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Humoral allogeneic immunity driven by anti-HLA donor-specific antibodies (DSAs) and antibody-mediated mediated rejection (ABMR) significantly impede prolonged survival of organ allografts after transplantation. Although the importance of T follicular helper (T(FH)) cells in controlling antibody responses has been long established, their role in directing DSA generation leading to ABMR was only recently appreciated in the clinical setting of organ transplantation. In this review, we provide a comprehensive summary of the current knowledge on the biology of human T(FH) cells as well as their circulating (cT(FH)) counterparts and describe their pivotal role in driving humoral alloimmunity. In addition, we discuss the intrinsic effects of current induction therapies and maintenance immunosuppressive drugs as well as of biotherapies on T(FH) cells; and provide future directions and novel opportunities of biotherapeutic targeting of T(FH) cells that have the potential of bringing the prophylactic and curative treatments of ABMR towards personalized and precision medicine.

Alloimmune responses driven by donor-specific antibodies (DSAs) can lead to antibody-mediated rejection (ABMR) in organ transplantation. Yet, the cellular states underlying alloreactive B cell responses and the molecular components controlling them remain unclear. Using high-dimensional profiling of B cells in a cohort of 96 kidney transplant recipients, we identified expanded numbers of CD27+CD21– activated memory (AM) B cells that expressed the transcription factor T-bet in patients who developed DSAs and progressed to ABMR. Notably, AM cells were less frequent in DSA+ABMR– patients and at baseline levels in DSA– patients. RNA-Seq analysis of AM cells in patients undergoing ABMR revealed these cells to be poised for plasma cell differentiation and to express restricted IGHV sequences reflective of clonal expansion. In addition to T-bet, AM cells manifested elevated expression of interferon regulatory factor 4 and Blimp1, and upon coculture with autologous T follicular helper cells, differentiated into DSA-producing plasma cells in an IL-21–dependent manner. The frequency of AM cells was correlated with the timing and severity of ABMR manifestations. Importantly, T-bet+ AM cells were detected within kidney allografts along with their restricted IGHV sequences. This study delineates a pivotal role for AM cells in promoting humoral responses and ABMR in organ transplantation and highlights them as important therapeutic targets.

BACKGROUND: Although antibody-mediated rejection (ABMR) has been long recognized as a leading cause of allograft failure after kidney transplantation, the cellular and molecular processes underlying the induction of deleterious donor-specific antibody (DSA) responses remain poorly understood. METHODS: Using high-dimensional flow cytometry, in vitro assays, and RNA sequencing, we concomitantly investigated the role of T follicular helper (T(FH)) cells and B cells during ABMR in 105 kidney transplant recipients. RESULTS: There were 54 patients without DSAs; of those with DSAs, ABMR emerged in 20 patients, but not in 31 patients. We identified proliferating populations of circulating T(FH) cells and activated B cells emerging in blood of patients undergoing ABMR. Although these circulating T(FH) cells comprised heterogeneous phenotypes, they were dominated by activated (ICOS(+)PD-1(+)) and early memory precursor (CCR7(+)CD127(+)) subsets, and were enriched for the transcription factors IRF4 and c-Maf. These circulating T(FH) cells produced large amounts of IL-21 upon stimulation with donor antigen and induced B cells to differentiate into antibody-secreting cells that produced DSAs. Combined analysis of the matched circulating T(FH) cell and activated B cell RNA-sequencing profiles identified highly coordinated transcriptional programs in circulating T(FH) cells and B cells among patients with ABMR, which markedly differed from those of patients who did not develop DSAs or ABMR. The timing of expansion of the distinctive circulating T(FH) cells and activated B cells paralleled emergence of DSAs in blood, and their magnitude was predictive of IgG3 DSA generation, more severe allograft injury, and higher rate of allograft loss. CONCLUSIONS: Patients undergoing ABMR may benefit from monitoring and therapeutic targeting of T(FH) cell–B cell interactions.

Introduction The cellular events that contribute to generation of donor-specific anti-HLA antibodies (DSA) post-kidney transplantation (KTx) are not well understood. Characterization of such mechanisms could allow tailoring of immunosuppression to benefit sensitized patients. Methods We prospectively monitored circulating T follicular helper (cTFH) cells in KTx recipients who received T-cell depleting (thymoglobulin, n = 54) or T-cell nondepleting (basiliximab, n = 20) induction therapy from pre-KTx to 1 year post-KTx and assessed their phenotypic changes due to induction and DSA occurrence, in addition to healthy controls (n = 13), for a total of 307 blood samples. Results Before KTx, patients displayed comparable levels of resting, central memory cTFH cells with similar polarization to those of healthy controls. Unlike basiliximab induction, thymoglobulin induction significantly depleted cTFH cells, triggered lymphopenia-induced proliferation that skewed cTFH cells toward increased Th1 polarization, effector memory, and elevated programmed cell death protein 1 (PD-1)int/hi expression, resembling activated phenotypes. Regardless of induction, patients who developed DSA post-KTx, harbored pre-KTx donor-reactive memory interleukin (IL)-21+ cTFH cells and showed higher % cTFH and lower % of T regulatory (TREG) cells post-KTx resulting in elevated cTFH:TREG ratio at DSA occurrence. Conclusion Induction therapy distinctly shapes cTFH cell phenotype post-KTx. Monitoring cTFH cells before and after KTx may help detect those patients prone to DSA generation post-KTx., Graphical abstract

