Maintenance Of CD8 Research Articles

AAV8-mediated Gene Transfer of Interleukin-4 to Endogenous β-Cells Prevents the Onset of Diabetes in NOD Mice

We have demonstrated the ability to deliver and express genes specifically in beta-cells for at least 6 months, using a murine insulin promoter (mIP) in a double-stranded, self-complementary AAV vector (dsAAV8-mIP). In this study, we evaluated the effects of dsAAV8-mIP-mediated delivery of interleukin 4 (mIL-4) to endogenous beta-cells in nonobese diabetic (NOD) mice. In 4-week-old NOD mice, the extent of gene transfer and expression in endogenous beta-cells after ip delivery of dsAAV8-mIP-enhanced green fluorescent protein (eGFP) was comparable to normal BALB/C mice. Further, after IP delivery of dsAAV8-mIP-IL4, expression of mIL-4 was detected in islets isolated from the treated mice and cultured. AAV8-mIP-mediated gene expression of mIL-4 in endogenous beta- cells of 4- and 8-week-old NOD mice prevented the onset of hyperglycemia in NOD mice and reduced the severity of insulitis. Moreover, expression of mIL-4 also maintained the level of CD4(+)CD25(+)FoxP3(+) cells, and adoptive transfer of splenocytes from nondiabetic dsAAV8-mIP-IL-4 mice to NODscid mice was able to block the diabetes induced by splenocytes co-adoptively transferred from nondiabetic dsAAV-mIP-eGFP mice. Taken together, these results demonstrate that local expression of mIL-4 in islets prevents islet destruction and blocks autoimmunity, partly through regulation of T-cell function.

Autophagy in CD4+ T-cell immunity and tolerance

Autophagy is a homeostatic process that enables eukaryotic cells to deliver cytoplasmic constituents for lysosomal degradation, to recycle nutrients and to survive during starvation. In addition to these primordial functions, autophagy has emerged as a key mechanism in orchestrating innate and adaptive immune responses to intracellular pathogens. Autophagy restricts viral infections as well as replication of intracellular bacteria and parasites and delivers pathogenic determinants for TLR stimulation and for MHC class II presentation to the adaptive immune system. Apart from its role in defense against pathogens, autophagy-mediated presentation of self-antigens in the steady state could have a crucial role in the induction and maintenance of CD4(+) T-cell tolerance. This review describes the mechanisms by which the immune system utilizes autophagic degradation of cytoplasmic material to regulate adaptive immune responses.

IL-15 as a mediator of CD4 + help for CD8 + T cell longevity and avoidance of TRAIL-mediated apoptosis

CD4+ helper T cells contribute to the induction and maintenance of antigen-specific CD8+ T cells. Their absence results in short-lived antigen-specific CD8+ T cells and defective secondary CD8+ T cell responses because of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Here, we show that IL-15 codelivered with vaccines can overcome CD4+ T cell deficiency for promoting longevity of antigen-specific CD8+ T cells and avoidance of TRAIL-mediated apoptosis. In both priming and secondary responses, IL-15 down-regulates proapoptotic Bax, an intermediate in TRAIL-mediated apoptosis, and increases anti-apoptotic Bcl-X(L) in CD8+ T cells. Thus, IL-15 is sufficient to mimic CD4+ T cell help. Antigen-specific CD4+ T cells induce dendritic cells (DCs) to produce IL-15. IL-15 is also necessary for optimal help, because helper cells do not deliver effective help through IL-15-/- DCs. Therefore, IL-15 codelivered with vaccines can overcome CD4+ helper T cell deficiency for induction of functionally efficient CD8+ T cells and maintenance of CD8+ cytotoxic T lymphocytes (CTLs), and IL-15 is probably one of the natural mediators of help. These findings suggest new vaccine strategies against infections and cancers, especially in individuals with CD4-deficiency.

