CD45RA CD62L Research Articles

Deep Immunophenotyping of Circulating T and B Cells in Relapsing Adult-Onset Still's Disease.

Adult-onset Still's disease (AOSD) is a complex systemic inflammatory disorder, categorized as an 'IL-1 driven' inflammasomapathy. Despite this, the interaction between T and B cells remains poorly understood. We conducted a study, enrolling 7 patients with relapsing AOSD and 15 healthy control subjects, utilizing deep flow cytometry analysis to examine peripheral blood T- and B-cell subsets. T-cell and B-cell subsets were significantly altered in patients with AOSD. Within CD4+ T cells, Th2 cells were decreased. Additionally, Th17 cell and follicular Th cell subsets were altered within CD45RA-CD62L+ and CD45RA-CD62L- Th cells in patients with AOSD compared to healthy controls. We identified changes in CD8+ T cell maturation and 'polarization' in AOSD patients, with an elevated presence of the TEMRA CD8+ T cell subset. Furthermore, the percentage of Tc1 cells was decreased, while the frequency of CCR6-CXCR3- Tc2 cells was elevated. Finally, we determined that the frequency of CD5+CD27- B cells was dramatically decreased in patients with AOSD compared to healthy controls. Further investigations on a large group of patients with AOSD are required to evaluate these adaptive immunity cells in the disease pathogenesis.

Imbalanced Immune Response of T-Cell and B-Cell Subsets in Patients with Moderate and Severe COVID-19.

Background: The immunological changes associated with COVID-19 are largely unknown. Methods: Patients with COVID-19 showing moderate (n = 18; SpO2 > 93%, respiratory rate > 22 per minute, CRP > 10 mg/L) and severe (n = 23; SpO2 < 93%, respiratory rate >30 per minute, PaO2/FiO2 ≤ 300 mmHg, permanent oxygen therapy, qSOFA > 2) infection, and 37 healthy donors (HD) were enrolled. Circulating T- and B-cell subsets were analyzed by flow cytometry. Results: CD4+Th cells were skewed toward Th2-like phenotypes within CD45RA+CD62L− (CM) and CD45RA–CD62L− (EM) cells in patients with severe COVID-19, while CM CCR6+ Th17-like cells were decreased if compared with HD. Within CM Th17-like cells “classical” Th17-like cells were increased and Th17.1-like cells were decreased in severe COVID-19 cases. Circulating CM follicular Th-like (Tfh) cells were decreased in all COVID-19 patients, and Tfh17-like cells represented the most predominant subset in severe COVID-19 cases. Both groups of patients showed increased levels of IgD-CD38++ B cells, while the levels of IgD+CD38− and IgD–CD38− were decreased. The frequency of IgD+CD27+ and IgD–CD27+ B cells was significantly reduced in severe COVID-19 cases. Conclusions: We showed an imbalance within almost all circulating memory Th subsets during acute COVID-19 and showed that altered Tfh polarization led to a dysregulated humoral immune response.

Comprehensive assessment of clinical and immune disorders in patients with acute concussion