Human regulatory dendritic cells (DCreg) were generated from CD14 immunobead-purified or elutriated monocytes in the presence of vitamin D3 and IL-10. They exhibited similar, low levels of costimulatory CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PD-L1) and IL-10 production compared to control immature DC (iDC). Following Toll-like receptor 4 ligation, unlike control iDC, DCreg resisted phenotypic and functional maturation and further upregulated PD-L1:CD86 expression. Whereas LPS-stimulated control iDC (mature DC; matDC) secreted pro-inflammatory tumor necrosis factor but no IL-10, the converse was observed for LPS-stimulated DCreg. DCreg weakly stimulated naive and memory allogeneic CD4+ and CD8+ T cell proliferation and IFNγ, IL-17A and perforin/granzyme B production in MLR. Their stimulatory function was enhanced however, by blocking PD-1 ligation. High-throughput T cell receptor (TCR) sequencing revealed that, among circulating T cell subsets, memory CD8+ T cells contained the most alloreactive TCR clonotypes and that, while matDC expanded these alloreactive memory CD8 TCR clonotypes, DCreg induced more attenuated responses. These findings demonstrate the feasibility of generating highly-purified GMP-grade DCreg for systemic infusion, their influence on the alloreactive T cell response, and a key mechanistic role of the PD1 pathway.

International audience; Background. The human regulatory macrophage (Mreg) has emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in solid organ transplant recipients. In this brief report, dehydrogenase/reductase 9 (DHRS9) is identified as a robust marker of human Mregs.Methods. The cognate antigen of a mouse monoclonal antibody raised against human Mregs was identified as DHRS9 by immunoprecipitation and MALDI-MS sequencing. Expression of DHRS9 within a panel of monocyte-derived macrophages was investigated by quantitative PCR, immunoblotting and flow cytometry.Results. DHRS9 expression discriminated human Mregs from a panel of in vitro derived macrophages in other polarisation states. Likewise, DHRS9 expression distinguished Mregs from a variety of human monocyte-derived tolerogenic antigen-presenting cells in current development as cell-based immunotherapies, including Tol-DC, Rapa-DC, DC-10, and PGE 2-induced myeloid-derived suppressor cells. A subpopulation of DHRS9-expressing human splenic macrophages was identified by immunohistochemistry. Expression of DHRS9 was acquired gradually during in vitro development of human Mregs from CD14 + monocytes and was further enhanced by IFN-γ treatment on day 6 of culture. Stimulating Mregs with 100 ng/mL lipopolysaccharide for 24 hours did not extinguish DHRS9 expression. Dhrs9 was not an informative marker of mouse Mregs. Conclusion. DHRS9 is a specific and stable marker of human Mregs.

The recently described T follicular helper (Tfh) cell is required for the production of high affinity antibody. After contact with follicular dendritic cells, Tfh cells move into the germinal centre and provide help to B cells both by direct B cell-T cell interaction and production of IL-21. This drives proliferation, differentiation, and affinity maturation of the B cells to produce plasma cells capable of secreting high-affinity antibody. Circulating Tfh cells are produced by movement of Tfh cells from lymph nodes after dendritic cell contact. A reduction of Tfh cell-associated molecules is linked with increased expression of other chemokine receptors to form Th1-, Th2-, and Th17-like Tfh cells. These circulating Tfh cells are able to help B cells in vitro and to move into target tissues to support antibody production. Alloantibody production is dependent on T-cell help via the indirect pathway. Antibody-mediated rejection is therefore dependent on Tfh cells. Animal data suggest that Tfh cells and B cells migrate to the allograft and are involved in alloantibody production within a tertiary lymphoid organ. There is some data supporting the same process within human allografts. The requirement for T-cell help provides a potential therapeutic target in the treatment of antibody-mediated rejection.

