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The high-affinity receptor for IgG and the T cell receptor-CD3 complex are associated with the gamma subunit of immunoglobulin Fc receptors which lead to immunocompromised mice when target-disrupted. The macrophages from these mice show inability to phagocytose antibody-coated particles and exhibit defective NK cell-mediated antibody-dependent cytotoxicity and mast cell-mediated allergic response. However, T cell development in these mice is not compromised by the absence of the gamma chain.

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8+ Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation. [ABSTRACT FROM AUTHOR]

Abstract: Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts. [Copyright &y& Elsevier]

Abstract: Alloantigen specific CD8 T suppressor cells can be generated in vitro either by multiple stimulations of CD3 T cells with allogeneic APC or by single stimulation in primary MLC containing recombinant ILT3.Fc protein. The aim of the present study was to determine whether multiple MLC stimulation induced in CD8+ CD28− T suppressor cells molecular changes that are similar to those observed in CD8 T suppressor cells from primary MLC containing ILT3.Fc protein. Our study demonstrates that the characteristic signatures of CD8 T suppressor cells, generated by either of these methods are the same consisting of up-regulation of the BCL6 transcriptional repressor and down-regulation of inflammatory microRNAs, miR-21, miR-30b, miR-146a, and miR-155 expression. In conclusion microRNAs which are increased under inflammatory conditions in activated CD4 and CD8 T cells with helper or cytotoxic function show low levels of expression in CD8 T cells which have acquired antigen-specific suppressor activity. [Copyright &y& Elsevier]

Aim The C1qScreen assay detects complement binding HLA antibodies. Recent studies in cardiac transplant recipients suggest that the presence of C1q positive donor specific antibodies (DSA) is associated with increased risk of antibody mediated rejection (AMR) early post-transplant. However, in some patients with documented AMR, C1q binding DSA are not detected suggesting poor sensitivity of the C1qScreen. In this study we compared the ability to detect DSA by the standard C1qScreen vs the anti-human globulin (AHG) enhanced C1q assay in a cohort of 37 heart transplant recipients with AMR. Methods 37 heart transplant recipients with biopsy proven AMR were studied. Sera were collected at the time of AMR and screened for the presence of DSA using the standard C1qScreen, the AHG enhanced C1q and the LABScreen single antigen bead (SAB)-IgG assay. The number of DSA detected and the average DSA MFI were compared. Results The C1qScreen failed to identify DSA in 14/37 (38%) of patients with biopsy-proven, SAB-IgG positive and/or C4d positive, AMR (Fig. 1). In contrast, the AHG enhanced C1q assay identified DSA in the majority of patients (32/37; 86.5%), approaching the rate of DSA detected by the SAB-IgG assay (34/37; 92%). The average number of DSA/patient identified was also improved with the AHG enhanced C1q assay (2.47 vs 1.56 DSA/patient; AHG enhanced C1q vs C1qScreen). Finally, the mean MFI of DSA was significantly increased (22,191.3 +/− 7454 AHG enhanced C1q vs 11,993.6 +/− 7608 C1qScreen; p 〈 0.001; Fig. 2) confirming that the sensitivity of the AHG enhanced C1q assay is improved. Conclusions The C1qScreen failed to detect DSA in a significant number of heart transplant recipients with biopsy proven AMR and thus, cannot be used to accurately detect pathogenic DSA. The AHG enhanced C1q assay was more sensitive, identifying DSA in the majority of AMR patients and improving detection of low level antibodies. Based on our findings, the AHG enhanced C1q assays may be useful in post-transplant monitoring to detect pathogenic DSA. [ABSTRACT FROM AUTHOR]

Aim The aim of this study was to evaluate the prognostic value of the C1q-SPA assay in recipients of kidney from deceased donors. Methods The study population consisted of 123 recipients transplanted at Columbia University Medical Center between January 1 and December 31, 2012. None of the patients showed DSA that could be detected by complement dependent lymphocytotoxicity. Sera obtained from each patient prior to transplantation were evaluated for donor-specific anti-HLA IgG antibody (DSA) by Luminex single-antigen bead (SAB) testing (SAB-IgG). SAB-IgG positive sera were further tested by Luminex SAB-based assay detecting C1q-binding HLA antibodies (SAB-C1q). Results The one year kidney allograft survival was 96.6% in this patient population. Four recipients had an episode of AMR although 27 showed DSA detectable by SAB-IgG. C1q testing shows DSA in 7 sera among the SAB-IgG positive results (7/27, 26%), 3 of which were in patients with AMR and 4 in patients without AMR. Conclusions There was a positive correlation between the presence of C1q detectable DSA in pre-transplantation sera and development of AMR. However, the correlation between SAB-IgG, SAB-C1q and rejection was weaker, suggesting the need of further evaluation of methods that can predict the risk of AMR, without affecting the patients’ chances of receiving a transplant. [ABSTRACT FROM AUTHOR]