Introduction. Craniocerebral trauma remains one of the most common forms of brain pathology. Its relevance is determined by both, high prevalence and significant financial costs associated with the treatment, and rehabilitation of injured. Mild forms predominate in the general structure of injury. Immune response is one of the manifestations characteristic for a complex of biochemical and pathophysiological reactions triggered in the brain in response to injury. At the same time, the subtle mechanisms of its functioning and their role in pathogenesis of diseases remain the subject of discussion. Our research was aimed to study the role of immune reactions in the pathogenesis of mild brain injury. Materials and methods. 22 patients with concussion (aged from 20 to 45) were examined. The control group included 37 healthy individuals aged 20 to 46. The examination included the collection of complaints, medical history, assessment of somatic and neurological status, neuropsychological testing. The object of laboratory research was venous blood. Attention was mainly paid to the T-helpers of central (CM, CD45RA–CD62L+) and effector (EM, CD45RA–CD62L–) memory, which were evaluated using multicolored cytometric analysis. Results. Patients presented complaints of general and cognitive nature upon admission to the hospital. Cerebellar lesion symptoms dominated while neurological status evaluation. Neuropsychological examination allowed us to detect neuro-dynamic and regulatory disorders predominance. While conducting the analysis of the main stages of cell maturation in the blood of patients with concussion, it was noted that there was a significant increase in both, relative and absolute content of central memory T-helpers compared to the control group. At the same time, the percentage of EM Th in those with injury was slightly reduced. Also, in patients with injured Th cells of central memory, the relative content of Th1 cells decreased and the relative content of Th17 cells increased. In addition, within the CM Th pool, there was an increase in the proportion of three types of Th17 — CCR6+DN Th17, Th17.1 and «classic» CCR4+CXCR3– Th17. Conclusion. Changes in T-helpers of central memory and T-helpers of peripheral memory subpopulation among CD3+CD4+ cells can be considered as a predictor of the course of concussion in the acute period. However, T cells involved in autoimmunity, do not necessarily reflect the immune system disorders. It is possible that the basis for the decrease in the Th1 level in the circulatory bed in patients with brain injury is the recruitment of immune cells. An increase in the proportion of Th17 was also detected among T-helpers of central memory patrolling the lymphoid tissue the other day after the brain injury. This circumstance may indicate the fact that in the early stages after the nervous tissue damage, processes leading to the formation of «pro-inflammatory» cells are triggered. It allows us to consider these lymphocytes as one of the main populations of immunocompetent cells possessing a neuro-destructive effect.

Human CD45RA− FoxP3hi Memory-Type Regulatory T Cells Show Distinct TCR Repertoires With Conventional T Cells and Play an Important Role in Controlling Early Immune Activation

Adoptive immunotherapy with regulatory T cells (Treg) is a new option to promote immune tolerance following solid organ transplantation (SOT). However, Treg from elderly patients awaiting transplantation are dominated by the CD45RA(-) CD62L(+) central memory type Treg subset (TregCM), and the yield of well-characterized and stable naïve Treg (TregN) is low. It is, therefore, important to determine whether these TregCM are derived from the thymus and express high stability, suppressive capacity and a broad antigen repertoire like TregN. In this study, we showed that TregCM use a different T cell receptor (TCR) repertoire from conventional T cells (Tconv), using next-generation sequencing of all 24 Vβ families, with an average depth of 534 677 sequences. This showed almost no contamination with induced Treg. Furthermore, TregCM showed enhanced suppressive activity on Tconv at early checkpoints of immune activation controlling activation markers expression and cytokine secretion, but comparable inhibition of proliferation. Following in vitro expansion under mTOR inhibition, TregCM expanded equally as well as TregN without losing their function. Despite relatively limited TCR repertoire, TregCM also showed specific alloresponse, although slightly reduced compared to TregN. These results support the therapeutic usefulness of manufacturing Treg products from CD45RA(-) CD62L(+) Treg-enriched starting material to be applied for adoptive Treg therapy.

Human Memory, but Not Naive, CD4+ T Cells Expressing Transcription Factor T-bet Might Drive Rapid Cytokine Production

We found that after stimulation for a few hours, memory but not naive CD4(+) T cells produced a large amount of IFN-γ; however, the mechanism of rapid response of memory CD4(+) T cells remains undefined. We compared the expression of transcription factors in resting or activated naive and memory CD4(+) T cells and found that T-bet, but not pSTAT-1 or pSTAT-4, was highly expressed in resting memory CD4(+) T cells and that phenotypic characteristics of T-bet(+)CD4(+) T cells were CD45RA(low)CD62L(low) CCR7(low). After short-term stimulation, purified memory CD4(+) T cells rapidly produced effector cytokines that were closely associated with the pre-existence of T-bet. By contrast, resting naive CD4(+) T cells did not express T-bet, and they produced cytokines only after sustained stimulation. Our further studies indicated that T-bet was expressed in the nuclei of resting memory CD4(+) T cells, which might have important implications for rapid IFN-γ production. Our results indicate that the pre-existence and nuclear mobilization of T-bet in resting memory CD4(+) T cells might be a possible transcriptional mechanism for rapid production of cytokines by human memory CD4(+) T cells.

Decreases in activated CD8+ T cells in patients with severe hepatitis B are related to outcomes.

