Immunosuppression withdrawal in living-donor renal transplant recipients following induction with antithymocyte globulin and rituximab: Results of a prospective clinical trial.

Durable tolerance in kidney transplant recipients remains an important but elusive goal. We hypothesized that adding B cell depletion to T cell depletion would generate an immune milieu postreconstitution dominated by immature transitional B cells, favoring tolerance. The Immune Tolerance Network ITN039ST Research Study of ATG and Rituximab in Renal Transplantation was a prospective multicenter pilot study of live donor kidney transplant recipients who received induction with rabbit antithymocyte globulin and rituximab and initiated immunosuppression (IS) withdrawal (ISW) at 26 weeks. The primary endpoint was freedom from rejection at 52 weeks post-ISW. Six of the 10 subjects successfully completed ISW. Of these 6 subjects, 4 restarted immunosuppressive medications due to acute rejection or recurrent disease, 1 remains IS-free for over 9 years, and 1 was lost to follow-up after being IS-free for 42 weeks. There were no cases of patient or graft loss. CD19+ B cell frequencies returned to predepletion levels by 26 weeks posttransplant; immunoglobulin D+CD27--naïve B cells predominated. In contrast, memory cells dominated the repopulation of the T cell compartment. A regimen of combined B and T cell depletion did not generate the tolerogenic B cell profile observed in preclinical studies and did not lead to durable tolerance in the majority of kidney transplant recipients.

Efficacy and Safety of Immunosuppression Withdrawal in Pediatric Liver Transplant Recipients: Moving Toward Personalized Management.

BACKGROUND AND AIMS: Tolerance is transplantation's holy grail, as it denotes allograft health without immunosuppression and its toxicities. Our aim was to determine, among stable long-term pediatric liver transplant recipients, the efficacy and safety of immunosuppression withdrawal to identify operational tolerance. APPROACH AND RESULTS: We conducted a multicenter, single-arm trial of immunosuppression withdrawal over 36-48 weeks. Liver tests were monitored biweekly (year 1), monthly (year 2), and bimonthly (years 3-4). For-cause biopsies were done at investigators' discretion but mandated when alanine aminotransferase or gamma glutamyltransferase exceeded 100 U/L. All subjects underwent final liver biopsy at trial end. The primary efficacy endpoint was operational tolerance, defined by strict biochemical and histological criteria 1 year after stopping immunosuppression. Among 88 subjects (median age 11 years; 39 boys; 57 deceased donor grafts), 33 (37.5%; 95% confidence interval [CI] 27.4%, 48.5%) were operationally tolerant, 16 were nontolerant by histology (met biochemical but failed histological criteria), and 39 were nontolerant by rejection. Rejection, predicted by subtle liver inflammation in trial entry biopsies, typically (n = 32) occurred at ≤32% of the trial-entry immunosuppression dose and was treated with corticosteroids (n = 32) and/or tacrolimus (n = 38) with resolution (liver tests within 1.5 times the baseline) for all but 1 subject. No death, graft loss, or chronic, severe, or refractory rejection occurred. Neither fibrosis stage nor the expression level of a rejection gene set increased over 4 years for either tolerant or nontolerant subjects. CONCLUSIONS: Immunosuppression withdrawal showed that 37.5% of selected pediatric liver-transplant recipients were operationally tolerant. Allograft histology did not deteriorate for either tolerant or nontolerant subjects. The timing and reversibility of failed withdrawal justifies future trials exploring the efficacy, safety, and potential benefits of immunosuppression minimization.

Immunosuppression Withdrawal in Liver Transplant Recipients on Sirolimus.

