Detection of Virus-Specific CD8+ T Cells With Cross-Reactivity Against Alloantigens: Potency and Flaws of Present Experimental Methods.

UNLABELLED: Virus-specific T cells have the intrinsic capacity to cross-react against allogeneic HLA antigens, a phenomenon known as heterologous immunity. In transplantation, these cells may contribute to the alloimmune response and negatively impact graft outcome. This study describes the various techniques that can be used to detect heterologous immune responses of virus-specific CD8(+) T cells against allogeneic HLA antigens. The strengths and weaknesses of the different approaches are discussed and illustrated by experimental data. METHODS: Mixed-lymphocyte reactions (MLRs) were performed to detect allo-HLA cross-reactivity of virus-specific CD8(+) T cells in total peripheral blood mononuclear cells. T-cell lines and clones were generated to confirm allo-HLA cross-reactivity by IFNγ production and cytotoxicity. In addition, the conventional MLR protocol was adjusted by introducing a 3-day resting phase and subsequent short restimulation with alloantigen or viral peptide, whereupon the expression of IFNγ, IL-2, CD107a, and CD137 was determined. RESULTS: The accuracy of conventional MLR is challenged by potential bystander activation. T-cell lines and clones can circumvent this issue, yet their generation is laborious and time-consuming. Using the adjusted MLR and restimulation protocol, we found that only truly cross-reactive T cells responded to re-encounter of alloantigen and viral peptide, whereas bystander-activated cells did not. CONCLUSIONS: The introduction of a restimulation phase improved the accuracy of the MLR as a screening tool for the detection of allo-HLA cross-reactivity by virus-specific CD8(+) T cells at bulk level. For detailed characterization of cross-reactive cells, T-cell lines and clones remain the golden standard.

Everolimus-treated renal transplant recipients have a more robust CMV-specific CD8+ T-cell response compared with cyclosporine- or mycophenolate-treated patients.

BACKGROUND: In renal transplant recipients, mammalian target of rapamycin (mTOR) inhibitors have been reported to protect against cytomegalovirus (CMV) disease. Here, we questioned whether mTOR inhibitors specifically influence human CMV-induced T-cell responses. METHODS: We studied renal transplant recipients treated with prednisolone, cyclosporine A (CsA), and mycophenolate sodium (MPS) for the first 6 months after transplantation followed by double therapy consisting of prednisolone/everolimus, which is an mTOR inhibitor (P/EVL; n=10), prednisolone/CsA (P/CsA; n=7), or prednisolone/MPS (P/MPS; n=9). All patients were CMV-IgG positive before transplantation. CMV reactivation was detectable in the first 6 months after transplantation and not thereafter. None of the patients included in this study suffered from CMV disease. Both CD27CD8 and CD27CD28CD4 effector-type T-cell counts, known to be associated with CMV infection, were measured before transplantation and at 6 and 24 months after transplantation. Additionally, we determined both number and function of CMV-specific CD8 T cells at these time points. RESULTS: The number of total CD8 T cells, CD27CD8 T cells, and CD28CD4 T cells increased significantly after switch to therapy with P/EVL but not after switch to P/CsA or P/MPS. Specifically, CMV-specific CD8 T-cell counts significantly increased after switch to therapy with P/EVL. Furthermore, the mTOR inhibitor sirolimus strongly inhibited alloresponses in vitro, whereas it did not affect CMV-specific responses. CONCLUSION: We observed a significant increase in (CMV-specific) effector-type CD8 and CD4 T-cell counts in everolimus-treated patients. These findings may at least in part explain the reported low incidence of CMV-related pathology in everolimus-treated patients.

Analysis of stem-cell-like properties of human CD161++IL-18Rα+ memory CD8+ T cells.

