Chris Callaghan

Germinal Center Alloantibody Responses Are Mediated Exclusively by Indirect-Pathway CD4 T Follicular Helper Cells

The durable alloantibody responses that develop in organ transplant patients indicate long-lived plasma cell output from T-dependent germinal centers (GCs), but which of the two pathways of CD4 T cell allorecognition is responsible for generating allospecific T follicular helper cells remains unclear. This was addressed by reconstituting T cell-deficient mice with monoclonal populations of TCR-transgenic CD4 T cells that recognized alloantigen only as conformationally intact protein (direct pathway) or only as self-restricted allopeptide (indirect pathway) and then assessing the alloantibody response to a heart graft. Recipients reconstituted with indirect-pathway CD4 T cells developed long-lasting IgG alloantibody responses, with splenic GCs and allospecific bone marrow plasma cells readily detectable 50 d after heart transplantation. Differentiation of the transferred CD4 T cells into T follicular helper cells was confirmed by follicular localization and by acquisition of signature phenotype. In contrast, IgG alloantibody was not detectable in recipient mice reconstituted with direct-pathway CD4 T cells. Neither prolongation of the response by preventing NK cell killing of donor dendritic cells nor prior immunization to develop CD4 T cell memory altered the inability of the direct pathway to provide allospecific B cell help. CD4 T cell help for GC alloantibody responses is provided exclusively via the indirect-allorecognition pathway.

Blocking lymphotoxin signaling abrogates the development of ectopic lymphoid tissue within cardiac allografts and inhibits effector antibody responses

Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody‐mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin‐β‐receptor (LTβR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal‐center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTβR‐Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs. 2.2 in control recipients; P=0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTβR‐Ig (median survival time: 18 vs. >50 d, respectively, P=0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.—Motallebzadeh, R., Rehakova, S., Conlon, T. M., Win, T. S., Callaghan, C. J., Goddard, M., Bolton, E. M., Ruddle, N. H., Bradley, J. A., Pettigrew, G. J. Blocking lymphotoxin signaling abrogates the development of ectopic lymphoid tissue within cardiac allografts and inhibits effector antibody responses. FASEB J. 26, 51–62 (2012). www.fasebj.org

Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1u recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1u recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.

Treatment of dialysis access-associated steal syndrome with the "revision using distal inflow" technique.

Purpose Dialysis access-associated steal syndrome (DASS) is a common, serious complication of antecubital fossa (ACF) arteriovenous fistulas (AVFs). We describe our experience of the “revision using distal inflow” (RUDI) technique for the treatment of DASS and review the literature. Methods Patients underwent fistula ligation at the anastomosis with re-establishment of inflow via the proximal radial or ulnar arteries using a venous interposition graft or venous collateral. A retrospective analysis of outcomes of all patients undergoing this procedure at our center was carried out. Results Seven patients with autogenous ACF AVFs underwent the RUDI procedure, four under local anesthesia. Interposition vein grafts were used in five patients, and inflow was achieved through the proximal radial artery in four cases. The median post-operative rise in digital systolic blood pressure was 65.5 mmHg. Follow-up at 7–36 months found that three fistulas had failed (one at 8 months, two within days), two patients had died with patent fistulas, one patient was transplanted with a functional AVF, and the remaining patient continues to dialyze through the fistula. No patients developed DASS postoperatively and no further interventions were required to maintain patency. Conclusions Although RUDI was successful at treating DASS, a high rate of AVF failure was seen. With technical modifications and further experience, RUDI may become a valuable tool in the surgical armamentarium.

