Absolute Number Of CD4 Research Articles

Starting T Cell and Cell Product Phenotype Are Associated with Durable Remission of Leukemia Following CD19 CAR-T Cell Immunotherapy

Chimeric antigen receptor (CAR) redirected T cells can induce remission in highly refractory leukemia and lymphoma subjects. Despite the potential of this emerging therapeutic modality, treatment with CD19 CAR T cells is not uniformly effective for remission induction, and remissions can be short-lived in a significant proportion of treated subjects.Here, we analyzed 43 pediatric and young adult subjects participating in the phase 1 trial (NCT02028455) and correlated their outcomes with in vivo performances of SCRI-CAR19v1(a CD19 specific CAR T cell product), as well as starting material (SM) T cell repertoire- and final cell product (FP) -intrinsic attributes. Subjects were allocated to the dysfunctional response group defined as early treatment failure (no remission or early disease progression after remission while still having CAR engraftment) (n=5) and the functional response group defined as those who obtained an MRD-negative remission that was sustained beyond 63 days (n=37).We found the magnitude of absolute CAR T cell engraftment area under the curve (AUC) was attenuated in the dysfunctional response group (AUC 150.3, range 0.54-752.8, Mann-Whitney p=0.0033) as compared to the functional response group (median AUC 1309, range 5.23-9537). The absolute number of CD8+CAR+ cells and CD4+CAR+ cells at peak engraftment was also significantly higher in the functional response versus the dysfunctional response. The phenotype of the CAR+ cells was analyzed at peak engraftment by multiparameter flow cytometry. CAR+ CD8+ cells from both groups had similar frequencies of PD-1+ cells, whereas the dysfunctional response group showed a significantly higher frequency of LAG-3+ T cells, both in the CAR+CD8+ cells and the CAR+CD4+ cells. A similar trend was seen with the expression of TIM-3.In order to assess whether T cell intrinsic factors contributed to therapeutic failure in these subjects, we studied T cell repertoire status in apheresis products and final expanded CAR T cell products. Several reports have shown that using SM rich in terminally differentiated cells result in CD19 CAR-T cell products having limited replicative capacity and attenuated ability to transition to long lived memory cells . When comparing apheresis derived SM from the functional and dysfunctional response subject groups by flow cytometry for markers associated with functional exhaustion (LAG-3, TIM-3, PD-1), we observed a significantly higher percentage of CD8+ T cells expressing PD-1 and LAG-3 in the dysfunctional response group compared to the functional response subjects (p=0.0266 and p=0.0052). We also observed a higher frequency of CD4+ cells expressing PD-1 in the dysfunctional group. There was no difference in the frequency of cells expressing CD45RA, CD45RO, CCR7, CD27, or in the frequency of cells expressing TNF-α, IFN-γ or IL-2 in response to CD3/CD28 stimulation in both CD4 and CD8 SM T cells between the groups.Lastly, using classification and regression tree analysis, subjects could be classified by the frequency of SM CD8+ T cells expressing LAG-3 and the frequency of SM CD8+ T cells capable of secreting TNF-α upon CD3/CD28 bead activation (r2=0.636). Subjects with fewer than 0.745% SM CD8+ T cells expressing LAG-3 were all in the functional response group (n=26/43). Subjects with equal or more than 0.745% SM CD8+ T cells expressing LAG-3 (n=16) could be further sub-divided into two groups: subjects with equal or more than 25.283% of CD8+ T cells expressing TNF-α were also functional responders (n=8/8), while subjects with fewer than 25.283% of CD8+ T cells expressing TNF-α were in majority in the dysfunctional response group (n=5/8). Of the three subjects from the functional response group with high frequencies of LAG3+ cells and low frequencies of TNF- α -secreting cells, all three had short duration BCA and relapsed within 6 months. The combination of elevated frequency of cells expressing LAG-3 and a reduced capacity to secrete cytokines in response to stimulation potential serve as biomarkers for patients with perturbated T cell repertoires that generate CAR T cell products with attenuated anti-leukemic potency.The parameters identified herein, should they be validated in larger trial cohorts, have the potential to prospectively identify patients at risk for initial therapeutic failure thus requiring alternative therapies from those who have an excellent prognosis. DisclosuresLi:Juno Therapeutics: Employment, Equity Ownership. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Ruxolitinib, a JAK1/JAK2 Selective Inhibitor Is Highly Efficacious in Corticosteroid Untreated and Refractory MHC-Mismatched Mouse Model of Acute GvHD

