Tetramer monitoring to assess risk factors for recurrent cytomegalovirus reactivation and reconstitution of antiviral immunity post allogeneic hematopoietic stem cell transplantation

S. Borchers, S. Luther, U. Lips, N. Hahn, J. Kontsendorn, M. Stadler, S. Buchholz, H. Diedrich, M. Eder, U. Koehl, A. Ganser, E. Mischak‐ Weissinger. Tetramer monitoring to assess risk factors for recurrent cytomegalovirus reactivation and reconstitution of antiviral immunity post allogeneic hematopoietic stem cell transplantation.
Transpl Infect Dis 2011: 13: 222–236. All rights reserved Background. Reactivation of cytomegalovirus (CMV) is a major cause of morbidity after allogeneic hematopoietic stem cell transplantation (HSCT). In healthy individuals, virus‐specific T cells (CMV‐CTL) control the reactivation of latent CMV. The monitoring of virus‐epitope‐binding CD8⁺ T cells using major histocompatibility complex‐I‐peptide complexes (tetramers) has recently been established, allowing assessment of the reconstitution of CMV‐CTL post HSCT. Patients and methods. In order to study immune reconstitution and reactivation control through CMV‐CTL, we regularly monitored all patients undergoing allogeneic HSCT in our department for 2 years, who matched at least 1 of 6 commercially available tetramers for common human leukocyte antigen (HLA) types. To verify risk factors for CMV reactivations in our cohorts, clinical characteristics of all patients transplanted within the last 10 years were included in statistical analyses determining the relative risk for single and recurrent CMV reactivations. Results. As expected, CMV serostatus, HLA match, and donor source significantly influenced the risk of recurrent CMV reactivation. Applying CMV‐CTL tetramer monitoring for 2 years allowed the monitoring of 114 (85%) of 134 patients, by testing a set of tetramers representing 6 epitopes from 3 different CMV proteins. The presence of CMV‐CTL before day +50 and their expansion post reactivation seem to protect against recurrent CMV reactivations. The mean number of CMV‐CTL by day +100 was >5‐fold higher in the recipient CMV‐positive/donor‐positive (R+/D+) group (91/μL) compared with the R+/D? (13/μL) and the R?/D+(2/μL) group. Seventy‐nine percent of patients from the R+/D+ setting recovered >10 CMV‐CTL per μL by day +100, while almost 50% of the other groups failed to mount a CMV‐specific response by that time (R+/D?: 58%; R?/D+: 43%). Conclusion. Tetramer monitoring can help to predict (recurrent) CMV reactivation and is a useful approach to monitor individual patients with increased risk for recurrent reactivation post HSCT; thus, it could help to identify patients in need of adoptive transfer of CMV‐CTL or to optimize the use of antiviral drugs.     

Pre‐transplant cytomegalovirus (CMV) serostatus remains the most important determinant of CMV reactivation after allogeneic hematopoietic stem cell transplantation in the era of surveillance and preemptive therapy