BACKGROUND AND AIMS: As conversion from calcineurin inhibitor to sirolimus (SRL), a mechanistic target of rapamycin inhibitor (mTOR-I), has been shown to enhance immunoregulatory profiles in liver transplant (LT) recipients (LTRs), mTOR-I therapy might allow for increased success of immunosuppression (IS) withdrawal. Our aim was to determine if operational tolerance could be observed in LTRs withdrawn from SRL and if blood/graft tolerance biomarkers were predictive of successful withdrawal. APPROACH AND RESULTS: We performed a prospective trial of SRL monotherapy withdrawal in nonimmune, nonviremic LTRs > 3 years post-LT. SRL was weaned over ~6 months, and biopsies were performed 12 months postweaning or at concern for acute rejection. Twenty-one LTRs consented; 6 were excluded due to subclinical acute rejection on baseline biopsy or other reasons, and 15 underwent weaning (age 61.3 ± 8.8 years; LT to SRL weaning 6.7 ± 3 years). Eight (53%) achieved operational tolerance (TOL). Of the 7 who were nontolerant (non-TOL), 6 had mild acute rejection on biopsy near the end of weaning or at study end; 1 was removed from the trial due to liver cancer recurrence. At baseline preweaning, there were statistically increased blood tolerogenic dendritic cells and cell phenotypes correlating with chronic antigen presentation in the TOL versus non-TOL groups. A previously identified biopsy gene signature accurately predicted TOL versus non-TOL in 12/14 LTRs before weaning. At study end, biopsy staining revealed statistically significant increases in antigen-presenting cell:leukocyte pairings, FOXP3+ /CD4+ T cells, Tbet+ /CD8+ T cells, and lobular dendritic cells in the non-TOL group. CONCLUSIONS: This study evaluated IS withdrawal directly from mTOR-I therapy in LTRs and achieved > 50% operational tolerance. Preweaning gene expression and peripheral blood mononuclear cell profiling may be useful as predictors of successful mTOR-I therapy withdrawal. NCT02062944.

IgG4 donor-specific HLA antibody profile is associated with subclinical rejection in stable pediatric liver recipients.

The impact of donor-specific HLA antibody (DSA) following liver transplantation remains controversial. We hypothesized DSA IgG subclass characteristics, compared to total DSA IgG, might correlate with specific histopathological phenotype(s) of subclinical graft injury. We therefore studied 129 stable, arguably "clinically ideal," pediatric liver recipients at the time of a screening biopsy to enter an immunosuppression withdrawal trial. Sixty-five (50%) subjects tested positive for class II DSA. IgG subclass profile was characterized by mean fluorescence intensity (MFI) and normalized subclass composition (>5%). A prominent IgG4 DSA profile was strongly correlated with greater HLA mismatch, a histopathological phenotype characterized by the presence of interface activity (with variable degrees of fibrosis), and a transcriptional profile of attenuated T cell-mediated rejection. Specifically, compared to those without class II DSA, those with IgG4 class II DSA MFI sum >2000 exhibited an odds ratio (OR) of 20.79 (95% confidence interval [CI] 4.38-98.69) and IgG4 subclass composition >5% exhibited an OR of 8.99 (95% CI 2.70-29.9). Our data suggest that IgG4 DSA may serve as a useful biomarker to identify, among clinically and biochemically stable liver transplant recipients, a subset with histological and transcriptional features indicative of an active, suboptimally controlled alloimmune response.

T-cell exhaustion correlates with improved outcomes in kidney transplant recipients.

Continuous antigen stimulation during chronic infection or malignancy can promote functional T cell silencing, a phenomenon called T cell exhaustion. The prevalence and impact of T cell exhaustion following organ transplantation, another immune stimulus with persistently high antigen load, are unknown. Here, we characterized serially collected peripheral blood mononuclear cells from 26 kidney transplant recipients using time-of-flight mass cytometry (CyTOF) to define distinct subsets of circulating exhausted T cells and their relationship to induction therapy and allograft function. We observed an increase in specific subsets of CD4+ and CD8+ exhausted T cells from pre-transplant to 6-months post-transplant, with greater increases in participants given anti-thymocyte globulin induction than in participants who received no induction or non-depleting induction. The percentages of exhausted T cells at 6 months correlated inversely with adenosine triphosphate (ATP) production (a surrogate of T cell function) and with allograft interstitial fibrosis. Guided by the CyTOF data, we delineated a PD-1+CD57- phenotype for CD4+ and CD8+ exhausted T cells, and confirmed that these cells have limited capacity for cytokine secretion and ATP production. In an independent cohort of 50 kidney transplant recipients, we confirmed the predicted increase of PD-1+CD57- exhausted T cells after lymphocyte-depleting induction therapy and its direct correlation with better allograft function. Our findings suggest that monitoring T cell exhaustion can be useful for post-transplant risk assessment and support the need to develop and test strategies aimed at augmenting T cell exhaustion following kidney transplantation.

Outcomes of immunosuppression minimization and withdrawal early after liver transplantation.

