The impact of regulatory T cells on the graft-versus-leukemia effect.

Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is the only curative therapy for many hematologic malignancies, whereby the Graft-versus-Leukemia (GVL) effect plays a pivotal role in controlling relapse. However, the success of GVL is hindered by Graft-versus-Host Disease (GVHD), where donor T cells attack healthy tissues in the recipient. The ability of natural regulatory T cells (Treg) to suppress immune responses has been exploited as a therapeutical option against GVHD. Still, it is crucial to evaluate if the ability of Treg to suppress GVHD does not compromise the benefits of GVL. Initial studies in animal models suggest that Treg can attenuate GVHD while preserving GVL, but results vary according to tumor type. Human trials using Treg as GVHD prophylaxis or treatment show promising results, emphasizing the importance of infusion timing and Treg/Tcon ratios. In this review, we discuss strategies that can be used aiming to enhance GVL post-Treg infusion and the proposed mechanisms for the maintenance of the GVL effect upon the adoptive Treg transfer. In order to optimize the therapeutic outcomes of Treg administration in allo-HSCT, future efforts should focus on refining Treg sources for infusion and evaluating their specificity for antigens mediating GVHD while preserving GVL responses.

Differential activation of basal and IL-7-induced PI3K/Akt/mTOR and JAK/STAT5 signaling distinguishes pediatric from adult acute lymphoblastic leukemia.

Not available.

BK virus-specific T-cell immune reconstitution after allogeneic hematopoietic cell transplantation.

Clinical disease caused by BK virus reactivation is a frequent complication of allogeneic hematopoietic cell transplantation (HCT). Because of the lack of effective antiviral agents, BK virus-specific T cells are emerging as a potential therapy for BK virus disease, but the immune response to BK virus after allogeneic HCT has not been well characterized. Our study describes reconstitution of BK virus-specific T-cell immunity in 77 adult patients after HCT. All patients had urinary symptoms, and urine was tested for BK virus replication; 33 patients were positive for BK virus (cases), and 44 were negative (controls). In BK virus cases, the median time to first positive test was 75 days (range, 2-511). BK virus cases had lower CD4 T-cell counts 3 to 9 months after transplant, but CD8 T-cell counts were similar in cases and controls. BK virus-specific T cells were identified by cytokine flow cytometry in cryopreserved samples collected prospectively. BK virus-specific CD4 T cells producing T helper 1 (Th1) cytokines recovered quickly after HCT. BK virus-specific T cells were detected more frequently in patients with BK virus reactivation at most time points, and CD4 T cells producing Th1 cytokines were more frequent than BK virus-specific cytolytic CD8 T cells. Early detection of interferon-γ+ and cytolytic BK virus-specific CD4 T cells was associated with lower rates of hematuria among cases. Overall, our study describes recovery of BK virus-specific T cells after HCT and the distinct roles for BK virus-specific T cells in the development and resolution of clinical symptoms.

Naive and Stem Cell Memory T Cell Subset Recovery Reveals Opposing Reconstitution Patterns in CD4 and CD8 T Cells in Chronic Graft vs. Host Disease.

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of hematological malignancies remains hampered by life-threatening chronic graft vs. host disease (cGVHD). Although multifactorial in nature, cGVHD has been associated with imbalances between effector and regulatory T cells (Treg). To further elucidate this issue, we performed a prospective analysis of patients undergoing unrelated donor allo-HSCT after a reduced intensity conditioning (RIC) regimen containing anti-thymocyte globulin (ATG) and the same GVHD prophylaxis, at a single institution. We studied T cell subset homeostasis over a 24-month follow-up after HSCT in a comparative analysis of patients with and without cGVHD. We also quantified naive and memory T cell subsets, proliferation and expression of the apoptosis-related proteins Bcl-2 and CD95. Finally, we assessed thymic function by T cell receptor excision circle (TREC) quantification and T cell receptor (TCR) diversity by TCRVß spectratyping. While the total number of conventional CD4 (Tcon) and CD8 T cells was similar between patient groups, Treg were decreased in cGVHD patients. Interestingly, we also observed divergent patterns of Naive and Stem Cell Memory (SCM) subset recovery in Treg and Tcon compared to CD8. Patients with cGVHD showed impaired recovery of Naive and SCM Tcon and Treg, but significantly increased frequencies and absolute numbers of Naive and SCM were observed in the CD8 pool. Markedly increased EMRA CD8 T cells were also noted in cGVHD. Taken together, these results suggest that Naive, SCM and EMRA CD8 play a role in the emergence of cGHVD. Reduced Naive and recent thymic emigrant Tcon and Treg in cGVHD was likely due to impaired thymic output, as it was accompanied by decreased CD4 TREC and TCR diversity. On the other hand, CD8 TCR diversity was similar between patient groups. Furthermore, no correlation was observed between CD8 TREC content and Naive CD8 numbers, suggesting limited thymic production of Naive CD8 T cells in patients after transplant, especially in those developing cGVHD. The mechanisms behind the opposing patterns of CD4 and CD8 subset cell recovery in cGVHD remain elusive, but may be linked to thymic damage associated with the conditioning regimen and/or acute GVHD.