B. George, N. Pati, N. Gilroy, M. Ratnamohan, G. Huang, I. Kerridge, M. Hertzberg, D. Gottlieb, K. Bradstock. Pre‐transplant cytomegalovirus (CMV) serostatus remains the most important determinant of CMV reactivation after allogeneic hematopoietic stem cell transplantation in the era of surveillance and preemptive therapy.
Transpl Infect Dis 2010: 12: 322–329. All rights reserved Abstract: Between January 2001 and June 2008, 315 adult patients (median age 43 years, range 16–65) including 203 males and 112 females undergoing hematopoietic stem cell transplantation (HSCT) had serial monitoring for cytomegalovirus (CMV) followed by initiation of preemptive therapy. The majority (62.1%) had a conventional myeloablative transplant with 116 (36.9%) having a reduced‐intensity conditioning (RIC) transplant, using either matched sibling/family (63.3%) or unrelated donors (36.7%). Graft source was peripheral blood stem cells in 257 (81.5%), bone marrow in 41 (13.1%), and cord blood in 16 (5.4%). T‐cell depletion with anti‐thymocyte globulin or alemtuzumab was used in 35%. Based upon CMV serostatus, patients were classified into low risk (donor [D]?/recipient [R]?), intermediate risk (D+/R?), or high risk (D?/R+ or D+/R+). Serial weekly monitoring for CMV viremia was performed using a qualitative polymerase chain reaction (PCR) and when positive, quantification was done using either pp65 antigen or a quantitative PCR. CMV reactivation was seen in 123 patients (39.1%) at a median of 50 days post HSCT (range 22–1978). CMV serostatus was the most important risk factor with incidence of 53% in the high‐risk group (53.3%) compared with 10.2% in the intermediate risk and 0% in the low‐risk group (P<0.0001). Other significant risk factors identified included use of alemtuzumab during conditioning (P=0.03), RIC transplants (P=0.06), and the presence of acute graft‐versus‐host disease (GVHD) (P<0.0001). On a multivariate analysis, CMV serostatus, RIC transplants, and acute GVHD remained independent predictors of CMV reactivation. All were treated with antiviral therapy with responses seen in 109 (88.6%). Sixteen patients (13%) developed CMV disease at a median of 59 days post HSCT (range 26 days–46 months), 8 of whom died. At a median follow up of 43 months (range 6–93), 166 patients (52.6%) are alive with a significantly higher survival among patients without CMV reactivation (57.2%) as compared with patients with CMV reactivation (45.5%; P=0.049). CMV reactivation and disease remains a major problem in high‐risk patients undergoing allogeneic HSCT. Novel prophylactic measures such as immunotherapy and drug prophylaxis need to be considered in this specific group of patients.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

Abstract

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28^? suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28^? T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28^? T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28⁻ suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28⁻ T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28⁻ T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

CD8+ T-cell immunity against cancer-testis antigens develops following allogeneic stem cell transplantation and reveals a potential mechanism for the graft-versus-leukemia effect

Background

Allogeneic stem cell transplantation is associated with a powerful ‘graft-versus-leukemia’ effect that is generally considered to result from an alloreactive T-cell immune response. However, disease remission can also be observed after syngeneic transplantation and we investigated whether a T-cell immune response to cancer-testis antigens can be detected in patients in the post-transplant period.

Design and Methods

The T-cell immune response against cancer-testis antigens was studied in a cohort of 41 patients who underwent allogeneic stem cell transplantation for the management of acute myeloid leukemia or multiple myeloma. The cytokine secretion assay was combined with magnetic selection to allow detection of an interferon-γ-secreting T-cell response to a panel of cancer-testis antigen peptides.

Results

A cancer-testis antigen-specific CD8⁺ T-cell immune response was observed in the peripheral blood of five patients with an average magnitude of 0.045% of the CD8⁺ T-cell repertoire. Four of these patients had undergone reduced intensity conditioning transplantation with alemtuzumab for the treatment of acute myeloid leukemia and three remain in long-term remission. T-cell immunity was focused against peptides derived from MAGE proteins and was markedly increased within the bone marrow.

Conclusions

Functional cancer-testis antigen-specific CD8⁺ T-cell immune responses develop in the early period following reduced intensity allogeneic stem cell transplantation and are preferentially localized to bone marrow. These immune responses are likely to contribute to the cellular basis of the graft-versus-leukemia effect.     

Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Leishmania infantum released proteins specifically regulate cytokine expression and production patterns by CD4+ and CD8+ T cells

Specific immune responses by CD4+ and CD8+ T cells, from two infected mice strains (BALB/c and C57BL/6), induced by High, Inter and Low protein fractions released by Leishmania infantum, were assessed through the evaluation of IL-12, IFN-gamma and IL-10 mRNA by real-time PCR and respective protein production by ELISA. During infection establishment, High and Inter fractions directed both mice strains T cells subsets to increase the production of IFN-gamma, associated to IL-12 release. Later on, parasite replication augmented in BALB/c and stabilised in C57BL/6 mice. Inter fraction induced CD4+ T cells to maintain IFN-gamma production, with the simultaneous release of IL-12 by both cell subsets in BALB/c mice and by CD8+ T cells in C57BL/6 mice. These observations suggested a prophylactic potential for Inter fraction which was able to induce Th1 response with IL-12 involvement, required for the maintenance of memory cells, in mice strains with different parasitic evolution.     