Purpose The short- and long-term effects of 90 Yttrium microspheres therapy for hepatocellular carcinoma (HCC) on peripheral blood lymphocytes are unknown and were therefore examined. Methods and Materials Ninety-two HCC patients were enrolled in a 90 Yttrium therapy study and routine blood counts were examined as part of standard clinical monitoring. Results We found an early, profound, and prolonged lymphopenia. In a subsequent cohort of 25 additional HCC patients, prospective flow cytometric immune-monitoring analysis was performed to identify specific changes on distinct lymphocyte subsets ( i.e. , CD3, CD4, CD8 T, and CD19 B lymphocytes) and NK cells absolute numbers, in addition to the granulocytes and platelets subsets. We found that the pretreatment lymphocyte subset absolute numbers (with the exception of NK cells) had a tendency to be lower compared with healthy control values, but no significant differences were detected between groups. Posttherapy follow-up revealed that overall, all lymphocyte subsets, except for NK cells, were significantly (>50% from pretherapy values), promptly (as early as 24 h) and persistently (up to 30 months) depleted post- 90 Yttrium microspheres therapy. In contrast, granulocytes increased rapidly (24 h) to compensate for lymphocyte depletion, and remained increased at 1-year after therapy. We further stratified patients into two groups, according to survival at 1 year. We found that lack of recovery of CD19, CD3, CD8, and especially CD4 T cells was linked to poor patient survival. No fungal or bacterial infections were noted during the 30-month follow-up period. Conclusions The results show that lymphocytes (and not granulocytes, platelets, or NK cells) are sensitive to hepatic arterial 90 Yttrium without associated clinical toxicity, and lack of lymphocyte recovery (possibly leading to dysregulation of adaptive cellular immunity) posttherapy indicates poor survival.

Serial EBV load monitoring of clinically asymptomatic pediatric thoracic organ transplant patients has identified three groups of children who exhibit undetectable (16,000 copies/ml) EBV loads in peripheral blood. Chronic high EBV load patients have a 45% rate of progression to late-onset posttransplant lymphoproliferative disorders. In this article, we report that asymptomatic patients carrying EBV loads (low and high) expressed increased frequencies of EBV-specific CD8+ T cells, as compared with patients with undetectable EBV loads. Although patients with low viral load displayed EBV-specific CD8+ T cells with moderate signs of activation (CD38+/−/CD127+/−), programmed death 1 upregulation and effective IFN-γ secretion, high EBV load carriers showed significant CD38+ upregulation, features of cellular exhaustion (programmed death 1+/CD127−) accompanied by a decline in IFN-γ release. Immunopolarization of EBV-specific CD8+ T cells was skewed from the expected type 1 (IFN-γ) toward type 0 (IFN-γ/IL-5) in patients, and Tr1 (IL-10) in high load carriers. These results indicate the importance of chronic EBV load and of the levels of antigenic pressure in shaping EBV-specific memory CD8+ T cells. Concomitant phenotypic and functional EBV monitoring is critical for identifying the complex “functional” versus “exhausted” signature of EBV-specific CD8+ T cells, with implications for immunologic monitoring in the clinic.

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.

Prolonged inhibition of the kinase, mammalian target of rapamycin (mTOR), during myeloid dendritic cell (DC) generation confers resistance to maturation. Recently, however, mTOR inhibition immediately before Toll-like receptor ligation has been found to exert proinflammatory effects on myeloid cells, notably enhanced IL-12p40/p70 production. We show, for the first time, that mouse or human DCs generated under mTOR inhibition exhibit markedly enhanced IL-12p70 production after lipopolysaccharide (LPS) stimulation, despite impaired costimulatory molecule expression and poor T-cell stimulatory ability. Consistent with this finding, we reveal that increased IL-12p40 production occurs predominantly in CD86lo immature DCs. High IL-12p40/p70 production by CD86lo DC resulted from failed down-regulation of glycogen synthase kinase-3 (GSK-3) activity and could not be ascribed to enhanced Akt function. Despite high IL-12p70 secretion, rapamycin-conditioned, LPS-stimulated DCs remained poor T-cell stimulators, failing to enhance allogeneic Th1 cell responses. We also report that inhibition of GSK-3 impedes the ability of LPS-stimulated DCs to induce forkhead box p3 in CD4+CD25− T cells, as does the absence of IL-12p40/p70. Thus, GSK-3 activity in DC is regulated via signaling linked to mTOR and modulates their capacity both to produce IL-12p40/p70 and induce forkhead box p3 in CD4+ T cells under inflammatory conditions.

As the target CD52 molecule is expressed on erythrocytes of most nonhuman primate strains, using alemtuzumab in these species would cause massive hemolysis. Six cynomolgus monkeys of Indonesian origin, screened by agglutination assay for absence of CD52 on erythrocytes, were administered alemtuzumab in a cumulative dose to a maximum of 60 mg/kg. In two monkeys, mycophenolate mofetil (MMF) was added as maintenance therapy. Complete depletion of T and B lymphocytes (99.5%) was achieved with 20 mg/kg alemtuzumab and was more profound than in monkeys treated with antithymocyte globulin (n = 5), as quantified by flow cytometry. Repopulation was suppressed by weekly injections of 10 mg/kg. Without MMF, repopulation of CD20(+)B cells and CD8(+)T cells was complete within 2 and 3 months, respectively, and repopulation of CD4(+)T cells was 67% after 1 year. MMF significantly delayed CD4(+)T-cell repopulation. Among repopulating CD4(+) and CD8(+) T cells, a phenotypic shift was observed from CD45RA(hi)CD62L(hi) naïve cells toward CD45RA(lo)CD62L(lo) effector memory cells. In lymph nodes, the depletion of naïve cells was more profound than of memory cells, which may have initiated a proliferation of memory cells. This model offers opportunities to investigate lymphocyte depletion/repopulation phenomena, as well as the efficacy of alemtuzumab in preclinical transplantation models.