Adult B-cell acute lymphoblastic leukemia cells display decreased PTEN activity and constitutive hyperactivation of PI3K/Akt pathway despite high PTEN protein levels.

Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.

Long-term immune reconstitution of naive and memory t cell pools after haploidentical hematopoietic stem cell transplantation.

Haploidentical hematopoietic stem cell transplantation (HSCT) constitutes an important alternative for patients lacking a human leukocyte antigen (HLA)-matched donor. Although the use of haploidentical donors is increasingly common, the long-term impact of generating a donor-derived immune system in the context of an HLA-mismatched thymic environment remains poorly characterized. We performed an in-depth assessment of immune reconstitution in a group of haploidentical HSCT recipients 4 to 6 years posttransplantation, in parallel with the respective parental donors and age-matched healthy control subjects. Our data show that the proportion of naive and memory subsets in the recipients, both within CD8(+) and CD4(+) T cells, more closely resembled that observed in age-matched control subjects than in the donors. HSCT recipients displayed relatively high signal-joint T cell-receptor excision circle levels and a high frequency of the recent thymic emigrant-enriched CD31(+) subset within naive CD4(+) and naive regulatory T cells. Moreover, CD8(+), CD4(+), and regulatory T cells from HSCT recipients displayed a diverse T cell repertoire. These results support a key role for thymic output in T cell reconstitution. Nevertheless, HSCT recipients had significantly shorter telomeres within a naive-enriched CD4(+) T cell population than age-matched control subjects, despite the similar telomere length observed within the most differentiated CD8(+) and CD4(+) T cell subsets. Overall, our data suggest that long-term immune reconstitution was successfully achieved after haploidentical HSCT, a process that appears to have largely relied on de novo T cell production.

Preserved CD4 T-cell telomere length during long-lasting HIV-2 infection.

HIV-2 infection features a much slower course than HIV-1 infection, often asymptomatic for over 20 years, without antiretroviral therapy (ART). Nevertheless, CD4 T cells progressively decline, in direct correlation with immune activation and cell cycling. We report, for the first time, preserved telomere length within naive and memory CD4 subsets in prolonged HIV-2 infection despite the increased CD4 turnover.

Reversible senescence in human CD4+CD45RA+CD27- memory T cells.

Persistent viral infections and inflammatory syndromes induce the accumulation of T cells with characteristics of terminal differentiation or senescence. However, the mechanism that regulates the end-stage differentiation of these cells is unclear. Human CD4(+) effector memory (EM) T cells (CD27(-)CD45RA(-)) and also EM T cells that re-express CD45RA (CD27(-)CD45RA(+); EMRA) have many characteristics of end-stage differentiation. These include the expression of surface KLRG1 and CD57, reduced replicative capacity, decreased survival, and high expression of nuclear γH2AX after TCR activation. A paradoxical observation was that although CD4(+) EMRA T cells exhibit defective telomerase activity after activation, they have significantly longer telomeres than central memory (CM)-like (CD27(+)CD45RA(-)) and EM (CD27(-)CD45RA(-)) CD4(+) T cells. This suggested that telomerase activity was actively inhibited in this population. Because proinflammatory cytokines such as TNF-α inhibited telomerase activity in T cells via a p38 MAPK pathway, we investigated the involvement of p38 signaling in CD4(+) EMRA T cells. We found that the expression of both total and phosphorylated p38 was highest in the EM and EMRA compared with that of other CD4(+) T cell subsets. Furthermore, the inhibition of p38 signaling, especially in CD4(+) EMRA T cells, significantly enhanced their telomerase activity and survival after TCR activation. Thus, activation of the p38 MAPK pathway is directly involved in certain senescence characteristics of highly differentiated CD4(+) T cells. In particular, CD4(+) EMRA T cells have features of telomere-independent senescence that are regulated by active cell signaling pathways that are reversible.

Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4+CD45RA+CD27+ T cells: the potential involvement of interleukin-7 in this process.

The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4+ T-cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA+ CD27+ CD4+ T cells over time. In contrast, the increase in CD45RA⁻ CD27⁻ and CD45RA+ CD27⁻ CD4+ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RA⁻ CD27⁻ and CD45RA+ CD27⁻ CD4+ T cells in CMV-seropositive donors are specific for this virus. CD45RA+ CD27⁻ CD4+ T cells have significantly reduced CD28, interleukin-7 receptor α (IL-7Rα) and Bcl-2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T-cell receptor activation compared with the other T-cell subsets in the same donors. Despite this, the CD45RA+ CD27⁻ subset is as multifunctional as the CD45RA⁻ D27+ and CD45RA⁻ CD27⁻ CD4+ T-cell subsets, indicating that they are not an exhausted population. In addition, CD45RA+ CD27⁻ CD4+ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4+ memory T cells re-expressing CD45RA can be generated from the CD45RA⁻ CD27+ population by the addition of IL-7 and during this process these cells down-regulated expression of IL-7R and Bcl-2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA+ CD27⁻ CD4+ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated.

KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative dysfunction of highly differentiated CD8+ T cells.

Highly differentiated CD8+CD28-CD27- T cells have short telomeres, defective telomerase activity, and reduced capacity for proliferation, indicating that they are close to replicative senescence. In addition, these cells express increased levels of the senescence-associated inhibitory receptor KLRG1 and have poor capacity for IL-2 synthesis and defective Akt (ser(473)) phosphorylation after activation. It is not known whether signaling via KLRG1 contributes to any of the attenuated differentiation-related functional changes in CD8+ T cells. To address this, we blocked KLRG1 signaling during T-cell receptor activation using antibodies against its major ligand, E-cadherin. This resulted in a significant enhancement of Akt (ser(473)) phosphorylation and T-cell receptor-induced proliferative activity of CD8+CD28-CD27- T cells. Furthermore, the increase of proliferation was directly linked to the Akt-mediated induction of cyclin D and E and reduction in the cyclin inhibitor p27 expression. In contrast, the reduced telomerase activity in highly differentiated CD8+CD28(-)CD27- T cells was not altered by KLRG1 blockade, indicating the involvement of other mechanisms. This is the first demonstration of a functional role for KLRG1 in primary human CD8+ T cells and highlights that certain functional defects that arise during progressive T-cell differentiation toward replicative senescence are maintained actively by inhibitory receptor signaling.

Prolonged exposure of naïve CD8+ T cells to interleukin-7 or interleukin-15 stimulates proliferation without differentiation or loss of telomere length.

Interleukin (IL)-7 and IL-15 are cytokines implicated in homeostatic control of the peripheral CD8 T-cell pool. We compared the effects of IL-7 and IL-15 on survival and proliferation of purified human CD8+ T-cell subsets. Low concentrations of either cytokine reduced the spontaneous apoptosis of all subsets, and enhancement of survival corresponded to the extent of Bcl-2 up-regulation. Surprisingly, although minimal proliferation of naïve CD8+ T cells was observed during the first week of culture with cytokines, a marked expansion of these cells occurred at later time points, particularly in response to IL-15. This occurred largely without phenotypic change or acquisition of effector function, indicating a dissociation of differentiation from proliferation. Notably, progression of naïve CD8+ T cells through several cell divisions resulted in up-regulation of telomerase and the maintenance of telomere length. These data show that IL-7 and IL-15 induce cell proliferation and rescue from apoptosis in a concentration, time and subset-dependent manner, and have implications for the homeostatic expansion of the naïve CD8+ T-cell pool.