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Regulated expression of human histocompatibility leukocyte antigen (HLA)-DO during antigen-dependent and antigen-independent phases of B cell development.

Chen X, Laur O, Kambayashi T, Li S, Bray RA, Weber DA, Karlsson L, Jensen PE - J. Exp. Med. (2002)

Bottom Line: In contrast, DO expression is initiated only after B cell development is complete.In all situations, there is a striking correlation between intracellular DO expression and cell surface class II-associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells.Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Human histocompatibility leukocyte antigen (HLA)-DO, a lysosomal resident major histocompatibility complex class II molecule expressed in B cells, has previously been shown to be a negative regulator of HLA-DM peptide loading function. We analyze the expression of DO in human peripheral blood, lymph node, tonsil, and bone marrow to determine if DO expression is modulated in the physiological setting. B cells, but not monocytes or monocyte-derived dendritic cells, are observed to express this protein. Preclearing experiments demonstrate that approximately 50% of HLA-DM is bound to DO in peripheral blood B cells. HLA-DM and HLA-DR expression is demonstrated early in B cell development, beginning at the pro-B stage in adult human bone marrow. In contrast, DO expression is initiated only after B cell development is complete. In all situations, there is a striking correlation between intracellular DO expression and cell surface class II-associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells. We report that the expression of DO is markedly downmodulated in human germinal center B cells. Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.

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Fraction of HLA-DM molecules bound to HLA-DO in B cells. (a) The top panels (lysates) show Western blot analysis of Raji B cell lysates before or after preclearing with anti-DO or control mAb, as indicated. The middle panels (IP) show Western blot analysis of immunoprecipitates generated with the anti-DO (DOB.L1) or control mAb. Blots were stained with mAb 4.7GS (DM) or DOB.L1 (DO). The bottom panels show DM Western blot analysis of titrations of Raji cell extracts depleted with anti–HLA-DO or with control antibody. Numbers indicate sample quantity (cell equivalents × 10−4) loaded in each lane. (b) Similar analysis with purified peripheral blood B cells.
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fig2: Fraction of HLA-DM molecules bound to HLA-DO in B cells. (a) The top panels (lysates) show Western blot analysis of Raji B cell lysates before or after preclearing with anti-DO or control mAb, as indicated. The middle panels (IP) show Western blot analysis of immunoprecipitates generated with the anti-DO (DOB.L1) or control mAb. Blots were stained with mAb 4.7GS (DM) or DOB.L1 (DO). The bottom panels show DM Western blot analysis of titrations of Raji cell extracts depleted with anti–HLA-DO or with control antibody. Numbers indicate sample quantity (cell equivalents × 10−4) loaded in each lane. (b) Similar analysis with purified peripheral blood B cells.

Mentions: Available evidence indicates that HLA-DO molecules are unstable in the absence of DM and that all DO present in post-Golgi compartments is stably bound to DM (17). Kropshofer et al. (23) reported that 60–70% of DM molecules in the WT-100 human B cell line are coprecipitated with mAb to DO. However, the fraction of DM molecules bound to DO in primary B cells has not been reported. This is an important issue when considering the potential biological roles for DO. If only a small fraction of DM molecules are occupied by DO, one must seriously consider the possibility that DO has some unique function other than inhibiting DM activity. We addressed this issue by preclearing detergent lysates of B cells with anti-DO mAb and measuring residual free DM in Western blots. As demonstrated in Fig. 2 a, a substantial fraction of total DM in the Raji B cell line is removed by depletion with antibodies to DO. Titration of lysates precleared with anti-DO versus control antibody demonstrated that ∼50% of DM in Raji cells is stably bound to DO. Similar experiments were performed with purified peripheral blood B cells, demonstrating that ∼50% of DM is stably bound to DO in these cells (Fig. 2 b). We conclude that a substantial fraction of DM is associated with DO in primary human B cells.