The Immune Tolerance Network ITN030ST A-WISH assessed immunosuppression withdrawal in liver transplant recipients with hepatitis C or nonimmune nonviral liver disease. Of 275 recipients enrolled before transplantation, 95 were randomly assigned 4:1 to withdrawal (n = 77) or maintenance (n = 18) 1- to 2-years posttransplant. Randomization eligibility criteria included stable immunosuppression monotherapy; adequate liver and kidney function; ≤Stage 2 Ishak fibrosis; and absence of rejection on biopsy. Immunosuppression withdrawal followed an 8-step reduction algorithm with ≥8 weeks per level. Fifty-two of 77 subjects (67.5%) reduced to ≤50% of baseline dose, and 10 of 77 (13.0%) discontinued all immunosuppression for ≥1 year. Acute rejection and/or abnormal liver tests were treated with increased immunosuppression; 5 of 32 rejection episodes required a methylprednisolone bolus. The composite end point (death or graft loss; grade 4 secondary malignancy or opportunistic infection; Ishak stage ≥3; or >25% decrease in glomerular filtration rate within 24 months of randomization) occurred in 12 of 66 (18%) and 4 of 13 (31%) subjects in the withdrawal and maintenance groups. Early immunosuppression minimization is feasible in selected liver recipients, while complete withdrawal is successful in only a small proportion. The composite end point comparison was inconclusive for noninferiority of the withdrawal to the maintenance group.

Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants.

BACKGROUND & AIMS: A substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles. METHODS: We conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data. RESULTS: The mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3. CONCLUSION: In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R.

Pediatric liver transplant recipients arguably have the most to gain and the most to lose from discontinuing immunosuppression (IS). Whereas IS undoubtedly exerts a cumulative toll, there is concern that insufficient or no IS may contribute to allograft deterioration. Twelve pediatric recipients of parental living donor liver grafts, identified as operationally tolerant through complete IS withdrawal (WISP-R; NCT00320606), were followed for a total of 5 years (1 year of IS withdrawal and 4 years off IS) with serial liver tests and autoantibody and alloantibody assessments. Liver biopsies were performed 2 and 4 years off IS, and, at these time points, immunoglobulin G (IgG) subclass and C1q binding activity for donor-specific antibodies (DSAs) were determined. There were no cases of chronic rejection, graft loss, or death. Allografts did not exhibit progressive increase in inflammation or fibrosis. Smooth-muscle actin expression by stellate cells and CD34 expression by liver sinusoidal endothelial cells remained stable, consistent with the absence of progressive graft injury. Three subjects never exhibited DSA. However, 3 subjects showed intermittent de novo class I DSA, 4 subjects showed persistent de novo class II DSA, and 5 subjects showed persistent preexisting class II DSA. Class II DSA was predominantly against donor DQ antigens, often of high mean fluorescence intensity, rarely of the IgG3 subclass, and often capable of binding C1q. CONCLUSION: Operationally tolerant pediatric liver transplant recipients maintain generally stable allograft histology in spite of apparently active humoral allo-immune responses. The absence of increased inflammation or progressive fibrosis suggests that a subset of liver allografts seem resistant to the chronic injury that is characteristic of antibody-mediated damage. (Hepatology 2017;65:647-660).

Regulatory T cell induction, migration, and function in transplantation.

Regulatory T cells (Treg) are important in maintaining immune homeostasis and in regulating a variety of immune responses, making them attractive targets for modulating immune-related diseases. Success in using induction or transfer of Treg in mice to mediate transplant tolerance suggests Treg-based therapies as mechanisms of long-term drug-free transplant tolerance in human patients. Although more work is needed, critical analyses suggest that key factors in Treg induction, migration, and function are important areas to concentrate investigative efforts and therapeutic development. Elucidation of basic biology will aid in translating data gleaned from mice to humans so that Treg therapies become a reality for patients.

IL-6 promotes cardiac graft rejection mediated by CD4+ cells.