Chimpanzee CD4+ T cells are relatively insensitive to HIV‐1 envelope‐mediated inhibition of CD154 up‐regulation

CD40-CD154 interaction forms a key event in regulation of crosstalk between dendritic cells and CD4 T cells. In human immunodeficiency virus (HIV)-1 infected patients CD154 expression is impaired, and the resulting loss of immune responsiveness by CD4+ T cells contributes to a progressive state of immunodeficiency in humans. Although chimpanzees are susceptible to chronic HIV-1/SIVcpz infection, they are relatively resistant to the onset of AIDS. This relative resistance is characterized by maintenance of CD4+ T cell populations and function, which is highly compromised in human patients. In our cohort of chronically HIV-1- and SIVcpz-infected chimpanzees, we demonstrated the capacity to produce IL-2, following CD3/CD28 stimulation, as well as preserved CD154 up-regulation. Cross-linking of CD4 with mAb was found to inhibit CD3/CD28-induced up-regulation of CD154 equally in chimpanzees and humans. However, specific cross-linking with trimeric recombinant HIV-1 gp140 revealed reduced sensitivity for inhibition of CD154 up-regulation in chimpanzees, requiring fourfold higher concentrations of viral protein. Chimpanzee CD4+ T cells are thus less sensitive to the immune-suppressive effect of low-dose HIV-1 envelope protein than human CD4+ T cells.

Modulation of CD4+ T cell function by HCV containing hepatocytes

Hepatitis C virus (HCV) infection is not cleared by the immune system in >70% of infected individuals. Viral persistence is correlated with a defect in T cell responses. CD4+ T cells are central in directing an effective immune response by supporting the activation and proliferation of B cells and maintenance of CD8+ effector function and memory response. We want to determine the impact that the HCV‐infected liver environment may have on CD4+ T cell responses. We utilized a co‐culture system that allows for direct contact between the hepatocytes and primary CD4+ T cells. A modified HCV cell culture system composed of a hepatoma cell line, Huh 7.5, containing full‐length HCV genome or the non‐structural portion of the genome only was used. Analysis of CD4+ T cell cytokine production indicates that the complete viral genome within hepatocytes has a suppressive effect on CD4+ T cell IFN‐γ production but no significant effect on IL‐10 production as measured by ELISA. In contrast, no significant difference in cytokine production was noted in cells that contained only the non‐structural portion of the HCV genome. Additionally, the inhibition of IFN‐γ was not detectable in CD8+ T cells. This data suggests that hepatocytes containing full‐length HCV genome have a specific immunosuppressive effect on CD4+ T cell function.

Understanding the dynamics of an endogenous ova specific CD8+ T‐cell response in a murine model of allergic airway disease

Elegant studies examining the endogenous antigen specific CD8+ T‐cell responses to bacterial infections have recently been published utilizing confocal imaging and tetramer technology. The current study was designed to elucidate the role of antigen specific CD8+ T‐cells in a well characterized murine model of allergic airway disease (AAD). In this AAD model acute aerosol exposure (3–7days) with 1% ovalbumin results in a clonal expansion of T and B‐cells in the local lung compartments (BAL, Lung tissue, HLN). Chronic aerosol exposure (42days) results in results in resolution of AAD. We have labeled this as local inhalational tolerance (LIT), which is characterized by the maintenance of CD8+ T cells. We hypothesis that antigen specific CD8+ T‐cells expand during the acute phase of the model and contribute to the development of AAD and are diminished at LIT. This makes them an important indicator of disease but suggests they have a limited role in the resolution of disease. Utilizing flow cytometry we have found an increase in tetramer positive CD8+ T cells in local lung compartments during AAD and a decrease during LIT. We are currently correlating this data using confocal microscopy.

