Characterization of eomesodermin and T-bet expression by allostimulated CD8+ T cells of healthy volunteers and kidney transplant patients in relation to graft outcome.

Memory T cell (Tmem) responses play a critical role in the outcome of allo-transplantation. While the role of the T-box transcription factor Eomesodermin (Eomes) in the maintenance of antigen-specific Tmem is well studied, little is known about Eomes+ CD8+ T cell responses after transplantation. We evaluated the phenotype and function of allo-reactive Eomes+ CD8+ T cells in healthy volunteers and kidney transplant patients and their relation to transplant outcome. High Eomes expression by steady-state CD8+ T cells correlated with effector and memory phenotype. Following allo-stimulation, the expression of both the T-box proteins Eomes and T-bet by proliferating cells increased significantly, where high expression of Eomes and T-bet correlated with higher incidence of allo-stimulated IFNγ+ TNFα+ CD8+ T cells. In patients with no subsequent rejection, Eomes but not T-bet expression by donor-stimulated CD8+ T cells, increased significantly after transplantation. This was characterized by increased Eomeshi T-bet-/lo and decreased Eomes-/lo T-bethi CD8+ T cell subsets, with no significant changes in the Eomeshi T-bethi CD8+ T cell subset. No upregulation of exhaustion markers programmed-death-1 (PD-1) and cytotoxic-T-lymphocyte-associated-antigen-4 (CTLA4) by donor-stimulated Eomes+ CD8+ T cells was observed. Before transplantation, in patients without rejection, there were higher incidences of Eomeshi T-bet-/lo , and lower incidences of Eomeshi T-bethi and Eomes-/lo T-bethi donor-stimulated CD8+ T cell subsets, compared to those with subsequent rejection. Overall, our findings indicate that high Eomes expression by allo-stimulated T-bet+ CD8+ T cells is associated with enhanced effector function, and that an elevated incidence of donor-stimulated CD8+ T cells co-expressing high levels of Eomes and T-bet before transplantation, may correlate with an increased incidence of acute cellular rejection.

Rapamycin augments human DC IL-12p70 and IL-27 secretion to promote allogeneic Type 1 polarization modulated by NK cells.

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.

Investigation of lymphocyte depletion and repopulation using alemtuzumab (Campath-1H) in cynomolgus monkeys.

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.

Contribution of naïve and memory T-cell populations to the human alloimmune response.

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.

EBV-specific CD8+ T cell reactivation in transplant patients results in expansion of CD8+ type-1 regulatory T cells.

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.

Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.

We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.

Ig-binding receptors on human NK cells as effector and regulatory surface molecules.

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.

Ex vivo generation of effective Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocytes from the peripheral blood of immunocompetent Epstein Barr virus-seronegative individuals.

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.

Ligand binding specificities and signal transduction pathways of Fc gamma receptor IIc isoforms: the CD32 isoforms expressed by human NK cells.

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.

Functional CD32 molecules on human NK 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.

Expression of functional CD32 molecules on human NK cells is determined by an allelic polymorphism of the FcgammaRIIC gene.

Human natural killer (NK) cells were thought to express only FcgammaRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcgammaR, ie, FcgammaRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcgammaRII from NK cells derived from several normal individuals that may represent four different products of the FcgammaRIIC gene. One transcript (IIc1) is identical with the already described FcgammaRIIc 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 FcgammaRIIc. 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 FcgammaRIIc isoforms on their NK cells.

Physical and functional association of Fc mu receptor on human natural killer cells with the zeta- and Fc epsilon RI gamma-chains and with src family protein tyrosine kinases.

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.

Divergent effects of Fc gamma RIIIA ligands on the functional activities of human natural killer cells in vitro.

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.

Natural killer (NK) activity in human responders and nonresponders to stimulation by anti-CD16 antibodies.

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.

Divergent signal transduction pathways and effects on natural killer cell functions induced by interaction of Fc receptors with physiologic ligands or antireceptor antibodies.

Natural killer (NK) cells express receptors that bind to the Fc portion of immunoglobulin molecules (FcR), and their function can potentially be modulated positively or negatively by such receptor:ligand interactions. This review discusses recent developments in the understanding of expression of various forms and isoforms of FcRs by NK cells, and the sequelae of binding of physiologic ligands. Particular attention is paid to the significance of FcR:Ig interactions in both activating and inactivating NK cells under various conditions.

Expression and function of Fc gamma RII on human natural killer cells.

In this report, we present data on the expression and function of Fc gamma RII (CD32) by natural killer (NK) cells. Highly enriched NK cell populations were isolated from peripheral blood lymphocytes by negative selection and consisted of > or = 95% CD3-/CD56+ cells. Flow cytometric analyses with anti-CD32 monoclonal antibodies (mAbs) demonstrated that a small proportion of NK cells were recognized by mAbs IV.3 and 41H16. Two-color flow cytometric analysis indicated coexpression of the epitope on NK cells recognized by both these mAbs. Verification of expression of CD32 on NK cells was obtained by demonstrating coexpression of CD32 on either CD16+ or CD56+ cells. The CD32+/CD16+ and CD32+/CD56+ cells represented approximately 7 and 3% of the total, respectively. CD32 transcripts were identified from highly purified NK cells using reverse transcription-polymerase chain reaction with CD32-specific primers, followed by Southern blotting. Enhanced chemiluminescence-Western blot (ECL-WB) analysis of lysates of purified NK cells indicated that mAb IV.3 recognized a molecule of approximately 40 kD. The Fc gamma RII on NK cells was able to transduce intracellular signals in several types of assay. Cross-linking of anti-CD32 resulted in a mobilization of intracellular Ca2+, although to a lesser extent than that induced by cross-linking CD16. Both mAbs IV.3 and 41H16 were found to be capable of inducing reverse antibody-dependent cellular cytotoxicity against FcR+ target cells (e.g. P815). These data represent the first direct description of the expression and function of Fc gamma RII on human NK cells.