Introduction:Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (REACH1: NCT02953678; REACH2: NCT02913261).Methods:Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells received from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the potent and selective JAK1/JAK2 inhibitor, ruxolitinib (60 mg/kg) twice daily. Engraftment was analyzed for the proportion of donor and host leukocytes (CD45, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analyses. Colon samples were sectioned at approximately 5 microns, and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3/STAT5, CD3+phosphoSTAT3/STAT5 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of ruxolitinib in corticosteroid refractory mice were evaluated by treating disease bearing BALB/c mice twice daily with an optimal oral dose of prednisolone (1 mg/kg) until the GvHD scores were comparable to vehicle treated mice. Steroid resistant animals were switched to ruxolitinib to determine the potential efficacy benefit in comparison to mice maintained on ruxolitinib treatment alone for the duration of the study.Results:Oral ruxolitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss, improving GvHD scores and had no detrimental effects on donor engraftment. Comparable efficacy was observed between prophylactic and therapeutic treatment with 60 mg/kg twice daily dosing regimens. Oral ruxolitinib administration in BALB/c mice achieved JAK1/JAK2 IC50 coverage for 8 h with 60 mg/kg twice daily. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Ruxolitinib (60 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen in groups treated with ruxolitinib, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with JAK/STAT pathway inhibition as measured by significant reductions in normalized phosphoSTAT3/STAT5 in T cells and colonic epithelial cells. Prednisolone was transiently effective until day 31 in reducing GvHD scores. Switching treatment regimens from prednisolone to ruxolitinib twice daily rapidly and significantly enhanced GvHD efficacy to a level comparable to ruxolitinib monotherapy. In a dose cessation and resumption paradigm, ruxolitinib restored efficacy when treatment was resumed on day 33 after being halted on day 24. In addition, ruxolitinib significantly enhanced the survival of the recipient BALB/c mice in comparison to vehicle treated animals.Conclusions:Ruxolitinib, a JAK1/JAK2 selective inhibitor, significantly ameliorated GvHD severity with no detrimental effects on engraftment in both a steroid untreated and a corticosteroid refractory MHC-mismatched model of acute GvHD. Efficacy was associated with JAK/STAT pathway inhibition in colon and target tissue infiltrating T-cells. In a dose cessation and resumption paradigm, ruxolitinib restored efficacy once treatment was resumed. In addition, ruxolitinib reduced disease burden and enhanced survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. DisclosuresJuvekar:Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

DNA Damage in Haemopoeitic Stem Cells Impacts on Neutrophil and Platelet Engraftment Following Autologous Transplantation

Background and Aim: Processing and cryopreservation is known to reduce the viability and absolute number of CD34+ haematopoetic stem cells (HSCs). DNA damage and subsequent apoptosis of stem cells may occur as a result of prior treatment regimens or from the accumulation of reactive oxygen species (ROS) during the freeze/thawing process. In our recent study we demonstrated that two distinct populations of HSCs are present in all cryopreserved patient samples, based on their ROS expression; these we defined as ROShigh and ROSlow. The significant correlation between the percentage of ROShigh HSCs in the graft and neutrophil recovery following transplant shown in our study suggests that some degree of ROS accumulation may play an important role in promoting neutrophil engraftment following autologous HSC transplantation (AHSCT) (Bai et al 2018). The current study examined the association between ROS levels and DNA damage in cryopreserved HSCs and whether these correlated with time to neutrophil and platelet engraftment following AHSCT.Methods: Cryopreserved HSC samples from 51 patients who underwent AHSCT were studied. HSC's were defined as CD45+/CD34+. DNA damage and intracellular ROS levels were assessed using an antibody against the phosphorylated form of histone H2Ax (γH2Ax) and 2‘,7‘-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Data acquisition and analysis were performed by flow cytometry.Results: We observed a proportion of HSC in each sample that expressed elevated levels of gH2Ax. The median percentage of γH2Ax expressing CD34+ cells was 54.6% (range 3.4 - 98.4%). We observed a strong correlation between ROShigh and γH2Ax levels in terms of the dose of cells/kg infused (p < 0.001, r = 0.72). The ratio of ROShigh/γH2Ax-expressing HSCs was inversely associated with delayed neutrophil engraftment (p = 0.03, r = -0.31).The cohort was divided into group 1 and group 2 based on ROShigh/γH2Ax ratios of >1 (n = 32) and ≤1 (n = 18) respectively. Median days to neutrophil engraftment were 12 (range 9-18), in group 1 and 15 (range 9-28) in group 2, (p = 0.007). Median days to platelet engraftment were 17 (range 10-28) in group 1 and 21.5 (range 14-55) in group 2, (p = 0.001). Median days to platelet count of >50 (plt50) was 18.5 (range 12-28) in group 1 and 22 (range 16-75) in group 2, (p = 0.001). In group 2, significant correlations were noted between the ROShigh/γH2Ax ratio and time to neutrophil engraftment (r = -0.54, p=0.025), plt20 (r = -0.64, p=0.004) and plt50 (r = -0.7, p = 0.002).Conclusion: Our data suggest that DNA damage in CD34+ and accumulation of ROS may impact upon the time to engraftment of HSCs. Calculating a ratio of ROShigh/γH2Ax expression may be useful in predicting engraftment time following autologous transplantation. DisclosuresNo relevant conflicts of interest to declare.