A vaccine strategy against AIDS: An HIV gp41 peptide immunization prevents NKp44L expression and CD4 + T cell depletion in SHIV-infected macaques

We previously showed that a gp41 peptide (3S) induces expression of a natural killer (NK) ligand (NKp44L) on CD4+ T cells during HIV-1 infection and that those cells are highly sensitive to NK lysis. In HIV-infected patients, anti-3S antibodies are associated with the maintenance of CD4+ T cell counts close to their baseline values, and CD4+ T cells decrease with the antibody titer. This study sought to determine whether anti-3S immunization could prevent NKp44L expression on these CD4+ T cells in vivo and inhibits the subsequent decline in CD4+ T cell counts by immunizing macaques with 3S and then infecting them with simian HIV(162P3). The results show that anti-3S antibodies inhibited NKp44L expression and NK activity and cytotoxicity. They also decreased the apoptosis rate of CD4+ T cells in peripheral blood and lymph nodes. These data raise questions about the pathogenesis of HIV and present opportunities for both preventive and therapeutic HIV vaccine strategies.

Effects of IL-7 on memory CD8+ T cell homeostasis are influenced by the timing of therapy in mice

IL-7 is integral to the generation and maintenance of CD8(+) T cell memory, and insufficient IL-7 is believed to limit survival and the persistence of memory CD8(+) T cells. Here, we show that during the mouse T cell response to lymphocytic choriomeningitis virus, IL-7 enhanced the number of memory CD8(+) T cells when its administration was restricted to the contraction phase of the response. Likewise, IL-7 administration during the contraction phase of the mouse T cell response to vaccinia virus or a DNA vaccine potentiated antigen-specific CD8(+) memory T cell proliferation and function. Qualitatively, CD8(+) T cells from IL-7-treated mice exhibited superior recall responses and improved viral control. IL-7 treatment during the memory phase stimulated a marked increase in the number of memory CD8(+) T cells, but the effects were transient. IL-7 therapy during contraction of the secondary CD8(+) T cell response also expanded the pool of memory CD8(+) T cells. Collectively, our studies show differential effects of IL-7 on memory CD8(+) T cell homeostasis and underscore the importance of the timing of IL-7 therapy to effectively improve CD8(+) T cell memory and protective immunity. These findings may have implications in the clinical use of IL-7 as an immunotherapeutic agent to bolster vaccine-induced CD8(+) T cell memory.

Identification of Phosphatidylinositol 4-Kinase Type II β as the First HLA Class II Associated Minor Histocompatibility Antigen Involved in Graft Versus Leukemia Reactivity.

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLI). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, mHag specific CD4+ T cells may selectively mediate Graft-versus-Leukemia (GvL) reactivity without Graft-versus-Host Disease (GvHD). Clinical studies have shown that adoptive transfer of CD4+ donor lymphocytes after HLA-matched alloSCT may lead to clinical remissions with a reduced incidence of GvHD, emphasizing the relevance of CD4+ T cells and HLA class II associated mHags for development of effective anti-tumor T cell therapies after alloSCT with a low risk for GvHD. The aim of this study was to identify the HLA class II associated mHag that is recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLA-matched alloSCT who developed strong GvL reactivity with mild GvHD of the skin after treatment with DLI. We previously developed recombinant bacteria cDNA expression libraries based on delivery of exogenous antigens for identification of HLA class II antigens and used this method for identification of the first autosomal HLA class II (HLA-DQB1*0603) associated mHag LB-PI4K2B-1S. LB-PI4K2B-1S has a population frequency of 40–50% and is encoded by the broadly-expressed phosphatidylinositol 4-kinase type II β gene. In the patient with CML, a polyclonal CD4+ T cell response against LB-PI4K2B-1S and simultaneous mHag specific CD8+ T cells were demonstrated. LB-PI4K2B-1S specific CD4+ T cells were shown to recognize the CD34+ CML cells of the patient as well as other leukemic cells. Recognition and lysis of normal hematopoietic cells by LB-PI4K2B-1S specific CD4+ T cells critically depended on the number of HLA-DQ molecules expressed at the cell surface and was restricted to high HLA-DQ-expressing B cells, mature dendritic cells (DC) and EBV-transformed B cells. HLA-DQ expression on T cells, PHA-stimulated blasts, monocytes and immature DC was absent or low and not sufficient for T cell recognition. We also demonstrated that HLA-DQ expression on normal cells of non-hematopoietic origin after extensive culturing with IFN-γ was moderately upregulated as compared to HLA-DR and -DP and not sufficient for recognition by LB-PI4K2B-1S specific CD4+ T cells. In conclusion, the data suggest that1.LB-PI4K2B-1S specific CD4+ T cells mediated tumor rejection by directly eliminating the malignant cells of the patient as effector cells and stimulating the induction and maintenance of CD8+ T cell immunity as helper cells, and2.HLA-DQ associated mHags may be appropriate targets for T cell therapies with the aim to selectively stimulate GvL after HLA-matched alloSCT with a low risk for GvHD.

