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Peripheral blood CD8αα+CD11c+MHC-II+CD3- cells attenuate autoimmune glomerulonephritis in rats.

Wu J, Zhou C, Robertson J, Carlock C, Lou YH - Kidney Int. (2013)

Bottom Line: Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25).When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats.Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic and Biomedical Sciences, SD, University of Texas Health Science Center at Houston, Houston, Texas, USA.

ABSTRACT
In an anti-glomerular basement membrane (GBM) glomerulonephritis (GN) model, GN-resistant Lewis rats naturally recover from early glomerular inflammation. Here we investigated recovery mechanisms for development of a potential immunotherapy for autoimmune GN. Our previous studies suggested that glomeruli-infiltrating leukocytes with a phenotype of CD8αα+CD11c+MHC-II+CD3- (GIL CD8αα+ cells) were responsible for recovery through induction of T-cell apoptosis. Now, we identified peripheral blood CD8αα+CD11c+MHC-II+CD3- cells (PBMC CD8αα+CD3- cells), which shared 9 markers with GIL CD8αα+ cells. Upon incubation, PBMC CD8αα+CD3- cells displayed a morphology resembling that of dendritic cells. Similar to GIL CD8αα+ cells, PBMC CD8αα+CD3- cells were capable of inducing T-cell apoptosis in vitro. Hence, PBMC CD8αα+CD3- cells were likely the precursor of GIL CD8αα+ cells. We next tested their potential in vivo function. PBMC CD8αα+CD3- cells were able to infiltrate inflamed but not normal glomeruli. Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25). When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats. Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats. Thus, PBMC CD8αα+CD3- cells of Lewis rats were able to terminate ongoing autoimmune inflammation in the glomeruli.

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Related in: MedlinePlus

Identification of PBMC CD8α+β−RT1B(MHC-II)+CD3− population and its expansion after immunization(a and b) Flow cytometry analyses on PBMC show a CD8α+CD3− population (red box in a) and a CD8α+RT1B+ population (red box in b). (c) FSC/SSC plot analysis of sorted PBMC CD8α+ cells shows two morphologically distinct populations (red and pink circle), which were CD8α+CD3− (right upper panel) and CD8α+CD3+ T cells (right lower panel), respectively. (d) Three color flow cytometry shows distinct populations of CD8α+β−CD3− cells (red dots) and CD8α+β+CD3+ T cells (blue dots) among sorted PBMC CD8α+ cells. A small CD8−CD3+ T cell population (green dots), probably CD4+ T cell contaminant, also can be seen. (e) Flow cytometry on PBMC shows a majority population of CD8α−CD94+ NK cells and a minor CD8α+CD94+ population (green box). (f) Expansion of the PBMC CD8α+CD3− population after immunization (left panel)(n=3 for normal and immunized group). Right two panels are representative flow cytometries on PBMC at d0 and d20 post immunization with pCol(28–40) for comparison of CD8α+CD3− populations (red box).
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Figure 1: Identification of PBMC CD8α+β−RT1B(MHC-II)+CD3− population and its expansion after immunization(a and b) Flow cytometry analyses on PBMC show a CD8α+CD3− population (red box in a) and a CD8α+RT1B+ population (red box in b). (c) FSC/SSC plot analysis of sorted PBMC CD8α+ cells shows two morphologically distinct populations (red and pink circle), which were CD8α+CD3− (right upper panel) and CD8α+CD3+ T cells (right lower panel), respectively. (d) Three color flow cytometry shows distinct populations of CD8α+β−CD3− cells (red dots) and CD8α+β+CD3+ T cells (blue dots) among sorted PBMC CD8α+ cells. A small CD8−CD3+ T cell population (green dots), probably CD4+ T cell contaminant, also can be seen. (e) Flow cytometry on PBMC shows a majority population of CD8α−CD94+ NK cells and a minor CD8α+CD94+ population (green box). (f) Expansion of the PBMC CD8α+CD3− population after immunization (left panel)(n=3 for normal and immunized group). Right two panels are representative flow cytometries on PBMC at d0 and d20 post immunization with pCol(28–40) for comparison of CD8α+CD3− populations (red box).