Regulation of Allograft Survival by Inhibitory FcγRIIb Signaling

Fcγ receptors (FcγR) provide important immunoregulation. Targeting inhibitory FcγRIIb may therefore prolong allograft survival, but its role in transplantation has not been addressed. FcγRIIb signaling was examined in murine models of acute or chronic cardiac allograft rejection by transplanting recipients that either lacked FcγRIIb expression (FcγRIIb−/−) or overexpressed FcγRIIb on B cells (B cell transgenic [BTG]). Acute heart allograft rejection occurred at the same tempo in FcγRIIb−/− C57BL/6 (B6) recipients as wild type recipients, with similar IgG alloantibody responses. In contrast, chronic rejection of MHC class II–mismatched bm12 cardiac allografts was accelerated in FcγRIIb−/− mice, with development of more severe transplant arteriopathy and markedly augmented effector autoantibody production. Autoantibody production was inhibited and rejection was delayed in BTG recipients. Similarly, whereas MHC class I–mismatched B6.Kd hearts survived indefinitely and remained disease free in B6 mice, much stronger alloantibody responses and progressive graft arteriopathy developed in FcγRIIb−/− recipients. Notably, FcγRIIb-mediated inhibition of B6.Kd heart graft rejection was abrogated by increasing T cell help through transfer of additional H2.Kd-specific CD4 T cells. Thus, inhibitory FcγRIIb signaling regulates chronic but not acute rejection, most likely because the supra-optimal helper CD4 T cell response in acute rejection overcomes FcγRIIb-mediated inhibition of the effector B cell population. Immunomodulation of FcγRIIb in clinical transplantation may hold potential for inhibiting progression of transplant arteriopathy and prolonging transplant survival.

Unlinked Memory Helper Responses Promote Long-Lasting Humoral Alloimmunity

Essential help for long-lived alloantibody responses is theoretically provided only by CD4 T cells that recognize target alloantigen, processed and presented by the allospecific B cell. We demonstrate that in an alloresponse to multiple MHC disparities, cognate help for class-switched alloantibody may also be provided by CD4 T cells specific for a second “helper” alloantigen. This response was much shorter-lived than when help was provided conventionally, by Th cell recognition of target alloantigen. Nevertheless, long-lasting humoral alloimmunity developed when T cell memory against the helper alloantigen was first generated. Costimulatory blockade abrogated alloantibody produced through naive Th cell recognition of target alloantigen but, crucially, blockade was ineffective when help was provided by memory responses to the accessory helper alloantigen. These results suggest that memory Th cell responses against previously encountered graft alloantigen may be the dominant mechanism for providing help to generate new specificities of alloantibody in transplant patients receiving immunosuppression.

Operative salvage of radiocephalic arteriovenous fistulas by formation of a proximal neoanastomosis

OBJECTIVE We examined the outcomes of radiocephalic arteriovenous fistulas salvaged by formation of a neoanastomosis in the proximal cephalic vein segment. METHODS Patients with a radiocephalic arteriovenous fistula revised by formation of a neoanastomosis in the proximal cephalic vein segment were identified from a prospectively maintained database and outcomes retrospectively analyzed. RESULTS Eighty patients had 81 radiocephalic arteriovenous fistulas revised by formation of a neoanastomosis in the proximal cephalic vein segment. Failure to mature was the indication for revision in 39 (48.1%), 17 (21.0%) were revised for poor flows during dialysis, and 25 (30.9%) were performed for thrombosis. Primary patency of the 81 neoanastomoses at 12, 24, and 36 months was 78.5%, 68.9%, and 54.9%, respectively. Compared with neoanastomoses that were performed on 50 immature radiocephalic arteriovenous fistulas, those performed on the 31 mature fistulas exhibited improved patency rates (P = .04). There was no difference in the primary patency of the neoanastomosis between those performed for 25 failed fistulas and 56 failing (but patent) fistulas (P = .15). There was one case (1.2%) each of bleeding, infection, and steal after neoanastomosis. Four patients (4.9%) required further interventions on their neoanastomoses. CONCLUSIONS Operative salvage of radiocephalic arteriovenous fistulas by formation of a neoanastomosis in the proximal cephalic vein segment demonstrates good patency and low complication rates and can be performed with reasonably good results in patients with failed or failing (but patent) radiocephalic arteriovenous fistulas. These patients should not automatically proceed to elbow fistula formation; rather, proximal neoanastomosis should be considered.

