Gamma-Chain Receptor Cytokines & PD-1 Manipulation to Restore HCV-Specific CD8+ T Cell Response during Chronic Hepatitis C.

Hepatitis C virus (HCV)-specific CD8+ T cell response is essential in natural HCV infection control, but it becomes exhausted during persistent infection. Nowadays, chronic HCV infection can be resolved by direct acting anti-viral treatment, but there are still some non-responders that could benefit from CD8+ T cell response restoration. To become fully reactive, T cell needs the complete release of T cell receptor (TCR) signalling but, during exhaustion this is blocked by the PD-1 effect on CD28 triggering. The T cell pool sensitive to PD-1 modulation is the progenitor subset but not the terminally differentiated effector population. Nevertheless, the blockade of PD-1/PD-L1 checkpoint cannot be always enough to restore this pool. This is due to the HCV ability to impair other co-stimulatory mechanisms and metabolic pathways and to induce a pro-apoptotic state besides the TCR signalling impairment. In this sense, gamma-chain receptor cytokines involved in memory generation and maintenance, such as low-level IL-2, IL-7, IL-15, and IL-21, might carry out a positive effect on metabolic reprogramming, apoptosis blockade and restoration of co-stimulatory signalling. This review sheds light on the role of combinatory immunotherapeutic strategies to restore a reactive anti-HCV T cell response based on the mixture of PD-1 blocking plus IL-2/IL-7/IL-15/IL-21 treatment.

Proteasome inhibition with bortezomib induces a therapeutically relevant depletion of plasma cells in SLE but does not target their precursors.

Long-lived plasma cells (PCs) not only provide protective humoral immunity, they are also an essential component of the autoreactive immunologic memory that may drive chronic immune responses in systemic autoimmunity, such as systemic lupus erythematosus (SLE). The therapeutic relevance of their targeting has been demonstrated in preclinical models and severe, treatment-refractory cases of autoimmune diseases using the proteasome inhibitor bortezomib. Herein, we describe in detail the dynamic serologic changes and effects on immune effector cells in eight SLE patients receiving a median two cycles of 1.3 mg/m2 intravenous bortezomib. Upon proteasome inhibition, immunoglobulin levels gradually declined by ∼30%, associated with a significant reduction of autoantibodies, and serum complement whereas B-cell activation factor levels increased. While proteasome inhibition was associated with a significant depletion of short- and long-lived PCs in peripheral blood and bone marrow by ∼50%, including those with a distinctly mature CD19- phenotype, their precursor B cells and T cells largely remained unaffected, resulting in a rapid repopulation of short-lived PCs after bortezomib withdrawal, accompanied by increasing autoantibody levels. Collectively, these findings identify proteasome inhibitors as a promising treatment option for refractory SLE, but also indicate that PC depletion needs to be combined with targeted B-cell therapies for sustained responses in systemic autoimmunity.

Efficacy of JAK/STAT pathway inhibition in murine xenograft models of early T-cell precursor (ETP) acute lymphoblastic leukemia.

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed.

Low density lipoprotein promotes human naive T cell differentiation to Th1 cells.

Oxidized LDL (oxLDL) in the arterial wall and its incorporation into foam cells leads to inflammation and nucleation of atherosclerotic plaque; this is opposed by HDL. OxLDL and HDL regulate activation of macrophages and endothelial cells, and study of T cell participation has been limited to mature, differentiated cells such as Th1 cells, which perpetuate atherogenesis by promoting cell-mediated responses and inflammation. Immature naïve T cells, just emerged from the thymus, have remained largely unstudied. We hypothesized that LDL and HDL provide selective modulation of immature naïve T cell differentiation and participation in plaque development. In our in vitro model, naïve cells become activated and differentiate to mature effector T cells that are Th1, Th2 or Treg cells. Addition of oxLDL favored differentiation to Th1 cells, reduced Th2 cell activity and prolonged cell survival. In contrast, HDL inhibited T cell proliferation and reduced cell survival. The data suggest a novel mechanism where oxLDL enhances differentiation of human naïve CD4+ T cells to Th1 cells capable of promoting inflammation and plaque progression, and this is opposed by HDL.