Many studies on T helper (Th)1, Th2, T regulatory and Th17 cells have been carried out in acute-on-chronic liver failure (ACLF). However, CD8(+) T cell, as a main participant in immune-mediated injuries and defense against microorganisms, has seldom been studied in ACLF. The purpose of this study was to investigate the CD8(+) T cell function, and the outcomes of patients with severe hepatitis [SH; serum bilirubin (SB) ≥ 10 mg/dl and prothrombin activity (PTA) < 60 %]. Thirty-six patients with chronic HBV-associated SH were included. Twenty normal chronic hepatitis B (CHB) patients (2 < SB < 10 (mg/dl) and PTA ≥ 60 %) and 28 healthy volunteers were enrolled as control groups. Twenty-six patients with SH were diagnosed with ACLF (SB ≥ 10 mg/dl and PTA ≤ 40 %). The non-recovered ACLFs (NR-ACLF) had higher HBV DNA loads than recovered ACLFs (R-ACLF) (6.03 ± 1.79 vs. 4.36 ± 1.61 (log10, IU/L)). The NR-ACLFs had the highest neutrophil:lymphocyte ratios (5.10 ± 2.37) (all P < 0.001; a = 0.05). The CHBs had higher perforin(+) and TCM (CD45RA(-)CD62L(hi)CCR7(+)) proportions [31.28 ± 19.51, 5.32 ± 3.57 (%)] compared to R-ACLFs (11.75 ± 15.35, 0.78 ± 0.76 (%); P = 0.004, 0.001, respectively), or NR-ACLFs (11.61 ± 5.79, 1.14 ± 0.67 (%); P = 0.006, 0.003). The non-ACLF SHs had higher CD38(+) proportions than R-ACLFs or NR-ACLFs (25.46 ± 8.02 vs. 16.24 ± 7.77 or 16.81 ± 6.30 (%), P = 0.039, 0.023). High neutrophil:lymphocyte ratios and a decrease in activated CD8(+) T cells may be related to poor outcomes in patients with SH.

Deficiency of CD8+ effector memory T cells is an early and persistent feature of multiple sclerosis

Background: Patients with multiple sclerosis (MS) have a deficiency of circulating CD8+ T cells, which might impair control of Epstein–Barr virus (EBV) and predispose to MS by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. Based on the expression of CD45RA and CD62L, CD4+ T cells and CD8+ T cells can be subdivided into four subsets with distinct homing and functional properties, namely: naïve, central memory, effector memory (EM) and effector memory re-expressing CD45RA (EMRA) cells. Objective: Our aim was to determine which memory subsets are involved in the CD8+ T cell deficiency and how these relate to clinical course. Methods: We used flow cytometry to analyze the memory phenotypes of T cells in the blood of 118 MS patients and 112 healthy subjects. Results: MS patients had a decreased frequency of EM (CD45RA–CD62L–) and EMRA (CD45RA+CD62L–) CD8+ T cells, which was present at the onset of disease and persisted throughout the clinical course. The frequencies of CD4+ EM and EMRA T cells were normal. Conclusion: Deficiency of effector memory CD8+ T cells is an early and persistent feature of MS and might underlie the impaired CD8+ T cell control of EBV.

AB0016 Association between the hla-b51 antigen and profiles of peripheral t cell subsets in tunisian patients with behcet’s disease

BackgroundBehçet’s disease (BD) is a multisystemic inflammatory disorder characterized by spontaneous remissions and relapses [1]. An association between Behçet’s disease and HLA-B51 has been regarded as the strongest evidence for...

Analysis of T lymphocyte absolute number and function in the early phase after haploidentical hematopoietic stem cell transplantation