CD161(++)IL-18Rα(+)CD8(+) human T cells have recently been identified as a new subset of memory cells but their exact role remains unclear. CD161(++)IL-18Rα(+)CD8(+), mucosal-associated invariant T cells express a semi-invariant TCR Vα7.2-Jα33, which recognizes the MHC-related protein 1. On the basis of properties including the expression of the ABC-B1 transporter, cKit expression and survival after chemotherapy, CD161(++)IL-18Rα(+)CD8(+) T cells have been designated as 'stem' cells. Here we analyse location and functional properties of CD161(++)IL-18Rα(+) CD8(+) T cells and question whether they have other traits that would mark them as genuine 'stem' cells. CD161(++)IL-18Rα(+)CD8(+) T cells were found in peripheral blood, spleen and bone marrow but interestingly hardly at all in lymph nodes (LNs), which may possibly be explained by the finding that these cells express a specific set of chemokine receptors that allows migration to inflamed tissue rather than to LNs. In addition to TCR ligation and co-stimulation, CD161(++)IL-18Rα(+) CD8(+) T cells require cytokines for proliferation. The CD161(++)IL-18Rα(+) CD8(+) pool contains cells reactive towards peptides, derived from both persisting and cleared viruses. Although CD161(++)IL-18Rα(+) CD8(+) T cells express the ABC-B1 transporter, they have shorter telomeres and less telomerase activity and do not express aldehyde dehydrogenase. Finally, CD161(++)IL-18Rα(+) CD8(+) T cells show similarities to terminally differentiated T cells, expressing IFNγ, KLRG1 and the transcription factor Blimp-1. In conclusion, CD161(++)IL-18Rα(+) CD8(+) T cells lack many features of typical 'stem' cells, but appear rather to be a subset of effector-type cells.

Expansion of effector T cells associated with decreased PD-1 expression in patients with indolent B cell lymphomas and chronic lymphocytic leukemia.

In patients with chronic lymphocytic leukemia (CLL), numbers of CD8 + CD45RA +/- CD27- effector T cells are expanded. We investigated whether this expansion is also present in other B cell malignancies and the possible mechanism underlying these changes. Whereas an increase in total CD4+and CD8+ T cell numbers was found only in CLL, numbers of CD4+ and CD8+ effector T cells were significantly increased in both CLL and indolent lymphoma, but not aggressive lymphoma and myeloma. Interestingly, PD-1 expression was decreased on effector T cells and inversely correlated with effector T cell numbers, suggesting a functional role for PD-1 in regulating T cell homeostasis. In vitro experiments revealed impaired up-regulation of PD-1 upon T cell activation in the presence of malignant but also healthy B cells. Our data suggest that in CLL and indolent lymphoma, the malignant B cells affect PD-1 expression on effector T cells, resulting in an expansion of these subsets.

Cytomegalovirus-induced effector T cells cause endothelial cell damage.

Human cytomegalovirus (CMV) infection has been linked to inflammatory diseases that involve vascular endothelial cell damage, but definitive proof for a direct cytopathic effect of CMV in these diseases is lacking. CMV infection is associated with a strong increase in both CD4(+) and CD8(+) T cells with constitutive effector functions that can perpetuate systemic inflammation. We investigated whether CMV-induced immune responses could lead to endothelial damage in humans. We found that terminally differentiated effector CD4(+) and CD8(+) T cells, formed during primary CMV infection and maintained throughout latency, express high levels of CX3CR1 and CXCR3. The ligands of these receptors, fractalkine and IP-10, respectively, are expressed by activated endothelial cells. Peripheral blood mononuclear cells (PBMC) stimulated with CMV antigen produced soluble factors that stimulated endothelial cells to produce both chemokines. Finally, effector cells migrated in a fractalkine- and IP-10-dependent fashion to activated endothelial cells and induced apoptosis in endothelial cells that were stimulated by supernatant from CMV-activated PBMC. Our findings offer an explanation for the accumulation of highly differentiated T cells near to the endothelium in CMV-infected individuals that may result in endothelial damage.

Circulating lymphocyte subsets in different clinical situations after renal transplantation.