Mixed leukocyte culture (MLC) is perhaps the most widely used of cellular assays. MLC has been used clinically for donor selection, predominantly for bone marrow transplantation. In contrast to MLC, which measures a bulk response, limiting dilution assay (LDA) is a quantitative tool. Mycoplasma contains the thymine kinase enzyme, which interferes with nucleic and amino acid metabolism and subsequently with DNA replication in the contaminated cell culture. In solid-organ transplantation, the histological analysis of allograft biopsies remains the "gold standard" for diagnosing rejection. Since the ultimate goal is the early detection of activated cells infiltrating the allograft and not in vitro priming, it also deals with methods that minimize the possibility of priming against the donor antigens in vitro. Several of the assays outlined in this chapter are being implemented in the clinical laboratory. Researchers compared helper and cytotoxic antirecipient T-cell frequencies in recipients who had received unrelated-donor bone marrow. They found that the helper T lymphocytes (HTLp) and cytotoxic T lymphocytes (CTLp) assays provided similar predictive information for outcome. The HTLp method is more rapid and less labor-intensive and, thus, may be more useful for donor selection in unrelated-donor bone marrow transplantation. A recent multicenter study evaluated the Cylex ImmuKnow assay for the measurement of immune response in immunosuppressed transplant recipients. The long-term goals of using the cellular assays described in this chapter are to assess accurately the changing immuno-logical statuses of transplant recipients, predict long-term graft outcome, and provide the immune response information needed to individualize immunosuppression therapy.

Immune responses to EBV in immunosuppressed (IS) solid organ transplant (SOTx) recipients have not been well characterized. Here we evaluate the phenotype and function of EBV-specific CD8+ T cells in peripheral blood isolated from "stable" IS SOTx recipients. The EBV-specific CD8+ T cell memory subset distribution in the peripheral blood of patients was examined by flow cytometric analysis using HLA-A2 tetramers incorporating BMLF1 (lytic), and LMP2 and EBNA3A (latent)-derived peptides, in conjunction with mAbs against the CD45RO, CD45RA, and CD62L markers. The ability of CD8+ T cells to produce IFN-gamma in response to the same EBV-derived peptides was measured by ELISPOT assay. Patients and healthy normal donors exhibited similar anti-EBV CD8+ T cell frequencies and specificities against the EBV epitopes evaluated. When compared to healthy normal donors, an overall significant expansion of the CD8+ T cell "effector memory" (CD45RO+/CD62L-) pool, including that of EBV "latent" (LMP2 and EBNA3A)-specific CD8+ T cells was detected in IS SOTx patients. However, the patients' EBV-specific CD8+ T cells showed decreased IFN-gamma production to the EBV-peptide stimulation. These results indicate that the impairment of EBV-specific CD8+ T cell activity is not due to clonal depletion, but is mainly due to impaired functional activation.

Pigs are considered a suitable source of cells and organs for xenotransplantation. All known strains of pigs contain porcine endogenous retrovirus (PERV) and PERV released by porcine cells may infect human cells in vitro and severe-combined immunodeficient (SCID) mice in vivo. Humanized SCID (hu-SCID) mice develop immune response to porcine antigens. Here we investigated PERV transmission in humanized SCID-beige mice using porcine peripheral blood mononuclear cells (PBMC) as the donor tissue (and the source of PERV). Mice were infused in the peritoneal cavity with 1.5-3.0 x 10(7) unfractionated human PBMC. Unfractionated porcine PBMC (1.5-3.0 x 10(7) cell/mouse) were infused to the mice simultaneously with human PBMC or 3 weeks after human PBMC infusion. The treated mice were monitored for weight and skin changes, donor cell chimerism, anti-pig antibodies and PERV transmission. All humanized mice tested 5-12 weeks after human PBMC transplantation were macrochimeric (up to 40% of cells in blood) for human cells, where 99% of the human cells were T-lymphocytes. Although human B lymphocytes were very rare in the blood of humanized mice at that point, the mice were positive for human anti-pig natural antibodies. The control SCID-beige mice or mice treated with porcine PBMC alone were negative for anti-porcine antibodies. Approximately 70% of the humanized mice treated with porcine PBMC were also microchimeric for porcine cells. Although some tissue samples of these mice were positive for PERV DNA in the absence of porcine DNA indicating PERV infection, the infection was non-productive as PERV transcripts were not detectable in those tissues. PERV infection of human and mouse cells in vitro by co-culturing with porcine PBMC was also non-productive. Humanized SCID-beige mice suffered weight loss and occasional minor skin changes due to graft vs. host disease caused by human PBMC but none of the mice showed observable effect attributable to the apparent PERV infection alone.