IL-6 mediates numerous immunologic effects relevant to transplant rejection; however, its specific contributions to these processes are not fully understood. To this end, we neutralized IL-6 in settings of acute cardiac allograft rejection associated with either CD8(+) or CD4(+) cell-dominant responses. In a setting of CD8(+) cell-dominant graft rejection, IL-6 neutralization delayed the onset of acute rejection while decreasing graft infiltrate and inverting anti-graft Th1/Th2 priming dominance in recipients. IL-6 neutralization markedly prolonged graft survival in the setting of CD4(+) cell-mediated acute rejection and was associated with decreased graft infiltrate, altered Th1 responses, and reduced serum alloantibody. Furthermore, in CD4(+) cell-dominated rejection, IL-6 neutralization was effective when anti-IL-6 administration was delayed by as many as 6 d posttransplant. Finally, IL-6-deficient graft recipients were protected from CD4(+) cell-dominant responses, suggesting that IL-6 production by graft recipients, rather than grafts, is necessary for this type of rejection. Collectively, these observations define IL-6 as a critical promoter of graft infiltration and a shaper of T cell lineage development in cardiac graft rejection. In light of these findings, the utility of therapeutics targeting IL-6 should be considered for preventing cardiac allograft rejection.

Anatomy of tolerance.

PURPOSE OF REVIEW: The mechanisms of tolerance induction and maintenance remain incompletely understood and have yet to be translated to clinical practice. Advances in imaging techniques have allowed precise examination of cell interactions in the lymph node, often in real time. Herein we review evidence that lymph node structure is dynamic and controls the character of the immune response in a multistep, multiplayer dance. T-cell responses in particular can be initiated or influenced in regions beyond the canonical T-cell zone. We propose that the cortical ridge is one such region required for induction and maintenance of tolerance. RECENT FINDINGS: Lymph node domains are more complex than T-cell and B-cell zones. Different domains are important for different types of immune responses. These domains are in part defined by dynamic, malleable physical structures that guide cell interactions and influence immune outcomes. SUMMARY: Further probing as to how lymph node stromal cells and fibers interact with and determine the character of immune responses should yield fundamental insights into tolerance and immunity. Manipulation of lymph node structure and associated unique cell types and molecules may allow therapeutic interventions in the tolerogenic process.

OX40 costimulation prevents allograft acceptance induced by CD40-CD40L blockade.

Disrupting the CD40-CD40L costimulation pathway promotes allograft acceptance in many settings. Herein, we demonstrate that stimulating OX40 overrides cardiac allograft acceptance induced by disrupting CD40-CD40L interactions. This effect of OX40 stimulation was dependent on CD4(+) T cells, which in turn provided help for CD8(+) T cells and B cells. Allograft rejection was associated with donor-reactive Th1 and Th2 responses and an unconventional granulocytic infiltrate and thrombosis of the arteries. Interestingly, OX40 stimulation induced a donor-reactive IgG class switch in the absence of CD40-CD40L interactions, and the timing of OX40 stimulation relative to transplantation affected the isotype of donor-reactive Ab produced. Inductive OX40 stimulation induced acute graft rejection, which correlated with both IgG1 and IgG2a deposition within the graft. Once graft acceptance was established following CD40-CD40L blockade, delayed OX40 stimulation did not induce acute allograft rejection despite priming of graft-reactive Th1 and Th2. Rather, chronic rejection was induced, which was characterized by IgG1 but not IgG2a deposition within the graft. These studies reveal both redundancy and key differences in function among costimulatory molecules that manifest in distinct pathologies of allograft rejection. These findings may help guide development of therapeutics aimed at promoting graft acceptance in transplant recipients.

Detection of alloreactive T cells from cryopreserved human peripheral blood mononuclear cells.

Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.

CD8+ Th17 mediate costimulation blockade-resistant allograft rejection in T-bet-deficient mice.

While studying Th responses induced by cardiac transplantation, we observed that mice deficient in the Th1 transcription factor T-bet (T-bet(-/-)) mount both Th1 and Th17 responses, whereas wild-type recipients mount only Th1 responses. Cells producing both IFN-gamma and IL-17 were readily detectable within the rejecting graft of T-bet(-/-) recipients, but were absent from the spleen, indicating that the in vivo microenvironment influences Th function. In addition, disrupting CD40-CD40L costimulatory interactions was highly effective at prolonging allograft survival in WT mice, but ineffective in T-bet(-/-) recipients. In this study, we report that CD8(+) Th17 mediate costimulation blockade-resistant rejection in T-bet(-/-) allograft recipients. Depleting CD8(+) cells or neutralizing IL-17 or the Th17-inducing cytokine IL-6 ablated the Th17 response and reversed costimulation blockade-resistant graft rejection. Neutralizing IL-4 in IFN-gamma(-/-) allograft recipients did not induce Th17, suggesting that T-bet, rather than IL-4 and IFN-gamma (known inhibitors of Th17), plays a critical role in negatively regulating Th17 in the transplant setting.

