Mouse anti Human CD169 antibody, clone 7-239 used for the evaluation of CD169 expression by monocyte-derived macrophages following exposure to ? interferon using flow cytometry and western blotting
Image caption:

Siglec-1 expression by MDMs is enhanced via IFN exposure and leads to VLP internalization.

(A) Representative Siglec-1 and tetherin surface expression of GM-CSF matured MDMs together with 24 h IFN alpha stimulation (1000 U/ml). (B) Western blot of Siglec-1 and actin upon incubation with increasing amounts of IFN alpha in GM-CSF derived MDMs. (C) Enhanced capture of HIV-1 Gag-EGFP VLPs by MDMs stimulated with 1000 U/ml IFN alpha as measured by p24 ELISA. (D) MDMs were incubated with 400 ng of sucrose-purified HIV-1 Gag-EGFP VLPs either treated with 1.0 U/ul neuraminidase, untreated or from 293T cultured in the presence of 10 ?M PDMP. Cell-associated HIV-1 p24 was quantified by ELISA.


From: Hammonds JE, Beeman N, Ding L, Takushi S, Francis AC, Wang J-J, et al. (2017)

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

PLoS Pathog 13(1): e1006181.

This image is from an open access article distributed under the terms of the Creative Commons Attribution License.

Mouse anti Human CD169 antibody, clone 7-239 used for the evaluation of CD169 expression by monocyte-derived macrophages following exposure to ? interferon immunofluorescence.

Image caption:

Siglec-1 expression by MDMs is enhanced via IFN exposure and leads to VLP internalization.

(E) Sucrose purified, HIV-1 Gag-EGFP VLPs treated with neuraminidase (NA) or untreated (F) were added to mature GM-CSF derived MDM cultures on day 8 for 6 hours. Cells were then washed, fixed, immunostained for Siglec-1 (red) and DAPI co-stained. Size bar = 21?m for (E) and 15?m for (F).


From: Hammonds JE, Beeman N, Ding L, Takushi S, Francis AC, Wang J-J, et al. (2017)

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

PLoS Pathog 13(1): e1006181.

This image is from an open access article distributed under the terms of the Creative Commons Attribution License.

Mouse anti Human CD169 antibody, clone 7-239 used for the evaluation of CD169 expression by monocyte-derived macrophages by immunofluorescence.

Image caption:

Time course of HIV-1 Gag-EGFP internalization within MDMs and colocalization with Siglec-1.

(A-D) 400 ng of sucrose-purified HIV-1 Gag-EGFP VLPs were added to MDM cultures and allowed to attach and be internalized from 10’ to 6 hours. At the indicated times, MDMs were washed, fixed in 4% PFA and immunostained with Siglec-1 (red, mAb clone 7–239) and DAPI co-stained. Shown are cells representative of the populations examined at each timepoint. Size bars = 10?m.


From: Hammonds JE, Beeman N, Ding L, Takushi S, Francis AC, Wang J-J, et al. (2017)

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

PLoS Pathog 13(1): e1006181.

This image is from an open access article distributed under the terms of the Creative Commons Attribution License.

Mouse anti Human CD169 antibody, clone 7-239 used for the evaluation of CD169 expression by monocyte-derived macrophages by western blotting.

Image caption:

Siglec-1 RNAi interference and competitive inhibition by a GM3 glycan mimetic inhibits HIV-1 VLP internalization by MDMs.

(A) MDMs were transfected with 60 nM control or Siglec-1 siRNA on day 8 after plating. Cell lysates were harvested and analyzed by Western blotting for Siglec-1 and actin expression. (B) MDMs were transfected with either control or Siglec-1 siRNA and 5 days later incubated with 400 ng of HIV-1 Gag-EGFP VLPs for 2 hr. Cells were washed and cell lysate p24 concentration determined by ELISA. (C) MDMs were pre-treated at RT with lactose or 3’-sialyllactose for 1 hour. Subsequently, MDMs were incubated with 400 ng of HIV-1 Gag-EGFP VLPs in the presence of compound for an additional hour. MDMs were then washed, cell lysates harvested and cell-associated p24 measured by ELISA. Error bars represent standard deviation; asterisks depict significant differences as measured by unpaired t-test.


From: Hammonds JE, Beeman N, Ding L, Takushi S, Francis AC, Wang J-J, et al. (2017)

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

PLoS Pathog 13(1): e1006181.

This image is from an open access article distributed under the terms of the Creative Commons Attribution License.

PE conjugated Mouse anti CD169 antibody, clone 7-239 used for the evaluation of CD169 expression on myeloid cell populations by flow cytometry. Mouse anti Human CD169 antibody, clone 7-239 used to block CD169 function on myeliod cells.