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.

Human monocytes have been grouped into classical (CD14++CD16−), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1β in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

Natural killer (NK) cells are important in host defense against viruses and tumors and can induce death of virally infected cells following engagement of cell surface receptors. Human NK cells express receptors for the Fc portion of IgG which stimulate antibody-dependent cell-mediated cytotoxicity and induce cytokine production. We have shown that NK cells from certain individuals can express, in addition to CD16 (FcgammaRIIIa), isoforms of CD32 (FcgammaRIIc1-4). Expression of CD32 on NK cells is dependent on an allelic polymorphism of the FcgammaRIIC gene. We analyzed the expression and function of CD32 on NK cells from 31 normal donors. Fourteen of the 31 (45%) donors expressed CD32 on their NK cells. Molecular characterization of FcgammaRIIc isoforms expressed by the CD32+ donors revealed that the majority of donors expressed the FcgammaRIIc1 isoform. Interestingly, 3 of the 14 positive donors did not express FcgammaRIIc1, and we identified a novel isoform, FcgammaRIIc5, expressed by these individuals. The expression of this isoform was correlated to a second allelic polymorphism that controls exon splicing. One of the three was found to express FcgammaRIIb on the NK cells. Biochemical analysis revealed that CD32+ donors of both types expressed a 40-kDa protein, specifically immunoprecipitated by anti-CD32 monoclonal antibodies. Functionally, only individuals expressing the FcgammaRIIc1 isoform were able to trigger reverse antibody-dependent cell-mediated cytotoxicity via CD32 whereas a CD32+ individual expressing the FcgammaRIIb isoform was unable to trigger this function. These results demonstrate that the presence of multiple allelic polymorphisms in the FcgammaRIIC gene determine the expression and function of CD32 on NK cells.

The receptors on human natural killer 9NK cells which can specifically bind the Fc portion of immunoglobulin molecules (Fc receptors) have been extensively studied. The best known and studied Fc receptor on human NK cells is FcgammaRIIIa. Interactions of NK cells with IgG antibodies via this receptor are well known to induce a signal transduction cascade and lead to antibody-dependent cell-mediated cytotoxicity (ADCC) as well as release of various cytokines. In addition, interactions with monomeric IgG and FcgammaRIIIa have been demonstrated, which result in negative regulation of NK activity and other immunomodulatory effects. Over the past several years, it has also become increasingly appreciated that human NK cells express a variety of other Fc receptors, including FcmuR, which also can mediate effector and immunoregulatory functions. Also, a novel form of FcgammaR has been demonstrated on human NK cells, termed FcgammaRIIc. Recent molecular studies have shown considerable polymorphism in the genes for FcgammaIIc and the functional consequences are being dissected. This appears to include cross-talk between FcgammaRIIIa and at least some forms of FcgammaRIIc, which may have important functional consequences.

Background Although readily accomplished from immunocompetent Epstein-Barr virus- (EBV) seropositive individuals, the effective ex vivo generation of EBV-specific cytotoxic T lymphocytes (CTL) from the peripheral blood mononuclear cells (PBMC) of EBV-seronegative subjects has proven to be a challenge. The focus of our study was to ascertain optimized culture conditions required for the ex vivo generation of EBV-reactive autologous CTL from the PBMC of EBV-seronegative volunteers. Method Freshly isolated PBMC obtained from immunocompetent EBV-seronegative and -seropositive individuals were used to generate EBV-specific autologous CTL lines using both conventional and a novel, modified ex vivo culture technique. Results In contrast to responses observed in EBV-seropositives after two to three rounds of ex vivo stimulation, gamma-irradiated autologous lymphoblastoid cell lines (LCL) were incapable of eliciting an effective anti-EBV cytotoxic response when freshly-isolated PBMC from EBV-seronegative individuals were used as responders. Under these culture conditions, CD4+ T cells with preferential expression of the Th2-type cytokine IL-4 were predominantly expanded in the PBMC obtained from EBV-seronegative individuals. However, the addition of recombinant human (rh) IL-12 during the primary phase of ex vivo stimulation resulted in augmentation of EBV-specific cytolysis of autologous LCL by CD8+ T cells. Furthermore, there was down-regulation in the secretion of IL-4 and up-regulation in that of the Th1-type cytokine IFN-gamma by responder CD4+ and CD8+ T cells. Conclusions Taken together these data suggest that the addition of rhIL-12 during the primary phase of ex vivo stimulation of freshly isolated PBMC from EBV-seronegative individuals results in skewing of the immune response predominantly towards a CD4+ Th1-type (IFN-gamma) with the generation of an efficacious CTL-mediated anti-EBV reactivity. This novel ex vivo approach for generating effective autologous EBV-specific CTL could be adopted to treat refractory post-transplant lymphoproliferative disorders, which may be encountered in EBV-seropositive-->EBV-seronegative organ transplant recipients. Additionally, these ex vivo generated anti-EBV T cells could also be infused perioperatively to enhance prophylactically immunity against EBV infection in high-risk EBV-seronegative organ allograft recipients.