IL-10 from marginal zone precursor B cells controls the differentiation of Th17, Tfh and Tfr cells in transplantation tolerance.

B cells are known to control CD4T cell differentiation in secondary lymphoid tissues. We hypothesized that IL-10 expression by marginal zone precursor (MZP) regulatory B cells controls the differentiation and positioning of effector and regulatory T cells during tolerization. Costimulatory blockade with donor-specific transfusion (DST) and anti-CD40L mAb in C57BL/6 mice induced tolerance to allogeneic cardiac allograft. B cell depletion or IL-10 deficiency in B cells prevented tolerance, resulting in decreased follicular regulatory CD4(+) T cells (Tfr) and increased IL-21 expression by T follicular helper (Tfh) cells in the B cell and T cell zones. IL-21 acted with IL-6 to induce CCR6(+) Th17 that caused rejection. Deficiency or blockade of IL-6, IL-21, IL-21R, or CCR6 prevented B cell depletion-induced acute cellular rejection; while agonistic mCCL20-Ig induced rejection. Adoptive transfer of IL-10(+/+) MZP in tolerogen treated CD19-Cre(+/-):IL-10(fl/fl) mice rescued the localization of Tfh and Tfr cells in the B cell follicle and prevented allograft rejection. MZP B cell IL-10 is necessary for tolerance and controls the differentiation and position of Th17, Tfh and Tfr cells in secondary lymphoid tissues. This has implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Lymph Node Stromal Fiber ER-TR7 Modulates CD4+ T Cell Lymph Node Trafficking and Transplant Tolerance.

BACKGROUND: Trafficking and differentiation of naive CD4+ and regulatory T cells (Treg) within the lymph node (LN) are integral for tolerance induction. The LN is comprised of stromal fibers that dictate lymphocyte migration and LN structure, organization, and microanatomic domains. Distribution of the stromal fiber ER-TR7 changes within the LN after antigenic challenge, but the contributions of ER-TR7 to the resulting immune response remain undefined. We hypothesized that these stromal fiber structural changes affect T cell fate and subsequently allograft survival. METHODS: C57BL/6 mice were left naive (untreated) or made immune or tolerant (donor-specific BALB/c splenocyte transfusion -/+ anti-CD40L monoclonal antibody), or made tolerant and received anti-ER-TR7 monoclonal antibody. Donor-specific T-cell migration was visualized by adoptive transfer of carboxyfluorescein diacetate, succinimidyl ester-labeled TEa T cell receptor transgenic CD4+ cells. Immunohistochemistry was performed on LNs to detect stromal fiber distribution, structure, CCL21 presence, and Treg and donor-specific cell location relative to high endothelial venules (HEV). Naive, tolerant, and tolerant + anti-ER-TR7 mice received BALB/c heterotopic cardiac allografts and graft survival was monitored. RESULTS: The ER-TR7 distribution changed after the induction of tolerance vs. immunity. Treating tolerant mice with anti-ER-TR7 altered HEV basement membrane structure and the distribution of CCL21 within the LN. These differences were mirrored by changes in the migration of naive and Treg cells within and surrounding the HEV. Anti-ER-TR7 prevented tolerance induction and resulted in allograft inflammation and rejection. CONCLUSIONS: These results identify ER-TR7 as an important component of LN structure in tolerance and a direct target for immune modulation.

Interleukin-10 From Marginal Zone Precursor B-Cell Subset Is Required for Costimulatory Blockade-Induced Transplantation Tolerance.