Phenotypic characterization of T and B lymphocytes allows the discrimination of functionally different subsets. Here, we questioned whether changes in peripheral lymphocyte subset distribution reflect specific clinical and histopathological entities after renal transplantation. Sixty-five renal transplant recipients with either histologically proven (sub)clinical acute rejection or chronic allograft dysfunction, or without abnormalities were studied for their peripheral lymphocyte subset composition and compared with 15 healthy control individuals. Naive, memory and effector CD8(+) T-cell counts were measured by staining for CD27, CD28 and CD45RO/RA. In addition, we studied the CD25(+) CD4(+) T-cell population for its composition regarding regulatory Foxp3(+) CD45RO(+) CD127(-) cells and activated CD45RO(+) CD127(+) cells. Naive, non-switched and switched memory B cells were defined by staining for IgD and CD27. We found a severe decrease in circulating effector-type CD8(+) T cells in recipients with chronic allograft dysfunction at 5 years after transplantation. Percentages of circulating CD25(+) CD127(low) CD4(+) regulatory T cells after transplantation were reduced, but we could not detect any change in the percentage of CD127(+) CD45RO(+) CD4(+) activated T cells in patients at any time or condition after renal transplantation. Regardless of clinical events, all renal transplant recipients showed decreased total B-cell counts and a more differentiated circulating B-cell pool than healthy individuals. The changes in lymphocyte subset distribution probably reflect the chronic antigenic stimulation that occurs in these transplant recipients. To determine the usefulness of lymphocyte subset-typing in clinical practice, large cohort studies are necessary.

Risk of Pneumocystis jiroveci pneumonia in patients long after renal transplantation.

BACKGROUND: Pneumocystis jiroveci pneumonia (PCP) is an important cause of morbidity and mortality in renal transplant recipients (RTRs). Chemoprophylaxis with trimethoprim/sulphamethoxazole is recommended during the early post-transplantation period, but the optimal duration has not been determined and a main drawback of chemoprophylaxis is the development of resistance of the commensal faecal flora. A cluster outbreak of PCP occurred in our outpatient Renal Transplant Unit. We aimed to investigate risk factors for PCP in RTRs to determine who should receive long-term chemoprophylaxis. METHODS: In a case-control study, we investigated common demographic variables and immunological parameters. Nine PCP cases diagnosed between August 2006 and April 2007 were matched with 18 control patients, who did not develop PCP, received their transplant in the same time-period and had a similar follow-up period with a comparable immunosuppressive drug regimen. RESULTS: The median time from transplantation to PCP was 19 months. We observed no significant differences in gender, age, donor type or number of rejections. In PCP cases, the median lymphocyte count just before PCP diagnosis was 0.49 (0.26-0.68), which was significantly reduced compared to the control patients after a similar follow-up period (median 1.36, 0.59-3.04, P = 0.002). This lymphocytopaenia was chronic and existed in most patients already for many months. CD4(+) T-cell counts were also significantly reduced in the PCP cases. We found no difference in the Th1, Th2 and Th17 subsets between PCP cases and control patients. CONCLUSION: Long-term prophylactic therapy for PCP may be indicated for RTR with persistent severe lymphocytopaenia.

Cytomegalovirus infection reduces telomere length of the circulating T cell pool.

Short telomeres of circulating leukocytes are a risk factor for age-related diseases, such as atherosclerosis, but the exact mechanisms generating variations in telomere length are unknown. We hypothesized that induction of differentiated T cells during chronic CMV infection would affect T cell telomere length. To test this, we measured the amount of differentiated T cells and telomere length of lymphocytes during primary CMV infection as well as CMV-seropositive and -seronegative healthy individuals. After primary CMV infection, we observed an increase in highly differentiated cells that coincided with a steep drop in telomere length. Moreover, we found in a cohort of 159 healthy individuals that telomere shortening was more rapid in CMV-seropositive individuals and correlated with the amount of differentiated T cells in both CD4(+) T cells and CD8(+) T cells. Finally, we found that telomere length measured in blood leukocytes is correlated with lymphocyte telomere length. Thus, CMV infection induces a strong decrease in T cell telomere length, which can be explained by changes in the composition of the circulating lymphocyte pool.