Microarray analysis reveals similarity between CD8+CD28- T cells from young and elderly persons, but not of CD8+CD28+ T cells

We isolated highly purified CD8⁺CD28⁺ and CD8⁺CD28⁻ T cell populations from healthy young and elderly persons for gene expression profiling using Affymetrix oligonucleotide microarrays. We demonstrate that the gene expression profile of CD8⁺CD28⁻ T cells is very similar in young and elderly persons. In contrast, CD8⁺CD28⁺ in elderly differ from CD8⁺CD28⁺ in young persons. Hierarchical clustering revealed that CD8⁺CD28⁺ in elderly are located between CD8⁺CD28⁺ in young and CD8⁺CD28⁻ (young and old) T cells regarding their differentiation state. Our study demonstrates a dichotomy of gene expression levels between CD8⁺CD28⁺ T cells in young and elderly persons but a similarity between CD8⁺CD28⁻ T cells in young and elderly persons. As CD8⁺CD28⁺ T cells from elderly and young persons are distinct due to a different composition of the population, these results suggest that the gene expression profile does not depend on chronological age but depends on the differentiation state of the individual cell types.     

The kinetics of immune reconstitution after cord blood transplantation and selected CD34+ stem cell transplantation in children: comparison with bone marrow transplantation

The present study compares immune reconstitution after allogeneic cord blood transplantation (CBT) and CD34+ stem cell transplantation (CD34-SCT) with that after bone marrow transplantation (BMT). Eighty-eight children who underwent CBT (20 patients), BMT (58), and CD34-SCT (10) were enrolled, and lymphocytes and T-, B-, and natural killer-lymphocyte subsets were monitored for more than 5 years after transplantation. CBT recipients showed significant ircreases in (1) total lymphocyte counts (P < .001), (2) CD4+/CD8+ cell ratios (P < .01), (3) CD4+ and CD4+CD45RA+ cells (P < .001), (4) CD8+CD11b+ cells (P < .001), and (5) CD19+ and CD19+CD5+ cells (P < .0001) and marked decreases in the frequencies of CD8+ and CD8+CD11b- cells (P < .0001). CD34-SCT recipients showed lower lymphocyte counts in the first 6 months and an emergence of lymphocyte and CD4+CD45RA+ cells at approximately 9 months and 1 year. Both CBT and CD34-SCT recipients showed increased frequencies of CD56+ cells at 1 month (CD34-SCT versus BMT, P < .001) but decreased frequencies after 6 months (CBT versus BMT, P < .001). Lymphoproliferative responses to exogenous interleukin 2 were constantly lower in CBT and CD34-SCT recipients than in BMT recipients. These results suggest that the delay in immune reconstitution after CBT in the early phase was mainly qualitative and related to the immaturity of cells, whereas the delay in CD34-SCT was mainly quantitative in the first several months.     

The CD4 molecule on CD8+ T lymphocytes directly enhances the immune response to viral and cellular antigens

CD8+ T lymphocytes play a major role in cellular-mediated immune responses to foreign antigen. We have previously demonstrated that costimulation of purified human CD8+ T cells induces de novo expression of the CD4 molecule and that ligation of CD4 on this cell type modulates CD8+ T cell activity in vitro. Herein, we investigate how the CD4 molecule expressed on murine CD8+ T cells contributes to CD8+ cell responses in vivo by employing adoptive transfer of CD8 cells from CD4 knockout mice into severe combined immunodeficient (SCID) recipients. Transfer of these cells into syngeneic SCID mice resulted in a decreased immune response to infection by lymphocytic choriomeningitis virus. These decreased responses occurred even in the presence of CD4+ T cells, indicating that this was truly a CD8-cell defect. Similarly, transfer of CD8+ T cells incapable of expressing CD4 into allogeneic SCID mice resulted in a decreased response to alloantigens compared with that of normal CD8+ T cells. Therefore, CD4 expression on CD8 T lymphocytes modulates cytotoxic T lymphocyte function and is critical in vivo for optimal cell-mediated immunity to viral and alloantigens.     

