL-4/STAT6/Gata3–induced Th2 differentiation. In addition, blockade of TIM-4 on previously activated DCs still leads to increased iTreg induction. iTregs induced under TIM-4 blockade have equivalent potency to control and, upon adoptive transfer, significantly prolong skin allograft survival in vivo. In RAG−/− recipients of skin allografts adoptively transferred with CD4+ T cells, we show that TIM-4 blockade in vivo is associated with a 3-fold prolongation in allograft survival. Furthermore, in this mouse model of skin transplantation, increased induction of allospecific iTregs and a reduction in T effector responses were observed, with decreased Th1 and Th2 responses. This enhanced allograft survival and protolerogenic skewing of the alloresponse is critically dependent on conversion of naive CD4+ to Tregs in vivo. Collectively, these studies identify blockade of DC-expressed TIM-4 as a novel strategy that holds the capacity to induce regulatory immunity in vivo.

Immune Profile of Pediatric Renal Transplant Recipients following Alemtuzumab Induction

The incidence of developing circulating anti-human leukocyte antigen antibodies and the kinetics of T cell depletion and recovery among pediatric renal transplant recipients who receive alemtuzumab induction therapy are unknown. In a collaborative endeavor to minimize maintenance immunosuppression in pediatric renal transplant recipients, we enrolled 35 participants from four centers and treated them with alemtuzumab induction therapy and a steroid-free, calcineurin-inhibitor-withdrawal maintenance regimen. At 3 months after transplant, there was greater depletion of CD4(+) than CD8(+) T cells within the total, naive, memory, and effector memory subsets, although depletion of the central memory subset was similar for CD4(+) and CD8(+) cells. Although CD8(+) T cells recovered faster than CD4(+) subsets overall, they failed to return to pretransplant levels by 24 months after transplant. There was no evidence for greater recovery of either CD4(+) or CD8(+) memory cells than naïve cells. Alemtuzumab relatively spared CD4(+)CD25(+)FoxP3(+) regulatory T cells, resulting in a rise in their numbers relative to total CD4(+) cells and a ratio that remained at least at pretransplant levels throughout the study period. Seven participants (20%) developed anti-human leukocyte antigen antibodies without adversely affecting allograft function or histology on 2-year biopsies. Long-term follow-up is underway to assess the potential benefits of this regimen in children.

The Role of Costimulatory Molecules in Directing the Functional Differentiation of Alloreactive T Helper Cells

Costimulatory molecules are a heterogenous group of cell surface molecules that act to amplify or counteract the initial activating signals provided to T cells from the T cell receptor following its interaction with an antigen/major histocompatibility complex, thereby influencing T cell differentiation and fate. Although costimulation was previously thought to be indispensable for T cell activation at all stages of development, it is now known that the requirements for costimulation, and the costimulatory molecules involved, vary according to the stage of T cell differentiation. The ability to influence T cell fate is of paramount interest in the field of transplantation as we seek therapeutic options that inhibit detrimental alloimmune responses whilst simultaneously promoting allograft tolerance. As with many immune mechanisms, there is a degree of functional overlap between certain costimulatory molecules, whereas some have diametrically opposite effects on different T cell subsets despite sharing common ligands. This is a critical point when considering these molecules as therapeutic targets in transplantation, as blockade of a costimulatory pathway, although desirable in itself, may prevent the ligation of an essential regulatory coinhibitory molecule. This review discusses the T helper cell lineages pertinent to transplantation and the costimulatory molecules involved in their differentiation.

Salt Accelerates Allograft Rejection through Serum- and Glucocorticoid-Regulated Kinase-1–Dependent Inhibition of Regulatory T Cells

A high-salt diet (HSD) in humans is linked to a number of complications, including hypertension and cardiovascular events. Whether a HSD affects the immune response in transplantation is unknown. Using a murine transplantation model, we investigated the effect of NaCl on the alloimmune response in vitro and in vivo. Incremental NaCl concentrations in vitro augmented T cell proliferation in the settings of both polyclonal and allospecific stimulation. Feeding a HSD to C57BL/6 wild-type recipients of bm12 allografts led to accelerated cardiac allograft rejection, despite similar mean BP and serum sodium levels in HSD and normal salt diet (NSD) groups. The accelerated rejection was associated with a reduction in the proportion of CD4(+)Foxp3(+) regulatory T cells (Tregs) and a significant decrease in Treg proliferation, leading to an increased ratio of antigen-experienced CD4(+) T cells to Tregs in mice recipients of a HSD compared with mice recipients of a NSD. Because serum- and glucocorticoid-regulated kinase-1 (SGK1) has been proposed as a potential target of salt in immune cells, we fed a HSD to CD4(Cre)SGK1(fl/fl) B6-transplanted recipients and observed abrogation of the deleterious effect of a HSD in the absence of SGK1 on CD4(+) cells. In summary, we show that NaCl negatively affects the regulatory balance of T cells in transplantation and precipitates rejection in an SGK1-dependent manner.

Jagged2-signaling promotes IL-6-dependent transplant rejection

The Notch pathway is an important intercellular signaling pathway that plays a major role in controlling cell fate. Accumulating evidence indicates that Notch and its ligands present on antigen‐presenting cells might be important mediators of T helper cell differentiation. In this study, we investigated the role of Jagged2 in murine cardiac transplantation by using a signaling Jagged2 mAb (Jag2) that activates recombinant signal‐binding protein‐Jκ. While administration of Jag2 mAb had little effect on graft survival in the fully allogeneic mismatched model BALB/c→B6, it hastened rejection in CD28‐deficient recipients. Similarly, Jag2 precipitated rejection in the bm12→B6 model. In this MHC class II‐mismatched model, allografts spontaneously survive for >56 days due to the emergence of Treg cells that inhibit the expansion of alloreactive T cells. The accelerated rejection was associated with upregulation of Th2 cytokines and proinflammatory cytokine IL‐6, despite expansion of Treg cells. Incubation of Treg cells with recombinant IL‐6 abrogated their inhibitory effects in vitro. Furthermore, neutralization of IL‐6 in vivo protected Jag2‐treated recipients from rejection and Jagged2 signaling was unable to further accelerate rejection in the absence of Treg cells. Our findings therefore suggest that Jagged2 signaling can affect graft acceptance by upregulation of IL‐6 and consequent resistance to Treg‐cell suppression.