In this issue

In this issue

Maintenance of CD4 + T cell TCR Vβ repertoire heterogeneity is characteristic of apathogenic SIV infection in non-human primate model of AIDS

A PCR-based assay was established to screen for potential changes in the heterogeneity of Vβ TCR expressing CD4 + T cells from rhesus macaques (RM) and sooty mangabeys (SM) prior to and following SIV infection. The rationale for these studies was to determine whether the progressive CD4 + T cell loss in SIV-infected disease susceptible RM and the moderate CD4 + T cell loss in disease resistant SM leads to the depletion of select Vβ TCR families of CD4 + T cells. Results show that whereas SIV infection leads to the loss of Vβ TCR heterogeneity in disease susceptible RM, the CD4 + T cells from SM retain their degree of Vβ TCR heterogeneity, suggesting that the mechanism(s) of SIV induced CD4 + T cell loss maybe distinct in these 2 species and contribute to the differences in the clinical outcome.

Human CD4+ central and effector memory T cells produce IL-21: effect on cytokine-driven proliferation of CD4+ T cell subsets

IL-21 regulates certain functions of T cells, B cells, NK cells and dendritic cells. Although activated CD4(+) T cells produce IL-21, data identifying the specific CD4(+) T cell subsets that produce IL-21 are conflicting. In a previous study, mouse IL-21 message was detected in T(H)2, whereas human IL-21 (hIL-21) message was found in both T(H)1 and follicular helper T cells. To identify the IL-21-secreting cell populations in human, we established a hybridoma cell line producing an anti-hIL-21 mAb. Intracellular hIL-21-staining experiments showed that hIL-21 was mainly expressed in activated CD4(+) central memory T cells and in activated CD4(+) effector memory T cells, but not in activated CD4(+) naive T cells. Moreover, IL-21 was produced upon activation by some IFN-gamma-producing T(H)1-polarized cells and some IL-17-producing T(H)17-polarized cells, but not by IL-4-producing T(H)2-polarized cells. These results suggest that specific CD4(+) T cell populations produce IL-21. In the functional analysis, we found that IL-21 significantly enhanced the cytokine-driven proliferation of CD4(+) helper T cells synergistically with IL-7 and IL-15 without T cell activation stimuli. Taken together, IL-21 produced from CD4(+) memory T cells may have a supportive role in the maintenance of CD4(+) T cell subsets.

Memory generation and maintenance of CD8+ T cell function during viral persistence.

During infection with viruses that establish latency, the immune system needs to maintain lifelong control of the infectious agent in the presence of persistent Ag. By using a gamma-herpesvirus (gammaHV) infection model, we demonstrate that a small number of virus-specific central-memory CD8+ T cells develop early during infection, and that virus-specific CD8+T cells maintain functional and protective capacities during chronic infection despite low-level Ag persistence. During the primary immune response, we show generation of CD8+ memory T cell precursors expressing lymphoid homing molecules (CCR7, L-selectin) and homeostatic cytokine receptors (IL-7alpha, IL-2/IL-15beta). During long-term persistent infection, central-memory cells constitute 20-50% of the virus-specific CD8+ T cell population and maintain the expression of L-selectin, CCR7, and IL-7R molecules. Functional analyses demonstrate that during viral persistence: 1) CD8+ T cells maintain TCR affinity for peptide/MHC complexes, 2) the functional avidity of CD8+ T cells measured as the capacity to produce IFN-gamma is preserved intact, and 3) virus-specific CD8+ T cells have in vivo killing capacity. Next, we demonstrate that at 8 mo post-virus inoculation, long-term CD8+ T cells are capable of mediating a protective recall response against the establishment of gammaHV68 splenic latency. These observations provide evidence that functional CD8+ memory T cells can be generated and maintained during low-load gammaHV68 persistence.

