Annette M. Jackson

A Change in the Structure of Vβ Chromatin Associated with TCR β Allelic Exclusion

To investigate chromatin control of TCR β rearrangement and allelic exclusion, we analyzed TCR β chromatin structure in double negative (DN) thymocytes, which are permissive for TCR β recombination, and in double positive (DP) thymocytes, which are postallelic exclusion and nonpermissive for Vβ to DβJβ recombination. Histone acetylation mapping and DNase I sensitivity studies indicate Vβ and DβJβ segments to be hyperacetylated and accessible in DN thymocytes. However, they are separated from each other by hypoacetylated and inaccessible trypsinogen chromatin. The transition from DN to DP is accompanied by selective down-regulation of Vβ acetylation and accessibility. The level of DP acetylation and accessibility is minimal for five of six Vβ segments studied but remains substantial for one. Hence, the observed changes in Vβ chromatin structure appear sufficient to account for allelic exclusion of many Vβ segments. They may contribute to, but not by themselves fully account for, allelic exclusion of others.

Regulation of T cell receptor beta allelic exclusion at a level beyond accessibility.

Allelic exclusion of Vβ-to-DJβ recombination depends on asynchronous rearrangement of alleles of the gene encoding T cell receptor β in double-negative thymocytes and feedback inhibition that is maintained in double-positive thymocytes. Feedback is thought to be enforced through downregulation of Vβ accessibility. In an attempt to override this negative regulation, we introduced the enhancer of the gene encoding T cell receptor α into the Vβ gene cluster downstream of Vβ12. In double-negative thymocytes, the introduced enhancer had no measurable effect on accessibility, but Vβ12 rearrangement was stimulated and Vβ12 allelic exclusion was partially subverted. In contrast, double-positive thymocytes showed increased Vβ transcription and accessibility, but feedback inhibition of Vβ-to-DJβ recombination remained intact. Our results indicate additional regulatory constraints on Vβ-to-DJβ recombination that operate beyond the accessibility barrier.

Turning T‐cell receptor β recombination on and off: more questions than answers

Summary:u2002 Successful V(D)J recombination at the T‐cell receptor β (Tcrb) locus is critical for early thymocyte development. The locus is subject to a host of regulatory mechanisms that impart a strict developmental order to Tcrb recombination events and that insure that Tcrb recombination occurs in an allelically excluded fashion. Progress has been made in the understanding of the cis‐acting control of Tcrb locus chromatin structure and the extent to which such accessibility control can account for the developmental regulation of Tcrb recombination. However, recent studies in our laboratory and elsewhere have made it abundantly clear that accessibility control is only part of the story, and multiple additional mechanisms impact both the developmental activation and inactivation of locus recombination events. Here we evaluate our current understanding of developmental regulation at the Tcrb locus. We highlight the many unresolved issues and we discuss how recent concepts emerging from studies of other antigen receptor loci may (or may not) help to resolve these issues.

Using donor exchange paradigms with desensitization to enhance transplant rates among highly sensitized patients.

Purpose of reviewMany sensitized patients have willing live donors but are unable to use them because of a human leukocyte antigen (HLA) incompatibility. The options for these patients include: remaining on the deceased-donor list, entering a kidney-paired donation scheme, or undergoing desensitization with high-dose IVIg or plasmapheresis and low-dose IVIg. Recent findingsMathematical simulations verified by actual data from several national kidney-paired donation (KPD) programs has shed light on which donor/recipient phenotypes are likely to benefit from each transplant modality. Pairs that are easy to match are likely to receive compatible kidneys in a KPD. Those who are hard to match may be better served by desensitization. The phenotype which is both hard to match and hard to desensitize due to board and strong HLA reactivity are most likely to be transplanted by a hybrid modality utilizing desensitization after identifying a more immunologically favorable donor in a KPD. SummaryRecent outcomes from desensitization in which starting donor-specific antibody strength is low have been very good. For broadly sensitized patients with a high-strength cross-match, searching for a better donor in a KPD pool can facilitate a safer, less expensive, and more successful desensitization treatment course.

Transplanting the highly sensitized patient: trials and tribulations.

Purpose of reviewHumoral sensitization to antigens of the human leukocyte antigen and ABO systems remains one of the largest barriers to further expansion in renal transplantation. This barrier translates into prolonged waiting time and a greater likelihood of death. The number of highly sensitized patients on the renal transplant waiting list continues to increase. This review focuses on the options available to these patients and speculates on future directions for incompatible transplantation. Recent findingsDesensitization protocols (to remove antibodies), kidney-paired donation (to circumvent antibodies) or a hybrid technique involving a combination of both have broadened the access to transplantation for patients disadvantaged by immunologic barriers. However, the risk of antibody-mediated rejection may be increased and warrants caution. Technical advances in antibody characterization using sensitive bead immunoassays and the C1q assay and therapeutic modalities such as complement inhibitors and proteasome inhibitors have been used to avoid or confront these antibody incompatibilities. SummaryA growing body of knowledge and literature indicates that these diagnostic and therapeutic modalities can facilitate a safer and more successful treatment course for these difficult-to-treat patients. Rigorous investigations into newer interventions will help in broadening the options for these patients and also expand the living donor pool.

