Loss Of CD4 Research Articles

CD4+ T cells in HIV infection show increased levels of expression of a receptor for vasoactive intestinal peptide, VPAC2

Immune activation is a strong predictor of disease outcome in HIV infection and promotes the loss of CD4+ T cells. The neuropeptide, vasoactive intestinal peptide (VIP), has immune-modulating properties with specific receptors identified on lymphocytes; VPAC1 and VPAC2. Studies have shown that VIP limits immune activation and apoptosis in T cells by decreasing the expression of the apoptosis signaling molecule Fas ligand (FasL). VIP receptor surface expression has not been investigated by flow cytometry in the context of HIV infection and may represent a novel target for immune-modulating therapy. Eighty-seven untreated HIV-infected individuals with CD4 counts >200 and 57 uninfected controls were recruited from a primary health clinic in Cape Town, South Africa. Flow cytometry was used to determine levels of expression of VPAC1 and VPAC2, as well as FasL on CD4+ T cells, and these results were correlated with the immune activation phenotype %CD38+CD8+ T cells. VPAC2 expression was significantly increased in the HIV group (mean %VPAC2+CD4+ cells 19.25 vs. control 12.56; p≤0.0001), but no difference in VPAC1 expression was observed. VPAC2 correlated positively with FasL (r=0.310; p=0.001), and there was a significant inverse correlation between FasL and the CD4 count (r=-0.211; p=0.013) and a direct correlation with %CD38+CD8+ T cells (r=0.39; p≤0.0001). Thus, higher levels of immune activation correlated with higher levels of the death-signaling FasL and lower CD4 counts. VPAC2 may provide a novel target for the selective limitation of CD4+ T-cell death in HIV infection.

Characterization of Mesenchymal Progenitor Cell Populations Directly Derived from Human Dermis

Mesenchymal stem cell (MSC) and progenitor cell (MPC) populations in human dermis remain poorly characterized, despite their importance to wound repair and the pathogenesis of many skin diseases. To identify MSC/MPC populations in human dermis we developed an 11-marker flow cytometry technique that enabled sorting of mesenchymal cell populations for functional assays, using adipose-derived stem cells (ASCs) from human adipose tissue as a positive control. Two populations of dermal cells had similar phenotypes to ASCs: both were CD34(+) CD73(+) CD105(-)/low, and lacked expression of c-kit (CD117) and hematopoietic or vascular markers (CD31, CD45, CD146, and HLA-DR). However, whereas ASCs were CD36(+/-) CD90(+), dermal mesenchymal progenitor cells (DMPCs) were split between a dominant CD36(-) CD90(+) population (DMPC1) and a small CD36(+) CD90(-) population (DMPC2). Both these populations were capable of differentiating into adipocytes, but only DMPC1 localized to a perivascular location, similar to that reported for ASCs. Re-gating of the flow cytometry data revealed that both DMPC1 and DMPC2 were part of CD45(-) CD73(+) CD146(-) populations with variable expression of CD34. This suggests that CD34 may not be a stable marker of DMPC populations in human dermis, consistent with data from MSCs in human bone marrow, and with the loss of CD34 we observed from both ASCs and DMPCs on cell culture. These data enable future study of DMPCs in health and disease, and may also explain why some mesenchymal cell lines derived from human dermis exhibit characteristics of MSCs.

Identification of Novel Thymic Epithelial Cell Subsets Whose Differentiation Is Regulated by RANKL and Traf6