This study was purposed to investigate the immune reconstitution of T-cells in patients who received haploidentical hematopoietic stem cell transplantation (hiHSCT). The peripheral blood was harvested from 22 patients before transplantation and at month 1, 3, 6 after hiHSCT. The proportions of T lymphocyte subtypes including CD3(+), CD4(+), CD8(+), CD45RO(+), and CD45RA(+)CD62L(+) were analyzed by flow cytometry, followed by the calculation of T cell numbers according to the amounts of peripheral blood leukocytes. Adenosine triphosphate (ATP) value in CD4(+) T cells was measured by ImmuKnow method to evaluate the function of lymphocytes. The results showed that the CD3(+) cell absolute value before transplantation was 833.75 ± 359.84/µl, but those values at month 1, 3, 6 after transplantation were 318.87 ± 266.71/µl, 1006.76 ± 512.32/µl and 1296.38 ± 958.77/µl respectively. The CD4(+) cell absolute value before transplantation was 336.99 ± 211.11/µl, but such values at month 1, 3, 6 after transplantation were 45.89 ± 44.21/µl, 142.97 ± 114.85/µl, and 181.78 ± 120.61/µl respectively. The CD8(+) cell absolute value before transplantation was 430.21 ± 159.48/µl, but those values at month 1, 3, 6 after transplantation were 230.44 ± 195.89/µl, 621.64 ± 318.83/µl, and 823.07 ± 633.55/µl respectively. The CD4(+)CD45RO(+) memory T cell absolute value before transplantation was 227.44 ± 73.34/µl, but such values at month 1, 3, 6 after transplantation were 43.47 ± 43.40/µl, 138.69 ± 110.17/µl, 147.73 ± 82.94/µl respectively. The CD8(+)CD45RO(+) memory T cell absolute value before transplantation was 212.70 ± 98.48/µl, but such values at month 1, 3, 6 after transplantation were 184.76 ± 168.65/µl, 445.90 ± 252.50/µl, 519.80 ± 475.53/µl respectively. CD4(+)CD45RA(+)CD62L(+) naive T cell number before transplantation was 68.94 ± 59.74/µl, but such cell numbers at month 1, 3, 6 after transplantation decreased to 2.44 ± 2.93/µl, 3.14 ± 3.48/µl, 23.22 ± 38.38/µl respectively. The CD8(+)CD45RA(+)CD62L(+) naive T cell absolute value before transplantation was 124.82 ± 60.95/µl, but those values at month 1, 3, 6 decreased to 19.37 ± 17.71/µl, 76.63 ± 50.85/µl, and 114.49 ± 174.29/µl respectively. The ATP value in CD4(+) T cells decreased to 210.19 ± 119.37 ng/ml at month 1 after transplantation and increased to 280.62 ± 110.03 ng/ml at month 3, and 357.28 ± 76.18 ng/ml at month 6 after transplantation. It is concluded that CD8(+) memory T cell reconstruction contributes critically to T cell recovery early after hiHSCT, while the thymic output function remains low. However, T cell function recovers to normal range at month 3 after transplantation.

THU0038 Abnormal Phenotypic T Cell Subsets in Peripheral Blood of Tunisian Patients with Behcet’s Disease

BackgroundBehçet’s disease (BD) is a chronic recurrent multisystemic inflammatory disorder of unknown origin. Immune-mediated mechanisms play a major role in the pathogenesis of the disease. BD is not considered to...

Regulatory T cells in kidney transplant recipients: the effect of induction immunosuppression therapy

Regulatory T cells have been suggested to down-regulate the alloimmune response. The aim of this prospective open study was to evaluate the effects of different inductive agents on peripheral blood regulatory T cells in kidney transplant patients and to analyse their association with short-term graft outcome. Regulatory and effector T cell numbers in peripheral blood were determined by flow cytometry in 71 prospectively followed kidney transplant recipients at postoperative day 0, 7, 14, 21, 28, 60 and 90. Patients were treated with a calcineurin inhibitor-based triple immunosuppression with polyclonal rabbit anti-thymocyte globulin (rATG, n = 28), basiliximab, the anti-CD25 monoclonal antibody (n = 18) or without induction (controls, n = 25). Flow cytometry data were correlated to rejection incidence. Compared to controls, CD4(+)CD25(+)FoxP3(+) regulatory T-cell expansion among CD4(+) T cells was noticed in the rATG group at all post-transplant time-points by Day 14 (P < 0.001). A significant decrease in Treg frequency (P < 0.001) and concurrently a transient increase of CD4(+)CD25(low/-)FoxP3(+) population were observed in basiliximab-treated patients 7-60 days post-transplantation. Biopsy-proven acute rejection occurred in 16.7% of controls, 10.7% of the rATG group and in 11.1% of the basiliximab group. Higher CD4(+)FoxP3(+)/CD8(+)CD45RA(+)CD62L(-) ratios were observed repeatedly in those patients after basiliximab induction who were rejection free (P < 0.01). In this study, the rATG induction therapy was associated with an expansion of regulatory cells. Sustained high CD4(+)FoxP3(+)/Teff ratios were associated with the absence of rejection after basiliximab induction.

