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Proper orchestration of T lymphocyte development is critical, as T cells underlie nearly all responses of the adaptive immune system. Developing thymocytes differentiate in response to environmental cues carried from cell surface receptors to the nucleus, shaping a distinct transcriptional program that defines their developmental outcome. Our recent work has identified a previously undescribed role for the vacuolar ATPase (V-ATPase) in facilitating the development of murine thymocytes progressing toward the CD4 + and CD8 + αβ T cell lineages. Vav1 Cre recombinase-mediated deletion of the a2 isoform of the V-ATPase (a2V) in mouse hematopoietic cells leads to a specific and profound loss of peripheral CD4 + and CD8 + αβ T cells. Utilizing T cell-restricted Lck Cre and CD4 Cre strains, we further traced this deficiency to the thymus and found that a2V plays a cell-intrinsic role throughout intrathymic development. Loss of a2V manifests as a partial obstruction in the double negative stage of T cell development, and later, a near complete failure of positive selection. These data deepen our understanding of the biological mechanisms that orchestrate T cell development and lend credence to the recent focus on V-ATPase as a potential chemotherapeutic target to combat proliferative potential in T cell lymphoblastic leukemias and autoimmune disease.

Human immunodeficiency virus (HIV) infection is characterized by a depletion of T cells. This depletion is caused both by the virus-induced death of infected T cells and by the death of uninfected cells (bystander depletion) by a mechanism which is largely uncharacterized. Regeneration and tolerance factor (RTF) is a subunit of the vacuolar ATPase and a protein that is involved with activation and apoptosis. Anti-RTF antibodies mediate apoptosis in T lymphocytes. When anti-RTF was added to lymphocytes from an HIV-positive individual, they underwent larger amounts of apoptosis than cells taken from healthy controls. When lymphocytes were examined by Western blotting, those from HIV-positive individuals exhibited increased levels of expression of the 50-kDa protein (P < 0.001). A 70-kDa protein was the predominant form of RTF in uninfected control lymphocytes, being expressed in 100% of individuals studied. The expression of the 50-kDa protein in HIV-positive individuals correlated with decreased absolute CD4 counts with a sensitivity of 92% and a positive predictive value of 86%. When uninfected lymphocytes were stimulated with anti-CD3 and anti-CD28, no RTF was detected during early stimulation but a 50-kDa protein was expressed during late stimulation. When the susceptibilities of the lymphocytes to anti-RTF-induced apoptosis were measured, they correlated with the size of the RTF protein expressed. The cells were not susceptible to apoptosis when the 70-kDa RTF was present but were susceptible when the 50-kDa RTF was present. We propose that the increase in the levels of the 50-kDa RTF on cells from HIV-positive individuals is important in preventing the cell from undergoing apoptosis.

Regeneration and tolerance factor (RTF) is a protein with immunosuppressive activity and is normally present in the thymus and placenta. RTF was measured in the livers of patients with regenerating nodules due to alcoholic cirrhosis and hepatitis C. RTF was expressed in the regenerating nodules of 26 patients with alcoholic cirrhosis. All patients with chronic hepatitis C without cirrhosis failed to express RTF. Flow cytometry revealed upregulation of RTF on the lymphocytes from alcoholic cirrhosis and downregulation in hepatitis C disease.

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.

Regeneration and tolerance factor (RTF) is a novel membrane protein that has a diverse expression pattern and immunoregulatory properties. RTF is expressed in vivo on the surface of individuals with B cell chronic lymphocytic leukemia and on activated T lymphocytes of HIV infected individuals as determined by their coexpression with CD38 and HLA-DR. The unique expression patterns of this protein in vivo lead us to investigate its expression in vitro. The activation of human PBMCs through the TCR, using anti-CD3 antibody and PMA, upregulated cell surface expression of RTF from 2. 3% to 91.2% (mean channel fluorescence [MCF] increased threefold). The activation of Jurkat T cells through the TCR upregulated surface expression of RTF from 8.3% (MCF-1.3) to 58.7% (MCF-13.1). The Jurkat T-cell line was used as a model system to explore RTF's role in cellular activation. Using the Jurkat T-cell model, we found anti-RTF antibody induces apoptosis. The addition of anti-RTF antibody increased annexin V binding by threefold compared with the IgG1 kappa isotype control antibody (p < 0.00002) and activated caspase 3. These data indicate that RTF is expressed during T-cell activation and may be associated with apoptosis.