Regulated expression of human histocompatibility leukocyte antigen (HLA)-DO during antigen-dependent and antigen-independent phases of B cell development.

Chen X, Laur O, Kambayashi T, Li S, Bray RA, Weber DA, Karlsson L, Jensen PE - J. Exp. Med. (2002)

Fraction of HLA-DM molecules bound to HLA-DO in B cells. (a) The top panels (lysates) show Western blot analysis of Raji B cell lysates before or after preclearing with anti-DO or control mAb, as indicated. The middle panels (IP) show Western blot analysis of immunoprecipitates generated with the anti-DO (DOB.L1) or control mAb. Blots were stained with mAb 4.7GS (DM) or DOB.L1 (DO). The bottom panels show DM Western blot analysis of titrations of Raji cell extracts depleted with anti–HLA-DO or with control antibody. Numbers indicate sample quantity (cell equivalents × 10−4) loaded in each lane. (b) Similar analysis with purified peripheral blood B cells.
© Copyright Policy
Related In: Results  -  Collection

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fig2: Fraction of HLA-DM molecules bound to HLA-DO in B cells. (a) The top panels (lysates) show Western blot analysis of Raji B cell lysates before or after preclearing with anti-DO or control mAb, as indicated. The middle panels (IP) show Western blot analysis of immunoprecipitates generated with the anti-DO (DOB.L1) or control mAb. Blots were stained with mAb 4.7GS (DM) or DOB.L1 (DO). The bottom panels show DM Western blot analysis of titrations of Raji cell extracts depleted with anti–HLA-DO or with control antibody. Numbers indicate sample quantity (cell equivalents × 10−4) loaded in each lane. (b) Similar analysis with purified peripheral blood B cells.
Mentions: Available evidence indicates that HLA-DO molecules are unstable in the absence of DM and that all DO present in post-Golgi compartments is stably bound to DM (17). Kropshofer et al. (23) reported that 60–70% of DM molecules in the WT-100 human B cell line are coprecipitated with mAb to DO. However, the fraction of DM molecules bound to DO in primary B cells has not been reported. This is an important issue when considering the potential biological roles for DO. If only a small fraction of DM molecules are occupied by DO, one must seriously consider the possibility that DO has some unique function other than inhibiting DM activity. We addressed this issue by preclearing detergent lysates of B cells with anti-DO mAb and measuring residual free DM in Western blots. As demonstrated in Fig. 2 a, a substantial fraction of total DM in the Raji B cell line is removed by depletion with antibodies to DO. Titration of lysates precleared with anti-DO versus control antibody demonstrated that ∼50% of DM in Raji cells is stably bound to DO. Similar experiments were performed with purified peripheral blood B cells, demonstrating that ∼50% of DM is stably bound to DO in these cells (Fig. 2 b). We conclude that a substantial fraction of DM is associated with DO in primary human B cells.

Bottom Line: In contrast, DO expression is initiated only after B cell development is complete.In all situations, there is a striking correlation between intracellular DO expression and cell surface class II-associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells.Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Human histocompatibility leukocyte antigen (HLA)-DO, a lysosomal resident major histocompatibility complex class II molecule expressed in B cells, has previously been shown to be a negative regulator of HLA-DM peptide loading function. We analyze the expression of DO in human peripheral blood, lymph node, tonsil, and bone marrow to determine if DO expression is modulated in the physiological setting. B cells, but not monocytes or monocyte-derived dendritic cells, are observed to express this protein. Preclearing experiments demonstrate that approximately 50% of HLA-DM is bound to DO in peripheral blood B cells. HLA-DM and HLA-DR expression is demonstrated early in B cell development, beginning at the pro-B stage in adult human bone marrow. In contrast, DO expression is initiated only after B cell development is complete. In all situations, there is a striking correlation between intracellular DO expression and cell surface class II-associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells. We report that the expression of DO is markedly downmodulated in human germinal center B cells. Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.

Show MeSH