Limits...
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.

Show MeSH

Related in: MedlinePlus

Coreceptor expression and lymphoid organ formation in humanized mice.(A) Representative FACS profile of human CCR5+CD4+ and CXCR4+CD4+ cells in spleen and lymph nodes, gate was set of human CD45 cells. Expression of CCR5 and CXCR4 were checked on CD14+ cells, gate was set on live human cell population. (B) Histology of lymphoid organs in CD34+ engrafted NSG mice. The lymphoid follicles mainly contained hCD45 cells. Spleen sections were stained with (a) anti-hCD45, (b) anti-hCD20 and (c) anti-hCD138. Lymph node sections were also stained with (d) anti-hCD45, (e) anti-hCD20 (f) anti-hCD3 (g,h) anti-hKi67 antibodies. Thymus section was stained with anti-CD3 antibody. (C) Proliferative responses of T cells were measured in nonstimulated cells, after stimulation with immobilized anti-CD3/anti-CD28 and anti-CD3+IL2 in spleen (white bars), lymph nodes (gray bars) and human PBMCs (black bars).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366932&req=5

pone-0038491-g003: Coreceptor expression and lymphoid organ formation in humanized mice.(A) Representative FACS profile of human CCR5+CD4+ and CXCR4+CD4+ cells in spleen and lymph nodes, gate was set of human CD45 cells. Expression of CCR5 and CXCR4 were checked on CD14+ cells, gate was set on live human cell population. (B) Histology of lymphoid organs in CD34+ engrafted NSG mice. The lymphoid follicles mainly contained hCD45 cells. Spleen sections were stained with (a) anti-hCD45, (b) anti-hCD20 and (c) anti-hCD138. Lymph node sections were also stained with (d) anti-hCD45, (e) anti-hCD20 (f) anti-hCD3 (g,h) anti-hKi67 antibodies. Thymus section was stained with anti-CD3 antibody. (C) Proliferative responses of T cells were measured in nonstimulated cells, after stimulation with immobilized anti-CD3/anti-CD28 and anti-CD3+IL2 in spleen (white bars), lymph nodes (gray bars) and human PBMCs (black bars).

Mentions: Next we studied the expression of HIV-1 co-receptors CXCR4 and CCR5 on human CD4+ T cells in the lymphoid organs of the humanized mice. CXCR4 was expressed on 78.11±17.13% (n = 4) CD4+ T cells in spleen and on 42.1±14.1% (n = 4) of CD4+ T cells in the lymph nodes (Fig. 3A). The proportion of CD4+ T cells positive for CCR5 was lower in both lymph nodes 6.51±2.13% and spleen 7.17±3.133%, indicative of a naïve state of T cells (Fig. 3A). Large proportions of CD14+ cells in the bone marrow were also positive for CCR5 and CXCR4 (Fig. 3A).


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)

Coreceptor expression and lymphoid organ formation in humanized mice.(A) Representative FACS profile of human CCR5+CD4+ and CXCR4+CD4+ cells in spleen and lymph nodes, gate was set of human CD45 cells. Expression of CCR5 and CXCR4 were checked on CD14+ cells, gate was set on live human cell population. (B) Histology of lymphoid organs in CD34+ engrafted NSG mice. The lymphoid follicles mainly contained hCD45 cells. Spleen sections were stained with (a) anti-hCD45, (b) anti-hCD20 and (c) anti-hCD138. Lymph node sections were also stained with (d) anti-hCD45, (e) anti-hCD20 (f) anti-hCD3 (g,h) anti-hKi67 antibodies. Thymus section was stained with anti-CD3 antibody. (C) Proliferative responses of T cells were measured in nonstimulated cells, after stimulation with immobilized anti-CD3/anti-CD28 and anti-CD3+IL2 in spleen (white bars), lymph nodes (gray bars) and human PBMCs (black bars).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366932&req=5

pone-0038491-g003: Coreceptor expression and lymphoid organ formation in humanized mice.(A) Representative FACS profile of human CCR5+CD4+ and CXCR4+CD4+ cells in spleen and lymph nodes, gate was set of human CD45 cells. Expression of CCR5 and CXCR4 were checked on CD14+ cells, gate was set on live human cell population. (B) Histology of lymphoid organs in CD34+ engrafted NSG mice. The lymphoid follicles mainly contained hCD45 cells. Spleen sections were stained with (a) anti-hCD45, (b) anti-hCD20 and (c) anti-hCD138. Lymph node sections were also stained with (d) anti-hCD45, (e) anti-hCD20 (f) anti-hCD3 (g,h) anti-hKi67 antibodies. Thymus section was stained with anti-CD3 antibody. (C) Proliferative responses of T cells were measured in nonstimulated cells, after stimulation with immobilized anti-CD3/anti-CD28 and anti-CD3+IL2 in spleen (white bars), lymph nodes (gray bars) and human PBMCs (black bars).
Mentions: Next we studied the expression of HIV-1 co-receptors CXCR4 and CCR5 on human CD4+ T cells in the lymphoid organs of the humanized mice. CXCR4 was expressed on 78.11±17.13% (n = 4) CD4+ T cells in spleen and on 42.1±14.1% (n = 4) of CD4+ T cells in the lymph nodes (Fig. 3A). The proportion of CD4+ T cells positive for CCR5 was lower in both lymph nodes 6.51±2.13% and spleen 7.17±3.133%, indicative of a naïve state of T cells (Fig. 3A). Large proportions of CD14+ cells in the bone marrow were also positive for CCR5 and CXCR4 (Fig. 3A).

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