Distinctive growth requirements and gene expression patterns distinguish progenitor B cells from pre-B cells.

Long-term bone marrow cultures have been useful in determining gene expression patterns in pre-B cells and in the identification of cytokines such as interleukin 7 (IL-7). We have developed a culture system to selectively grow populations of B lineage restricted progenitors (pro-B cells) from murine bone marrow. Pro-B cells do not grow in response to IL-7, Steel locus factor (SLF), or a combination of the two. c-kit, the SLF receptor, and the IL-7 receptor are both expressed by pro-B cells, indicating that the lack of response is not simply due to the absence of receptors. Furthermore, SLF is not necessary for the growth of pro-B cells since they could be expanded on a stromal line derived from Steel mice that produces no SLF. IL-7 responsiveness in pre-B cells is associated with an increase in n-myc expression and is correlated with immunoglobulin (Ig) gene rearrangements. Although members of the ets family of transcription factors and the Pim-1 kinase are expressed by pro-B cells, n-myc is not expressed. Pro-B cells maintain Ig genes in the germline configuration, which is correlated with a low level of recombination activating genes 1 and 2 (Rag-1 and 2) mRNA expression, but high expression of sterile mu and terminal deoxynucleotidyl transferase. Pro-B cells are unable to grow separated from the stromal layer by a porous membrane, indicating that stromal contact is required for growth. These results suggest that pro-B cells are dependent on alternative growth signals derived from bone marrow stroma and can be distinguished from pre-B cells by specific patterns of gene expression.

Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA).

The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation.

Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice.

The cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk, formerly bpk or atk), is crucial for B cell development. Loss of kinase activity results in the human immunodeficiency, X-linked agammaglobulinemia, characterized by a failure to produce B cells. In the murine X-linked immunodeficiency (XID), B cells are present but respond abnormally to activating signals. The Btk gene, btk, was mapped to the xid region of the mouse X chromosome by interspecific backcross analysis. A single conserved residue within the amino terminal unique region of Btk was mutated in XID mice. This change in xid probably interferes with normal B cell signaling mediated by Btk protein interactions.

Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia.

We identified TMPRSS2 as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metastasis. Using specific monoclonal antibodies, we show that the TMPRSS2-encoded serine protease is expressed as a Mr 70,000 full-length form and a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, suggesting that it occurs as a result of autocleavage. Mutational analysis reveals the cleavage site to be at Arg-255. A consequence of autocatalytic cleavage is the secretion of the protease domain into the media by TMPRSS2-expressing prostate cancer cells and into the sera of prostate tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression detected in seven additional normal tissues. These data demonstrate that TMPRSS2 is a secreted protease that is highly expressed in prostate and prostate cancer, making it a potential target for cancer therapy and diagnosis.

Immunotherapy of established tumors in mice by intratumoral injection of interleukin-2 plasmid DNA: induction of CD8+ T-cell immunity.

Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice. Tumor regression was observed in 60-90% of mice that were injected i.t. for 4 days with IL-2 plasmid DNA complexed with the cationic lipid DMRIE/DOPE ((+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propa naminium bromide/dioleoylphosphatidylethanolamine). The mice remained tumor-free until the conclusion of the study, which was 4 months after tumor challenge. In a rechallenge experiment, mice that were rendered tumor-free for 6 months by IL-2 plasmid DNA treatment rejected a subsequent challenge of Renca cells but could not reject a challenge with the unrelated, syngeneic CT-26 tumor. Spleen cells from cured mice contained Renca-specific cytotoxic T lymphocytes, and adoptive transfer of mixed lymphocyte cultures into naive mice at 2 days after challenge with Renca cells prevented tumor growth. In vivo depletion of T-cell subsets at the time of i.t. injection with IL-2 plasmid DNA demonstrated that CD8+ T cells, but not CD4+ T cells, were the primary effectors of the antitumor response.

Suppression of mixed lymphocyte reactivity by murine bone-marrow-derived suppressor factor--inhibition of proliferation due to a deficit in IL-2 production.

Murine bone marrow natural suppressor cells constitutively produce a soluble factor, BDSF, that suppresses murine Ab responses in vitro. BDSF is recovered in supernatants of cultured BM cells and resides in an MW range between 1-10 kD. We now demonstrate that BDSF suppresses proliferation in the mixed lymphocyte reaction (MLR). In order to elucidate the potential mechanism of action, BDSF was tested for the ability to interfere with IL-2-mediated T cell proliferation. BDSF was not able to inhibit IL-2-driven proliferation of the cell lines CTLL-2 and HT-2. However, examination of MLR culture supernatants revealed that suppression of proliferation by BDSF was associated with decreased levels of IL-2 production. Furthermore, responsiveness of BDSF-treated cultures could be reconstituted by adding exogenous IL-2, at the time of plating. The results suggest that hyporesponsiveness in the presence of BDSF is due to a deficit in IL-2 production, preventing expansion of Ag-stimulated cells.