Thymic epithelial cells (TECs) are critical for the normal development and function of the thymus. Here, we examined the developmental stages of TECs using quantitative assessment of the cortical and medullary markers Keratin 5 and Keratin 8 (K5 and K8) respectively, in normal and gain/loss of function mutant animals. Gain of function mice overexpressed RANKL in T cells, whereas loss of function animals lacked expression of Traf6 in TECs (Traf6ΔTEC). Assessment of K5 and K8 expression in conjunction with other TEC markers in wild type mice identified novel cortical and medullary TEC populations, expressing different combinations of these markers. RANKL overexpression led to expansion of all medullary TECs (mTECs) and enlargement of the thymic medulla. This in turn associated with a block in thymocyte development and loss of CD4+CD8+, CD4+ and CD8+ thymocytes. In contrast, Traf6 deletion inhibited the production of most TEC populations including cortical TECs (cTECs), defined by absence of UEA-1 binding and LY51 expression, but had no apparent effect on thymocyte development. These results reveal a large degree of heterogeneity within the TEC compartment and the existence of several populations exhibiting concomitant expression of cortical, medullary and epithelial markers and whose production is regulated by RANKL and Traf6.

Immunophenotypic and molecular features of ‘cuplike’ acute myeloid leukemias

Nuclear invaginations, also referred to as fishmouth or cuplike nuclei, have long been identified in microgranular APL, myelomonocytic and monocytic AMLs. More recently, this typical morphological feature has been associated with NPM1 and FLT3 mutations, as well as with the lack of CD34 and HLA-DR expression. In this study, we retrospectively analyzed the morphologic, immunophenotypic, cytogenetic, and molecular features of 68 patients with AML. A cuplike nuclear invagination was detected in more than 10% of blast cells in 15 (22%) cases. Our data show that a cuplike morphology is associated with FLT3-ITD positivity, as well as with the loss of CD34 and HLA-DR expression. The results were not significantly modified when a higher cutoff of cuplike cells was used. Our results are not sufficient to suggest that cuplike AML could represent a distinct subtype, but further investigations could yield a better characterization of this feature in patients with AML.

A Parameter for IL-10 and TGF-ß Mediated Regulation of HIV-1 Specific T Cell Activation Provides Novel Information and Relates to Progression Markers

HIV replication is only partially controlled by HIV-specific activated effector T cells in chronic HIV infection and strategies are warranted to improve their efficacy. Chronic T cell activation is generally accompanied by regulation of antigen-specific T cell responses which may impair an effective control of chronic infections. The impact of HIV-induced T cell regulation on individual patients’ disease progression is largely unknown, since classical T cell activation assays reflect net activation with regulation as unknown contributing factor. We here explore a quantitative parameter for antigen-induced cytokine-mediated regulation (RAC) of HIV-specific effector T cell activation by functional antibody-blockade of IL-10 and transforming growth factor-β. HIV Env- and Gag-specific T cell activation and RAC were estimated in peripheral blood mononuclear cells from 30 treatment-naïve asymptomatic HIV-infected progressors (CD4 count 472/µl, HIV RNA 37500 copies/ml) stimulated with overlapping peptide panels for 6 days. RAC was estimated from differences in T cell activation between normal and blocked cultures, and related to annual CD4 loss, immune activation (CD38) and microbial translocation (plasma lipopolysaccharides). RAC was heterogeneously distributed between individual patients and the two HIV antigens. Notably, RAC did not correlate to corresponding classical activation. Env RAC correlated with CD38 and CD4 loss rates (r> = 0.37, p = <0.046) whereas classical Gag activation tended to correlate with HIV RNA (r = −0.35, p = 0.06). 14 patients (47%) with low RAC’s to both Env and Gag had higher CD8 counts (p = 0.014) and trends towards lower annual CD4 loss (p = 0.056) and later start with antiretroviral treatment (p = 0.07) than the others. In contrast, patients with high RAC to both Env and Gag (n = 8) had higher annual CD4 loss (p = 0.034) and lower CD8 counts (p = 0.014). RAC to Env and Gag was not predicted by classical activation parameters and may thus provide additional information on HIV-specific immunity. RAC and other assessments of regulation deserve further in-depth exploration.