Two separate defects affecting true naive or virtual memory T cell precursors combine to reduce naive T cell responses with aging.

Naive T cell responses are eroded with aging. We and others have recently shown that unimmunized old mice lose ≥ 70% of Ag-specific CD8 T cell precursors and that many of the remaining precursors acquire a virtual (central) memory (VM; CD44(hi)CD62L(hi)) phenotype. In this study, we demonstrate that unimmunized TCR transgenic (TCRTg) mice also undergo massive VM conversion with age, exhibiting rapid effector function upon both TCR and cytokine triggering. Age-related VM conversion in TCRTg mice directly depended on replacement of the original TCRTg specificity by endogenous TCRα rearrangements, indicating that TCR signals must be critical in VM conversion. Importantly, we found that VM conversion had adverse functional effects in both old wild-type and old TCRTg mice; that is, old VM, but not old true naive, T cells exhibited blunted TCR-mediated, but not IL-15-mediated, proliferation. This selective proliferative senescence correlated with increased apoptosis in old VM cells in response to peptide, but decreased apoptosis in response to homeostatic cytokines IL-7 and IL-15. Our results identify TCR as the key factor in differential maintenance and function of Ag-specific precursors in unimmunized mice with aging, and they demonstrate that two separate age-related defects--drastic reduction in true naive T cell precursors and impaired proliferative capacity of their VM cousins--combine to reduce naive T cell responses with aging.

A dynamic dual role of IL-2 signaling in the two-step differentiation process of adaptive regulatory T cells.

The molecular mechanism of the extrathymic generation of adaptive, or inducible, CD4(+)Foxp3(+) regulatory T cells (iTregs) remains incompletely defined. We show that exposure of splenic CD4(+)CD25(+)Foxp3(-) cells to IL-2, but not other common γ-chain cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ∼10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4(+)CD25(+)Foxp3(-) iTreg precursors. In this study, to further define the role of IL-2 in the formation of iTreg precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg differentiation model. During the initial "conditioning" step, CD4(+)CD25(-)Foxp3(-) naive T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3-Stat5 was required during this step to generate CD4(+)CD25(+)Foxp3(-) cells containing iTreg precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg precursors. This two-step method generated a large number of iTregs with relatively stable expression of Foxp3, which were able to prevent CD4(+)CD45RB(high) cell-mediated colitis in Rag1(-/-) mice. In consideration of this information, whereas initial inhibition of IL-2 signaling upon T cell priming generates iTreg precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg differentiation and may facilitate the therapeutic use of iTregs in immune disorders.

Interleukin-7 supports survival of T-cell receptor-ß-expressing CD4(-) CD8(-) double-negative thymocytes.

Among the milestones that occur during T-cell development in the thymus is the expression of T-cell receptor-ß (TCR-ß) and the formation of the pre-TCR complex. Signals emanating from the pre-TCR trigger survival, proliferation and differentiation of T-cell precursors. Although the pre-TCR is essential for these cell outcomes, other receptors, such as Notch and CXCR4, also contribute. Whether interleukin-7 (IL-7) participates in promoting the survival or proliferation of pre-TCR-expressing cells is controversial. We used in vitro and in vivo models of T-cell development to examine the function of IL-7 in TCR-ß-expressing thymocytes. Culturing TCR-ß-expressing CD4(-) CD8(-) double-negative thymocytes in an in vitro model of T-cell development revealed that IL-7 reduced the frequency of CD4(+) CD8(+) double-positive thymocytes at the time of harvest. The mechanism for this change in the percentage of double-positive cells was that IL-7 promoted the survival of thymocytes that had not yet differentiated. By preserving the double-negative population, IL-7 reduced the frequency of double-positive thymocytes. Interleukin-7 was not required for proliferation in the in vitro system. To follow this observation, we examined mice lacking CD127 (IL-7Rα). In addition to the known effect of CD127 deficiency on T-cell development before TCR-ß expression, CD127 deficiency also impaired the development of TCR-ß-expressing double-negative thymocytes. Specifically, we found that Bcl-2 expression and cell cycle progression were reduced in TCR-ß-expressing double-negative thymocytes in mice lacking CD127. We conclude that IL-7 continues to function after TCR-ß is expressed by promoting the survival of TCR-ß-expressing double-negative thymocytes.