An expression cDNA library was constructed from the helper T cell hybridoma, A.1.1, which has been shown to produce constitutively proteins involved in the down regulation of the immune response. From this library we identified and characterized a cDNA clone, J6B7, by screening with a polyclonal antibody specific for secreted immune regulatory proteins. The mRNA for J6B7 is expressed specifically in some T cells, but not in the thymoma BW5147 or liver cells. J6B7 is 2937 nucleotides in length and contains one open reading frame encoding for a peptide of predicted Mr of 98,042. The nucleotide and deduced amino acid sequences of J6B7 did not reveal significant homology to any published sequences. Hybridization and translation experiments reveal that the J6B7 can hybrid select mRNA from total RNA isolated from either A.1.1 cells or thymic tissue which can be translated in vitro to a peptide which is bound by a monoclonal antibody (mAb) specific for antigenic determinant(s) shared by immune regulatory proteins. Furthermore, the in vitro translated proteins obtained from A.1.1 cells and thymus showed significant suppression of a mixed lymphocyte reaction (MLR) in a dose dependent manner, reaching maximum suppression of 71% and 89%, respectively. These results suggest that the cDNA, J6B7, codes for an immune regulatory protein.

A two color flow cytometry crossmatch (FCXM) was used to evaluate the induction of anti-lymphocyte antibodies in 34 women undergoing immunotherapy for recurrent spontaneous abortions. All women had anti-lymphocyte antibodies that reacted with T-cells when analyzed by FCXM. However, inhibition of the binding of anti-CD3 to paternal CD3 lymphocytes in the presence of maternal antipaternal lymphocyte antiserum was found for some couples following lymphocyte immunotherapy for spontaneous recurrent abortions. Ten couples who had another spontaneous abortion following immunotherapy showed inhibition. In contrast, eight couples who did not show inhibition of the binding of anti-CD3 T lymphocytes to paternal lymphocytes by maternal anti-lymphocyte antiserum had live births. Women of the remaining 16 couples were either pregnant and awaiting birth or not pregnant. Thus, by FCXM it may be possible to predict those couples who will have successful pregnancies following this treatment.

T lymphocyte antigen binding molecules are distinct from B cell immunoglobulins. These proteins were produced by an antigen-specific T cell hybrid as monoclonal products and these cells were successfully grown as an ascites. The T cell antigen binding proteins in ascites were harvested and purified in milligram amounts. The procedure we describe should provide a valuable source of large quantities of pure T lymphocyte antigen binding molecules. This method will allow for studies into their mechanisms of function in regulation of the immune response, biochemistry and molecular biology as B cell myelomas and monoclones provided for the study of immunoglobulins.

Cell surface-radioiodinated proteins of a murine T-cell hybrid specific for and able to bind azobenzenearsonate were isolated by adsorption to Sepharose beads conjugated with a rabbit antiserum to murine T-cell antigen-binding molecules. These isolated proteins, Mr 72,000, were found to bind specifically azobenzenearsonate while proteins isolated in this manner from the tumor parent BW5147 did not bind azobenzenearsonate. Similar cell surface proteins were isolated by affinity for antigen and immunoprecipitated with an antiserum to T-cell antigen-binding molecules. The results suggest that antigen-binding T cells express T-cell antigen-binding molecules as membrane receptors for antigen.

The T-cell hybridoma 51H7D specifically binds the hapten azobenzenearsonate (ABA). An antiserum (anti-PCIF) specific for antigenic determinants shared by antigen-binding molecules of T cells (TABM) was used to precipitate polyribosomes containing mRNA for TABM from this T-cell hybridoma. The resultant mRNA was translated in vitro. The translated product (TrP51H7D) bound specifically ABA and was bound by both anti-PCIF and anti-T-cell suppressor factor. The parent lymphoma BW5147 yielded a similar translated product with the same antiserum used to isolate specific mRNA containing polysomes. This product (TrPBW) was bound by the antiserum used but did not bind ABA. The specific translated protein from both cells had a pI of approximately equal to 5.0, and apparent molecular weight of 71,000 (reduced) or 145,000 (nonreduced). Both protein products, when treated with guanidine to break down all noncovalent bonds, revealed an elemental peptide of Mr 23,500. The cDNA made from the isolated mRNA had 600-900 bases. mRNA of this size is expected for a protein of Mr 25,000. Our data indicate that a TABM specific for ABA is composed of peptides of Mr 23,500.

T-cell antigen binding molecules (TABM) specific for trinitrophenol (TNP), oxazalone, azobenzenearsonate or sheep erythrocytes were purified by affinity to antigen, adsorption to monoclonal antibodies to antigen binding molecules or were synthesized by translation of immunopurified mRNA for TABM in vitro. These molecules and a T-cell line, BW5147, membrane protein bound by rabbit antibodies to TABM were radiolabeled by 125I, digested with Staphylococcus V8 protease, and peptides of the proteolytic digest were resolved by 2D-gel peptide mapping. Comparison of the peptide maps of these proteins and amino acid analysis of T-cell antigen binding molecules specific for TNP or sheep erythrocytes indicate similarities and distinctions suggesting variable and constant domains in these molecules.