Aim: Exosomes may contribute to cell-cell communication at the level of immunological synapses or at a distance, by delivering mRNA and miRNA modifying the recipient cell protein production and gene expression. The present study aims to determine whether the effect of ILT3Fc on allostimulated CD8 T cells is accompanied by changes in the miRNA content of exosomes generated during allostimulation. Methods: Exosomes were isolated from culture supernatants of MLC in which CD3 T cells had been stimulated with allogeneic APC in the presence or absence of ILT3Fc. The identity and relative quantity of the exosomal miRNA content was determined by RT-PCR. The inhibitory or inflammatory properties of these exosomes were evaluated by 24h pretreatment of sorted CD8 Ts from MLC containing ILT3Fc. The CD8 T cells were then tested for the effect on the proliferation of naive, syngeneic T cells primed in response to the same stimulating APC. Results: CD8 Ts primed in the presence of ILT3Fc inhibit syngeneic CD4 T cell responses by >80%. When CD8 Ts are pretreated with inflammatory exosomes, and are used in MLC at lower than 1:1 ratios to respoding Th cells, they lose inhibitory activity. These exosomes contain miRNA-21, -30b, -146a, -155, -395, and Let7a, known to be increased in T cells during activation. In contrast, exosomes obtained from T cells primed in the presence of ILT3Fc, do not abolish CD8 Ts inhibitory potential, suggesting that ILT3Fc blocks the release into the medium or alters the RNA content of inflammatory exosomes. Conclusions: Micro RNA contained by exosomes released from alloactivated T cells enhance T helper activity even in the presence of T suppressor cells. This suggests the potential use of exosomal inflammatory miRNA as a monitor of cellular immune responses against the graft. Furthermore, such exosomes may be of use in eliciting immune T cell responses against tumors or microbial agents. [Copyright &y& Elsevier]

Aim: The aim of this study was to establish whether the presence of DSA detected by Luminex based solid-phase assay (SPA) correlates with the cross-match results performed by complement-dependent cytotoxicity (CDC) in deceased donor kidney transplantation. Methods: Between January 1, 2012 and December 31, 2012, lymphocytes from a total of 398 deceased donors were cross-matched by our laboratory. For each donor, sera from an average of 6.24 patients were selected for direct cross-matching by CDC, amounting to a total of 2,485 cross-matches. Pre-transplant sera were screened by CDC on HLA reference panel, and by LABScreen using HLA class I and class II antigen coated beads. Results: Of the 2,485 patients cross-matched, 870 had DSA and should have been excluded as potential transplant recipients on this basis. However, 39.3% (342/870) of the DSA positive population showed no cytotoxic antibodies to the putative donor reflecting the non-Complement fixing nature of the antibodies detected by SPA and/or their low affinity for their target antigen. Since 60.7% (528/870) of patients with DSA showed positive CDC cross-matches, there was still a highly significant correlation (P<0.0001) between SPA (virtual) and cytotoxicity (actual) cross-matches. Of the 398 deceased donors which we have cross-matched, 123 were allocated to our program. The mean age of recipients at time of transplant was 55 years old (range: 22-78). While all 123 recipients were cross match negative by CDC, 27 showed SPA detectable DSA. The overall one-year graft survival was 96.6%. Two grafts were lost for non-immunologic reasons by patients without DSA (N=96) and two kidneys were rejected by patients with DSA (N=27). (P=NS). Conclusions: In this study, we have shown that 39.3% of patients with DSA would have been denied the transplant if it was solely based on the virtual cross-match result. Furthermore, there was no statistically significant difference between graft function at one year in patients with or without DSA. [Copyright &y& Elsevier]