Nitric oxide and glucocorticoids synergize in inducing apoptosis of CD4⁺8⁺ thymocytes: implications for 'Death by Neglect' and T-cell function.

Thymic epithelial cells (TECs) play a central role in T-cell development by presenting self-antigens on MHC proteins. Double-positive (DP) thymocytes that fail to interact with TEC via their TCR die by 'Death by Neglect'. We demonstrated a role for TEC-derived glucocorticoids (GCs) in this process. In a previous study, we used an in vitro system recapitulating Death by Neglect, to demonstrate the involvement of nitric oxide (NO) and inducible NO synthase (iNOS) in this process. In this study, we show that NO synergizes with GCs to induce apoptosis of DP thymocytes in a fetal thymic organ culture. Also, DP thymocytes from iNOS⁻/⁻ mice are less sensitive to GC-induced apoptosis. Furthermore, the number of DP thymocytes in iNOS⁻/⁻ mice is higher than in wild-type mice, suggesting a role for NO in Death by Neglect. This phenomenon effects T-cell function profoundly: iNOS⁻/⁻ T cells do not respond to TCR-mediated activation signals, measured by up-regulation of CD69, IL-2R and IFNγ secretion. This failure to activate is a result of TCR incompetence because iNO⁻/⁻ T cells respond to TCR-independent stimuli (phorbol myristate acetate and calcium ionophore). This study suggests that NO and GCs synergize to execute TEC-induced death of DP thymocytes.

Successful vaccination induces multifunctional memory T-cell precursors associated with early control of hepatitis C virus.

BACKGROUND & AIMS: T cells are an important component for development of a vaccine against hepatitis C virus (HCV), but little is known about the features of successful vaccine-induced T cells. METHODS: We compared the phenotype, function, and kinetics of vaccine-induced and infection-induced T cells in chimpanzees with HCV infection using multicolor flow cytometry and real-time polymerase chain reaction. RESULTS: In chimpanzees successfully vaccinated with recombinant adenovirus and DNA against HCV NS3-5, HCV-specific T cells appeared earlier, maintained better functionality, and persisted at higher frequencies for a longer time after HCV challenge, than those of mock-vaccinated chimpanzees. Vaccine-induced T cells displayed higher levels of CD127, a marker of memory precursors, and lower levels of programmed death-1 (PD-1) than infection-induced T cells. Vaccine-induced, but not infection-induced, T cells were multifunctional; their ability to secrete interferon gamma and tumor necrosis factor α correlated with early expression of CD127 but not PD-1. Based on a comparison of vaccine-induced and infection-induced T cells from the same chimpanzee, the CD127(+) memory precursor phenotype was induced by the vaccine itself rather than by low viremia. In contrast, induction of PD-1 correlated with viremia, and levels of intrahepatic PD-1, PD-L1, and 2,5-OAS-1 messenger RNAs correlated with peak titers of HCV. CONCLUSIONS: Compared with infection, vaccination-induced HCV-specific CD127(+) T cells with high functionality that persisted at higher levels for a longer time. Control of viremia prevented up-regulation of PD-1 on T cells and induction of PD-1, PD-L1, and 2,5-OAS-1 in the liver. Early development of a memory T-cell phenotype and, via control of viremia, attenuation of the inhibitory PD1-PD-L1 pathway might be necessary components of successful vaccine-induced protection against HCV.

Differential IL-7 responses in developing human thymocytes.