The Majority of CD4 + T-Cell Depletion during Acute Simian-Human Immunodeficiency Virus SHIV89.6P Infection Occurs in Uninfected Cells

Untreated human immunodeficiency virus (HIV) infection is characterized by depletion of CD4(+) T cells, ultimately leading to the impairment of host immune defenses and death. HIV-infected CD4(+) T cells die from direct virus-induced apoptosis and CD8 T-cell-mediated elimination, but a broader and more profound depletion occurs in uninfected CD4(+) T cells via multiple indirect effects of infection. We fit mathematical models to data from experiments that tested an HIV eradication strategy in which five macaques with a proportion of CD4(+) T cells resistant to simian-human immunodeficiency virus (SHIV) entry were challenged with SHIV89.6P, a highly pathogenic dual-tropic chimeric SIV-HIV viral strain that results in rapid loss of both SHIV-susceptible and SHIV-resistant CD4(+) T cells. Our results suggest that uninfected (bystander) cell death accounts for the majority of CD4(+) T-lymphocyte loss, with at least 60% and 99% of CD4(+) T cell death occurring in uninfected cells during acute and established infection, respectively. Mechanisms to limit the profound indirect killing effects associated with HIV infection may be associated with immune preservation and improved long-term survival. HIV infection induces a massive depletion of CD4(+) T cells, leading to profound immunodeficiency, opportunistic infections, and eventually death. While HIV induces apoptosis (programmed cell death) by directly entering and replicating in CD4(+) T cells, uninfected CD4(+) T cells also undergo apoptosis due to ongoing toxic inflammation in the region of infection. In this paper, we use mathematical models in conjunction with data from simian-human immunodeficiency virus SHIV89.6P infection in macaques (a model of HIV infection in humans) to estimate the percentage of cell death that occurs in uninfected cells during the initial period of infection. We reveal that the vast majority of cell death occurs in these cells, which are not infected. The "bystander effects" that lead to enormous reductions in the number of uninfected CD4(+) T cells may be a target for future interventions that aim to limit the extent of damage caused by HIV.

Hypopigmented mycosis fungoides in Saudi Arabia, epidemiological and pathological study

IntroductionMycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma. Hypopigmented MF is one of the less common variants of MF. Although rare, the exact frequency of HMF is still unknown. Materials and methodsWe performed a retrospective review of 17 cases of hypopigmented mycosis fungoides at two major tertiary centers in Saudi Arabia from the period 2010–2013. We described the clinical and important histological features of these cases. CD3, CD4, CD8, and CD7 stains performed for these cases have also been reviewed. ResultsThere were 5 male and 11 female patients. The age of onset of disease ranged between 5 and 60 with a mean of 17years. All the patients presented with hypopigmented scaly patches. Most common sites of distribution of the lesions were upper and lower extremities (100%) then the trunk (76%). The presence of lymphocytes at the dermoepidermal junction was the most frequent finding seen in 100% cases. Basilar predilection of the lymphocytes was seen in 75.5%. CD8 positivity in epidermal and dermal lymphocytes was seen in 14/17 biopsies. Focal positivity CD4 of the dermal lymphocytes was evident in 5/17. Loss of CD7 is noted in 14/17 biopsies.

Immunosuppression Induced by HIV Infection

Immunosuppression due to HIV infection is a resultant of several factors including defective innate signaling pathways, increased viral replication and virus load, gradual loss of peripheral CD4+T cells and depletion of T lymphocytes at mucosal sites that collectively lead to progressive immune deficiency and AIDS development. T cells derived from HIV patients were anergic and failed to mount a robust adaptive immune response. The host, on the other hand, becomes vulnerable to opportunistic infections such as tuberculosis and AIDS-related cancer such as Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer with remarkable level of immunosuppression. Elucidation of the various mechanisms involved in these immunologic perturbations is necessary to understand HIV pathogenesis for an effective immunotherapy.

Relationships between CD4+ Counts and the Presence of Oral Lesions in Human Immunodeficiency Virus Positive Women in Nigeria.