Image caption:

Siglec-1 mediates HIV-1 capture by IFN?-treated myeloid cells. A. Representative profiles of Siglec-1 staining in distinct myeloid cells cultured with or without 1000 U/ml of IFN? and assessed by FACS. Staining of matched-isotype control is also shown. The mean fold increase in fluorescence after IFN&apha; treatment of cells derived from three donors is shown in red numbers. B. Mean number of Siglec-1 antibody binding sites per cell displayed by different myeloid cells exposed to 1000 U/ml of IFN? for 48 h and assessed by quantitative FACS analysis. Data show mean values and SEM from four experiments including cells from 12 donors. C. Comparative binding of HIV-1NL4–3 to different myeloid cells ly exposed to 1000 U/ml of IFN? for 48 h. Cells were cultured with HIV-1NL4–3 for 4 h at 4°C, washed and lysed to measure p24Gag by ELISA. Data show mean values and SEM from two experiments including cells from six donors. D. Relative binding of HIV-1NL4–3 by different IFN?-treated myeloid cells that had been pre-incubated with 10 ug/ml of the indicated mAbs before HIV-1 exposure for 4 h at 4°C as described in C. To compare the effect of the mAbs in different myeloid cells, values were normalized to the level of HIV-1 binding by mock-treated cells (set to 100%). Data show mean values and SEM from two experiments including cells from six donors. Statistical differences were assessed with a paired t test in B and C, and with a one sample t-test in D.


From: Pino M, Erkizia I, Benet S, Erikson E, Fernández-Figueras MT, Guerrero D, Dalmau J, Ouchi D, Rausell A, Ciuffi A, Keppler OT, Telenti A, Kräusslich HG, Martinez-Picado J, Izquierdo-Useros N.
HIV-1 immune activation induces Siglec-1 expression and enhances viral trans-infection in blood and tissue myeloid cells.

Retrovirology. 2015 May 7;12:37.

This image is from an open access article distributed under the terms of the Creative Commons Attribution License.

PE conjugatedMouse anti Human CD169 antibody, clone 7-239 used to block CD169 function on myeloid cells.

Image caption:

Siglec-1 mediates HIV-1 uptake into a storage compartment and enhances HIV-1 trans-infection specially in IFN?-treated monocytes and DCs. A. Uptake of HIV-1NL4–3 by different myeloid cells exposed to IFN?. Cells were cultured with HIV-1 to measure p24Gag by ELISA. Mean values and SEM from four experiments include cells from 12 donors. B. Fold change in HIV-1NL4–3 uptake of cells treated with bafilomycin A1 compared to untreated cells. Mean values and SEM include cells from three donors. C. Relative uptake of HIV-1NL4–3 by IFN?-treated myeloid cells pre-incubated with the indicated mAbs. Values are normalized to the level of HIV-1 uptake by mock-treated cells (set at 100%). Mean values and SEM from two experiments include cells from six donors. D. Confocal microscopy analysis of different IFN?-treated myeloid cells pulsed with HIV-1Cherry and stained for Siglec-1 (Alexa 488), HLA-DR (Alexa 647) and DAPI. (Top) Representative viral pattern for each kind of myeloid cell analyzed, showing maximum fluorescence intensity of four channels. (Bottom) Percentage of myeloid cells with distinct viral patterns: random distribution, polarized accumulation, and sac-like compartment formation, as illustrated in the left drawing. Mean values of 50 cells from two different donors are shown. E. HIV-1 transmission from IFN?-treated myeloid cells to a luciferase reporter CD4+ cell line. HIV-1 infection was determined by induced luciferase activity in relative light units (RLUs). Mean values and SEM from four experiments include cells from 12 donors. F. Relative HIV-1 transmission from IFN?-treated myeloid cells pre-incubated with the indicated mAbs. Values are normalized to the level of HIV-1 trans-infected by mock-treated cells. Mean values and SEM from two experiments include cells from six donors. Statistical differences were assessed with a paired t test in A and E, and with a one sample t-test in B, C and F.


From: Pino M, Erkizia I, Benet S, Erikson E, Fernández-Figueras MT, Guerrero D, Dalmau J, Ouchi D, Rausell A, Ciuffi A, Keppler OT, Telenti A, Kräusslich HG, Martinez-Picado J, Izquierdo-Useros N.

HIV-1 immune activation induces Siglec-1 expression and enhances viral trans-infection in blood and tissue myeloid cells.

Retrovirology. 2015 May 7;12:37.

This image is from an open access article distributed under the terms of the Creative Commons Attribution License.