Comparison of leucocyte profiles between healthy children and those with asymptomatic and symptomatic Plasmodium falciparum infections

Abstract Background The immune mechanisms that determine whether a Plasmodium falciparum infection would be symptomatic or asymptomatic are not fully understood. Several studies have been carried out to characterize the associations between disease outcomes and leucocyte numbers. However, the majority of these studies have been conducted in adults with acute uncomplicated malaria, despite children being the most vulnerable group. Methods Peripheral blood leucocyte subpopulations were characterized in children with acute uncomplicated (symptomatic; n = 25) or asymptomatic (n = 67) P. falciparum malaria, as well as malaria-free (uninfected) children (n = 16) from Obom, a sub-district of Accra, Ghana. Leucocyte subpopulations were enumerated by flow cytometry and correlated with two measures of parasite load: (a) plasma levels of P. falciparum histidine-rich protein 2 (PfHRP2) as a proxy for parasite biomass and (b) peripheral blood parasite densities determined by microscopy. Results In children with symptomatic P. falciparum infections, the proportions and absolute cell counts of total (CD3 +) T cells, CD4 + T cells, CD8 + T cells, CD19 + B cells and CD11c + dendritic cells (DCs) were significantly lower as compared to asymptomatic P. falciparum-infected and uninfected children. Notably, CD15 + neutrophil proportions and cell counts were significantly increased in symptomatic children. There was no significant difference in the proportions and absolute counts of CD14 + monocytes amongst the three study groups. As expected, measures of parasite load were significantly higher in symptomatic cases. Remarkably, PfHRP2 levels and parasite densities negatively correlated with both the proportions and absolute numbers of peripheral leucocyte subsets: CD3 + T, CD4 + T, CD8 + T, CD19 + B, CD56 + NK, γδ + T and CD11c + cells. In contrast, both PfHRP2 levels and parasite densities positively correlated with the proportions and absolute numbers of CD15 + cells. Conclusions Symptomatic P. falciparum infection is correlated with an increase in the levels of peripheral blood neutrophils, indicating a role for this cell type in disease pathogenesis. Parasite load is a key determinant of peripheral cell numbers during malaria infections.

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Lymphocyte aggregation in response to adrenergic stimulation

Blood ◽

10.1182/blood.v71.5.1470.1470 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1470-1474 ◽

Cited By ~ 5

Author(s):

DE Hammerschmidt ◽

C Jeanneret ◽

M Husak ◽

M Lobell ◽

HS Jacob

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Binding Studies ◽

Beta Adrenergic ◽

Cell Counts ◽

Significant Difference ◽

Induced Aggregation

Abstract A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

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Early Recovery of Thymus-Derived Naïve T Cells in Pediatric Patients (pts) Treated with Non-Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation (NMSCT) for Cancer.

Blood ◽

10.1182/blood.v108.11.310.310 ◽

2006 ◽

Vol 108 (11) ◽

pp. 310-310

Author(s):