Mentions: GIL CD8αα+ cells are derived from bone marrow, suggesting that the cells migrate into inflamed glomeruli through peripheral blood.15 We first searched for CD8α+CD3− non-T cells among peripheral blood mononuclear cells (PBMC) in LEW rats. A CD8α+CD3− population (approximately 2.9% of PBMC) was observed (Figure 1a). Another analysis showed a CD8α+RT1B(MHC-II)+ of a similar size (2.7%)(Figure 1b). Because CD8+ T cells do not express MHC-II, the above CD8α+CD3− population was determined to be RT1B+. Next, we sorted whole PBMC CD8α+ cells (both CD3+ and CD3−). Based on the FSC and SSC plot of flow cytometry, CD8α+CD3− cells formed a cluster, which was distinguishable from CD8+ T cells (Figure 1c). A previous study has reported CD8+ regulatory T cells in a LEW rat model for Heymann nephritis.17 Thus, we further tested the above PBMC CD8α+ cells for their CD8β and CD3 expression using three color flow cytometry. The PBMC CD8α+ cells contained two distinct populations: CD8α+β+CD3+ T cells and CD8α+β−CD3− non-T cells (Figure 1d). Since the CD8α+β−CD3− cell population did not express CD8β, they were designated as PBMC CD8αα+CD3− cells. A subset of NK cells is CD8+. Using CD94 as an NK marker, we showed that the majority of CD94+ NK cells were CD8α− with a minor CD94+CD8α+ population, which was approximately 0.7% of total PBMC (Figure 1e). Thus, the CD94+CD8α+ NK cells could be a small portion, but not all of the CD8αα+CD3−cells. In summary, our preliminary observations of PBMC suggested that there was a CD8αα+RT1.B+CD3−CD94−CD3− non-NK non-T cell population among PBMC, which formed a morphologically identical population. However, this was not conclusive. Therefore, we purified this population for further characterization.


Peripheral blood CD8αα+CD11c+MHC-II+CD3- cells attenuate autoimmune glomerulonephritis in rats.

Wu J, Zhou C, Robertson J, Carlock C, Lou YH - Kidney Int. (2013)

Identification of PBMC CD8α+β−RT1B(MHC-II)+CD3− population and its expansion after immunization(a and b) Flow cytometry analyses on PBMC show a CD8α+CD3− population (red box in a) and a CD8α+RT1B+ population (red box in b). (c) FSC/SSC plot analysis of sorted PBMC CD8α+ cells shows two morphologically distinct populations (red and pink circle), which were CD8α+CD3− (right upper panel) and CD8α+CD3+ T cells (right lower panel), respectively. (d) Three color flow cytometry shows distinct populations of CD8α+β−CD3− cells (red dots) and CD8α+β+CD3+ T cells (blue dots) among sorted PBMC CD8α+ cells. A small CD8−CD3+ T cell population (green dots), probably CD4+ T cell contaminant, also can be seen. (e) Flow cytometry on PBMC shows a majority population of CD8α−CD94+ NK cells and a minor CD8α+CD94+ population (green box). (f) Expansion of the PBMC CD8α+CD3− population after immunization (left panel)(n=3 for normal and immunized group). Right two panels are representative flow cytometries on PBMC at d0 and d20 post immunization with pCol(28–40) for comparison of CD8α+CD3− populations (red box).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4008668&req=5