Itacitinib, a JAK1 Selective Inhibitor Preserves Graft-Versus-Leukemia (GVL), Enhances Survival and Is Highly Efficacious in a MHC-Mismatched Mouse Model of Acute GvHD

Introduction:Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK1/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (GRAVITAS-301: NCT03139604; GRAVITAS-119: NCT03320642).Methods:Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the selective JAK1 inhibitor, itacitinib (60 mg/kg or 120 mg/kg twice daily). Engraftment was analyzed for the proportion of donor and host leukocytes (CD45+, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analysis. Colon samples were sectioned and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3 and CD3+phosphoSTAT3 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of itacitinib on preservation of Graft-versus-Leukemia (GVL) were evaluated by injecting BALB/c mice with A20 lymphoma cells that are of H-2Kd phenotype along with combination of splenocytes and T cell depleted bone marrow from C57BL/6 (H-2Kb) mice.Results:Itacitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss and improving GvHD scores. Itacitinib did not significantly impact donor engraftment as determined by CD45+/H-2Kb quantification by flow cytometry. Similar efficacy was observed with 60 mg/kg versus 120 mg/kg twice daily dosing regimens. Oral itacitinib administration achieved JAK1 IC50 coverage for 4 h and 12 h at 60 mg/kg twice daily and 120 mg/kg twice daily, respectively. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Itacitinib (120 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen with itacitinib treatment, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with significant JAK1/STAT3 inhibition as measured by reductions in normalized pSTAT3 in T cells and colonic epithelial cells. Itacitinib treatment did not negatively impact GVL responses, as evidence by T cell mediated reduction of tumor burden. Furthermore, itacitinib treatment enhanced the survival of the recipient BALB/c mice in comparison to the vehicle treated animals.Conclusions:Itacitinib, a selective JAK1 inhibitor ameliorated GvHD severity when administered prophylactically or therapeutically and had no detrimental effects on engraftment and preservation of GVL. Furthermore, itacitinib inhibited JAK1/STAT3 activation in diseased colon tissue and infiltrating T-cells, and reduced disease burden and improved survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. DisclosuresJuvekar:Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

Resolvin D1 treatment on goblet cell mucin and immune responses in the chronic allergic eye disease (AED) model.

Severe, chronic eye allergy is an understudied, vision-threatening condition. Treatments remain limited. We used a mouse model of severe allergic eye disease (AED) to determine if topical application of the pro-resolution mediator Resolvin D1 (RvD1) terminates the response. AED was induced by injection of ovalbumin (OVA) followed by topical challenge of OVA daily. RvD1 was applied topically prior to OVA. Clinical symptoms were scored. Eye washes were assayed for MUC5AC. After 7 days, eyes were removed and the number of goblet cells, T helper cell responses and presence of immune cells in draining lymph nodes and conjunctiva determined. Topical RvD1 treatment significantly reduced symptoms of AED. RvD1 did not alter the systemic type 2 immune response in the lymph nodes. AED increased the total amount of goblet cell mucin secretion, but not the number of goblet cells. RvD1 prevented this increase, but did not alter goblet cell number. Absolute numbers of CD4+ T cells, total CD11b+ myeloid cells, eosinophils, neutrophils, and monocytes, but not macrophages increased in AED versus RvD1 treated mice. We conclude that topical application of RvD1 reduced the ocular allergic response by a local actions in conjunctival immune response and a decrease in goblet cell mucin secretion.

Tolerogenic dendritic cells induced the enrichment of CD4+Foxp3+ regulatory T cells via TGF-β in mesenteric lymph nodes of murine LPS-induced tolerance model

Endotoxin tolerance is an important state for the prevention of lethal infection and inflammatory response, which is closely associated with the participation of innate immune cells. Moreover, mesenteric lymph nodes (MLNs)-resident immune cells, such as CD4+Foxp3+ regulatory T (Treg) cells and dendritic cells, play important roles in the maintenance of peripheral immune tolerance. However, the potential roles of these cells in MLNs in the development of endotoxin tolerance remain largely unknown. Recent research work showed that CD4+Foxp3+ Treg cells contributed to the development of endotoxin tolerance. Here, we further analyzed the possible change on CD4+Foxp3+Tregs population in MLNs in murine LPS-induced endotoxin tolerance model. Our data showed that the proportion and absolute number of CD4+Foxp3+Tregs, expressing altered levels of CTLA4 and GITR, significantly increased in MLNs of murine LPS-induced tolerance model. Moreover, the expression level of TGF-β in MLNs also increased obviously. Furthermore, TGF-β blockade could obviously reduce the proportion and absolute number of CD4+Foxp3+Tregs in MLNs and subsequently impair the protection effect against LPS rechallenge. Of note, we found that tolerogenic dendritic cell (Tol-DC), expressing lower levels of MHC-II and CD86 molecules, dominantly secreted TGF-β in MLNs in murine LPS-induced tolerance model. In all, our data provided an unknown phenomenon that the total cell number of CD4+Foxp3+Tregs significantly increased in MLNs in endotoxin tolerance, which was related to MLN-resident TGF-β secreting CD11c+DCs, providing a new fundamental basis for the understanding on the potential roles of MLN-resident immune cells in the development of endotoxin tolerance.