Terry J. Fry ◽

Alison R. Rager ◽

Frances Hakim ◽

Cynthia Love ◽

Paula Layton ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

High Risk ◽

Peripheral Blood ◽

T Cell Subsets ◽

Immune Recovery ◽

Early Recovery ◽

Thymic Function ◽

Cell Counts ◽

Increased Risk

Abstract Background: Current SCT approaches consistently achieve rapid donor myeloid engraftment, but delayed immune recovery remains a significant obstacle and results in increased risk of infection and relapse. T cells are regenerated via 2 pathways, thymus-derived and peripheral expansion, processes for which IL-7 is critical. We postulated that non-myeloablative pre-transplant conditioning might preserve thymic function in pediatric SCT recipients thus enhancing thymus-derived naïve T cell regeneration. Methods: We analyzed T cell subsets, T cell receptor excision circles (TREC), and IL-7 levels in peripheral blood after SCT in 21 pediatric pts with high-risk malignancies (median age 14, range 4–21). Fludarabine-based induction chemotherapy was administered for disease control and targeted CD4 count reduction. Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) × 4 days plus melphalan (100 mg/m2 × 1 dose in sarcoma pts). Grafts consisted of G-CSF mobilized unmodified peripheral blood stem cells from 5–6/6 HLA-matched first-degree relatives (median CD34 dose 11.7 × 10E6/kg, range 4.4–19.1; median CD3 dose 416 × 10E6/kg, range 228–815). Cyclosporine was used for GVHD prophylaxis. Results: Donor-derived engraftment was rapid (absolute neutrophil count > 500/uL median day 9, range 8–11). Complete donor lymphoid chimerism (>95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all by day 28. Immune recovery was brisk and sustained. Substantial numbers of naïve (CD45RA+/CD62L+) CD4+ and CD8+ T-cells were detected at day 28 (Fig 1). There was a steady increase in TREC from 3 to 12 months consistent with early, robust thymic-dependant T cell generation (Fig 2). This was not seen in adult pts treated on a parallel trial (data not shown). IL-7 levels were elevated and inversely correlated with T cell counts (r=−0.56, p<0.0001). Conclusions: Targeted immune depletion and NMSCT results in rapid, sustained immune reconstitution in pediatric pts with malignancy. Preserved thymic function appears to contribute to naïve T cell recovery in this setting. We postulate that non-myeloablative conditioning is thymus sparing and that this, in combination with immune depletion-induced IL-7 elevation, promotes early thymic-derived lymphoid recovery. This approach may serve as a strategy to overcome the prolonged immunodeficiency commonly encountered after allogeneic SCT in pediatrics and might be used as a platform to direct allogeneic anti-tumor immune responses in high-risk childhood cancers. Figure 1 Figure 1. Figure 2 Figure 2.

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Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method

Blood ◽

10.1182/blood.v83.5.1268.bloodjournal8351268 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1268-1277 ◽

Cited By ~ 1

Author(s):

M Carbonari ◽

M Cibati ◽

M Cherchi ◽

D Sbarigia ◽

AM Pesce ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Hiv Infection ◽

Peripheral Blood ◽

Peripheral Blood Leukocytes ◽

Blood Leukocytes ◽

Cell Counts ◽

Immunodeficiency Virus

We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/microL) than in patients at earlier stages (> 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.

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NKG2C+CD57+ Natural Killer Cells May Play an Important Role in Maintaining Virus-Specific Immune Reconstitution Post-Allogeneic HSCT

Blood ◽

10.1182/blood-2018-99-110779 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3327-3327

Author(s):

Debora Queiros ◽

Susanne Luther-Wolf ◽

Eva M Weissinger ◽

Arnold Ganser

Keyword(s):

T Cells ◽

Healthy Volunteers ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Immune Reconstitution ◽