Interleukin (IL)-7 is a factor essential for mouse and human thymopoiesis. Mouse thymocytes have altered sensitivities to IL-7 at different developmental stages. CD4/CD8 double positive (DP) mouse thymocytes are shielded from the influence of IL-7 because of loss of CD127 (IL-7Ralpha). In this study, we assessed IL-7 receptor expression and IL-7 signaling in human thymocytes. We found human DP cells to be severely limited in their ability to phosphorylate STAT-5 in response to IL-7. The relative expression levels of the IL-7-inducible proteins Bcl-2 and Mcl-1 were also lower in human DP cells, consistent with a stage-specific decrease in IL-7 responsiveness. IL-7 responses were restored in a subset of cells that matured past the DP stage. Unlike the regulation of IL-7 signaling in mouse thymocytes, loss of IL-7 signaling in human DP cells was not due to absence of CD127, but instead correlated with downregulation of CD132 (common gamma chain).

[Effect of rapamycin in inducing naïve murine effector T cell convert to regulatory T cell].

OBJECTIVE: To study the effect of rapamycin in inducing naïve murine effector T cell (Teff) convert to regulatory T cell (Treg) in vitro. METHODS: The forkhead box protein 3 (FoxP3) negative Teff were isolated and purified from the spleen and lymph node of C57 BL/6 murines aged 6-8 weeks, then Teff were cultured in three groups with mature dendritic cells (mDC), B cells, and plate coated Anti-CD3. In addition, the control wells and the test wells were prepared in each group, rapamycin were not added in the control wells but added in the test wells with concentrations of 1, 10, 50, and 100 nmol/L. Percentages of FoxP3 positive Treg were examined by flow cytometry after 4 days in Anti-CD3 group and after 6 days in the other two groups. RESULTS: As shown by the flow cytometry, the percentages of FoxP3 positive Treg were as follows in three group: in the mDC group, it was 0.01% in the control well and 0.39%, 0.47%, 0.34%, and 0.26% in test wells; in B cell group, it was 0.01% in the control wells and 5.56%, 5.89%, 7.15%, and 4.72% in the test wells; in Anti-CD3 group, it was 0.93% in the control wells and 1.35%, 1.07%, 1.02%, and 1.19% in test wells. No significant difference was found between the test wells and control wells in the mDC group and Anti-CD3 group; however, the percentages of FoxP3 positive Treg was significantly different between the test wells and control wells in the B cell group (P < 0.01). CONCLUSION: When B cell is acted as the antigen-presenting cell, rapamycin can effectively induce Teff convert to Treg in vitro.

Inhibition of deoxynucleoside kinases in human thymocytes prevents dATP accumulation and induction of apoptosis.

Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.

Proteasome inhibition drastically but reversibly impairs murine lymphocyte development.

The proteasome inhibitor bortezomib, which induces cell death in various cancer cell lines including lymphatic neoplasias, has recently been approved for the treatment of relapsed multiple myeloma. Important mechanisms of proteasome inhibitor-mediated tumor cell death are the inhibition of NF-kappaB activation and induction of the terminal unfolded protein response (UPR). However, little is known about effects of bortezomib on developing and mature lymphocytes. Therefore, Balb/C mice were injected with bortezomib and lymphocyte subsets were analyzed. This treatment resulted in dramatically decreased numbers of T and B lymphocyte precursors, while mature lymphocytes were only partially affected. Thymocytes were almost depleted 3 days after a single bortezomib injection, pro-B and pre-B cells already after 2 days. Thymocytes and B cell precursors recovered within 2 weeks. The decreased numbers of developing lymphocytes were due to apoptotic cell death accompanied by strongly increased caspase 3/7 activity. Within 8 h after bortezomib injection, there was a strong induction of heat shock protein 70 and C/EBP homologous protein in bone marrow B cells, indicating endoplasmic reticulum stress and activation of the terminal UPR, respectively. Hence, induction of apoptosis by proteasome inhibition can dramatically affect lymphocyte development, a fact which has important implications for the clinical use of bortezomib, especially in situations with ongoing lymphopoiesis.