Association Between Programed Cell Death-1 and CD4+ T Cell Alterations in Different Phases of Ischemic Stroke Patients.

Objective: We aimed to analyze alterations in T cell subgroups during different post-ischemic stroke (IS) phases to explore the possible mechanisms underlying stroke-induced immune depression (SIID).Methods: Sixty-four IS patients who met the entry criteria were divided into three groups: an acute phase group, a sub-acute phase group and a stable phase group. Fourteen healthy individuals were selected as normal controls. The phenotype distribution of T cells in patient peripheral blood was analyzed, and the immune checkpoint receptors programed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) were detected in different T cell phenotypes.Results: Compared with the control group, the absolute number of CD4+ T cells and CD4+ T central memory (TCM) cells was significantly increased in the acute phase group but decreased in the sub-acute phase and stable phase groups compared with that in the acute phase group. PD-1 expression in CD4+ T cells in the stable phase group showed a significant increase compared with that in the acute phase group. The expression of PD-1 on CD4+ TCM cells and CD4+ T effector memory (TEM) cells showed significant decreases in the acute phase compared with control cells; however, in the sub-acute phase and the stable phase, PD-1 expression was significantly increased compared with that in the acute phase.Conclusions: T cell dysfunction, especially CD4+ T cell dysfunction, occurred during different IS phases. PD-1 was highly expressed in CD4+ T cells of different phenotypes after the acute phase and was associated with alterations in CD4+ T cells. Particularly, PD-1 was negatively correlated with the absolute number of TCM cells among different CD4+ T cell phenotypes, which may be one of the possible mechanisms of SIID.

Altered levels of memory T cell subsets and common γc cytokines in Strongyloides stercoralis infection and partial reversal following anthelmintic treatment

BackgroundCD4+ and CD8+ T cells are central players in immunity to helminth infections. However, the role of T cell subsets in human helminth infections is not well understood. In addition, the common γc cytokines, IL-2, IL-4, IL-7, IL-9 and IL-15 play an important role in the maintenance of these CD4+ and CD8+ T cell subsets.MethodsTo examine the major T cell subsets and their association with the common γc cytokines, the absolute numbers of CD4+ and CD8+ naïve, central memory, effector memory and effector cells and the plasma levels of IL-2, IL-4, IL-7, IL-9 and IL-15 were measured in Strongyloides stercoralis (Ss) infected (INF, n = 60), helminth—uninfected (UN, n = 58) and in post treatment INF individuals.ResultsSs infection is characterized by significantly increased absolute numbers of naïve and decreased absolute numbers of central and effector memory CD4+ T cells in comparison to UN individuals. No significant difference in the numbers of CD8+ T cell subsets was observed between the groups. The numbers of naïve cells and central memory CD4+ T cells were significantly reversed after anthelmintic treatment. Circulating levels of IL-2, IL-7 and IL-15 were significantly diminished, whereas the levels of IL-4 and IL-9 were significantly increased in INF compared to UN individuals. Following anthelminthic treatment, IL-2, IL-7 and IL-15 levels were significantly increased, while IL-4 and IL-9 levels were significantly decreased. Our data also showed a significant positive correlation between the levels of IL-7 and the numbers of central and effector memory CD4+ T cells.ConclusionSs infection is characterized by alterations in the absolute numbers of CD4+ T cell subsets and altered levels of common γc cytokines IL-2, IL-4, IL-7, IL-9 and IL-15; alterations which are partially reversed after anthelmintic treatment.

Pro-Inflammatory Th1 and Th17 Cells Are Suppressed During Human Experimental Endotoxemia Whereas Anti-Inflammatory IL-10 Producing T-Cells Are Unaffected.