Blood Samples ◽

Allogeneic Hsct ◽

Cell Counts ◽

Cmv Reactivation

Abstract Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for malignant hematological diseases in adults. Due to the delayed immune reconstitution after HSCT, human cytomegalovirus (CMV) can reactivate, leading to prolonged hospitalization and increased morbidity and even mortality. Natural Killer (NK) cells have recently been described to undergo persistent reconfiguration in response to CMV-reactivation. Here we analyzed the presence and expansion of CMV-specific NK cells in patients after allogeneic HSCT. Methods: A multicolor flow cytometry panel for monitoring the CMV-specific NK cell (NKG2C+CD57+) reconstitution and expression of activating receptors was established. Reconstitution of CMV-specific NK cells was assessed in peripheral blood samples from 67 CMV-seropositive patients. The samples were collected and analyzed between day 0 and 100 post-HSCT at intervals of 7-10 days. Monitoring of CMV-reactivation by CMV-pp65 expression and reconstitution of CMV-specific T cells (CMV-CTLs) was done routinely in our laboratory, using 7 commercially available, certified CMV-tetramers, allowing for comparison of CMV-CTL and NKG2C+CD57+ NK cells. For further immunological tests, PBMCs from CMV-seropositive healthy volunteers were isolated by density gradient centrifugation. NK cells were negatively selected by magnetic bead separation. Additional purification of NKG2C+CD57+ NK cellswas achieved by cell sorting. Selected NK cells were expanded by co-culture with irradiated allogeneic PBMCs as feeder cells and the medium was supplemented with PHA and IL-2. Expanded CD57+NKG2C+ NK cells were KIR-typed. Results: Our patient cohort consisted of 67 patients after allogeneic HSCT with a median age of 59 years (range: 20-75). Forty-two patients (62.7%) were transplanted for acute leukemia, 54 (80.6%) received reduced intensity conditioning (RIC) and 62 (92.5%) received anti-thymocyte-antibodies globulin (ATG). GvHD-prophylaxis was cyclosporine A (CsA) in combination with mycophenolate motefil (MMF) for 82.1% of the patients and 77.6% were transplanted from matched donors. Thirty-three (49.2%) patients reactivated CMV (median age: 59.5 years, range 28-75; median day of reactivation: 38 days post-HSCT, range: 19-54). A significant increase in the absolute cell counts of NKG2C+CD57+ NK cells was observed after CMV reactivation, when compared to patients who did not reactivate CMV (p<0.0001). Interestingly, we observed a decreased expression of the CD8-molecule on NK cells during CMV-reactivation. CD8-expression on NK cells was previously described to be associated with a more cytotoxic phenotype of NK cells, this decrease may be a consequence of apoptosis following lytic activity. Monitoring for an additional activation marker, NKG2D, showed a significant increased expression after CMV reactivation (p=0.006), demonstrating not only the activating regulation of NK cells, but also, the co-stimulatory effects on T cell proliferation and cytokine production. Remarkably, when comparing NKG2C+CD57+ NK cells with CMV-specific T cells (Figure 1), both cell populations show similar kinetics of expansion, with an increase in the absolute cell counts during and after CMV-reactivation. NKG2C+CD57+ preliminary expansion-studies were performed using peripheral blood samples from CMV-seropositive healthy volunteers. After two weeks in culture, an expansion of up to 3100-fold was achieved. Further studies to assess the proliferative capacity of NKG2C+CD57+ subpopulation and its functional properties post-HSCT, are ongoing. In addition, an extensive panel of cytokines and chemokines excreted by the NKG2C+CD57+cells will be studied in order to evaluate their recruitment ability of other cell-types. Conclusion: Taken together, our results indicate that NK cells undergo a dynamic modulation and expansion of this population occurs in response to CMV-reactivation. Additionally, NKG2C+CD57+ NK cells may substitute for missing CMV-specific T cells shortly after HSCT and may play an important role in sustaining the immune reconstitution after CMV-reactivation. This study shows that NKG2C+CD57+ NK cells can be selected and expanded in vitro, holding promise for adoptive transfer in patients with recurrent CMV-reactivations. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

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The Effect of CCCP on Mitophagy in CD4+CD25+ Regulatory T Cells in Human Peripheral Blood

10.21203/rs.3.rs-38918/v1 ◽

2020 ◽

Author(s):

Yu-Jie Yang ◽

Md Rezaul Karim ◽

Jang Yuan ◽

Xiao-Qian Peng ◽

Pei Zeng ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Peripheral Blood ◽

Mitochondrial Membrane ◽

Human Peripheral Blood ◽

Treg Cells ◽

Oxygen Species ◽

Orange Yellow ◽

Significant Difference

Abstract Objective: To investigate the effects and mechanisms of different concentrations of CCCP on mitophagy in human peripheral blood regulatory T cells. Methods: Tregs were isolated, identified, and then grouped, treating with CCCP at a concentration of 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM for 24h in an incubator. Flow cytometry detected the reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial quality, and fluorescence microscopy observed the co-localization of mitochondria and lysosomes in each group. Results: The purity of CD4+CD25+Tregs was (93.36 ± 1.87) %. With the increase of CCCP concentration, the ROS level gradually increased, while the MMP decreased gradually. About the mitochondria and lysosome fusion, the fluorescence intensity of orange (yellow) was the highest when the concentration of CCCP was in the range of 5-10 μM while decreased with the CCCP concentration continually increasing. The mitochondrial quality decreased with the increase of CCCP concentration. However, there was no significant difference between groups C, D and E. The mitochondrial quality of groups F and G were significantly lower than that of group E. Conclusions: With the concentration of CCCP gradually increased, the level of ROS in Treg cells increased, and MMP decreased, which promoted the mitophagy, mitochondrial quality maintains homeostasis. When ROS accumulated, and MMP decreased significantly, the mitophagy was inhibited, and the mitochondrial quality decreased significantly.