Background:Oral lesions are common findings in human immunodeficiency virus (HIV) infection. The main factor associated with the development of oral lesions is damage to the immune system, specifically loss of CD4+ lymphocytes, which are involved in cell-mediated immunity.Aim:This study was aimed to determine the association of oral lesions in HIV/acquired immune deficiency syndrome women patients with the level of immune suppression as measured by the CD4+ counts.Subjects and Methods:This was a prospective cross-sectional study with a study population of 191 consecutive female patients seen at the University of Benin Teaching Hospital, Nigeria. Ethical clearance was obtained from the institution of study and informed consent was given by every participant. HIV sero-status was determined for all patients. CD4+ count was analyzed for both the HIV+ and HIV– women with oral lesions. The relationships between oral lesions and CD4+ cell count were investigated.Result:About 56.0% (107/191) of the 191 women studied were HIV positive. Age range for the HIV positive women was 18-50 years with a mean age (standard deviation) of 36 (9.2) years. The most common oral lesion observed in the HIV positive women was pseudomembranous candidiasis accounting for 34.6% (37/107). About 68.4% (67/98) of the oral lesions occurred at CD4+ count < 200 cells/ml. Chi-square revealed statistically significant association between the presence of oral lesions and CD4+ count in HIV infected women (P = 0.03).Conclusion:As the CD4+ count was decreasing the presence of oral lesions was increasing in the study. The presence of pseudomembranous candidiasis was found to be significantly associated with CD4+ count level < 200 cells/ml. This association of oral candidiasis with CD4+ cell counts could be used as additional markers of immunosuppression and progression of HIV infection, particularly in a developing country like Nigeria where CD4+ count cannot be determined routinely.

Loss of CD34 and high IGF2 are associated with malignant transformation in solitary fibrous tumors

The aim of this study was to characterize the subgroups of solitary fibrous tumor (SFT) and to investigate the expression of different biomarkers including CD34 and IGF2 in malignant transformation.Two hundred and ninety-four (294) SFTs from a single German consultation center of soft tissue tumors were categorized into the new proposal of SFT designation. We found the fibrous variant in 223 (75.9%), the cellular variant in 65 (22.1%), the fat forming variant in 4 (1.4%), and the giant cell-rich variant in 2 (0.6%) cases. Anatomical location, size, mitotic index, necrosis, cellularity, collagenous ropes, and growth pattern of the vessels were recorded. Criteria of malignancy were found in 68 (23%) tumors. Expression of IGF2, IGF1R, CD34, BCL2, CD99, SMA, S100, PanCK, and Ki67 was analyzed immunohistochemically. Low expression of CD34 and high expression of IGF2 were significantly associated with malignant transformation and the metastatic rate. Moreover the presence of necrosis showed the most significant p-value (p<0.004).Of all SFTs, the fibrous variant is the most common, followed by the cellular variant. The fat-forming and giant cell-rich variants are very rare. Low expression of CD34 and high expression of IGF2 are significantly associated with malignant transformation, and might be an interesting target of individualized therapy.

P53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes

Cellular senescence contributes to aging and decline in tissue function. p53 isoform switching regulates replicative senescence in cultured fibroblasts and is associated with tumor progression. Here, we found that the endogenous p53 isoforms Δ133p53 and p53β are physiological regulators of proliferation and senescence in human T lymphocytes in vivo. Peripheral blood CD8+ T lymphocytes collected from healthy donors displayed an age-dependent accumulation of senescent cells (CD28-CD57+) with decreased Δ133p53 and increased p53β expression. Human lung tumor-associated CD8+ T lymphocytes also harbored senescent cells. Cultured CD8+ blood T lymphocytes underwent replicative senescence that was associated with loss of CD28 and Δ133p53 protein. In poorly proliferative, Δ133p53-low CD8+CD28- cells, reconstituted expression of either Δ133p53 or CD28 upregulated endogenous expression of each other, which restored cell proliferation, extended replicative lifespan and rescued senescence phenotypes. Conversely, Δ133p53 knockdown or p53β overexpression in CD8+CD28+ cells inhibited cell proliferation and induced senescence. This study establishes a role for Δ133p53 and p53β in regulation of cellular proliferation and senescence in vivo. Furthermore, Δ133p53-induced restoration of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection.