Sepsis is one of the leading causes of the deaths in hospitals. During sepsis, patients are exposed to endotoxemia, which may contribute to the dysregulation of the immune system frequently observed in sepsis. This dysregulation leads to impaired pro-inflammatory responses and may increase the risk for secondary infections in sepsis. The experimental human endotoxemia model is widely used as a model system to study the acute effects of endotoxemia. Under physiological circumstances, the immune system is tightly regulated. Effector T-cells exert pro-inflammatory function and are restrained by regulatory T-cells (Tregs), which modulate pro-inflammatory effector responses. Endotoxemia may induce inadequate Treg activity or render effector T-cells dysfunctional. It was the aim of the study to investigate effector T-cell and Treg responses in an experimental human endotoxemia model. In a cross-over designed placebo-controlled study, 20 healthy male volunteers received an intravenous injection of either lipopolysaccharide (LPS) (0.8 ng/kg body weight) or a placebo (saline 0.9%). CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and intracellular cytokine profiles were measured with flow cytometry at baseline and at repeated points after LPS/placebo injection. Complete blood cell counts were obtained with an automated hematology analyzer and cytokines were quantified by ELISA. Circulating neutrophils were significantly increased 2 h after LPS injection (p < 0.001) while absolute number of CD3+ T-cells, CD4+ T-cells, and CD8+ T-cells decreased (p < 0.001). Effector T-helper-cells (THs) showed a significant-but transient-decrease of pro-inflammatory IFNγ, interleukin (IL)-2, TNFα, and IL-17A production after LPS injection (p < 0.001). In contrast, the frequency of Treg and the capacity to produce IL-10 were unchanged (p = 0.21). Effector THs fail to produce pro-inflammatory Th1-/Th17-associated cytokines after LPS challenge. In contrast, IL-10 production by Treg is not affected. Thus, endotoxemia-induced suppression of pro-inflammatory THs might be considered as a contributing factor to immunoparalysis in sepsis.

Enhanced expression of PD-1 and other activation markers by CD4+ T cells of young but not old patients with metastatic melanoma

The biological behavior of melanoma is unfavorable in the elderly when compared to young subjects. We hypothesized that differences in T-cell responses might underlie the distinct behavior of melanoma in young and old melanoma patients. Therefore, we investigated the circulating T-cell compartment of 34 patients with metastatic melanoma and 42 controls, which were classified as either young or old. Absolute numbers of CD4+ T cells were decreased in young and old melanoma patients when compared to the age-matched control groups. Percentages of naive and memory CD4+ T cells were not different when comparing old melanoma patients to age-matched controls. Percentages of memory CD4+ T cells tended to be increased in young melanoma patients compared to young controls. Proportions of naive CD4+ T cells were lower in young patients than in age-matched controls, and actually comparable to those in old patients and controls. This was accompanied with increased percentages of memory CD4+ T cells expressing HLA-DR, Ki-67, and PD-1 in young melanoma patients in comparison to the age-matched controls, but not in old patients. Proportions of CD45RA−FOXP3high memory regulatory T cells were increased in young and old melanoma patients when compared to their age-matched controls, whereas those of CD45RA+FOXP3low naive regulatory T cells were similar. We observed no clear modulation of the circulating CD8+ T-cell repertoire in melanoma patients. In conclusion, we show that CD4+ T cells of young melanoma patients show signs of activation, whereas these signs are less clear in CD4+ T cells of old patients.

Expansion of circulating T follicular helper cells is associated with disease progression in HIV-infected individuals.

T follicular helper (Tfh) cells within germinal centers (GC) of lymphoid tissue play an important role in HIV infection. Recently, circulating Tfh cells have been described, which share phenotypic and functional characteristics with GC Tfh cells. This study aimed to investigate the effect of HIV infection on four circulating Tfh subsets, including CD4+CXCR5+, CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, and CD4+CXCR5+ICOS+PD-1+ cells. Peripheral blood samples were collected from 33 HIV-infected individuals and 21 healthy controls. The frequency and absolute number of CD3, CD4 and CD8 cells were detected by flow cytometry. The frequency of circulating Tfh cell subsets was also determined by flow cytometry. The correlation between the frequency of Tfh subsets and CD4 T cells counts was assessed by Pearson correlation analysis. There was no significant difference in the frequency of peripheral CD4+CXCR5+ Tfh cells between HIV-infected individuals and healthy controls. However, the percentages of circulating CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, and CD4+CXCR5+ICOS+PD-1+ Tfh cells were significantly higher in individuals with HIV infection than those of healthy controls. Furthermore, the percentage of CD4+CXCR5+PD-1+ Tfh cells showed negative correlation with CD4 T cell counts in HIV-infected individuals. Our results suggested the potential involvement of circulating CD4+CXCR5+PD-1+ Tfh cells during the development of HIV infection.

CD4+CD25Hi FoxP3+ regulatory T cells in long-term cardiac xenotransplantation.

CD4+CD25Hi FoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long-term baboon recipients who have received genetically modified cardiac xenografts from pig donors. Heterotopic cardiac xenotransplantation was performed on baboons using hearts obtained from GTKO.hCD46 (n=8) and GTKO.hCD46.TBM (n=5) genetically modified pigs. Modified immunosuppression regimen included antithymocyte globulin (ATG), anti-CD20, mycophenolate mofetil (MMF), cobra venom factor (CVF), and costimulation blockade (anti-CD154/anti-CD40 monoclonal antibody). FACS analysis was performed on PBLs labeled with anti-human CD4, CD25, and FoxP3 monoclonal antibodies (mAb) to analyze the percentage of Treg cells in six baboons that survived longer than 2months (range: 42-945days) after receiving a pig cardiac xenograft. Total WBC count was low due to immunosuppression in baboons who received cardiac xenograft from GTKO.hCD46 and GTKO.hCD46.hTBM donor pigs. However, absolute numbers of CD4+CD25Hi FoxP3 Treg cells in PBLs of long-term xenograft cardiac xenograft surviving baboon recipients were found to be increased (15.13±1.50 vs 7.38±2.92; P<.018) as compared to naïve or pre-transplant baboons. Xenograft rejection in these animals was correlated with decreased numbers of regulatory T cells. Our results suggest that regulatory T (Treg) cells may contribute to preventing or delaying xenograft rejection by controlling the activation and expansion of donor-reactive T cells, thereby masking the antidonor immune response, leading to long-term survival of cardiac xenografts.