Human natural killer (NK) cells were thought to express only FcγRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcγR, ie, FcγRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcγRII from NK cells derived from several normal individuals that may represent four different products of the FcγRIIC gene. One transcript (IIc1) is identical with the already described FcγRIIc form. The other three (IIc2-IIc4) appear to represent unique, alternatively spliced products of the same gene, and include a possible soluble form. Analyses of the full-length clones have revealed an allelic polymorphism in the first extracellular exon, resulting in either a functional open reading frame isoform or a null allele. Stable transfection experiments enabled us to determine a unique binding pattern of anti-CD32 monoclonal antibodies to FcγRIIc. Further analyses of NK-cell preparations revealed heterogeneity in CD32 expression, ranging from donors lacking CD32 expression to donors expressing high levels of CD32 that were capable of triggering cytotoxicity. Differences in expression were correlated with the presence or absence of null alleles. These data show that certain individuals express high levels of functional FcγRIIc isoforms on their NK cells.

Organ transplant recipients face life-long immunosuppression and consequently are at high risk of comorbidities. Occasionally, kidney transplant recipients develop a state of targeted immune quiescence (operational tolerance) against an HLA-mismatched graft, allowing them to withdraw all immunosuppression and retain stable graft function while resuming immune responses to third-party antigens. Methods to better understand and monitor this state of alloimmune quiescence by transcriptional profiling may reveal a gene signature that identifies patients for whom immunosuppression could be titrated to reduce patient and graft morbidities. Therefore, we investigated 571 unique peripheral blood samples from 348 HLA-mismatched renal transplant recipients and 101 nontransplant controls in a four-stage study including microarray, quantitative PCR, and flow cytometry analyses. We report a refined and highly validated (area under the curve, 0.95; 95% confidence interval, 0.92 to 0.97) peripheral blood three-gene assay (KLF6, BNC2, CYP1B1) to detect the state of operational tolerance by quantitative PCR. The frequency of predicted alloimmune quiescence in stable renal transplant patients receiving long-term immunosuppression (n=150) was 7.3% by the three-gene assay. Targeted cell sorting of peripheral blood from operationally tolerant patients showed a significant shift in the ratio of circulating monocyte-derived dendritic cells with significantly different expression of the genes constituting the three-gene assay. Our results suggest that incorporation of patient screening by specific cellular and gene expression assays may support the safety of drug minimization trials and protocols.

The Fc gamma receptor (R)IIIA (CD 16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via Fc gamma RIIIA, purified NK cells were cultured for 2-3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to Fc gamma RIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2R alpha, ICAM-1), cytotoxicity, cytokine production (IL-1 beta, IFN-gamma and TNF-alpha) and release of soluble IL-2R. Thus, mIgG binding to Fc gamma RIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2R alpha expression, cell proliferation and cytokine release.

Mammalian target of rapamycin kinase inhibitor (mTORi) rapamycin (RAPA) use in transplantation can lead to inflammatory complications in some patients. Our goal was to better understand how mTORi-exposed human monocyte-derived dendritic cells (DC) stimulated with pro-inflammatory cytokines shape T cell allo-immunity. RAPA-conditioned-DC (RAPA-DC) displayed a more immature phenotype than untreated, control (CTRL)-DC. However, subsequent exposure of RAPA-DC to an inflammatory cytokine cocktail (ICC) plus IFN-γ induced a mature Type-1 promoting phenotype, consisting of elevated HLA-DR and co-stimulatory molecules, augmented IL-12p70 and IL-27 production, but decreased IL-10 secretion compared to CTRL-DC. Co-culture of mature (m)RAPA-DC with allogeneic peripheral blood mononuclear cells resulted in significantly increased Type-1 (IFN-γ) responses by T cells. Moreover, NK cells acted as innate modulators that conveyed activating cell-to-cell contact signals in addition to helper (IFN-γ) and/or regulatory (IL-10) soluble cytokines. We conclude that production of IL12-p70, IL-27 and low IL-10 by RAPA-DC allowed us to elucidate how these cytokines as well as NK-DC interaction shapes T cell allo-immunity. Thus, lack of inhibitory NK cell function during allo-specific T cell activation by human ICC + IFN-γ-stimulated RAPA-DC may represent an unwanted effector mechanism that may underlie RAPA-induced inflammatory events in transplant patients undergoing microbial infection or allograft rejection.

Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes.We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied.We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4 memory T cells, and decreased proportion of CD4 regulatory T cells. Reactive patients exhibited higher proportions of CD4 effector memory T cells (TEM) and CD8 terminally differentiated TEM (TEMRA), with greater CD4 TEM and CD8 TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8 TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8 T cells.Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

EBV-immortalized B-lymphoblastoid cell lines are used as models for cellular transformation and as antigen-presenting cells in immunological assays. LCLs vary in surface markers and other phenotypic properties, but it is not known how this heterogeneity relates to the EBV life cycle. To explore correlations, we examined 62 LCLs for cellular and viral phenotypes. LCLs generated from pediatric and adult donors could similarly be categorized as either low in EBV copy number or fluctuating within a high range. High-copy status accompanied higher lytic viral gene expression and lower latent gene expression. Inhibiting lytic EBV replication did not affect cellular phenotype or lytic switch protein expression, indicating that an LCL's lytic permissivity was a stable property. Among the cellular genes overexpressed in permissive LCLs were unfolded protein response genes and plasma cell markers. Among genes overexpressed in non-permissive LCLs were transcription factors involved in maintaining B cell lineage, in particular EBF1. This study suggests previously undetected mechanisms by which cellular pathways influence the lytic reactivation of EBV.

T-cell alloimmunity plays a dominant role in allograft rejection. The precise contribution of naïve and memory T cells to this response however remains unclear. To address this question, we established an ex vivo flow-cytometric assay that simultaneously measures proliferation, precursor frequency and effector molecule (IFNgamma, granzyme B/perforin) production of alloreactive T cells. By applying this assay to peripheral blood mononuclear cells from healthy volunteers, we demonstrate that the CD4+ and CD8+ populations mount similar proliferative responses and contain comparable frequencies of alloreactive precursors. Effector molecule expression, however, was significantly higher among CD8+ T cells. Analysis of sorted naïve and memory T cells showed that alloreactive precursors were equally present in both populations. The CD8+ effector and terminally differentiated effector memory subsets contained the highest proportion of granzyme B/perforin after allostimulation, suggesting that these cells present a significant threat to transplanted organs. Finally, we demonstrate that virus-specific lymphocytes contribute significantly to the alloresponse in certain responder-stimulator HLA combinations, underscoring the importance of T-cell cross-reactivity in alloimmunity. These results provide a quantitative assessment of the roles of naïve and memory T-cell subsets in the normal human alloimmune response and establish a platform for measuring T-cell alloreactivity pre- and posttransplantation.

Polyomavirus BK (BKV) infections are increasingly recognized. The development of immune-monitoring strategies against BKV requires definition of antigenic epitopes. Bioinformatic algorithms were used to identify 60 BKV large T-antigen (LT-Ag) peptides predicted to bind HLA class I alleles. In vitro peptide binding was used to select a subset of 19 peptides for interferon (IFN)-gamma ELISPOT assays in 13 healthy subjects and 12 kidney transplant recipients. Four A01-, nine A03-, and five A24-binding immunogenic peptides were identified in 1 to 3 (14-67%) tested subjects in each group. BKV epitope sequences were identical to homologous JC virus sequences for 3 of 19 peptides and homologous SV40 sequences for 5 of 19 peptides. Homology modeling localized these epitopes to the helicase, origin of DNA binding, or J domains, respectively. In conclusion, we have identified multiple 9-mer BKV LT-Ag-derived immunogenic epitopes that bind HLA-A01, -A03, or -A24 molecules. Sequence alignments indicate that two epitopes, FLICKGVNK and RYWLFKGPI, are common to BKV, JC virus, and SV40 virus.

Posttransplantation lymphoproliferative disorders (PTLD) are life-threatening complications of solid organ transplantation, triggered by EBV infection in chronically immunosuppressed (IS) patients. Our goal is to establish DC-based protocols for adoptive immunotherapy of refractory PTLD, while understanding how the immunosuppressive drug environment may subvert DC-EBV-specific T cell interactions. Type-1 CD8(+) T cells are critical for efficient immune surveillance and control of EBV infection, whereas type-2 or Treg/type-3 responses may provide an environment conductive to disease progression. We have recently reported that chronic IS inhibits DC function in transplant patients. Here, we have analyzed the comparative ability of mature, type-1 polarized DCs (i.e. DC1) generated from quiescent transplant patients or healthy controls, to boost type-1 EBV-specific CD8(+) T cells in vitro. Our results show that unlike healthy controls, where DC1 loaded with MHC class I EBV peptides preferentially reactivate specific type-1 CD8(+) T cells, DC1 generated from transplant patients reactivate EBV-specific CD8(+) T cells that produce both IFN-gamma and IL-10, up-regulate FOXP3 mRNA, and suppress noncognate CD4(+) T-cell proliferation via cell-cell contact. These data support a novel regulatory pathway for anti-EBV T-cell-mediated responses in IS transplant patients, with implications for the design of adoptive immunotherapies in this setting.