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Lymphocyte aggregation in response to adrenergic stimulation

Blood ◽

10.1182/blood.v71.5.1470.bloodjournal7151470 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1470-1474

Author(s):

DE Hammerschmidt ◽

C Jeanneret ◽

M Husak ◽

M Lobell ◽

HS Jacob

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Binding Studies ◽

Beta Adrenergic ◽

Cell Counts ◽

Significant Difference ◽

Induced Aggregation

A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

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γδ+ T Cells Preferentially Respond to Live Rather than Killed Malaria Parasites

Infection and Immunity ◽

10.1128/iai.66.5.2393-2398.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2393-2398 ◽

Cited By ~ 35

Author(s):

Martin Waterfall ◽

Antony Black ◽

Eleanor Riley

Keyword(s):

T Cells ◽

Plasmodium Falciparum ◽

Peripheral Blood ◽

Interleukin 2 ◽

Γδ T Cells ◽

Feeder Cells ◽

Parasite Culture ◽

Recombinant Interleukin 2 ◽

Culture Supernatants

ABSTRACT We have compared the in vitro responses of peripheral blood T cells from malaria-unexposed donors to live Plasmodium falciparumschizonts, freeze-thawed schizont extracts (P. falciparumschizont extracts [PfSE]), and parasite culture supernatants. We show that the cells responding to PfSE and parasite culture supernatants are predominantly CD4+ TCRαβ+ while in the presence of live schizonts there is an additional activation of TCRγδ+ cells. Activation of TCRγδ+cells in response to PfSE was seen only when irradiated autologous feeder cells or recombinant interleukin-2 (IL-2) was added to the cultures. Live schizonts but not PfSE induced significant IL-2 production in vitro in the first 5 days after stimulation, suggesting that induction of early IL-2 by live parasites may contribute to the marked activation of the TCRγδ+ population.

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Circulating immune markers predict the therapeutic effect in primary lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e21203 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e21203-e21203

Author(s):

Liangliang Xu ◽

Jitian Zhang ◽

Li Yang ◽

Guangqiang Shao ◽

Taiyang Liuru ◽

...

Keyword(s):

Lung Cancer ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Blood Lymphocyte ◽

Lymphocyte Subpopulations ◽

Significant Difference

e21203 Background: Radiotherapy (RT), surgical resection (SR), and immunotherapy (IT) as main therapies in lung cancer have either suppressive or stimulatory effects on the immune system. It’s still unclear the mechanism involved in the systemic changes of immune cells in the blood. Peripheral blood lymphocyte subpopulations were useful markers for evaluating immune response in tumor patients. Hence, we aimed to systematically investigate the alteration of lymphocyte subpopulations during the local therapies to evaluate antitumor treatment effects. Methods: Blood samples were obtained EDTA coated tubes and then centrifuged gently for white blood cell separation. The white blood cells in 10% DMSO and 90% FBS were frozen slowly in -80°C refrigerator. The following fluorochrome-conjugated surface and nuclear antibodies were used in the lymphocyte subtyping: CD11b, CD45, CD19, CD3, CD56, CD4, CD8a, CD25,CD127 and FOXP3. The staining cells were detected in the BD FACS machine and data were analyzed by the paired T-test. The percentage of Lymphocytes, Myeloid cells, B cells, T cells, Treg, CD8+ T cells, CD4+ T cells, NK cells, and NKT were examined. Results: Between July 2019 and January 2020, a total of 176 patients eligible, including 135 RT patients and 29 SR patients,12 IT patients, with both blood collection with both Pre, During and End therapies. Before local therapies, the percentage of total T cells in the RT group was significantly higher than SR (RT v.s SR mean:64.1 v.s 55.3, P = 0.02) while CD8+ T cells (RT v.s SR mean:28.2 v.s 34.5, P = 0.04)and Tregs (RT v.s SR mean:0.0 v.s 0.1, P = 0.055) were lower. The baseline level of T cells and their subtypes showed a significant difference in these two group patients. After local therapies, myeloid cells, lymphocytes, CD4+ T cells, CD8+ T cells, NK cells were significant different. There is no significant difference due to the smaller number of IT patients. In the RT group, lymphocytes (Pre-RT v.s End-RT mean:75.2 v.s 54.3, P = 0.004) and B cells (Pre-RT v.s End-RT mean:12.6 v.s 8.0, P = 0.03) were significantly decreased while other subpopulations didn’t show any significant difference after RT. Interestingly, in the SR group, there was a significant increase in CD4+ T cells (mean:59.0 v.s 62.1, p = 0.02) a trend of reduction in CD8+ T cells (mean:34.5 v.s 32.0, p = 0.055) after SR. In addition, there was an increased trend of Tregs after IT. Conclusions: There are some different patterns of distribution in subtypes of leukocytes in operable and inoperable patients and between different therapies. All RT, SR and IT changed the distribution of peripheral blood lymphocyte subpopulations. Further validation study is warranted to validate our findings particularly in circulating lymphocytes and B cells as a marker to evaluate immune status after RT, CD4+ T cells and CD8+ T cells after SR, Tregs after IT, as well as their relationship with tumor microenvironment and implication for personalized care.