Identification and Characterization of Stimulator of Interferon Genes As a Robust Adjuvant Target for Early Life Immunization.

Immunization is key to preventing infectious diseases, a leading cause of death early in life. However, due to age-specific immunity, vaccines often demonstrate reduced efficacy in newborns and young infants as compared to adults. Here, we combined in vitro and in vivo approaches to identify adjuvant candidates for early life immunization. We employed newborn and adult bone marrow-derived dendritic cells (BMDCs) to perform a screening of pattern recognition receptor agonists and found that the stimulator of interferon genes ligand 2′3′-cGAMP (hereafter cGAMP) induces a comparable expression of surface maturation markers in newborn and adult BMDCs. Then, we utilized the trivalent recombinant hemagglutinin (rHA) influenza vaccine, Flublok, as a model antigen to investigate the role of cGAMP in adult and early life immunization. cGAMP adjuvantation alone could increase rHA-specific antibody titers in adult but not newborn mice. Remarkably, as compared to alum or cGAMP alone, immunization with cGAMP formulated with alum (Alhydrogel) enhanced newborn rHA-specific IgG2a/c titers ~400-fold, an antibody subclass associated with the development of IFNγ-driven type 1 immunity in vivo and endowed with higher effector functions, by 42 days of life. Highlighting the amenability for successful vaccine formulation and delivery, we next confirmed that cGAMP adsorbs onto alum in vitro. Accordingly, immunization early in life with (cGAMP+alum) promoted IFNγ production by CD4+ T cells and increased the proportions and absolute numbers of CD4+ CXCR5+ PD-1+ T follicular helper and germinal center (GC) GL-7+ CD138+ B cells, suggesting an enhancement of the GC reaction. Adjuvantation effects were apparently specific for IgG2a/c isotype switching without effect on antibody affinity maturation, as there was no effect on rHA-specific IgG avidity. Overall, our studies suggest that cGAMP when formulated with alum may represent an effective adjuvantation system to foster humoral and cellular aspects of type 1 immunity for early life immunization.

Value of CD3+CD4+ T Cell Count in Prediction of Viral Infection after Hematopoietic Stem Cell Transplantation in Patients Severe Aplastic Anemia

To explore the value of CD3+CD4+ T cell count in prediction of viral infections after allogeneic hematopoietic stem cell transplantation(allo-HSCT) in the patients with severe aplastic anemia(SAA). A total of 78 SAA patients with allo-HSCT in Guangzhou First People's Hospital from January 2014 to July 2016 were enrolled in this study. The absolute numbers of CD3+CD4+T cells were measured by flow cytometry at 1,2,3,6, and 12 month after allo-HSCT. According to the cell counts, the patients were divided into 3 groups: i.e. <50/µl (n=120), 50-100/µl(n=48) and >100/µl(n=123)groups. The infection incidences of human cytomegalovirus (HCMV) and Epstein-Barr virus(EBV) within 2 weeks around each time point were compared between different groups. According the counts of CD3+CD4+T cells at 3 months after-transplant, these patients were divided into 2 groups, i.e.>100/µl (n=30) and ≤100/µl (n=48). The incidences and duration of HCMV and EBV infection, overall survival rate were compared between 2 groups. The incidences of CMV and EBV infection significantly decreased in CD3+CD4+ T cell >100/µl group as compared with <50/µl and 50-100/µl groups. At 3 months after-transplant, there was lower incidence rates of CMV disease, EBV infection, shorter durations of CMV infection and better survival in CD3+CD4+ T cell >100/µl group as compared with ≤100/µl group. CD3+CD4+ T cell count is a good predictor for CMV and EBV infection after allo-HSCT in SAA patients. There are low risk of infe-ctions from CMV and EBV when CD3+CD4+ T cell count >100/µl in any time after transplant, which means lower occurrence of CMV and EBV infection and better survival when CD3+CD4+ T cell counts is >100/µl in 3 months after transplant in SAA patients.