Separation of allostimulatory and natural suppressor/stem cell functions of murine bone marrow--implications for bone marrow transplantation.

Natural suppressor (NS) activity is defined by the ability of a null cell population to inhibit antigenic and mitogenic activation of lymphoid cells. We had previously demonstrated that murine and human bone marrow was a rich source of NS activity and could be enriched in a population of large, low-density cells after counterflow centrifugal elutriation (CCE). In this report we confirm previous findings in the mixed lymphocyte reaction (MLR) that NS activity is an endogenous function of BM and is not present in peripheral immune tissues. Furthermore, suppression by BM NS cells in the MLR is not MHC restricted, which distinguishes NS activity from veto activity also associated with BM. To enrich for NS activity, BM cells were separated into 3 fractions according to size and density by CCE. Fraction (F) 1 contained small, high-density cells; F2 contained cells of intermediate size and density; and F3 comprised large, low-density cells. Addition of CCE-enriched fractions to the MLR revealed potent NS activity associated with F3, consistent with our previous findings in the Ab response. CCE-enriched fractions of BM cells were also used as stimulators in an MLR to determine which fraction, if any, could support alloantigen-induced proliferation. The use of unfractionated C57Bl/6 (H-2b) BM cells as stimulators for BALB/c (H-2d) splenic responders resulted in little detectable proliferation as compared with that induced by C57Bl/6 splenic stimulators. However, when elutriated fractions were used as stimulators, F1 BM cells induced a significant proliferative response, albeit to a lesser magnitude than spleen cells. In order to determine which fraction contained hematopoietic progenitors, CCE-separated cells were assayed for granulocyte/macrophage colony formation (CFU-GM). F3 BM was enriched five-fold for CFU-GM progenitors as compared with unfractionated BM, while progenitors were virtually absent in F1 and F2 BM. The results suggest that CCE may represent an effective way of removing potential alloantigen-presenting cells (F1) from BM, while retaining hematopoietic progenitors and NS activity (F3). The implications of the findings as they relate to allogeneic BM transplantation are discussed.

Further characterization of murine bone marrow-derived natural suppressor cells. Potential relationships between NS and NK/LAK activities.

Murine bone marrow (BM) cells regulate a variety of immune responses via an endogenous natural suppressor (NS) activity. We demonstrate that BM-derived NS activity resides in an enriched fraction of large, low-density cells which have a high proliferative rate. Complement-dependent lysis of BM cells by antibody directed against markers of Veto and NK/LAK cells had no effect on NS activity. The BM of SCID mice and their littermate C.B-17 possessed normal NS activity. Conversely, the BM of NK-deficient C57 beige mice displayed reduced NS activity as compared to normal C57 black mice. Long-term BM cultures (LTBMC) generated in medium containing supernatants of Con A-stimulated (CAS) rat spleen cells resulted in the emergence of a population of cells which possessed NS activity greater than that of fresh BM cells. The LTBMC were also potent effectors of NK activity, as compared to fresh BM, which had little NK activity. Thus, while NS, NK/LAK, and Veto cells are all nonspecific effectors of immune suppression, the exact relationship between them is not clear.

The immunoregulatory role of bone marrow. IV. Role of an immunoenhancing glycoprotein derived from murine bone marrow.

Bone marrow-enhancing factor (B-EF) is the spontaneous product of whole bone marrow cells cultured in serum-free medium for a short term (24-48 hr). The factor is prepared by ultrafiltration of BMC supernates to yield a preparation with a MW of greater than 10,000. Production of the factor is not dependent upon antigenic or mitogenic stimulation of BMC, but is inhibited by treatment of BMC with cycloheximide. B-EF augments the in vitro primary PFC response to SRBC, as well as in vitro secondary IgM and IgG PFC responses to SRBC. Enhancement by B-EF is antigen dependent, genetically nonrestricted, and maximal when present at the initiation of culture. B-EF cannot induce a polyclonal antibody response like the polyclonal activator LPS. B-EF is directly mitogenic for thymocytes, bone marrow, and whole spleen cells, but fails to act as a costimulator of thymocyte proliferation in the presence of Con A. B-EF cannot support the growth of the IL-2-dependent cell line CTLL-2. Since B-EF has not been purified, the supernatant may contain more than one activity. The factor is heat labile at 65 degrees C and is sensitive to enzymatic digestion with trypsin and neuraminidase; this implies that B-EF may be a glycoprotein.

Identification of a human bone marrow-derived immunoenhancing factor, BDEF.

