Geetha Chalasani

Regulatory dendritic cell infusion prolongs kidney allograft survival in nonhuman primates.

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine‐mobilized donor blood monocytes in vitamin D3 and IL‐10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand‐1 (B7‐H1), and were resistant to pro‐inflammatory cytokine‐induced maturation. They were infused intravenously (3.5–10 × 106/kg), together with the B7‐CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day −2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg‐treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor‐specific alloantibody levels. Immunologic monitoring also revealed regulation of donor‐reactive memory CD95+ T cells and reduced memory/regulatory T cell ratios in DCreg‐treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell‐ and Ab‐mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

B cells help alloreactive T cells differentiate into memory T cells

B cells are recognized as effector cells in allograft rejection that are dependent upon T cell help to produce alloantibodies causing graft injury. It is not known if B cells can also help T cells differentiate into memory cells in the alloimmune response. We found that in B‐cell‐deficient hosts, differentiation of alloreactive T cells into effectors was intact whereas their development into memory T cells was impaired. To test if B cell help for T cells was required for their continued differentiation into memory T cells, activated T cells were sorted from alloimmunized mice and transferred either with or without B cells into naïve adoptive hosts. Activated T cells cotransferred with B cells gave rise to more memory T cells than those transferred without B cells and upon recall, mediated accelerated rejection of skin allografts. Cotransfer of B cells led to increased memory T cells by enhancing activated CD4 T‐cell proliferation and activated CD8 T‐cell survival. These results indicate that B cells help alloreactive T‐cell differentiation, proliferation and survival to generate optimal numbers of functional memory T cells.

The yin and yang of B cells in graft rejection and tolerance

Various lineages of B cells are being increasingly recognized as important players in the etiology and prognosis of both acute and chronic graft rejection. The role of immature, chronically activated B cells, as efficient antigen-presenting cells, supporting recalcitrant cell-mediated graft rejection and late lineage B cells driving humoral rejections, is being increasingly recognized. This review captures the recent literature on this subject and discusses the various roles of the B cell in renal graft rejection and conversely, also in graft tolerance, both in animal and human studies. In addition, novel therapies targeting specific B-cell lineages in graft rejection are also discussed, with a view to developing more targeted therapies for graft rejection.

Exosomes released from Mycoplasma infected tumor cells activate inhibitory B cells.

Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.

The Roles of CD8 Central and Effector Memory T-Cell Subsets in Allograft Rejection

The contribution of secondary lymphoid tissue‐homing central memory T cells (TCM) and peripheral tissue‐homing effector memory T cells (TEM) to allograft rejection is not known. We tested whether TEM is the principal subset responsible for allograft rejection due to the nonlymphoid location of target antigens. Skin allograft rejection was studied after transferring either CD8 TCM or TEM to wild‐type mice and to mice that lack secondary lymphoid tissues. We found that CD8 TCM and TEM were equally effective at rejecting allografts in wild‐type hosts. However, CD8 TEM were significantly better than TCM at rejecting allografts in the absence of secondary lymphoid tissues. CD8 TCM were dependent upon secondary lymphoid tissues more than TEM for optimal differentiation into effectors that migrate into the allograft. Recall of either CD8 TCM or TEM led to accumulation of TEM after allograft rejection. These findings indicate that either CD8 TCM or TEM mediate allograft rejection but TEM have an advantage over TCM in immune surveillance of peripheral tissues, including transplanted organs.

The Polyomavirus BK Large T-Antigen-Derived Peptide Elicits an HLA-DR Promiscuous and Polyfunctional CD4+ T-Cell Response

ABSTRACT BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4+ T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4+ T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols.

Innate immunity alone is not sufficient for chronic rejection but predisposes healed allografts to T cell-mediated pathology.