Immunologic and Clinical Responses To a CD20-Targeted Immunocytokine, DI-Leu16-IL2, In Relapsed Non-Hodgkin Lymphoma

BackgroundTumor-targeted antibody-cytokine fusion proteins, known as immunocytokines (ICKs) have potent antitumor activity in several preclinical tumor models. We have engineered an anti-CD20-interleukin 2 (IL-2) ICK based on the Leu16 anti-CD20 antibody with de-immunized variable (V) region and junction between the heavy (H) chain constant region and IL-2 (DI-Leu16-IL2). In-vitro studies of DI-Leu16-IL2 have demonstrated CD20 targeting, IL2 bioactivities, and antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In a SCID mouse model, DI-Leu16-IL2 showed superior anti-lymphoma effects over rituximab or rituximab combined with systemic IL-2. MethodsWe have initiated a pilot trial of DI-Leu16-IL20 in CD20+ B-cell non-Hodgkin lymphoma (NHL) under an IRB-approved protocol (clinicaltrials.gov: NCT00720135). A total of 7 patients have been treated with DI-Leu16-IL20 at 0.5mg/m2. The first 2 cases received DI-Leu16-IL2 by intravenous infusion on days 2, 4 and 23, 25 after peripheral B cell depletion with low-dose rituximab (50mg/m2) on days 1 and 22. Five remaining patients received DI-Leu16-IL2 weekly for 4 weeks (days 2, 9, 16, 23) after B cell depletion with low-dose rituximab on days 1, 8, 15, and/or 22 as necessary, based on serum rituximab levels. Clinical ResponsesThe treatment has been generally well tolerated with the majority of adverse events (AE) grade 1 or 2. Non-DLT grade 3 AE included one case of hyperglycemia, infection (herpes zoster), and prolonged QTc. One patient developed grade 3 hyponatremia, which was the only DLT in the study to date. One patient with diffuse large B cell NHL of the lower extremity achieved a CR after 2 cycles (significant PR after cycle 1; Figure 1). Another patient showed a temporal increase in FDG uptake on PET after cycle 1, which decreased after cycle 2 in 2 of the 3 responding lymph nodes, suggesting a local immunologic reaction with corresponding FDG uptake (immune-mediated FDG flare). A lymph node biopsy demonstrated a dense infiltration of T cells. One patient who had her residual tumor biopsied after DI-Leu16-IL2 treatment demonstrated loss of CD20 expression.Pre-ICK B Cell Depletion and Pharmacologic Data: We have successfully maintained minimal or absent peripheral blood B cells, with a level-guided dosing of low-dose rituximab. The Cmax ranged between 75 and 125 ng/ml following each infusion of DI-Leu16-IL2. The half life was 6-8 hours and did not change significantly between doses. We have detected no anti-DI-Leu16-IL2 antibodies in any of the 7 patients treated on this protocol. [Display omitted] Immunologic ResponsesDI-Leu16-IL2 was highly immunologically active. There was a significant increase in local infiltrating T cells (predominantly CD8 T cells) in lymph node samples taken after DI-Leu16-IL2 in 3 of the 4 cases whose samples were available. Soluble IL2 receptor levels spiked with each DI-Leu16-IL2 infusion, up to 3 times above baseline. Among the cytokines and chemokines we evaluated using the Luminex platform, IL-5, IL-7, IL-10, IP10, IL-15, and IFN-gamma demonstrated high spikes (>2-fold increase) within 12 hours from each DI-Leu16-IL2 infusion, while IL-1, IL-12, MIG, TNF-alpha, IFN-alpha, and GCSF levels had a lesser degree of spikes (<2-fold increase). The data on the CR patient seem to show higher peak levels of IL15, IL7, and IP10, while MCP1, IL5, IL10, and MCP appeared lower compared with others. Flow cytometry-based cellular analyses showed no significant increase in regulatory T cells or myeloid-derived suppressor cells. Peripheral blood T cells were analyzed for TCR CDR3 sequence, and demonstrated oligoclonal T cell expansions, suggestive of antigen-driven T cell expansion. ConclusionThis study demonstrated the clinical and immunologic data consistent with a proof-of-principle that DI-Leu16-IL2 is capable of inducing both local and systemic immune responses at a dose 0.5mg/m2, and was associated with a CR in a patient who had highly refractory NHL. A multi-center phase I trial of subcutaneously administered DI-Leu16-IL2 is currently underway. Disclosures:Gillies: Provenance Biopharmaceuticals Corp (employment and equity ownership), Alopexx Oncology (Consultancy) : Consultancy, Employment, Equity Ownership.

