Iulia Popescu

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 (IFNγ, 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.

Decreased NKp46 and NKG2D and elevated PD-1 are associated with altered NK-cell function in pediatric transplant patients with PTLD

Post‐transplantation lymphoproliferative disorders (PTLD) are life‐threatening complications of organ transplantation caused by EBV infection and the use of chronic immunosuppression. While T‐cell impairment is known to play a critical role in the immunopathogenesis of EBV complications post‐transplantation, the role of NK cells is still under investigation. Here, we have characterized NK‐cell phenotype and function in peripheral blood from asymptomatic pediatric thoracic transplant patients, patients with PTLD, and healthy controls. Overall, asymptomatic pediatric solid organ transplant (Tx) patients presented significant expansion of the CD56brightCD16± subset and displayed effective NK‐cell function, while PTLD patients accumulated CD56dimCD16− and CD56−CD16+ NK‐cell subsets. In addition, NK cells from PTLD patients down‐regulated NKp46 and NKG2D, and significantly up‐regulated PD‐1. These phenotypic changes were associated with NK functional impairment, resembling cellular exhaustion. Disrupting PD‐1 inhibitory pathway improved IFN‐γ release, but did not enhance cytotoxicity in PTLD patients, suggesting that these defects were partially PD‐1 independent. Our results indicate the important role of NK cells during EBV surveillance post‐transplantation, with implications for the immunopathogenesis of EBV complications, and suggest that monitoring NK cells in transplant patients may hold clinical value.

Human NK cells express Fc receptors for IgA which mediate signal transduction and target cell killing

Receptors for the Fc region of IgG (FcγRIIIa, FcγRIIc) and IgM (FcμR) were previously described on NK cells. In this work the expression of Fc receptors for IgA (FcαR) on humanNK cells and the signaling events were investigated. The FcαR was demonstrated by flow cytometry using secretory IgA (sIgA) and anti‐human IgA antibody. The percentage of NK cells (CD3–CD56+CD16+) expressing FcαR ranged between 55.7% and 95.7%, with a mean ± SD of 75.2±11.8. The association constant and the number of 125I‐labeled sIgA (125I‐sIgA) molecules bound per cell, calculated by Scatchard analysis, were 2×107 M–1 and 1.7×104, respectively. The binding specificity was proved by inhibition experiments. Cold sIgA but not IgA Fab fragments were able to inhibit 125I‐sIgA binding in a concentration‐dependent manner. Binding of sIgA to NK cells was neither inhibited by anti‐mannose receptor antibody, nor by L‐fucose, D‐galactose, D‐glucose, D‐mannose or N‐acetyl‐D‐glucosamine. Pretreatment of NK cells with polymeric IgA inhibited their capacity to kill 51Cr‐labeled K562 target cells by 34.8%, whereas with monomeric IgA only by 13.1%. Ligand‐induced clustering of the FcαR resulted in activation of tyrosine kinases Lck, Syk and phosphatidylinositol 3‐kinase. The present studies support the concept that human NK cells bind preferentially sIgA and polymeric IgA with moderate affinity via FcαR, which is different from the FcαRI/CD89 and other carbohydrate‐recognizing receptors like mannose receptor/CD206. This novel structure mediates signal transduction and cell killing.

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‐γ 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.

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

We recently reported that human NK cells express, in addition to CD16 [Fcγ receptor (FcγR) IIIA], a second type of FcγR, namely CD32 (FcγRII). Molecular characterization of CD32 transcripts expressed by highly purified NK cells revealed that they predominantly express products of the FcγRIIC gene. Using stable Jurkat transfectants we have analyzed the functional properties of two FcγRIIc‐specific isoforms isolated from NK cells, namely FcγRIIc1 and FcγRIIc3, 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 FcγRIIb isoforms. Immunoprecipitation studies of FcγRIIc isoforms expressed in Jurkat cells revealed a protein of around 40 kDa for FcγRIIc1, and a protein of around 32 kDa for FcγRIIc3. Signal transduction studies performed on FcγRIIc1‐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 ζ chain was not found to functionally associate with FcγRIIc1. In contrast, FcγRIIc3 transfectants showed an impaired ability of this molecule to mobilize Ca2+, but activation of Lck was detected following activation via FcγRIIc3. These studies demonstrate the functional activity of FcγRIIc isoforms and suggest that the presence of CD32, in addition to CD16, on NK cells may have functional relevance.

Ex Vivo Priming of Naïve T Cells Into EBV-Specific Th1/Tc1 Effector Cells by Mature Autologous DC Loaded with Apoptotic/Necrotic LCL

Posttransplant lymphoproliferative disorders (PTLDs) represent life‐threatening complications of bone marrow and solid organ transplantation (SOTx). These are B‐cell malignancies triggered by Epstein‐Barr Virus (EBV) infection in chronically immunosuppressed (IS) recipients. Immunosuppressed EBV seronegative (EBV−) organ recipients are at highest risk of developing PTLD owing to the lack of anti‐EBV memory T cells to control subsequent EBV challenges. Our aim is to establish effective anti‐EBV T‐cell generation protocols for prevention or treatment of PTLD encountered in SOTx. We have used autologous dendritic cells (DCs) loaded with apoptotic/necrotic lymphoblastoid cell lines (LCLs) to evaluate the ability of such an approach to activate naïve T cells in vitro. In EBV− individuals, both CD8+ and CD4+ T‐cell responses were amplified by this approach, as detected by IFN‐γ ELISPOT and cytotoxicity assays. The CD8+ T cells were poly‐specific anti‐EBNA3 A, ‐LMP2 and ‐BMLF1, with uniform reversion to a CD45RO+/RA‐phenotype, decreased CD62L expression, and up‐regulation of the activation markers CD28 and CD69. Addition of rhIL‐12 improved anti‐viral T‐cell responses and reduced the functional differences observed between EBV+ and EBV− responders. In conclusion, the DC/LCL method promotes cross‐presentation of EBV‐associated epitopes and may serve as an effective protocol for the adoptive immunotherapy of PTLD in EBV− SOTx patients.