Mycophenolate mofetil inhibits T-cell proliferation in kidney transplant recipients without lowering intracellular dGTP and GTP.

To study if mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), indeed inhibits T-cell proliferation in kidney transplant recipients by lowering intracellular deoxyguanosine triphosphate (dGTP) and guanosine triphosphate (GTP) levels. Blood was drawn from 11 kidney transplant recipients. Ex vivo T-cell proliferation was measured by stimulation with phytohemagglutin (PHA) and anti-CD3 monoclonal antibody (mAb). Plasma MPA levels and intracellular dGTP and GTP in peripheral blood mononuclear cells were measured. MMF induces a significant decrease in T-lymphocyte proliferation at all time points (i.e. 24 h, 10 days and 8 weeks) after stimulation with both PHA (P = 0.001, 0.002 and 0.013 respectively) and anti-CD3 mAb (P = 0.004, 0.004 and 0.005 respectively). There was no significant change in intracellular dGTP (P = 0.31, 0.16 and 0.35) or GTP levels (P = 0.99, 0.32 and 0.49) between baseline and day 1, day 10 or week 8. All MPA levels were above the minimal required concentration for the inhibition of lymphocyte proliferation. MMF inhibits T-lymphocyte proliferation in kidney transplant recipients without lowering intracellular dGTP or GTP levels. This suggests another mechanism underlying its immunosuppressive capacity.

Rapamycin enhances the number of alloantigen-induced human CD103+CD8+ regulatory T cells in vitro.

BACKGROUND: Regulatory T cells (T(reg) cells) may be operational in both the induction and maintenance of transplantation tolerance. We recently showed that alloantigen-induced CD103+ CD8+ T cells strongly suppressed T-cell proliferation in mixed lymphocyte culture (MLC) via a contact-dependent mechanism. CD103 directs T lymphocytes to their ligand E-cadherin, which is expressed on renal tubular epithelial cells, and CD103+ CD8+ T cells have been described to be present in late renal allograft rejection. METHODS: We studied the influence of prednisolone, cyclosporin, tacrolimus, CD25 monoclonal antibodies, rapamycin, and mycophenolate mofetil (MMF) on the development and functional activity of alloantigen-activated CD103+ CD8+ T cells in MLC. RESULTS: Calcineurin inhibitors, MMF, and CD25mAb did not influence the number of CD103 expressing CD8+ T cells. In contrast, corticosteroids diminished CD103 expression on alloactivated CD8+ T cells, which appeared to be caused by their inhibitory action on myeloid dendritic cells. Addition of rapamycin to allocultures led to an increased percentage of CD103+ CD8+ alloreactive T cells. Moreover, in the presence of rapamycin, these cells tended to show higher suppressive capacity. CONCLUSIONS: Alloreactive CD103+ CD8+ T(reg) cells may expand and exert their suppressive function during immunosuppressive treatment with rapamycin. These data are relevant in the design of immunosuppressive drug regimens intended to induce and/or maintain transplantation tolerance.

Rapamycin does not induce anergy but inhibits expansion and differentiation of alloreactive human T cells.