BACKGROUND: Blocking CD40-CD40L costimulatory signals induces transplantation tolerance. Although B-cell depletion prevents alloantibody formation, nonhumoral functions of B cells in tolerance have not been well characterized. We investigated whether specific subsets of B cell or B cell-derived interleukin (IL)-10 contribute to tolerance. METHODS: Wild type C57BL/6, or B cell-specific interleukin (IL)-10 (CD19-Cre::IL-10) mice, received vascularized BALB/c cardiac allografts. BALB/c donor-specific splenocyte transfusion and anti-CD40L monoclonal antibody were used as tolerogen. B cells were depleted with antimouse CD20 monoclonal antibody. Various B-cell subsets were purified and characterized by flow cytometry, reverse transcription polymerase chain reaction, and adoptive transfer. RESULTS: B-cell depletion prevented costimulatory blockade-induced allogeneic tolerance. Costimulatory blockade increased IL-10 in marginal zone precursor (MZP) B cells, but not other subsets. In particular, costimulatory blockade did not change other previously defined regulatory B-cell subsets (Breg), including CD5CD1d Breg or expression of TIM1 or TIM4 on these Breg or other Breg cell subsets. Costimulatory blockade also induced IL-21R expression in MZP B cells, and IL-21R MZP B cells expressed even more IL-10. B-cell depletion or IL-10 deficiency in B cells prevented tolerance in a cardiac allograft model, resulting in rapid acute cardiac allograft rejection. Adoptive transfer of wild type MZP B cells but not other subsets to B cell-specific IL-10 deficient mice prevented graft rejection. CONCLUSIONS: CD40 costimulatory blockade induces MZP B cell IL-10 which is necessary for tolerance. These observations have implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Laminins affect T cell trafficking and allograft fate.

Lymph nodes (LNs) are integral sites for the generation of immune tolerance, migration of CD4⁺ T cells, and induction of Tregs. Despite the importance of LNs in regulation of inflammatory responses, the LN-specific factors that regulate T cell migration and the precise LN structural domains in which differentiation occurs remain undefined. Using intravital and fluorescent microscopy, we found that alloreactive T cells traffic distinctly into the tolerant LN and colocalize in exclusive regions with alloantigen-presenting cells, a process required for Treg induction. Extracellular matrix proteins, including those of the laminin family, formed regions within the LN that were permissive for colocalization of alloantigen-presenting cells, alloreactive T cells, and Tregs. We identified unique expression patterns of laminin proteins in high endothelial venule basement membranes and the cortical ridge that correlated with alloantigen-specific immunity or immune tolerance. The ratio of laminin α4 to laminin α5 was greater in domains within tolerant LNs, compared with immune LNs, and blocking laminin α4 function or inducing laminin α5 overexpression disrupted T cell and DC localization and transmigration through tolerant LNs. Furthermore, reducing α4 laminin circumvented tolerance induction and induced cardiac allograft inflammation and rejection in murine models. This work identifies laminins as potential targets for immune modulation.

Tolerance and lymphoid organ structure and function.

This issue of Frontiers in Immunologic Tolerance explores barriers to tolerance from a variety of views of cells, molecules, and processes of the immune system. Our laboratory has spent over a decade focused on the migration of the cells of the immune system, and dissecting the signals that determine how and where effector and suppressive regulatory T cells traffic from one site to another in order to reject or protect allografts. These studies have led us to a greater appreciation of the anatomic structure of the immune system, and the realization that the path taken by lymphocytes during the course of the immune response to implanted organs determines the final outcome. In particular, the structures, microanatomic domains, and the cells and molecules that lymphocytes encounter during their transit through blood, tissues, lymphatics, and secondary lymphoid organs are powerful determinants for whether tolerance is achieved. Thus, the understanding of complex cellular and molecular processes of tolerance will not come from "96-well plate immunology," but from an integrated understanding of the temporal and spatial changes that occur during the response to the allograft. The study of the precise positioning and movement of cells in lymphoid organs has been difficult since it is hard to visualize cells within their three-dimensional setting; instead techniques have tended to be dominated by two-dimensional renderings, although advanced confocal and two-photon systems are changing this view. It is difficult to precisely modify key molecules and events in lymphoid organs, so that existing knockouts, transgenics, inhibitors, and activators have global and pleiotropic effects, rather than precise anatomically restricted influences. Lastly, there are no well-defined postal codes or tracking systems for leukocytes, so that while we can usually track cells from point A to point B, it is exponentially more difficult or even impossible to track them to point C and beyond. We believe this represents one of the fundamental barriers to understanding the immune system and devising therapeutic approaches that take into account anatomy and structure as major controlling principles of tolerance.

Th17 cells and transplant acceptance.

The discovery of Th17 cells has revealed a novel pathway of T-cell maturation. As with Th1 and Th2 lineages, Th17 cells promote graft pathology. However, a growing body of evidence indicates that Th17 cells may exhibit resistance to current methods of immunosuppression. Identification of this lineage provides an additional and challenging target for promoting graft acceptance.