Figure 1: Identification of PBMC CD8α+β−RT1B(MHC-II)+CD3− population and its expansion after immunization(a and b) Flow cytometry analyses on PBMC show a CD8α+CD3− population (red box in a) and a CD8α+RT1B+ population (red box in b). (c) FSC/SSC plot analysis of sorted PBMC CD8α+ cells shows two morphologically distinct populations (red and pink circle), which were CD8α+CD3− (right upper panel) and CD8α+CD3+ T cells (right lower panel), respectively. (d) Three color flow cytometry shows distinct populations of CD8α+β−CD3− cells (red dots) and CD8α+β+CD3+ T cells (blue dots) among sorted PBMC CD8α+ cells. A small CD8−CD3+ T cell population (green dots), probably CD4+ T cell contaminant, also can be seen. (e) Flow cytometry on PBMC shows a majority population of CD8α−CD94+ NK cells and a minor CD8α+CD94+ population (green box). (f) Expansion of the PBMC CD8α+CD3− population after immunization (left panel)(n=3 for normal and immunized group). Right two panels are representative flow cytometries on PBMC at d0 and d20 post immunization with pCol(28–40) for comparison of CD8α+CD3− populations (red box).
Mentions: GIL CD8αα+ cells are derived from bone marrow, suggesting that the cells migrate into inflamed glomeruli through peripheral blood.15 We first searched for CD8α+CD3− non-T cells among peripheral blood mononuclear cells (PBMC) in LEW rats. A CD8α+CD3− population (approximately 2.9% of PBMC) was observed (Figure 1a). Another analysis showed a CD8α+RT1B(MHC-II)+ of a similar size (2.7%)(Figure 1b). Because CD8+ T cells do not express MHC-II, the above CD8α+CD3− population was determined to be RT1B+. Next, we sorted whole PBMC CD8α+ cells (both CD3+ and CD3−). Based on the FSC and SSC plot of flow cytometry, CD8α+CD3− cells formed a cluster, which was distinguishable from CD8+ T cells (Figure 1c). A previous study has reported CD8+ regulatory T cells in a LEW rat model for Heymann nephritis.17 Thus, we further tested the above PBMC CD8α+ cells for their CD8β and CD3 expression using three color flow cytometry. The PBMC CD8α+ cells contained two distinct populations: CD8α+β+CD3+ T cells and CD8α+β−CD3− non-T cells (Figure 1d). Since the CD8α+β−CD3− cell population did not express CD8β, they were designated as PBMC CD8αα+CD3− cells. A subset of NK cells is CD8+. Using CD94 as an NK marker, we showed that the majority of CD94+ NK cells were CD8α− with a minor CD94+CD8α+ population, which was approximately 0.7% of total PBMC (Figure 1e). Thus, the CD94+CD8α+ NK cells could be a small portion, but not all of the CD8αα+CD3−cells. In summary, our preliminary observations of PBMC suggested that there was a CD8αα+RT1.B+CD3−CD94−CD3− non-NK non-T cell population among PBMC, which formed a morphologically identical population. However, this was not conclusive. Therefore, we purified this population for further characterization.

Bottom Line: Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25).When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats.Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic and Biomedical Sciences, SD, University of Texas Health Science Center at Houston, Houston, Texas, USA.

ABSTRACT
In an anti-glomerular basement membrane (GBM) glomerulonephritis (GN) model, GN-resistant Lewis rats naturally recover from early glomerular inflammation. Here we investigated recovery mechanisms for development of a potential immunotherapy for autoimmune GN. Our previous studies suggested that glomeruli-infiltrating leukocytes with a phenotype of CD8αα+CD11c+MHC-II+CD3- (GIL CD8αα+ cells) were responsible for recovery through induction of T-cell apoptosis. Now, we identified peripheral blood CD8αα+CD11c+MHC-II+CD3- cells (PBMC CD8αα+CD3- cells), which shared 9 markers with GIL CD8αα+ cells. Upon incubation, PBMC CD8αα+CD3- cells displayed a morphology resembling that of dendritic cells. Similar to GIL CD8αα+ cells, PBMC CD8αα+CD3- cells were capable of inducing T-cell apoptosis in vitro. Hence, PBMC CD8αα+CD3- cells were likely the precursor of GIL CD8αα+ cells. We next tested their potential in vivo function. PBMC CD8αα+CD3- cells were able to infiltrate inflamed but not normal glomeruli. Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25). When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats. Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats. Thus, PBMC CD8αα+CD3- cells of Lewis rats were able to terminate ongoing autoimmune inflammation in the glomeruli.

Show MeSH
Related in: MedlinePlus