BACKGROUND Acute allograft rejection is dependent on adaptive immunity, but it is unclear whether the same is true for chronic rejection. Here we asked whether innate immunity alone is sufficient for causing chronic rejection of mouse cardiac allografts. METHODS We transplanted primarily vascularized cardiac grafts to recombinase activating gene-knockout (RAG(-/-)) mice that lack T and B cells but have an intact innate immune system. Recipients were left unmanipulated, received adjuvants that stimulate innate immunity, or were reconstituted with B-1 lymphocytes to generate natural IgM antibodies. In a second model, we transplanted cardiac allografts to mice that lack secondary lymphoid tissues (splenectomized aly/aly recipients) and studied the effect of NK cell inactivation on T cell-mediated chronic rejection. RESULTS Acute cardiac allograft rejection was not observed in any of the recipients. Histological analysis of allografts harvested 50 to 90 days after transplantation to RAG(-/-) mice failed to identify chronic vascular or parenchymal changes beyond those observed in control syngeneic grafts. Chronic rejection of cardiac allografts parked in splenectomized aly/aly mice was observed only after the transfer of exogenously activated T cells. NK inactivation throughout the experiment, or during the parking period alone, reduced the severity of T cell-dependent chronic rejection. CONCLUSIONS The innate immune system alone is not sufficient for causing chronic rejection. NK cells predispose healed allografts to T cell-dependent chronic rejection and may contribute to chronic allograft pathology.

Type I Interferons Are Not Critical for Skin Allograft Rejection or the Generation of Donor‐Specific CD8+ Memory T Cells

Type I interferons (IFN‐I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN‐I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor‐specific CD8+ T‐cell responses in mice that lack the IFN‐I receptor (IFN‐IR−/−). We found that IFN‐IR−/− mice reject fully allogeneic wild‐type skin grafts at the same rate as wild‐type recipients. Similarly, allograft rejection was not delayed if IFN‐IR−/− male skin was transplanted to syngeneic IFN‐IR−/− female mice. Quantitation of the male (H‐Y)‐specific CD8+ T‐cell response in these mice revealed normal generation of donor‐specific CD8+ effector T cells but fourfold reduction in CD8+ memory T cells. Memory CD8+ T cells generated in the absence of IFN‐IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN‐IR−/− mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN‐1. Our findings indicate that IFN‐I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor‐specific CD8+ T cells into long‐lived, functional memory T cells.

Role of secondary lymphoid tissues in primary and memory T-cell responses to a transplanted organ

Secondary lymphoid tissues are the hub of adaptive immune responses wherein rare cognate lymphocytes encounter dendritic cells bearing antigen from peripheral tissues and differentiate into effector and memory cells that eliminate antigen. It is accepted that immune responses against microbial and tumor antigens are initiated within secondary lymphoid tissues. There is less agreement on whether the same principle applies to immune responses to a transplanted organ because an allograft expresses foreign major histocompatibility complex and contains donor antigen presenting cells that could activate T cells directly in situ leading to rejection. Recent studies confirm that although naïve T cells can be primed within the allograft, their differentiation to effect rejection is dependent on secondary lymphoid tissues. Antigen-experienced memory T cells, unlike Naïve T cells, function largely independent of secondary lymphoid tissues to cause allograft rejection. In an alloimmune response, secondary lymphoid tissues support not only immune activation but also immune regulation essential for allograft survival. Here, we will review recent findings and discuss the role of secondary lymphoid tissues in primary and memory alloimmune responses.

Non-Antibody Mediated Roles of B Cells in Allograft Survival

Antibody production is unquestionably a key effector function of B cells that remains a formidable barrier against long-term graft survival. However, emerging evidence indicates that B cells play a key role in shaping the effector responses by mechanisms that extend beyond their function as antibody producing cells. B cell depletion in transplant recipients has resulted in paradoxical outcomes of increased graft rejection versus improved graft function, implying that B cells function as both enhancers and regulators of the alloimmune response. Based on findings from animal and human studies, we address mechanisms by which B cells modulate the immune response and highlight their role in promoting allograft rejection or tolerance.