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An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγ mice allows HIV replication and development of anti-HIV immune responses.

Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, Vandergeeten C, Delacroix L, Van Gulck E, Vanham G, de Leval L, Rahmouni S, Moutschen M - PLoS ONE (2012)

Bottom Line: In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice.Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days.We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and infectious diseases unit GIGA-I3, University of Liege, Liege, Belgium.

ABSTRACT
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ() (NSG) and NOD/SCID/IL2Rγ() (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

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HIV induced immune activation and expression levels of multiple activation markers on CD4+ and CD8+.Blood samples were obtained from mice before and 6 week after HIV-1 JRCSF infection. (A) Beta-2-microglobline levels were measured using ELISA before (Pre) and after HIV-infection (Post) (n = 4) and at same time intervals in engrafted but noninfected mice. (B) CD69 (C) HLA-DR (D) PD-1 and (E) CD27 (F) CCR5 levels were evaluated on CD4+ and CD8+ cells. (G) LPS levels in humanized mice 2 and 8 weeks post-HIV infection. Each circle and square represent one mouse at the indicated time point. Means are shown in solid lines. P values were determined by unpaired student’s t-tests. ** indicates a P value<0.001 and *** indicates a P value<0.0001.
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pone-0038491-g005: HIV induced immune activation and expression levels of multiple activation markers on CD4+ and CD8+.Blood samples were obtained from mice before and 6 week after HIV-1 JRCSF infection. (A) Beta-2-microglobline levels were measured using ELISA before (Pre) and after HIV-infection (Post) (n = 4) and at same time intervals in engrafted but noninfected mice. (B) CD69 (C) HLA-DR (D) PD-1 and (E) CD27 (F) CCR5 levels were evaluated on CD4+ and CD8+ cells. (G) LPS levels in humanized mice 2 and 8 weeks post-HIV infection. Each circle and square represent one mouse at the indicated time point. Means are shown in solid lines. P values were determined by unpaired student’s t-tests. ** indicates a P value<0.001 and *** indicates a P value<0.0001.

Mentions: Aspecific activation of the innate immune system has been described in HIV-1 infection and possibly drives subsequent activation of the adaptive immune response. Levels of non-specific immune activation marker such as β2-microglobulin have been correlated with disease progression [18]. In humanized mice, HIV infection induced a strong increase of β2-microglobulin from 0.42±0.09 mg/l at day 0 to 5.41±0.29 mg/l (n = 4). Aging in non-infected mice was associated with a moderate increase of β2-microglobulin but this marker remained much lower than in the infected animals (Fig. 5A). Next we studied the expression of early activation markers CD69 and HLA-DR on CD4 and CD8 T cells of humanized mice after HIV infection. The percentage of CD4+ T cells expressing CD69 increased from 0.67±0.29% to 4.55±1.58% (n = 4) whereas it went from 0.30±0.21% to 8.33±1.38% (n = 4) for CD8+ T cells (Fig. 5B). The increase in HLA-DR expression after HIV infection was even more pronounced. The expression of HLA-DR was observed on 2.50±0.81% of CD4+ T cells and 3.00±0.80% of CD8+ T cells before infection and jumped to 39.28±2.90% (n = 4) and 49.98±2.24% (n = 4) respectively for CD4+ and CD8+ T cells after infection (Fig. 5C). Some activation markers are specifically linked to impaired T cells functions. This is the case for the inhibitory receptor PD-1 (Programmed Death 1), which is a marker for T cell exhaustion [19]. After infection with HIV-JRCSF, there was a strong increase of the percentage of T cells positive for PD-1 in comparison with non-infected counterparts. This was true for CD4+ (16.50±1.84% (n = 4) vs. 1.29±0.89% (n = 4)) as well as for CD8+ T cells (22.25±1.37% (n = 4) vs. 2.59±2.34% (n = 4)) (Fig. 5D). CD27, a receptor involved in co-stimulation, is down regulated with advancing cell differentiation from early-differentiated CD27+ memory T cells to late differentiated CD27− memory T cells. The loss of CD27 on T cells is classically viewed as marker of immune senescence and occurs in HIV infection. In HIV-infected NSG humanized mice, we observed down-regulation of CD27 expression in CD4+ (from 91.25±1.70% positive before infection to 82.00±2.94% at week 6) and CD8+ T cells (from 91.25±1.89% to 77.50±3.11%) (Fig. 5E).