EBV-specific CD8+ T cells from asymptomatic pediatric thoracic transplant patients carrying chronic high EBV loads display contrasting features: activated phenotype and exhausted function.

Serial EBV load monitoring of clinically asymptomatic pediatric thoracic organ transplant patients has identified three groups of children who exhibit undetectable (<100 copies/ml), chronic low (100–16,000 copies/ml), or chronic high (>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.

Activation-induced Cell Death Drives Profound Lung CD4+ T-Cell Depletion in HIV-associated Chronic Obstructive Pulmonary Disease

RATIONALE As overall survival improves, individuals with HIV infection become susceptible to other chronic diseases, including accelerated chronic obstructive pulmonary disease (COPD). OBJECTIVES To determine whether individuals with HIV-associated COPD exhibit dysregulated lung mucosal T-cell immunity compared with control subjects. METHODS Using flow cytometry, we evaluated peripheral blood and lung mucosal T-cell immunity in 14 HIV(+)COPD(+), 13 HIV(+)COPD(-), and 7 HIV(-)COPD(+) individuals. MEASUREMENTS AND MAIN RESULTS HIV(+)COPD(+) individuals demonstrated profound CD4(+) T-cell depletion with reduced CD4/CD8 T-cell ratios in bronchoalveolar lavage-derived lung mononuclear cells, not observed in peripheral blood mononuclear cells, and diminished CD4(+) T cell absolute numbers, compared with control subjects. Furthermore, HIV(+)COPD(+) individuals demonstrated decreased pulmonary HIV-specific and staphylococcal enterotoxin B-reactive CD4(+) memory responses, including loss of multifunctionality, compared with HIV(+)COPD(-) control subjects. In contrast, lung mucosal HIV-specific CD8(+) T-cell responses were preserved. Lung CD4(+) T cells from HIV(+)COPD(+) individuals expressed increased surface Fas death receptor (CD95) and programmed death-1, but similar bronchoalveolar lavage viral loads as control subjects. However, programmed death-1 expression inversely correlated with HIV-specific lung CD4(+)IFN-γ(+) T-cell responses, suggesting functional exhaustion. Moreover, lung CD4(+) T cells from HIV(+)COPD(+) patients demonstrated increased basal and HIV antigen-induced expression of the early apoptosis marker annexin V compared with control subjects, which was significantly attenuated with anti-Fas blockade. Lastly, lung mucosal, but not blood, CD4(+)/CD8(+) ratios from HIV(+) patients significantly correlated with the FEV1, but not in HIV(-)COPD(+) patients. CONCLUSIONS Together, our results provide evidence for profound lung mucosal CD4(+) T-cell depletion via a Fas-dependent activation-induced cell death mechanism, along with impaired HIV-specific CD4(+) immunity as immunologic features of HIV-associated COPD.

The impact of EBV load on T cell immunity in pediatric thoracic transplant recipients

Background. Immunologic monitoring of pediatric transplant (Tx) recipients, who are at increased risk of Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disease, is an important goal in clinical transplantation. Here, we investigated the impact of EBV load on T-cell immunity from pediatric Tx recipients, using clinically applicable tests for improved assessment of T-cell immune competence. Methods. Thirty-five asymptomatic pediatric thoracic Tx patients were categorized into three groups according to their EBV load levels as follows: undetectable viral load (UVL), chronic low viral load (LVL) and chronic high viral load (HVL). Global and EBV-specific T-cell immunity were assessed by ATP release using Cylex Immuknow and T Cell Memory™ assays. Results. UVL patients exhibited normal ATP release to Concanavalin A (ConA) and phytohemagglutinin (PHA; 190±86 ng/mL, 328±163 ng/mL) and detectable EBV-specific (37±34 ng/mL) ATP responses. LVL patients displayed significantly stronger responses to ConA (373±174 ng/mL), PHA (498±196 ng/mL) and EBV (152±179 ng/mL), when compared with UVL or to HVL patients (ConA 185±114 ng/mL, PHA 318±173 ng/mL, and EBV 33±42 ng/mL). Moreover, HVL patients displayed significant inverse correlation between CD4+ T-cell ATP levels and EBV loads. Conclusions. Evaluation of global and EBV-specific T-cell immunity provides a rapid assessment of patients’ immune competence. It is still unclear whether selective oversuppressed ATP release by CD4+ T cells reflects HVL patients at risk of posttransplant lymphoproliferative disease. Further longitudinal studies will determine the importance of Immuknow test in identifying asymptomatic HVL patients vulnerable to EBV complications.

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.