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Relation of CD4+CD25+ Regulatory T Cells to Immune Status in Patients with HIV Infection.

Blood ◽

10.1182/blood.v104.11.3106.3106 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3106-3106

Author(s):

Sachi Tsunemi ◽

Tsuyoshi Iwasaki ◽

Takehito Imado ◽

Satoshi Higasa ◽

Eizo Kakishita ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Hiv Infection ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

Healthy Controls ◽

Cell Counts ◽

Hiv 1

Abstract Human immunodeficiency virus (HIV) infection is characterized by marked defects in CD4+ helper T cell (Th) functions that commonly progress to a substantial decline in peripheral CD4+ T cell counts. However, the mechanisms responsible for the loss of Th functions in HIV-infected patients independent of CD4+ T cell counts remains unclear. CD4+CD25+ regulatory T cells (T Reg) are essential for down-regulation of both autoreactive and alloreactive T cells. Therefore, we decided to investigate the role of T Reg in immune status of HIV-infected patients. We examined the expression of cell surface CD25, cytoplasmic IL-4 and cytoplasmic IFN-gamma in peripheral blood CD4+ T cells from both healthy controls (n=9) and HIV-infected patients (n=43). We also compared T Reg functions between the 2 groups. CD4+CD25+ T Reg isolated from both HIV-infected patients and healthy controls strongly expressed CD45RO, HLA-DR, and FoxP3, and suppressed the proliferation of CD4+CD25− T cells, suggesting that CD4+CD25+ T cells from both healthy controls and HIV-infected patients possess phenotypic and functional characteristics of Treg. CD4+CD25high T cells are a subset of circulating CD4+CD25+ T cells in normal humans and exhibit strong in vitro regulatory functions similar to those reported for murine CD4+CD25+ T Reg. We measured the frequency of CD4+CD25high T Reg by analysis of surface CD25 on CD4+ T cells in peripheral blood samples. We also examined Th1 and Th2 frequencies by analysis of cytoplasmic IFN-gamma and IL-4 levels in CD4+ T cells. T Reg from HIV-infected patients with detectable plasma HIV-1 RNA showed a statistically significant increase in CD4+CD25high cell frequency (p<0.05) compared to healthy controls, with T Reg frequencies inversely proportional to CD4+ T cell numbers (p<0.01). However, in HIV-infected patients with undetectable plasma HIV-RNA, frequencies of CD4+CD25high T Reg were not increased and not related to CD4+ T cell numbers. In both HIV-infected patient groups, T Reg frequency was inversely related to Th1 frequency (detectable: p<0.05, undetectable: p<0.001), but positively related to Th2 frequency (detectable: p<0.01, undetectable: p<0.001). Our results indicate that increased frequencies of peripheral blood T Reg were related to disease progression as measured by detectable plasma HIV-1 RNA, decreased peripheral blood CD4+ T cell counts, and polarization toward Th2 immune responses in HIV-infected patients. HIV infection may lead to induction of T reg that inhibit antiviral immune responses, resulting in the progression of the disease. Manipulation of T Reg could help restore antiviral immune responses in HIV infection, and prevent the progression of HIV infection.

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Rag1 Deficiency Does Not Affect Myelodysplasia but Leads to More Rapid Leukemic Transformation in a Mouse Model of MDS.