BACKGROUND: Studies in mice have shown that rapamycin inhibits cell cycle progression and promotes the development of clonal anergy. We here addressed the question if rapamycin can induce anergy of human T cells and studied the effects of rapamycin on activation, proliferation and expression of cytotoxic effector molecules of alloresponsive T cells in mixed lymphocyte cultures. METHODS: Peripheral blood mononuclear cells from healthy individuals were labeled with CFSE to monitor subsequent cell divisions. Cells were cocultured with allogeneic irradiated cells in the presence or absence of rapamycin. Flowcytometric analysis was performed after staining for surface CD4, CD8, and CD25 and for intracellular perforin, granzyme B, active caspase-3, and TGF-beta. Bio-Plex cytokine assay was done to measure the secretion of IL-2, IL-4, IL-10, and IFN-gamma. RESULTS: Addition of rapamycin at a final concentration of 10 ng/ml strongly decreased precursor frequencies of alloreactive CD4+ and CD8+ T cells. However, when these cells were washed and subsequently specifically restimulated in the absence of rapamycin, the proliferative capacity appeared normal. Next to lowering precursor frequencies, rapamycin also inhibited T cell expansion by inducing apoptosis in divided alloreactive CD4+ and CD8+ T cells. Rapamycin did not interfere with the formation of CD25brightCD4+ T cells during allogeneic stimulation and did not inhibit their suppressive function. Furthermore, the drug decreased production of effector molecules perforin and granzyme B by alloreactive T cells and diminished alloreactive cytotoxicity. CONCLUSION: Our data show that rapamycin strongly inhibits proliferation and effector functions of alloreactive T cells in vitro, but does not induce alloantigen specific nonresponsiveness.

The granzyme B inhibitor SERPINB9 (protease inhibitor 9) circulates in blood and increases on primary cytomegalovirus infection after renal transplantation.

SERPINB9 is the only known human intracellular inhibitor of granzyme B (GrB), the effector molecule in immunity against cytomegalovirus (CMV) and in renal allograft rejection. Therefore, using specific enzyme-linked immunosorbent assays, we addressed the presence of circulating SERPINB9 during primary CMV infection, subclinical rejection, acute rejection, and uncomplicated posttransplantation course. Soluble (s) SERPINB9 circulates in blood and increases on primary CMV infection. This increase was significantly higher in symptomatic than in asymptomatic patients. In contrast, sSERPINB9 levels did not change in response to subclinical or acute rejection. We demonstrated the presence of circulating sSERPINB9/sGrB complexes, which suggests that SERPINB9 has extracellular functions as well.

IL-7 receptor alpha chain expression distinguishes functional subsets of virus-specific human CD8+ T cells.

Virus-specific CD8+ T cells emerge after infection with herpesviruses and maintain latency to these persistent pathogens. It has been demonstrated that murine memory CD8+ T-cell precursors specific for acute lymphocytic choriomeningitis virus express interleukin-7 receptor alpha (IL-7Ralpha), and IL-7 is involved in maintaining memory populations after the clearance of antigen. To investigate whether human CD8+ T cells reactive toward persistent viruses are maintained similarly, we analyzed IL-7Ralpha expression and function on these virus-specific cells. During primary infection, all cytomegalovirus (CMV)-specific CD8+ T cells and most Epstein-Barr virus (EBV)-specific CD8+ T cells lacked IL-7Ralpha expression. Only some virus-specific T cells expressed IL-7Ralpha late after viral replication became undetectable. CD8+ T cells specific for cleared viruses, influenza (FLU), and respiratory syncytial virus (RSV) all expressed IL-7Ralpha. Remarkably, the percentage of IL-7Ralpha- CMV-specific T cells correlated with the height of viral replication in the acute phase. Virus-specific IL-7Ralpha+ cells proliferated vigorously in response to IL-7, IL-15, or peptide, whereas IL-7Ralpha- cells required both peptide and helper-cell activation or IL-2 or IL-15 for optimal expansion. Our data suggest that although IL-7 is essential for the maintenance of memory cells in the absence of antigen, CD8+ T cells specific for latent viruses need T-cell receptor activation plus helper factors to persist.

Suppression extends to major histocompatibility antigens linked to tolerizing minor histocompatibility antigens, but not the other way round.