The Chromosome 21 Kinase DYRK1A Controls Cell Cycle Exit and Survival During Lymphoid Development and Is a Novel Therapeutic Target In Acute Lymphoblastic Leukemia

The dual specificity tyrosine-regulated kinase 1A (DYRK1A) phosphorylates a growing number of proteins to influence various aspects of cell biology, including survival, apoptosis, proliferation, and neuronal development and function. Encoded on human chromosome 21, the DYRK1A locus resides in the Down syndrome critical region (DSCR) and has been implicated in the pathogenesis of neurocognitive deficits that result from trisomy 21. We recently demonstrated a prominent tumor-promoting role for DYRK1A in acute megakaryoblastic leukemia in children with DS (DS-AMKL). This discovery prompted us to ask whether DYRK1A is important for normal hematopoiesis, and if additional types of leukemia result from enhanced DYRK1A activity.Apart from our study in DS-AMKL, the involvement of DYRK1A in normal and malignant hematopoiesis has not been described. Dyrk1a germline knockout mice die mid-gestation; therefore, we generated conditional knockout (CKO) mice to allow inducible deletion of the kinase in hematopoietic cells with Mx1-Cre. In the bone marrow, CKO mice exhibit significant reductions of the hematopoietic stem cell-enriched LSK and common lymphoid progenitor populations (5-fold and >20-fold reductions vs. controls, respectively). In B cell lineage, CKO mice have 2-fold reductions of small pre-B and immature B cells, and a 5-fold reduction of mature recirculating B cells. In the thymus, CKO mice show dramatic loss of CD4+/CD8+double positive cells (DP, >30-fold fewer at 4 weeks). Furthermore, DYRK1A-deficient cells fail to reconstitute lymphoid cells in competitive bone marrow transplantation experiments and give rise to virtually no pre-B cell colonies in ex vivo assays. In contrast to these lymphoid defects, myeloid development is largely normal, with slightly increased numbers of erythroid precursors, granulocytes, and monocytes.To understand the mechanisms by which DYRK1A controls lymphopoiesis, we performed microwestern blot arrays. This approach allowed us to simultaneously screen >200 antibodies to identify differentially expressed proteins between CKO and control lymphoid cells. We found increased levels of cell cycle regulatory proteins, such as CDK1, suggesting that DYRK1A modulates cell cycle status during lymphoid development. Indeed, cell cycle analysis revealed that there were 20-30% fewer quiescent (G0) cells among CKO small pre-B cells and DP thymocytes, two developmental stages characterized by exit from the cell cycle following a proliferative burst. Among its targets, DYRK1A is known to contribute to DREAM (DP, Rb-like, E2F, and MuvB) complex assembly via phosphorylation of LIN52. The DREAM complex, not previously described in hematopoiesis, promotes quiescence by repressing the transcription of numerous cell cycle-associated genes during G0. RNA sequencing analysis in flow-purified G0 cells confirmed that DREAM complex targets are markedly de-repressed in the affected CKO populations, highlighting the importance of DYRK1A/DREAM complex interaction during lymphoid cell cycle exit.Both B- and T-lineage ALL express high levels of DYRK1A relative to other tumor types. Having observed that DYRK1A is essential for lymphoid cells, we next asked whether targeting DYRK1A with small molecule inhibitors would be an effective therapy for ALL. We found that EHT 1610, a highly selective DYRK1 inhibitor (Leblond B., Casagrande A.-S., Désiré L., Foucourt A., Besson T., DYRK1 inhibitors and uses thereof, WO 2013026806, 2013.02.28), dose-dependently induced apoptosis in B- and T-ALL cell lines and primary human pediatric ALL samples. Thus far, all cell lines and all primary samples tested were exquisitely sensitive to EHT 1610. Moreover, EHT 1610 induced apoptosis of primary ALL cells that were resistant to cytarabine, suggesting that DYRK1A inhibitors may be used in combination with standard ALL therapies for refractory or relapsed cases. Together, our data establish novel essential roles for DYRK1A in both normal and malignant lymphoid development and provide rationale for the design of DYRK1A-targeted ALL therapies. Disclosures:Leblond:Diaxonhit Therapeutics: Employment. Casagrande:Diaxonhit Therapeutics: Employment. Désiré:Diaxonhit Therapeutics: Employment. Crispino:Sanofi: Research Funding.