CLINICO-LABORATORY INDICES CHARACTERIZING THE IMMUNE SYSTEM OF CHICKENPOX PATIENTS

The aim of the study was the evaluation of the function of the immune system in chicken pox patients based on clinical and laboratory data. There were examined 75 patients. The study on phenotypic composition of cells in innate immunity was executed with the method of flow cytometry. A detailed study of the immune system in chickenpox patients revealed the alteration in the composition of the subpopulation of T-lymphocytes, with declining populations of T-helper cells, namely, the lowering of relative and absolute number of CD3+ CD4+ and an increase in the relative number of CD3+CD8+ lymphocytes, which is indicative of immunosuppression. No changes according to the number of NK cells, which must actively respond to viral infection (the final stage of activation of cell growth with an increase in their number in peripheral blood). The absence of their increase is indicative of the irresponsive immunity in chickenpox patients

Identification of the genetic determinants for the frequency of regulatory T cells using system genetics

Abstract Regulatory T (Treg) cells are indispensable components of immune regulation contributing to the maintenance of respiratory tolerance and preventing autoimmunity and exaggerated immune responses. The frequency and absolute number of Treg cells in lymphoid and non-lymphoid tissues are tightly regulated through incompletely understood mechanisms. In this study, we sought to understand the pulmonary immune regulation through identification of the genetic determinants of the frequency and absolute number of CD4+FOXP3+T cells in mice using a genome-wide association study (GWAS) approach. We quantified the frequency and absolute number of CD4+FOXP3+T cells in the lungs and spleen of 102 inbred strains from the hybrid mouse diversity panel under steady state conditions and after exposure to house dust mite (HDM). Among strains of mice examined, the absolute number of pulmonary CD4+FOXP3+ T cells varied by 312-fold at the steady state and 210-fold after HDM exposure. A GWAS analysis results revealed several loci that contribute to the regulation of CD4+FOXP3+ T cells in the lungs and spleen of mice, including a locus on chromosome 5 harboring the homeodomain-only protein (Hopx) gene. Characterization of Hopx−/− mice validated the GWAS findings by demonstrating that the frequency and number of CD4+FOXP3+ T cells are higher in the lungs of Hopx−/− compared to WT mice at the steady state and after HDM exposure. We found that in contrast to the lungs, the number of CD4+FOXP3+ T cells is lower in the spleen of Hopx−/− compared to WT mice at the steady state. Our findings suggest that Hopx is a novel genetic determinant of the frequency and number of CD4+FOXP3+ T cells in mice with opposing directional effects in mucosal and secondary lymphoid organs.

Rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, reduces but does not prevent, the pulmonary CD4+ effector T-cell response in a murine model of asthma

Abstract Nuclear factor (NF)-κB is regarded as a promising target for the development of a novel therapeutic strategy in asthma. Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists inhibit the activity of NF-κB. Therefore, these agents are potent therapeutic agents for the treatment of asthma. It was found that rosiglitazone (ROS), a PPAR-γ agonist, attenuated airway inflammation in a murine model of asthma. The present study attempts to shed light on the involvement of CD4+ effector (Teff) and regulatory (Treg) T cells in the anti-asthmatic action of ROS in ovalbumin (OVA)-induced airway allergic inflammation (AAI) in BALB/c mice. We studied the effect of a 10-day treatment with ROS on the development of AAI and the absolute counts of Treg (Foxp3+CD25+CD4+) cells and activated Teff (Foxp3−CD25+CD4+) cells in the mediastinal lymph nodes (MLNs) and lungs of OVA-sensitized/challenged mice. The severity of AAI was only reduced, but not abolished, by the treatment with ROS. The absolute number of CD4+ Treg and Teff cells was increased in the MLNs and lungs of OVA-sensitized/challenged mice treated with the vehicle as compared to healthy mice. No differences were found in the count of CD4+ Treg cells between vehicle- and ROS-treated OVA-sensitized/challenged mice. The treatment with ROS reduced, but did not fully prevent, the OVA-induced increase in the count of activated CD4+ Teff cells in the MLNs and lungs. The results suggest that the impairment of the activation and clonal expansion of CD4+ Teff cells in the MLNs is involved in the anti-asthmatic action of PPAR-γ agonists. This effect does not seem to be mediated through increased recruitment of CD4+ Treg cells into the MLNs and lungs and/or local generation of Treg cells.

Characteristics of CD8+ stem memory T cells in patients with chronic HIV-1 infection during antiretroviral therapy

Objective To investigate the characteristics of CD8+ stem memory T cells (CD8+ Tscm) in patients with chronic HIV-1 infection before and after antiretroviral therapy (ART) and to analyze their associations with progression of HIV-1 infection. Methods Thirty-six patients with chronic HIV-1 infection and 20 healthy subjects were enrolled in this study. Flow cytometry was performed to detect the percentages and absolute numbers of CD8+ Tscm in patients with chronic HIV-1 infection before and after antiretroviral therapy (ART) as well as in healthy subjects. Correlation analysis was used to demonstrate the relationships between CD8+ Tscm and markers for progression of HIV-1 infection (CD4+ T cell count, HIV-1 viral load and level of activated T cells). Results The percentages and the absolute numbers of CD8+ Tscm in patients with chronic HIV-1 infection had no significant change before and after ART. They were respectively positively correlated with the percentages and the absolute numbers of CD4+ Tscm. The percentage of CD8+ Tscm was proportional to the percentage of CD8+ central memory T cells (CD8+ Tcm), but was inversely proportional to the percentage of CD8+ effector memory T cells (CD8+ Tem). In addition, the percentages of CD8+ Tscm in patients with HIV-1 infection were negatively correlated with the viral loads before ART. Conclusion CD8+ Tscm are responsible for maintaining the homeostasis of other CD8+ T cell subsets. CD8+ Tscm play an important role in inhibiting viral replication. Key words: HIV-1 chronic infection; Antiretroviral therapy; CD8+ Tscm