FoxP3+ regulatory T cells essentially contribute to peripheral CD8+ T-cell tolerance induced by steady-state dendritic cells

Peripheral T-cell tolerance is thought to significantly contribute to the prevention of autoimmunity, and it has been shown that antigen-presenting steady-state dendritic cells efficiently induce peripheral tolerance. We previously showed that dendritic-cell–induced tolerance is a T-cell–intrinsic process that depends on coinhibitory molecules such as programmed death-1. Here we specifically analyze the involvement of FoxP3⁺ regulatory T cells, which are known to be important for maintenance of self-tolerance. We show that antigen presentation by steady-state dendritic cells failed to induce peripheral tolerance in the absence of FoxP3⁺ regulatory T cells but induced protective CD8⁺ T-cell–mediated immunity instead. Regulatory T-cell–depleted mice had massively increased numbers of dendritic cells in lymph nodes. Dendritic cells isolated from mice without regulatory T cells had up-regulated costimulatory molecules and showed stronger T-cell stimulatory capacity ex vivo, suggesting that regulatory T cells contribute to peripheral tolerance by keeping the dendritic cells in an immature state. Using blocking antibodies, we demonstrate that CTLA-4 but not IL-10 is necessary for control of dendritic cells by regulatory T cells.     

Effects of baicalin in CD4 + CD29 + T cell subsets of ulcerative colitis patients

AIM: To evaluate the role of baicalin in ulcerative colitis (UC) with regard to the CD4⁺CD29⁺ T helper cell, its surface markers and serum inflammatory cytokines.METHODS: Flow cytometry was used to detect the percentage of CD4⁺CD29⁺ cells in patients with UC. Real time polymerase chain reaction was used to detect expression of GATA-3, forkhead box P3, T-box expressed in T cells (T-bet), and retinoic acid-related orphan nuclear hormone receptor C (RORC). Western blotting was used to analyze expression of nuclear factor-κB (NF-κB) p65, phosphorylation of NF-κB (p-NF-κB) p65, STAT4, p-STAT4, STAT6 and p-STAT6. The concentrations of interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-6, IL-10 and TGF-β in serum were determined by ELISA assay.RESULTS: The percentages of CD4⁺CD29⁺ T cells were lower in treatment with 40 and 20 μmol/L baicalin than in the treatment of no baicalin. Treatment with 40 or 20 μmol/L baicalin significantly upregulated expression of IL-4, TGF-β1 and IL-10, increased p-STAT6/STAT6 ratio, but downregulated expression of IFN-γ, IL-5, IL-6, RORC, Foxp3 and T-bet, and decreased ratios of T-bet/GATA-3, p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin.CONCLUSION: The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4⁺CD29⁺ cells and modulating immunosuppressive pathways.     

Low serum transferrin levels in HFE C282Y homozygous subjects are associated with low CD8(+) T lymphocyte numbers

Hereditary hemochromatosis (HH) is a genetic iron overload disease, in the majority of cases associated with homozygosity for the C282Y mutation of the HFE gene. In spite of this genetic homogeneity, there is a great clinical heterogeneity among HH patients. Low CD8(+) lymphocyte numbers have been associated with a more severe expression of iron overload in HH patients, and in experimental models of iron overload. HH patients present low serum transferrin levels. Transferrin is an indispensable resource for lymphopoiesis. Lymphocyte homeostasis follows general ecology rules of population dynamics that involve competition for limiting resources. In the present study, we questioned whether transferrin levels could be associated with the anomalies seen previously in lymphocyte subset numbers in HH patients. Transferrin levels, total and subset T lymphocyte counts were done in 426 apparently healthy subjects genotyped for HFE. All HFE C282Y carriers presented significantly lower serum transferrin levels than the wild type group, a difference that could not be explained solely by the degree of iron overload. Significant differences were also seen in transferrin levels between males and females, with females presenting higher average serum Transferrin levels. In the population of subjects with Transferrin levels lower than 248 mg/dl, a positive correlation was seen between the peripheral CD8(+) lymphocyte numbers and serum transferrin levels (R(2) = 2.41; r = 0.16; P = 0.018). To test the possible limiting resource effect of transferrin, the correlation between transferrin levels and CD8(+) lymphocyte numbers was scrutinized in 34 HH patients, homozygous for the C282Y mutation. In the homozygous males, where the lowest average transferrin levels were seen, another highly significant correlation was observed between Transferrin levels and CD8(+) numbers. This correlation points to a possible role of transferrin as a limiting resource for MHC class I dependent lymphocyte proliferation, an effect that was not observed in C282Y homozygous female patients.     