Mesenchymal stromal cells derived from CD271+ bone marrow mononuclear cells exert potent allosuppressive properties

BACKGROUND AIMS. Because data on the immunosuppressive effect of different subsets of mesenchymal stromal cells (MSC) are sparse, we investigated the molecular and cellular mechanisms underlying the allosuppressive effect of MSC generated from bone marrow CD271(+) cells (CD271-MSC) and asked whether this potential is comparable with that of MSC generated through plastic adherence (PA-MSC). METHODS. The immunosuppressive effect of CD271-MSC on the allogeneic reaction was investigated by mixed lymphocyte reaction (MLR). RESULTS. CD271-MSC significantly suppressed the alloantigen-induced proliferation of mononuclear cells (MNC) of two HLA-disparate donors at all MSC:MNC ratios, 1:1, 1:2 and 1:10. They also demonstrated a significantly higher allosuppression than PA-MSC at an MSC:MNC ratio of 1:1. This inhibitory effect was associated with significantly elevated levels of prostaglandin E2 (PGE2) at ratios of 1:1 and 1:2 (about 4-fold), but not at a ratio of 1:10. Indomethacin, and inhibitor of cyclooxygenase-1 and 2 necessary for the biosynthesis of PGE2, mitigated suppressive effects of CD271-MSC only at a ratio of 1:1, indicating that PGE2 is not involved in MSC-mediated inhibition when allogeneic MNC are in excess. The increase of PGE2 was associated with a significant decrease of pro-inflammatory cytokine levels (interferon-gamma and tumor necrosis-alpha), while no changes in levels of interleukin-10, soluble HLA-G and nitric oxide were observed. In addition, CD271-MSC induced an expansion of highly suppressive naive CD4(+)CD25(high)CD45RA(+)CD62L(+) T-regulatory cells, which may extend their allosuppressive effect. CONCLUSIONS. Our data suggest that CD271-MSC exert potent allosuppressive properties and therefore can be used as a reasonable alternative to PA-MSC for the treatment of patients with graft-versus-host disease.

Age-Related Differences in Naturally Acquired T Cell Memory to Plasmodium falciparum Merozoite Surface Protein 1

Naturally acquired immunity to Plasmodium falciparum malaria in malaria holoendemic areas is characterized by the gradual, age-related development of protection against high-density parasitemia and clinical malaria. Animal studies, and less commonly, observations of humans with malaria, suggest that T-cell responses are important in the development and maintenance of this immunity, which is mediated primarily by antibodies that slow repeated cycles of merozoites through erythrocytes. To advance our rather limited knowledge on human T-cell immunity to blood stage malaria infection, we evaluated CD4 and CD8 T-cell effector memory subset responses to the 42 kDa C-terminal fragment of Merozoite Surface Protein 1 (MSP142), a malaria vaccine candidate, by 49 healthy 0.5 to ≥18 year old residents of a holoendemic area in western Kenya. The proportion of individuals with peripheral blood mononuclear cell MSP142 driven IFN-γ ELISPOT responses increased from 20% (2/20) among 0.5–1 year old children to 90% (9/10) of adults ≥18 years (P = 0.01); parallel increases in the magnitude of IFN-γ responses were observed across all age groups (0.5, 1, 2, 5 and ≥18 years, P = 0.001). Less than 1% of total CD4 and CD8 T-cells from both children and adults produced IFN-γ in response to MSP142. However, adults had higher proportions of MSP142 driven IFN-γ secreting CD4 and CD8 effector memory (CD45RA− CD62L−) T-cells than children (CD4: 50.9% vs. 28.8%, P = 0.036; CD8: 52.1% vs. 18.3%, respectively P = 0.009). In contrast, MSP142 driven IFN-γ secreting naïve-like, transitional (CD45RA+ CD62L+) CD4 and CD8 cells were the predominant T-cell subset among children with significantly fewer of these cells in adults (CD4: 34.9% vs. 5.1%, P = 0.002; CD8: 47.0% vs. 20.5%, respectively, P = 0.030). These data support the concept that meaningful age-related differences exist in the quality of T-cell immunity to malaria antigens such as MSP1.