Differential role of IL‐2R signaling for CD8+ T cell responses in acute and chronic viral infections

IL-2 is a cytokine with multiple and even divergent functions; it has been described as a key cytokine for in vitro T cell proliferation but is also essential for down-regulating T cell responses by inducing activation-induced cell death as well as regulatory T cells. The in vivo analysis of IL-2 function in regulating specific T cell responses has been hampered by the fact that mice deficient in IL-2 or its receptors develop lymphoproliferative diseases and/or autoimmunity. Here we generated chimeric mice harboring both IL-2R-competent and IL-2R-deficient T cells and assessed CD8+ T cell induction, function and maintenance after acute or persistent viral infections. Induction and maintenance of CD8+ T cells were relatively independent of IL-2R signaling during acute/resolved viral infection. In marked contrast, IL-2 was crucial for secondary expansion of memory CD8+ T cells and for the maintenance of virus-specific CD8+ T cells during persistent viral infections. Thus, depending on the chronicity of antigen exposure, IL-2R signaling is either essential or largely dispensable for induction and maintenance of virus-specific CD8+ T cell responses.

Anti-B7-1/B7-2 mAb treatment enhanced anti-tumor immunity in TRAMP mice (50.14)

Abstract Immune tolerance is the major mechanism applied by the immune system to discriminate between self and non-self. There is sufficient evidence to show that CD4+CD25+ Treg cells play an important role in the maintenance of peripheral tolerance. Accumulating evidence suggests that CD4+CD25+ Treg cells also suppress tumor antigen-specific immunity in tumor-bearing mice and in humans. Therefore, breaking Treg-mediated peripheral tolerance to tumor antigens may enhance anti-tumor immunity. B7:CD28 interactions are needed for both the development and maintenance of CD4+CD25+ Treg cells. This raises the possibility that blocking the B7 signal may break tumor tolerance by reducing the number of CD4+CD25+ Treg cells. Here we show that administration of anti B7-1/B7-2 mAbs either at a beginning stage or at an intermediate stage of prostate cancer development significantly enhanced anti-tumor immunity by postponing the disease process. Anti-tumor immunity was improved after inhibiting the generation of CD4+CD25+ regulatory T cells in thymus and periphery without a hint of autoimmune disease. Our results demonstrate that blocking the costimulatory pathway with anti B7-1/B7-2 antibodies can enhance anti-tumor immunity.

Convergence of TCR and cytokine signaling leads to FOXO3a phosphorylation and drives the survival of CD4+ central memory T cells

The molecular events involved in the establishment and maintenance of CD4+ central memory and effector memory T cells (TCM and TEM, respectively) are poorly understood. In this study, we demonstrate that ex vivo isolated TCM are more resistant to both spontaneous and Fas-induced apoptosis than TEM and have an increased capacity to proliferate and persist in vitro. Using global gene expression profiling, single cell proteomics, and functional assays, we show that the survival of CD4+ TCM depends, at least in part, on the activation and phosphorylation of signal transducer and activator of transcription 5a (STAT5a) and forkhead box O3a (FOXO3a). TCM showed a significant increase in the levels of phosphorylation of STAT5a compared with TEM in response to both IL-2 (P < 0.04) and IL-7 (P < 0.002); the latter is well known for its capacity to enhance T cell survival. Moreover, ex vivo TCM express higher levels of the transcriptionally inactive phosphorylated forms of FOXO3a and concomitantly lower levels of the proapoptotic FOXO3a target, Bim. Experiments aimed at blocking FOXO3a phosphorylation confirmed the role of this phosphoprotein in protecting TCM from apoptosis. Our results provide, for the first time in humans, an insight into molecular mechanisms that could be responsible for the longevity and persistence of CD4+ TCM.