Loss of memory CD4+ T-cells in semi-wild mandrills (Mandrillus sphinx) naturally infected with species-specific simian immunodeficiency virus SIVmnd-1

Simian immunodeficiency virus (SIV) infection is found in a number of African primate species and is thought to be generally non-pathogenic. However, studies of wild primates are limited to two species, with SIV infection appearing to have a considerably different outcome in each. Further examination of SIV-infected primates exposed to their natural environment is therefore warranted. We performed a large cross-sectional study of a cohort of semi-wild mandrills with naturally occurring SIV infection, including 39 SIV-negative and 33 species-specific SIVmnd-1-infected animals. This study was distinguished from previous reports by considerably greater sample size, examination of exclusively naturally infected animals in semi-wild conditions and consideration of simian T-lymphotropic virus (STLV) status in addition to SIVmnd-1 infection. We found that SIVmnd-1 infection was associated with a significant and progressive loss of memory CD4+ T-cells. Limited but significant increases in markers of immune activation in the T-cell populations, significant increases in plasma neopterin and changes to B-cell subsets were also observed in SIV-infected animals. However, no increase in plasma soluble CD14 was observed. Histological examination of peripheral lymph nodes suggested that SIVmnd-1 infection was not associated with a significant disruption of the lymph node architecture. Whilst this species has evolved numerous strategies to resist the development of AIDS, significant effects of SIV infection could be observed when examined in a natural environment. STLVmnd-1 infection also had significant effects on some markers relevant to understanding SIV infection and thus should be considered in studies of SIV infection of African primates where present.

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7

BackgroundCD7 expression is found on ~30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7+) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation. MethodsKG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7+, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCβII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA. ResultsSECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCβII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression. ConclusionThese findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis. General SignificanceThis work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.

Abstract A12: Small molecule inhibitor of BCL2 in combination with RCHOP is effective in CD20 negative DLBCL.

Abstract Rituximab alone or in combination with chemotherapeutics is the first-line therapy for diffuse large B cell lymphomas (DLBCL). Although the complete response rate is quite impressive, vast majority of patient presents recurrent disease. The association between CD20 expression and clinical outcome in patients strongly suggests that reduced CD20 expression leads to inferior response to RCHOP (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone). In order to understand how loss of CD20 leads to development of RCHOP resistance, we developed rituximab resistant DoHH2 model in vivo by chronic exposure to rituximab. Characterization of several resistant in vivo xenografts revealed one model that maintained resistance to an acute dose of rituximab and demonstrated loss of CD20. Further characterization of the model demonstrated a loss of CD20 is associated with over expression of BCL2 and BIM. In vivo efficacy studies showed resistant line is insensitive to acute dose of RCHOP and treatment with an inhibitor of BCL2 (ABT199) in combination with chemotherapy resulted in better efficacy than RCHOP alone. We have identified an in vivo model of DLBCL where loss of CD20 and over expression of anti-apoptotic protein BCL2 leads to RCHOP resistance. These data suggest the addition of BCL2 inhibitor to chemotherapy might be effective in treating CD20 negative lymphomas. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A12. Citation Format: Chaitali Dutta, Michele Mayo, Mike Collins, Christopher Brueckner, Deborah Lawson, Zhongwu Lai, Larry Bao, Corinne Reimer. Small molecule inhibitor of BCL2 in combination with RCHOP is effective in CD20 negative DLBCL. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A12.