BK virus (BKV) infections after renal transplantation are increasingly recognized. Development of immune monitoring strategies against BKV requires definition of antigenic epitopes. Hence, T cells from HLA-A02-positive healthy subjects and kidney transplant recipients were stimulated by BKV lysate pulsed on mature autologous dendritic cells and screened against four different T antigen peptides or against BKV lysate. IFN-gamma production was measured by ELISPOT assays. The peptide BKV362-371 (MLTERFNHIL) was naturally processed and recognized by five of six healthy subjects (39 +/- 11 IFN-gamma spots/100,000 cells) and five of seven kidney transplant recipients (21 +/- 12 IFN-gamma spots). Less frequent and weaker CD8+ T-cell responses were detected against three other peptides. Thus, BKV large T antigen is a target for CD8+ T-cell immunity. T-antigen-specific T-cytotoxic cells circulate in healthy blood donors, implying that transient expression of T antigen presumably occurs at sites of viral latency and helps maintain a constant pool of circulating CD8+ T memory cells.

Human NK cells are large granular lymphocytes that kill neoplastic or virally infected targets using perforin-dependent mechanisms. CD16 or FcgammaRIII is one of the cell surface molecules that can trigger the killing machinery following binding of the Fc portion of IgG to the receptor: a mechanism known as antibody dependent cell-mediated cytotoxicity (ADCC). We have recently shown that some individuals express an additional FcgammaR on their NK cells, CD32 or FcgammaRII. This receptor has now been characterized at the molecular, biochemical and functional level. The present review outlines our findings to date on the features of this novel receptor. These findings suggest that the presence of a functional FcgammaRII on the surface of NK cells could have important clinical consequences in both tumor immunotherapy and autoimmune disease.

Various anti-Fc gamma RIII (CD16) monoclonal antibodies (mAbs) are shown here to have positive or negative modulatory effects on human NK cells. Thus, 3G8 mAb (IgG1) triggered a dose-dependent augmentation of NK activity in 67% (23/34) of individuals tested, who were designated as responders. All four IgG1 anti-CD16 mAb tested (BL-LGL/1, B73.1, Leu11c, and 3G8) were stimulatory for NK cells isolated from responders, whereas six non-IgG1 anti-CD16 mAbs were either inhibitory or had no significant effects on NK activity. The upregulation of NK activity in responders was not attributable to an increase in either the conjugate formation or the delivery of the lethal hit to target cells. This mAb-mediated up-regulation of NK activity was shown to be associated with a recycling capacity higher than that of controls and with enhanced release of cytokines by activated NK cells. Anti-CD16 mAb inhibited binding of either monomeric or polymeric IgG to Fc gamma RIIIA on NK cells. Also, mAb 3G8 or its F(ab')2 fragments decreased or reversed inhibition of NK activity induced by monomeric IgG (mIgG). Our data indicate that regulation of NK activity via the Fc gamma RIIIA is influenced by dose-dependent interactions between cytophilic mIgG and anti-CD16 mAb of IgG1 isotype.

We recently reported that human NK cells express, in addition to CD16 [Fcgamma receptor (FcgammaR) IIIA], a second type of FcgammaR, namely CD32 (FcgammaRII). Molecular characterization of CD32 transcripts expressed by highly purified NK cells revealed that they predominantly express products of the FcgammaRIIC gene. Using stable Jurkat transfectants we have analyzed the functional properties of two FcgammaRIIc-specific isoforms isolated from NK cells, namely FcgammaRIIc1 and FcgammaRIIc3, which differ in their cytoplasmic tails. The ligand binding specificity for both murine and human IgG isotypes was found to be similar to that observed for FcgammaRIIb isoforms. Immunoprecipitation studies of FcgammaRIIc isoforms expressed in Jurkat cells revealed a protein of around 40 kDa for FcgammaRIIc1, and a protein of around 32 kDa for FcgammaRIIc3. Signal transduction studies performed on FcgammaRIIc1-expressing Jurkat cells indicated that this molecule is functional, i. e. capable of Ca2+ mobilization and activation of Lck, Zap-70 and Syk protein tyrosine kinases, although the CD3 zeta chain was not found to functionally associate with FcgammaRIIc1. In contrast, FcgammaRIIc3 transfectants showed an impaired ability of this molecule to mobilize Ca2+, but activation of Lck was detected following activation via FcgammaRIIc3. These studies demonstrate the functional activity of FcgammaRIIc isoforms and suggest that the presence of CD32, in addition to CD16, on NK cells may have functional relevance.