Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

We used a systems biological approach to study innate and adaptive responses to influenza vaccination in humans, during 3 consecutive influenza seasons. Healthy adults were vaccinated with inactivated (TIV) or live attenuated (LAIV) influenza vaccines. TIV induced greater antibody titers and enhanced numbers of plasmablasts than LAIV. In TIV vaccinees, early molecular signatures correlated with, and accurately predicted, later antibody titers in two independent trials. Interestingly, the expression of Calcium/calmodulin-dependent kinase IV (CamkIV) at day 3 was inversely correlated with later antibody titers. Vaccination of CamkIV −/− mice with TIV induced enhanced antigen-specific antibody titers, demonstrating an unappreciated role for CaMKIV in the regulation of antibody responses. Thus systems approaches can predict immunogenicity, and reveal new mechanistic insights about vaccines.

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Many strategies have been proposed to induce tolerance to transplanted tissue in rodents; however, few if any have shown equal efficacy when tested in nonhuman primate transplant models. We hypothesized that a critical distinction between specific pathogen-free mice and nonhuman primates or human patients is their acquired immune history. Here, we show that a heterologous immune response — specifically, virally induced alloreactive memory — is a potent barrier to tolerance induction. A critical threshold of memory T cells is needed to promote rejection, and CD8+ “central” memory T cells are primarily responsible. Finally, treatment with deoxyspergualin, an inhibitor of NF-κB translocation, together with costimulation blockade, synergistically impairs memory T cell activation and promotes antigen-specific tolerance of memory. These data offer a potential explanation for the difficulty encountered when inducing tolerance in nonhuman primates and human patients and provide insight into the signaling pathways essential for memory T cell activation and function

Renal transplant patients sensitized to HLA antigens comprise nearly one-third of the UNOS wait-list and receive 14% of deceased donor (DD) transplants, a rate half that of unsensitized patients. Between 1999 and 2003, we performed 492 adult renal transplants from DD; 120 patients (∼25%) had a panel reactive antibody (PRA) of >30%, with nearly half (n = 58) having a PRA of >80%. Our approach is based upon highresolution solid-phase HLA antibody analysis to identify class I/II antibodies and a 'virtual crossmatch' to predict compatible donor/recipient combinations. Recipients are excluded from the United Network for Organ Sharing match run if donors possess unacceptable antigens. Thus, when sensitized patients appear on the match run, they have a high probability of a negative final crossmatch. Here, we describe our 5-year experience with this approach. Five-year graft survival ranged from 66% to 70% among unsensitized (n = 272), moderately sensitized (PRA < 30%, n = 100) and highly sensitized (>30% PRA; n = 120) patients, equal to the average national graft survival (65.7%). The application of this approach (the Emory Algorithm) provides a logical and systematic approach to improve the access of sensitized patients to DD organs and promote more equitable allocation to a highly disadvantaged group of patients awaiting renal transplantation.

For patients with chronic renal and liver diseases, simultaneous liver and kidney transplantation (SLKT) is the best therapeutic option. The role of a pretransplant donor-specific antibody (DSA) in SLKT is unclear. We report the results of a retrospective review from 7/08 to 10/09 of SLKT at our institution. Monitoring of DSA was performed using single antigen bead assay. Between 7/08 and 10/09, there were six SLKT who had preformed DSA and positive XM (four class I and II DSA, one class I DSA only, one class II only). One-year patient and renal graft survival was 83%. Death-censored liver allograft survival was 100%. Acute humoral rejection (AHR) of the kidney occurred in 66% (three with both class I and II DSA and one with only class II DSA) of patients. In those with AHR, class I antibodies were rapidly cleared (p < 0.01) while class II antibodies persisted (p = 0.25). All patients who had humoral rejection of their kidney had preformed anticlass II antibodies. Liver allografts may not be fully protective of the renal allograft, especially with pre-existing MHC class II DSA. Long-term and careful follow-up will be critical to determine the impact of DSA on both allografts. Key words: Antibody-mediated rejection, kidney allograft, liver allograft Abbreviations: SLKT, simultaneous liver kidney transplantation; DSA, donor-specific antibody; AHR, acute humoral rejection; DTT, dithiothreitol; cMCF, delta mean channel fluorescence; MESF, molecules of equivalent soluble fluorescence; MFI, mean fluroescence intensity; AMR, antibody-mediated rejection; HLA, human leukocyte antigen; UNOS, United Network for Organ Sharing; MELD, modified end-stage liver disease; MHC, major histocompatibility complex; IVIG, intravenous immunoglobulin G.

Summary The growing success of solid organ transplantation poses unique challenges for the implementation of effective immunization strategies. Although live attenuated vaccines have proven benefits for the general population, immunosuppressed patients are at risk for unique complications such as infection from the vaccine because of lack of both clearance and containment of a live attenuated virus. Moreover, while vaccination strategies using killed organisms or purified peptides are believed to be safe for immunosuppressed patients, they may have reduced efficacy in this population. The current lack of knowledge of the basic safety and efficacy of vaccination strategies in the immunosuppressed has limited the development of guidelines regarding vaccination in this population. Recent fears of influenza pandemics and potential attacks by weaponized pathogens such as smallpox heighten the need for increased knowledge. Herein, we review the current understanding of the effects of immunosuppressants on the immune system and the ability of the suppressed immune system to respond to vaccination. This review highlights the need for systematic, longitudinal studies in both humans and nonhuman primates to understand better the defects in innate and adaptive immunity in transplant recipients, thereby aiding the development of strategies to vaccinate these individuals.

We used the cell-attached patch clamp technique to investigate the interaction ofexogenous prostaglandins (PG), intracellular ICa2"I , and protein kinase C (PKC) on the high selectivity, 4 pS Na' channel found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) cultures grown on collagen supports with 1.5 MiM aldosterone. Application of 0.5 ,M PGE2 to the basolateral membrane decreased mean NP0 (number of channels times the open probability) for apical Na' channels by 46.5% (n = 9). There was no consistent change in NP0 after apical 0.5 MtM PGE2 (n = 12) or after apical or Invest. 1992Invest. . 90:1328Invest. -1334