Resistance to Infection, Early and Persistent Suppression of Simian Immunodeficiency Virus SIVmac251Viremia, and Significant Reduction of Tissue Viral Burden after Mucosal Vaccination in Female Rhesus Macaques

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIVmac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIVmac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8(+) T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4(+) central memory and CD4(+)/α4β7(+) T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4(+), and CD8(+) T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.

Loss of CCR4 antigen expression after mogamulizumab therapy in a case of adult T‐cell leukaemia‐lymphoma

Loss of <scp>CCR</scp>4 antigen expression after mogamulizumab therapy in a case of adult <scp>T</scp>‐cell leukaemia‐lymphoma

Imbalances of Gut-Homing CD4+ T-Cell Subsets in HIV-1–Infected Chinese Patients

Full reconstitution of CD4 T cells in both peripheral blood and mucosal tissues is a desirable goal of treating AIDS patients. To date, few studies have investigated the potential role of gut-homing CD4 T-cell subsets as biomarkers in assisting Asian populations infected with HIV-1. A large cross-sectional study was conducted among Chinese patients with focus on the frequency, absolute number, and ratio of gut-homing Th1, Th17, and Treg subsets in 3 groups of age- and gender-matched study subjects: healthy donors, untreated AIDS patients, and antiretroviral therapy (ART)-treated patients with sustained undetectable viral load. HIV-1 chronic infection resulted in positively correlated loss of total and gut-homing CD4 T cells (P < 0.001) among patients compared with healthy controls. Profiles of T-cell subsets, however, were different between total and gut-homing CD4 T cells in terms of frequency and absolute number. ART partially restored the frequencies of gut-homing Th1, Th17, and Treg cells but the lost number of gut-homing Th17 cells was found not easily reversible. These changes together with an increased frequency of gut-homing CD4 Treg cells led to dual imbalances of gut-homing Th1/Treg and Th17/Treg ratios, which were negatively correlated with viral load (P = 0.014 and P < 0.001) and hardly restored even by prolonged ART. Our findings provide new insights into the investigation of gut-homing Th1/Treg and Th17/Treg imbalances in AIDS patients, which may have potential implications on the reconstitution of mucosal CD4 T cells.

Selective depletion of FOXP3high cells by Fas–Fas-L–induced apoptosis occurs in CD4+CD25+-enriched populations during repeated expansion

Background aimsExpansion of anti-CD25 bead-isolated human Tregs culture has paradoxically resulted in reduced suppressive activity, but the mechanism(s) responsible for these observations are poorly defined. MethodsMagnetic-bead isolated human CD25+ cells were expanded with anti-CD3/CD28 beads and high doses of rhIL-2. Detection of Fas and Fas ligand (Fas-L) expression, activation of Caspase 8, cell proliferation and cytokine production was evaluated by multi-color fluorescence-activated cell sorting analysis. The role of Fas–Fas-L–mediated cell death was dissected through the use of agonist or antagonist monoclonal antibodies directed at Fas and Fas-L. ResultsRepeated expansion of bead-enriched CD4+CD25+ cells generated a cellular product with markedly reduced suppressive activity and with significantly increased CD8+ T cells and CD4+ T cells producing interferon-γ and/or interleukin-2. We showed that Fas–Fas-L–mediated apoptosis of CD4+FOXP3high cells and rapid cell-cycling of CD8+ T cells were collectively responsible for the reduced proportion of CD4+FOXP3high cells in expanded cultures. The depletion of CD4+FOXP3high cells and activation of Caspase 8 in CD4+FOXP3high cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4+FOXP3high cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies. ConclusionsTaken together, the data show that Fas–Fas-L–mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.