In this report we describe the production and biological activity of human bone marrow-derived enhancing factor (BDEF). This factor is the constitutive product of cultured human BMC and could initially be recovered by ultrafiltration of cell-free BM supernatants to yield a crude fraction of Mr greater than 10,000 Da. This preparation can enhance the Ab response of human tonsillar cells, as well as murine spleen cells, to SRBC. HPLC fractionation of BM supernatants enriches for enhancing activity in a peak with an approximate Mr of 60 kDa. PAGE gel analysis reveals two protein bands which migrate to this area, one of 60 kDa, and a slightly smaller protein at 55 kDa. Antibodies generated against the above two proteins were shown to be specific by Western blotting and could recognize the native BM proteins as determined by ELISA. The antibodies were used to affinity purify the respective proteins, p60 and p55. The BM protein p60, but not p55, was able to enhance Ab synthesis in vitro and was also mitogenic for murine BMC and thymocytes. The addition of anti-p60 Ab to human tonsillar cells cultured with SRBC and human BDEF preparations resulted in abrogation of enhancement. These findings support the notion that the BM protein p60 is BDEF and that it may represent a novel enhancing molecule produced by normal human BM.

Target antigens for prostate cancer immunotherapy.

The detection and treatment of prostate cancer has been markedly improved by the use of Prostate-Specific Antigen (PSA) as a serological biomarker for disease. However, even after surgical intervention and hormone ablation therapy, a significant proportion of patients progress to advanced metastatic disease, for which there is no cure. An important goal has become the identification of antigens in advanced stage prostate cancer that represent targets for therapy. Recently, great progress has been made to utilize immunological therapies to treat cancer. Monoclonal antibody therapy has been successfully approved for the treatment of breast cancer and B-cell lymphoma, and multiple clinical trails are currently in progress in a variety of cancers, including prostate cancer. Pre-clinical and clinical studies are also underway to evaluate cancer vaccine approaches directed against antigens that are highly expressed in prostate and other cancers. This article describes several target antigens expressed in prostate cancer and immunological approaches directed against them that may be effective for treating prostate cancer patients.

Alternative signals to RAS for hematopoietic transformation by the BCR-ABL oncogene.

Biological function of the BCR-ABL oncogene is dependent on its activated tyrosine kinase. Mutations that inactivate the SRC homology 2 (SH2) domain, the GRB2-binding site in BCR, or the major autophosphorylation site of the kinase domain selectively disrupt downstream signaling but not tyrosine kinase activity. Despite a loss of fibroblast transformation activity, all three mutants retain the ability to render hematopoietic cell lines growth factor independent and transform primary bone marrow cells in vitro. In vivo tests of malignant potential reveal a most critical role for signals dependent on the BCR-ABL SH2 domain. The efficiency of both fibroblast and hematopoietic transformation by BCR-ABL is strongly affected by increased dosage of the SHC adapter protein, which can connect tyrosine kinase signals to RAS. The BCR-ABL oncogene activates multiple alternative pathways to RAS for hematopoietic transformation.

Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts.

Prostate stem-cell antigen (PSCA) is a cell-surface antigen expressed in normal prostate and overexpressed in prostate cancer tissues. PSCA expression is detected in over 80% of patients with local disease, and elevated levels of PSCA are correlated with increased tumor stage, grade, and androgen independence, including high expression in bone metastases. We evaluated the therapeutic efficacy of anti-PSCA mAbs in human prostate cancer xenograft mouse models by using the androgen-dependent LAPC-9 xenograft and the androgen-independent recombinant cell line PC3-PSCA. Two different anti-PSCA mAbs, 1G8 (IgG1kappa) and 3C5 (IgG2akappa), inhibited formation of s.c. and orthotopic xenograft tumors in a dose-dependent manner. Furthermore, administration of anti-PSCA mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies suggest PSCA as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-PSCA mAbs for the treatment of local and metastatic prostate cancer.

Cell-mediated cytotoxicity results from, but may not be critical for, primary allograft rejection.

Tumor cells insensitive to lysis through the Fas and TNF pathways were injected either subcutaneously or into the peritoneal cavities of allogeneic perforin-less (P0) and perforin wild-type (P2) mice. In three of four cases, the tumors were rejected equally rapidly in both strains of mice. Rejection was accompanied by vigorous in vitro cytotoxicity in P2, but not in P0 mice. The rapid clearance of allografted cells in mice where all three known cytolytic pathways are seriously compromised raises important questions about the involvement of cell-mediated cytotoxicity, as defined by current assay techniques, in primary allograft rejection.

Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia.

We describe a novel cytoplasmic tyrosine kinase, termed BPK (B cell progenitor kinase), which is expressed in all stages of the B lineage and in myeloid cells. BPK has classic SH1, SH2, and SH3 domains, but lacks myristylation signals and a regulatory phosphorylation site corresponding to tyrosine 527 of c-src. BPK has a long, basic amino-terminal region upstream of the SH3 domain. BPK was evaluated as a candidate for human X-linked agammaglobulinemia (XLA), an inherited immunodeficiency characterized by a severe deficit of B and plasma cells and profound hypogammaglobulinemia. BPK mapped to within 100 kb of a probe defining the polymorphism most closely linked to XLA at DXS178. Reduction in or the absence of BPK mRNA, protein expression, and kinase activity was observed in XLA pre-B and B cell lines. BPK is likely the XLA gene and functions in pathways critical to B cell expansion.

STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors.

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell-cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.