Immunohematologic Reconstitution in Pediatric Patients after T Cell-Depleted HLA-Haploidentical Stem Cell Transplantation for Thalassemia

To analyze immunohematologic reconstitution, particularly of natural killer (NK) cells, we evaluated 13 β-thalassemia patients after 20 and 60 days and 1 year posttransplantation with T cell-depleted HLA-haploidentical stem cells. We assessed lymphocyte and bone marrow (BM) progenitor cell phenotype and differentiation capacity, spontaneous BM cytokine production, stromal cells, and stromal cell interleukin (IL)-7 production. A reduced clonogenic capability manifested at day +20. Patients had significantly lower CD4(+) T cells versus controls, mainly in the CD45RA(+)CD62L(+) subset. NKs were among the first lymphocytes to repopulate the peripheral blood. At day +60, an increase in primitive BM progenitor cells paralleled small increases in CD4(+), naïve CD4(+), and thymic naïve Th cells. A significant increase in CD4(+) and CD8(+) markers paralleled an increase in CD3⁻CD16(+) NKs, especially with full engraftment. In patients with stable mixed chimerism we observed very low levels of CD3(+) donor chimerism early after transplant that increased over time, but a stable population of high donor NK cells, suggesting a role of these cells on donor engraftment. Stromal cells secreted less IL-7 and displayed "macrophage-like" morphology. Patients initially manifested impaired stem/progenitor cell growth and differentiation capacity in parallel with altered T cell homeostasis and a reduced T cell naïve compartment. We hypothesize that T cell compartment damage partly arises from altered new T cell production from the hematopoietic stem/progenitor cells under stromal cytokine influence. NNK subset analysis might be useful for determining transplant outcome.

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

Evidence is presented that thermal or oxidizing stress-activated DC interact with CD4(+) T cells to induce and maintain a TCR-independent homeostatic memory circuit. Stress-activated DC expressed endogenous intra-cellular and cell surface HSP70. The NF-kappaB signalling pathway was activated and led to the expression of membrane-associated IL-15 molecules. These interacted with the IL-15 receptor complex on CD4(+) T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T-cell proliferation and IFN-gamma production. CD40 ligand on CD4(+) T cells in turn re-activated CD40 molecules on DC, inducing DC maturation and IL-15 expression thereby maintaining the feedback circuit. The proliferating CD4(+) T cells were characterized as CD45RA(-) CD62L(+) central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC-class-II-TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC-class II antibodies. These findings suggest that stress can activate a DC-CD4(+) T-cell interacting circuit, which may be responsible for maintaining a homeostatic antigen-independent memory.

Investigation of Lymphocyte Depletion and Repopulation Using Alemtuzumab (Campath-1H) in Cynomolgus Monkeys

As the target CD52 molecule is expressed on erythrocytes of most nonhuman primate strains, using alemtuzumab in these species would cause massive hemolysis. Six cynomolgus monkeys of Indonesian origin, screened by agglutination assay for absence of CD52 on erythrocytes, were administered alemtuzumab in a cumulative dose to a maximum of 60 mg/kg. In two monkeys, mycophenolate mofetil (MMF) was added as maintenance therapy. Complete depletion of T and B lymphocytes (>99.5%) was achieved with 20 mg/kg alemtuzumab and was more profound than in monkeys treated with antithymocyte globulin (n = 5), as quantified by flow cytometry. Repopulation was suppressed by weekly injections of 10 mg/kg. Without MMF, repopulation of CD20(+)B cells and CD8(+)T cells was complete within 2 and 3 months, respectively, and repopulation of CD4(+)T cells was 67% after 1 year. MMF significantly delayed CD4(+)T-cell repopulation. Among repopulating CD4(+) and CD8(+) T cells, a phenotypic shift was observed from CD45RA(hi)CD62L(hi) naïve cells toward CD45RA(lo)CD62L(lo) effector memory cells. In lymph nodes, the depletion of naïve cells was more profound than of memory cells, which may have initiated a proliferation of memory cells. This model offers opportunities to investigate lymphocyte depletion/repopulation phenomena, as well as the efficacy of alemtuzumab in preclinical transplantation models.