'Active suppression', a mechanism of transplantation tolerance, can spread to newly introduced minor antigens once these antigens are linked to tolerizing antigens. We explored whether this suppression can extend to major histocompatibility (MHC) antigens and whether this phenomenon can be demonstrated once tolerance is induced to a MHC antigen. Mice were tolerized using donor bone marrow plus CD4 and CD8 monoclonal antibodies. The following strain combinations were used: AKR (H-2k) into CBA (H-2k), a multiple minor difference and B6 (H-2b) into B6(bm12) (H-2b), a MHC class II difference. Tolerance was tested by a donorskingraft. CBA mice tolerant to AKR received a second skin carrying either AKR antigens plus additional multiple minor antigens [F1(AKRxBalb.K)] or carrying additional minors and a MHC class I antigen (B10.AKM-H2M). B6(bm12) (H-2b) tolerant to B6 (H-2b) were grafted with skin from a Balb.B donor (Balb minors linked to the tolerizing class II antigen) or from a B10.A(3R) strain (a MHC class I antigen linked to the tolerizing class II antigen). CBA mice tolerant to AKR accepted F1(AKRxBalb.K) skin, whereas F1(CBAxBalb.K) were rejected. Rejection of B10.AKM/H2M skin by tolerant mice was delayed as compared with nontolerant mice. Tolerant and nontolerant B6(bm12) mice rejected Balb.B skin and B10.A(3R) skin within the same time. Thus, in this model, suppression was linked to minors. Alloreactivity against minors and majors could be suppressed. Suppression linked to a class II antigen could not be demonstrated.

The size and phenotype of virus-specific T cell populations is determined by repetitive antigenic stimulation and environmental cytokines.

Based on the expression of the TNFR SFP CD27, two Ag-primed CD8(+) T cell subsets can be discerned in the circulation of healthy individuals: CD27(+) T cells that produce a variety of cytokines but do not display immediate cytolytic activity; and cytotoxic CD27(-) T cells, which secrete only IFN-gamma and TNF-alpha. The mechanism that controls the generation of these different phenotypes is unknown. We show that CMV reactivation not only increases the number of virus-specific T cells but also induces their transition from a CD27(+) to a CD27(-) phenotype. In support of a relation between pool size and phenotype in a cohort of latently infected individuals, the number of Ag-specific CD27(-) CD8(+) T cells was found to be linearly related to the total number of CMV-specific CD8(+) T cells. In vitro studies revealed that the acquisition of the CD27(-) phenotype on CMV-specific T cells depended on the interaction of CD27 with its cellular ligand, CD70. Expression of CD70 was proportional to the amount of antigenic stimulation and blocked by the CD4(+) T cell-derived cytokine IL-21. Thus, induction of CD70, which may vary in distinct viral infections, appears to be a key factor in determining the size and phenotype of the CMV-specific T cell population in latently infected individuals.

Effects of CD25 monoclonal antibody on proliferative and effector functions of alloactivated human T cells in vitro.

Prophylactic treatment with CD25 mAb has led to a significant decrease of acute rejection rates after renal transplantation. However, despite its inhibitory effect on T cell proliferation and effector functions, rejections still occur. To obtain more insight in persistent alloreactivity, we evaluated the effects of the chimeric IgG1kappa CD25 mAb Basiliximab on proliferation and differentiation of alloactivated T cells from healthy individuals in vitro. Moreover, the capacity of other members of the common cytokine-receptor gamma-chain family to overcome the inhibitory effects of CD25 mAb was studied. The CD25 mAb appeared to limit expansion of alloreactive lymphocytes rather than blocking entry into cell cycle, and it did so irrespective of the previous antigen experience of the cells. Both CD4+ and CD8+ alloresponsive lymphocytes showed diminished intracellular expression of IFN-gamma, TNF-alpha, perforin and granzyme B. Remarkably, cytotoxicity was completely abolished. IL-7, IL-15 and IL-21 could bypass the inhibitory effects of the CD25 mAb on both proliferation and cytotoxicity. In conclusion, persistent alloreactivity in the presence of therapeutic concentrations of CD25 mAb may be caused by alloreactive T cells that still produce cytokines that can damage the allograft. In addition, other members of the common cytokine-receptor gamma-chain family can rescue the proliferative and cytotoxic activity of these alloreactive T cells.