Blood ◽

10.1182/blood.v114.22.2784.2784 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2784-2784

Author(s):

Sheryl M Gough ◽

Yang Jo Chung ◽

Peter D. Aplan

Keyword(s):

Bone Marrow ◽

T Cells ◽

Peripheral Blood ◽

Cytotoxic T Cells ◽

Immune Surveillance ◽

Severe Anemia ◽

Control Groups ◽

Leukemic Transformation ◽

Significant Difference

Abstract Abstract 2784 Poster Board II-760 MDS comprises a premalignant heterogeneous group of clonal stem cell disorders that also show bone marrow dysplasia and which often evolve to acute myeloid leukemia (AML). Aplastic anemia (AA) patients also share the bone marrow failure, anemia and resulting peripheral blood cytopenias of MDS. AA is thought to be caused by an oligoclonal expansion of cytotoxic T-cells that target haematopoietic stem and progenitor cells. The severe anemia and leucopenia characteristic of both diseases is relieved in AA patients and some MDS patients by immunosuppressive therapy, supporting the role of cytotoxic T-cells in the etiology of AA. However, the role of the lymphocytes in progressive MDS remains unclear. MDS has been associated with a number of genetic aberrations, including chromosomal translocations involving the NUP98 gene. Using mice that express a NUP98-HOXD13 (NHD13) transgene, previously shown to manifest the same clinical symptoms as those of MDS patients, we have followed a cohort of NHD13/Rag1−/− mice to determine if the absence of lymphocytes, especially T cells, might 1) diminish the severity of the MDS, or 2) effect transformation and/or survival in the NHD13 mice, as would be predicted by an “immune surveillance” hypothesis of malignant transformation. Serial CBCs at two month time intervals were used to evaluate the extent of anemia and leucopenia in NHD13+ /Rag1+/+ and NHD13/Rag1−/−, as well as WT/Rag1+/− and WT/Rag1−/− control groups over a 15 month period. NHD13/Rag1−/− mice were generated by crossing the NHD13+ (C57BL/6) with the B6;129S7-Rag1tm1Mom/J mouse, and housed in a Specific Pathogen-Free (SPF) environment. Mice were euthanized and analyzed when CBCs indicated severe anemia/leucopenia or leukemic transformation, or when determined to be unwell (hunched, immobile, dyspnea) by observation. Flow cytometry, histology and genomic analyses further determined leukemia subtype, extent of infiltration and leukemia clonality. NHD13+ /Rag1+/+ and NHD13/Rag1−/− mice showed no significant differences at any two month time-point in hemoglobin (Hg), mean corpuscular volume (MCV), or platelet levels, and progressive MDS occurred in both groups. Consistent with previous studies, and excluding cases that showed evident transformation to acute leukemia, NHD13+ /Rag1+/+ mice showed low WBC, neutrophil and lymphocyte numbers, which were not significantly different from the NHD13/Rag1−/− mice. NHD13/Rag1−/− mice did however show a significantly reduced survival when compared with the NHD13+ /Rag1+/+ mice (Log-rank test, p = 0.0135), and survival medians of 11 and 13 months, respectively. Incidence of leukemic transformation was increased in the NHD13/Rag1−/− compared with the NHD13+ /Rag1+/+ mice (p=0.0079). A range of leukemia subtypes was observed in both the NHD13+ /Rag1+/+ and NHD13/Rag1−/− mice, including myeloid, B-cell, T-cell, and erythroid leukemias. In the SPF environment provided, the WT/Rag1+/− and WT/Rag1−/− control groups showed no significant difference in survival rates. Serial CBC data indicated that there was no significant difference in the timing or degree of peripheral blood cytopenias between the NHD13+ /Rag1+/+ and NHD13/Rag1−/− mice, supporting the conclusion that absence of lymphocytes does not lead to improvement in the peripheral blood cytopenias caused by the NHD13 transgene. This observation suggests that the NHD13 transgene does not produce MDS caused by an autoimmune phenomenon. The poorer survival and increased frequency of leukemic transformation in the NHD13/Rag1−/− mice suggests that lymphocytes might play a role in the evolution of MDS to AML in the NHD13 mouse model, and supports the ‘immune surveillance' hypothesis. Disclosures: No relevant conflicts of interest to declare.

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