Reference ranges and age-related changes of peripheral blood lymphocyte subsets in Chinese healthy adults

This study was performed to build region-specific reference ranges of peripheral blood lymphocyte subsets for Chinese healthy adults from the young to the elderly and analyze the trends of changes in lymphocyte subsets for evaluating the impact of age on the values. 151 healthy adults aged 19-86 were recruited based on the SENIEUR protocol. Three sets of reference ranges were finally built applicable for the healthy young (19-44 years), middle-aged (45-64 years) and elder adults ([Symbol: see text]65). Comparisons in parameters among the three cohorts showed that a statistically significant increase in CD16CD56+ NK cell was observed between the middle-aged and elder cohorts, whereas for the majority of the parameters, a significant decline was observed between the young and the middle-aged cohorts. Further results showed that inverse correlations were observed between the age and CD19(+) B, CD3(+) T, CD3(+)CD4(+) T, CD4(+)CD45RA(+)CD62L(+) naïve T cell and CD4(+)CD28(+)/CD4(+), while the positive one was identified between the age and the NK cell. These significant changes of the most of immune parameters provided evidence for immunosenescence. Notably, T cell activation markers of CD8(+)CD38(+) and CD8(+)HLA-DR(+) showed reverse trends of association with age, which provides a clue for further researches on the mechanisms underlying the paradoxical clinical presentation of the elder patients.

Expression of CD62L on Donor CD4+ T Cells in Allografts: Correlation with Graft-Versus-Host Disease after Unmanipulated Allogeneic Blood and Marrow Transplantation

The aim of this study was to investigate the association of donor CD4(+) T cells expressing CD62L with transplant outcomes. We report a prospective analysis of 31 patients who were treated with a Bu/Cy regimen, followed by unmanipulated blood and marrow transplantation. Median number (range) of CD4(+)CD62L(+), CD4(+)CD45RA(+)CD62L(+), and CD4(+)CD45RO(+)CD62L(+) cells infused were 0.31(0.05-1.10)x10(8)/kg, 0.22(0.03-0.95)x 10(8)/kg, and 0.17(0.01-0.81)x10(8)/kg, respectively. The incidence of grades II to IV aGVHD was 36%. In a multivariate analysis, infusion of >0.22 x 10(8) CD4(+)CD45RA(+)CD62L(+) cells infused/kg increased the risk of grades II to IV aGVHD (HR = 4.741, 95% CI = 1.037-21.662, P = 0.045). Thirteen of 31 patients experienced cGVHD, the risk of cGVHD was increased in patients receiving >0.45 x 10(8) CD4(+)CD45RA(+) cells infused/kg (HR = 4.614, 95% CI = 1.265-16.829, P = 0.021). Our results suggest that a high cell dose of CD4(+)CD45RA(+)CD62L(+) cells increase the incidence of grades II-IV aGVHD. A high number of CD4(+)CD45RA(+) cells infused were associated with increased risk of cGVHD in our transplant settings.

Neem leaf glycoprotein directs T-bet–associated type 1 immune commitment

Neem leaf glycoprotein (NLGP)–mediated immune activation and associated immune polarization was studied. NLGP–induced activation is reflected in upregulation of early activation marker CD69 on lymphocytes, monocytes, and dendritic cells. Activation is also denoted by CD45RO enhancement, with a decrease in CD45RA phenotype and CD62L (L-selectin). NLGP-activated T cells secrete greater amount of signature T-helper (Th)1 cytokines interferon-γ and a lower amount of the Th2 cytokine interleukin (IL)–4. Similar type 1 directiveness is also observed in antigen-presenting monocytes and dendritic cells by upregulation of IL-12, tumor necrosis factor –α and downregulation of IL-10. Creation of the type 1 microenvironment is also assisted by NLGP-induced downregulation of FoxP3 + T-Reg cells. A type 1–specific transcription factor, T-bet, is upregulated in circulating immune cells after their stimulation with NLGP. In the creation of type 1 immune network, increased phosphorylation of STAT1 and STAT4 with decreased phosphorylation of STAT3 might have significance. We conclude that NLGP may be effective in maintaining normal immune homeostasis by upregulating type 1 response in immunosuppressed hosts, which may have significant role in the induction of host protective antitumor functions.

Requirements for Retroviral Targeting of a Suicide Gene to Alloreactive Memory Stem T Cells for Adoptive Immunotherapy of Leukemia.

In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.