Kinetics of interferon‐gamma producing cytomegalovirus (CMV)‐specific CD4+ and CD8+ T lymphocytes and the risk of subsequent CMV viremia after allogeneic hematopoietic stem cell transplantation

Background. Deficiencies in cytomegalovirus (CMV)‐specific T lymphocytes impair the immunologic response against CMV reactivation after allogeneic hematopoietic stem cell transplantation (HSCT). Methods. A time‐dependent analysis was conducted to determine the association between the percentages and kinetics of interferon‐gamma‐producing CMV‐specific CD4+ and CD8+ T lymphocytes and CMV viremia among 30 allogeneic HSCT recipients. Results. Higher percentages of CD4+ T lymphocytes activated with CMVpp65 (hazard ratio [HR]: 2.06; 95% confidence interval [95% CI]: 1.18–3.6; P=0.011) and CMV lysate (HR: 1.18; 95% CI: 0.99–1.42; P=0.072), and higher percentages of CD8+ T lymphocytes activated by CMV immediate early‐1 (HR: 1.2; 95% CI: 1.01–1.43; P=0.038) and CMVpp65 (HR: 1.12; 95% CI: 1.0–1.27; P=0.060) were associated with time‐to‐CMV viremia. Furthermore, a higher degree in the decline of CMV lysate‐activated CD4+ T lymphocytes (HR: 1.14; 95% CI: 0.96–1.36; P=0.125) and CMVpp65‐activated CD8+ T lymphocytes (HR: 1.36; 95% CI: 1.03–1.78; P=0.031) was suggestive of or significantly associated with time‐to‐CMV viremia. Conclusions. Higher levels of CMV‐specific CD4+ and CD8+ T lymphocytes were associated with subsequent CMV viremia after HSCT. The association between CMV viremia and the degree of decline in CMV‐specific T lymphocytes suggests that severe disruption in homeostatic CMV‐specific immune environment contributes to the immunopathogenesis of CMV after allogeneic HSCT.     

Specific, functional effector/memory CD8+ T cells are found in the liver post-vaccination

BACKGROUND: The liver efficiently eliminates activated CD8+ T blasts. It is unknown if vaccine-primed CD8+ T blasts migrate to and establish functional CD8+ T cell immunity in the liver post-immunization. AIMS: We tested, if functional CD8+ T cell populations can be detected in the liver post-vaccination. METHODS: Murine CD8+ T cells with different epitope/restriction specificities were primed by intramuscular injection of protein- or DNA-based vaccines. The kinetics of appearance in the liver, as well as the surface phenotype and functional competence of intrahepatic, specific CD8+ T cell populations was tested. RESULTS: High numbers of specific CD8+ T cells appear in the liver after vaccination that are activated (CD69+ CD44+), express effector functions (CD27lo/CD28lo phenotype, interferon gamma secretion, specific cytolytic reactivity), but show no evidence of apoptosis (annexin V-, B220lo, similar numbers/kinetics in primed, congenic lpr/lpr mice). Specific CD8+ T cells from the liver adoptively transferred into a na?ve, syngeneic host successfully reconstitute specific CD8+ T cell immunity. CONCLUSIONS: Specific, functionally competent CD8+ effector/memory T cell populations are established in the liver for months post-vaccination.