Allele-Specific Regulation of TCRβ Variable Gene Segment Chromatin Structure

Allelic exclusion of the murine Tcrb locus is imposed at the level of recombination and restricts each cell to produce one functional VDJβ rearrangement. Allelic exclusion is achieved through asynchronous Vβ to DJβ recombination as well as feedback inhibition that terminates recombination once a functional rearrangement has occurred. Because the accessibility of Vβ gene segment chromatin is diminished as thymocytes undergo allelic exclusion at the CD4−CD8− (double-negative) to CD4+CD8+ (double-positive) transition, chromatin regulation was thought to be an important component of the feedback inhibition process. However, previous studies of chromatin regulation addressed the status of Tcrb alleles using genetic models in which both alleles remained in a germline configuration. Under physiological conditions, developing thymocytes would undergo Vβ to DJβ recombination on one or both alleles before the enforcement of feedback. On rearranged alleles, Vβ gene segments that in germline configuration are regulated independently of the Tcrb enhancer are now brought into its proximity. We show in this study that in contrast to Vβ segments on a nonrearranged allele, those situated upstream of a functionally rearranged Vβ segment are contained in active chromatin as judged by histone H3 acetylation, histone H3 lysine 4 (K4) methylation, and germline transcription. Nevertheless, these Vβ gene segments remain refractory to recombination in double-positive thymocytes. These results suggest that a unique feedback mechanism may operate independent of chromatin structure to inhibit Vβ to DJβ recombination after the double-negative stage of thymocyte development.

Anti-Angiotensin II Type 1 Receptor and Anti-Endothelial Cell Antibodies: A Cross-Sectional Analysis of Pathological Findings in Allograft Biopsies

Background This is a cross-sectional study designed to evaluate the histologic characteristics of graft injury in the presence of anti-angiotensin II type 1 receptor antibody (AT1R-Ab) and anti-endothelial cell antibody (AECA). Methods Non-HLA antibody testing was included in the posttransplant evaluation for 70 kidney recipients. Biopsies were performed for cause for 47 patients and as protocol for the remaining 23 patients. Biopsy-proven rejection was defined according to the Banff 2009-2013 criteria. AT1R-Ab was measured on an ELISA platform. Patients were divided into 3 groups based on AT1R-Ab levels (>17, 10-17, and <10 U/ml). AECA was evaluated using an endothelial cell crossmatch (ECXM) in patients whose HLA antibody level was insufficient to cause a positive flow cytometric crossmatch. Results AT1R-Ab levels were higher in patients diagnosed with antibody mediated rejection compared to those with no rejection (P = 0.004). Glomerulitis (g) and peritubular capillaritis (ptc) scores were independently correlated with increased AT1R-Ab concentrations in the presence or absence of HLA-DSA (P = 0.007 and 0.03 for g scores; p = 0.005 and 0.03 for ptc scores). Patients with a positive ECXM had higher AT1R-Ab levels compared to those with a negative ECXM (P = 0.005). Microcirculation inflammation (MCI = g + ptc score) was higher in patients with a positive ECXM and with AT1R-Ab >17 U/ml, although this did not reach statistical significance (P = 0.07). Conclusions The data show an association between non-HLA antibodies detected in the ECXM and AT1R ELISA and microvascular injury observed in antibody mediated rejection.

Peripheral blood allogeneic microchimerism in lung and cardiac allograft recipients

We have been investigating two parameters, donor antigen‐specific hyporeactivity and peripheral blood allogeneic microchimerism, to determine whether these parameters will predict a chronic rejection‐free state and which recipients may be candidates for steroid withdrawal. We have identified donor antigen‐specific hyporeactivity for 33% (16/48) of lung and 23% (11/47) of heart recipients. For both organ groups, the hyporeactive subgroup experienced a lower incidence of chronic rejection. The probability of donor antigen‐specific hyporeactivity predicting a chronic rejection‐free state is 100% for lung and 91% for heart recipients. We have identified peripheral blood allogeneic microchimerism for 77% (20/26) of lung and 36% (9/25) of heart recipients tested at 12–18 months posttransplant. Donor antigen‐specific hyporeactivity correlates with a critical level of donor cells in lung recipients; the probability of high peripheral blood allogeneic microchimerism levels predicting a chronic rejection‐free state in lung recipients is 100%. The results in heart recipients are not as clear with a short‐, but not long‐term, trend of higher chimerism levels correlating with the development of donor antigen‐specific hyporeactivity. These results illustrate the usefulness of immune parameters to predict long‐term graft outcome in an organ‐specific manner. J. Leukoc. Biol. 66: 306–309; 1999.

A GPS for finding the route to transplantation for the sensitized patient

Purpose of reviewTo identify factors that affect the choice of route to renal transplantation for the sensitized patient. The evolution of protocols for transplanting sensitized patients has been desensitization (DES), paired donation, and most recently, paired donation combined with DES. Use of these protocols has revealed various factors that influence which route is the most likely to work for a given patient. Recent findingsThe data indicate that patient blood type and HLA sensitization have the dominant influence on what route is best for a patient but numerous other factors, particularly the number, HLA type, and ABO type of donors a patient brings to a program will also affect the likelihood of transplantation. The distribution of these factors among patients transplanted or unable to find a compatible donor can be used to calculate the probability of transplantation via paired donation. SummaryKidney paired donation with or without DES provides benefits that cannot be achieved with DES alone. However, DES may provide the fastest route to transplantation.

A Role for MAPK in Feedback Inhibition of Tcrb Recombination

The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4−CD8− double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4+CD8+ double-positive (DP) differentiation, proliferation, and feedback inhibition of further Vβ to DJβ rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of Vβ gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of Vβ chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vβ chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vβ segment accessibility and normal feedback inhibition of the Vβ to DJβ rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.