Dimethyl Fumarate Selectively Reduces Memory T Cells and Shifts the Balance between Th1/Th17 and Th2 in Multiple Sclerosis Patients.

Dimethyl fumarate (DMF; trade name Tecfidera) is an oral formulation of the fumaric acid ester that is Food and Drug Administration approved for treatment of relapsing-remitting multiple sclerosis. To better understand the therapeutic effects of Tecfidera and its rare side effect of progressive multifocal leukoencephalopathy, we conducted cross-sectional and longitudinal studies by immunophenotyping cells from peripheral blood (particularly T lymphocytes) derived from untreated and 4-6 and 18-26 mo Tecfidera-treated stable relapsing-remitting multiple sclerosis patients using multiparametric flow cytometry. The absolute numbers of CD4 and CD8 T cells were significantly decreased and the CD4/CD8 ratio was increased with DMF treatment. The proportions of both effector memory T cells and central memory T cells were reduced, whereas naive T cells increased in treated patients. T cell activation was reduced with DMF treatment, especially among effector memory T cells and effector memory RA T cells. Th subsets Th1 (CXCR3+), Th17 (CCR6+), and particularly those expressing both CXCR3 and CD161 were reduced most significantly, whereas the anti-inflammatory Th2 subset (CCR3+) was increased after DMF treatment. A corresponding increase in IL-4 and decrease in IFN-γ and IL-17-expressing CD4+ T cells were observed in DMF-treated patients. DMF in vitro treatment also led to increased T cell apoptosis and decreased activation, proliferation, reactive oxygen species, and CCR7 expression. Our results suggest that DMF acts on specific memory and effector T cell subsets by limiting their survival, proliferation, activation, and cytokine production. Monitoring these subsets could help to evaluate the efficacy and safety of DMF treatment.

Clinical Significance of Detecting CD4+ T Cells in Peripheral Blood of Patients with Follicular lymphoma

To investigate the changes of CD4+ T lymphocytes in peripheral blood of patients with follicular lymphoma and its clinical significance. Blood samples were collected for detection of whole blood cells, including absolute monocyte count (AMC), absolute lymphocyte count (ALC), hemoglobin (Hb), platelet count (Plt). Age, sex, pathological grade, number of involved lymph nodes, bone marrow involvement (BMI), Ann Arbor stage, B symptoms, serum lactate dehydrogenase (LDH) and serum β-2 microglobulin (β2-MG) were recorded, the prognostic stratification was performed by using FLIPI and FLIPI-2. The T lymphocyte subsets were analyzed by flow cytometry, including the absolute number of CD4+ T lymphocytes (ACD4C) and the absolute number of CD8+ T lymphocytes (ACD8C). Patients were with higher Ann Arbor stage, Hb<120 g/L, LDH greater than the upper limit of normal, the number of lymph nodes were involved> 4, the bone marrow was involvement, β2-MG levels were high FLIPI score and FLIPI-2 score, AMC level was higher (P<0.05). There were no significant differences in ACD4C levels among different groups. Patients with AMC≥0.89×109/L showed a shorter progression-free survival (PFS) and a shorter overall survival time (OS) (P=0.010,0.002) as compared with patients with AMC<0.89×109/L. The patients with ACD4C>0.16×109/L had longer progression-free survival and overall survival time, as compared with patients with ACD4C ≤0.16×109/L (P=0.016,0.012). Low ACD4C and high AMC related with shorter PFS and OS (P=0.013, 0.020). Univariate Cox regression analysis showed that age (P=0.026), bone marrow involvement (P=0.017), elevated LDH (P=0.001), β2-MG (P=0.014), FLIPI and FLIP2 score (P= 0.004 and 0.000) related with a shorter PFS. Multivariable Cox regression analysis showed that Hb (P=0.015), elevated LDH (P=0.003), β2-MG (P=0.045), bone marrow involvement (P=0.016) and FLIPI-2 score(P=0.003) related with short OS. ACD4C ≤0.16×109/L was a factor influencing prognosis of FL patients (PFS and OS) (P<0.05). Low ACD4C levels relatees with poor prognosis of patients with FL, and the ACD4C levels may be an important predictor for FL disease and prognosis.