Early Outcomes of the New UK Deceased Donor Kidney Fast-Track Offering Scheme

Background The UK Kidney Fast-Track Scheme (KFTS) was introduced in 2012 to identify kidneys at high risk of discard and to rapidly facilitate transplantation. A retrospective analysis of kidneys transplanted through the KFTS was undertaken. Methods UK Transplant Registry data were collected on deceased donor kidneys implanted between November 1, 2012, and April 30, 2015, (donation after brain death [DBD] donors) and March 1, 2013, and April 30, 2015 (donation after circulatory death [DCD] donors). Posttransplant outcomes included 1-year estimated glomerular filtration rate and death-censored graft survival (DCGS). Results Over the study period, 523 deceased donor kidneys were transplanted through the KFTS and 4174 via the standard National Kidney Allocation Scheme (NKAS). Kidneys in the KFTS were more likely to be from older diabetic donors, had a higher frequency of poor ex vivo perfusion, had longer cold ischemic times, and were transplanted into older recipients. One-year DCGS of KFTS and NKAS DBD donor kidneys was similar (94% vs 95%; P = 0.70), but for DCD donor kidneys, DCGS was lower in those allocated via the KFTS (91% versus 95%; P = 0.04). Median 1-year estimated glomerular filtration rate for DBD donor kidneys was lower in those allocated via the KFTS (49 vs 52 mL/min per 1.73 m2; P = 0.01), but for DCD kidneys, there was no difference (45 vs 48 mL/min per 1.73 m2; P = 0.10). Conclusions Although KFTS kidneys have less favorable donor, graft, and recipient risk factors than NKAS kidneys, short-term graft and patient outcomes are acceptable. National schemes that identify and rapidly offer kidneys at high risk of discard may contribute to minimizing the unnecessary discard of organs.

The Utility of Routine Ultrasound Imaging after Elective Transplant Ureteric Stent Removal

Background. Ureteric stent insertion during kidney transplantation reduces the incidence of major urological complications (MUCs). We evaluated whether routine poststent removal graft ultrasonography (PSRGU) was useful in detecting MUCs before they became clinically or biochemically apparent. Methods. A retrospective analysis was undertaken of clinical outcomes following elective stent removals from adult single renal transplant recipients (sRTRs) at our centre between 1 January 2011 and 31 December 2013. Results. Elective stent removal was performed for 338 sRTRs. Of these patients, 222 had routine PSRGU (median (IQR) days after stent removal = 18 (11–31)), 79 had urgent PSRGU due to clinical or biochemical indications, 12 had CT imaging, and 25 had no further renal imaging. Of the 222 sRTRs who underwent routine PSRGU, 210 (94.6%) had no change of management, three (1.4%) required repeat imaging only, and eight patients (3.6%) had incidental (nonureteric) findings. One patient (0.5%) had nephrostomy insertion as a result of routine PSRGU findings, but no ureteric stenosis was identified. Of 79 patients having urgent PSRGU after elective stent removal, three patients required transplant ureteric reimplantation. Conclusions. This analysis found no evidence that routine PSRGU at two to three weeks after elective stent removal provides any added value beyond standard clinical and biochemical monitoring.

Beyond Poiseuille: Preservation Fluid Flow in an Experimental Model

Poiseuilles equation describes the relationship between fluid viscosity, pressure, tubing diameter, and flow, yet it is not known if cold organ perfusion systems follow this equation. We investigated these relationships in an ex vivo model and aimed to offer some rationale for equipment selection. Increasing the cannula size from 14 to 20 Fr increased flow rate by a mean (SD) of 13 (12)%. Marshalls hyperosmolar citrate was three times less viscous than UW solution, but flows were only 45% faster. Doubling the bag pressure led to a mean (SD) flow rate increase of only 19 (13)%, not twice the rate. When external pressure devices were used, 100 mmHg of continuous pressure increased flow by a mean (SD) of 43 (17)% when compared to the same pressure applied initially only. Poiseuilles equation was not followed; this is most likely due to “slipping” of preservation fluid within the plastic tubing. Cannula size made little difference over the ranges examined; flows are primarily determined by bag pressure and fluid viscosity. External infusor devices require continuous pressurisation to deliver high flow. Future studies examining the impact of perfusion variables on graft outcomes should include detailed equipment descriptions.