An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγ mice allows HIV replication and development of anti-HIV immune responses.

Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, Vandergeeten C, Delacroix L, Van Gulck E, Vanham G, de Leval L, Rahmouni S, Moutschen M - PLoS ONE (2012)

HIV induced immune activation and expression levels of multiple activation markers on CD4+ and CD8+.Blood samples were obtained from mice before and 6 week after HIV-1 JRCSF infection. (A) Beta-2-microglobline levels were measured using ELISA before (Pre) and after HIV-infection (Post) (n = 4) and at same time intervals in engrafted but noninfected mice. (B) CD69 (C) HLA-DR (D) PD-1 and (E) CD27 (F) CCR5 levels were evaluated on CD4+ and CD8+ cells. (G) LPS levels in humanized mice 2 and 8 weeks post-HIV infection. Each circle and square represent one mouse at the indicated time point. Means are shown in solid lines. P values were determined by unpaired student’s t-tests. ** indicates a P value<0.001 and *** indicates a P value<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366932&req=5

pone-0038491-g005: HIV induced immune activation and expression levels of multiple activation markers on CD4+ and CD8+.Blood samples were obtained from mice before and 6 week after HIV-1 JRCSF infection. (A) Beta-2-microglobline levels were measured using ELISA before (Pre) and after HIV-infection (Post) (n = 4) and at same time intervals in engrafted but noninfected mice. (B) CD69 (C) HLA-DR (D) PD-1 and (E) CD27 (F) CCR5 levels were evaluated on CD4+ and CD8+ cells. (G) LPS levels in humanized mice 2 and 8 weeks post-HIV infection. Each circle and square represent one mouse at the indicated time point. Means are shown in solid lines. P values were determined by unpaired student’s t-tests. ** indicates a P value<0.001 and *** indicates a P value<0.0001.
Mentions: Aspecific activation of the innate immune system has been described in HIV-1 infection and possibly drives subsequent activation of the adaptive immune response. Levels of non-specific immune activation marker such as β2-microglobulin have been correlated with disease progression [18]. In humanized mice, HIV infection induced a strong increase of β2-microglobulin from 0.42±0.09 mg/l at day 0 to 5.41±0.29 mg/l (n = 4). Aging in non-infected mice was associated with a moderate increase of β2-microglobulin but this marker remained much lower than in the infected animals (Fig. 5A). Next we studied the expression of early activation markers CD69 and HLA-DR on CD4 and CD8 T cells of humanized mice after HIV infection. The percentage of CD4+ T cells expressing CD69 increased from 0.67±0.29% to 4.55±1.58% (n = 4) whereas it went from 0.30±0.21% to 8.33±1.38% (n = 4) for CD8+ T cells (Fig. 5B). The increase in HLA-DR expression after HIV infection was even more pronounced. The expression of HLA-DR was observed on 2.50±0.81% of CD4+ T cells and 3.00±0.80% of CD8+ T cells before infection and jumped to 39.28±2.90% (n = 4) and 49.98±2.24% (n = 4) respectively for CD4+ and CD8+ T cells after infection (Fig. 5C). Some activation markers are specifically linked to impaired T cells functions. This is the case for the inhibitory receptor PD-1 (Programmed Death 1), which is a marker for T cell exhaustion [19]. After infection with HIV-JRCSF, there was a strong increase of the percentage of T cells positive for PD-1 in comparison with non-infected counterparts. This was true for CD4+ (16.50±1.84% (n = 4) vs. 1.29±0.89% (n = 4)) as well as for CD8+ T cells (22.25±1.37% (n = 4) vs. 2.59±2.34% (n = 4)) (Fig. 5D). CD27, a receptor involved in co-stimulation, is down regulated with advancing cell differentiation from early-differentiated CD27+ memory T cells to late differentiated CD27− memory T cells. The loss of CD27 on T cells is classically viewed as marker of immune senescence and occurs in HIV infection. In HIV-infected NSG humanized mice, we observed down-regulation of CD27 expression in CD4+ (from 91.25±1.70% positive before infection to 82.00±2.94% at week 6) and CD8+ T cells (from 91.25±1.89% to 77.50±3.11%) (Fig. 5E).

Bottom Line: In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice.Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days.We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and infectious diseases unit GIGA-I3, University of Liege, Liege, Belgium.

ABSTRACT
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ() (NSG) and NOD/SCID/IL2Rγ() (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

Show MeSH
Related in: MedlinePlus