Maintenance of CD8+T-cell anergy by CD4+CD25+ regulatory T cells in chronic graft-versus-host disease

In a murine model of systemic lupus erythematosus (SLE)-like chronic graft-versus-host disease (cGVHD), donor CD8+ T cells rapidly fall into anergy to host cells, while donor CD4+ T cells hyperactivate B cells and break B-cell tolerance to self-Ags in the recipient mouse. The functional recovery of donor CD8+ T cells can result in the conversion of cGVHD to acute GVHD (aGVHD), indicating that donor CD8+ T-cell anergy is a restriction factor in the development of cGVHD. In this report, we present evidence that donor CD4+CD25+ regulatory T cells (Treg cells) are critical in maintaining the donor CD8+ T-cell anergy and thus suppressing the development of aGVHD in mice that are naturally prone to cGVHD. Our results provide a novel insight into the role of Treg cells in determining cGVHD versus aGVHD.

IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

Establishment and recall of CD8+ T‐cell memory in a model of localized transient infection

The influenza A virus model of localized, transient respiratory infection provides a well-defined experimental system for dissecting the induction and maintenance of CD8+ T-cell memory. This review focuses on quantitative and qualitative aspects of the prominent D(b)NP366- and D(b)PA224-specific CD8+ T-cell responses in virus-infected B6 mice. The different virus-specific effector and memory sets are compared by phenotypic [CD62L, interleukin-7 receptor-alpha (IL-7Ralpha), and IL-15Rbeta expression] and functional [interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and IL-2 production] analyses. Most clonotypes [defined by T-cell receptor (TCR) CDR3beta sequence] generated during the acute phase of infection survive into memory, with those expressing the more consensus 'canonical' TCRs being the major contributors to the recall response. The extent of clonal expansion and the size of memory CD8+ T-cell populations has been characterized for mice challenged with either wildtype or mutant viruses, where broadly equivalent D(b)NP366 and D(b)PA224 expression was achieved by disabling the peptides in their native configuration, then expressing them in the viral neuraminidase protein. Combining the clonotypic and antigen dose analyses led to a somewhat mechanistic conclusion that the magnitude of any virus-specific CD8+ T-cell response will be a direct function of antigen dose and the size of the naïve or memory CD8+ T-cell precursor pool.

CD8 + T-Cell Subpopulations in Human Abdominal Aortic Aneurysm Lesion

To the Editor: We read with interest the article by Duftner et al1 reporting the prevalence of peripheral interferon-γ (IFN-γ)–producing CD4+CD28− and CD8+CD28− T cells in patients with small abdominal aortic aneurysm (AAA). Along with the recent description that Th1-type immune responses predominate in human end-stage AAA lesion,2,3 their observation further supports preference toward polarized type 1 T-cell responses in aneurysm disease. The potential involvement of Th1 cells in the pathogenesis of the disorder is also suggested by the convincing demonstration that absence of CD4+ T cells or targeted deletion of IFN-γ prevents the induction of experimental AAA in a calcium chloride–induced mouse model,4 AAA formation being reconstituted by administration of IFN-γ into CD4−/− mice or infusion of competent splenocytes from wild-type mice into IFN-γ−/− mice. In their study, Duftner et al further established that both circulating CD4+CD28− and CD8+CD28− T cells are highly differentiated cells that display extensive CD45RO to CD45RA reversion and produce large amounts of IFN-γ and perforin. Surprisingly, low percentages of CD8+CD28− T cells were identified in AAA tissue sections using immunohistochemistry compared with flow cytometric analysis of peripheral blood mononuclear cells. In a series of our …