Autologous B-cell Lines Research Articles

Retroviral transduction of IL2RG into CD34+ cells from X-linked severe combined immunodeficiency patients permits human T- and B-cell development in sheep chimeras

AbstractX-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain of cytokine receptors, γc. Because bone marrow transplantation (BMT) for XSCID does not provide complete immune reconstitution for many patients and because of the natural selective advantage conferred on lymphoid progenitors by the expression of normal γc, XSCID is a good candidate disease for therapeutic retroviral gene transfer to hematopoietic stem cells. We studied XSCID patients who have persistent defects in B-cell and/or combined B- and T-cell function despite having received T cell–depleted haploidentical BMT. We compared transduction of autologous B-cell lines and granulocyte colony-stimulating factor–mobilized peripheral CD34+ cells from these patients using an MFGS retrovirus vector containing the γc gene IL2RG pseudotyped with amphotropic, gibbon ape leukemia virus, or RD114 envelopes. Transduced B-cell lines and peripheral CD34+ cells demonstrated provirus integration and new cell-surface γc expression. The chimeric sheep model was exploited to test development of XSCID CD34+ cells into mature myeloid and lymphoid lineages. Transduced and untransduced XSCID CD34+ cells injected into developing sheep fetuses gave rise to myeloid cells. However, only transduced γc+ progenitors from XSCID patients developed into T and B cells. These results suggest that gene transfer to autologous peripheral CD34+ cells using MFGS-gc retrovirus may benefit XSCID patients with persistent T- and B-cell deficits despite prior BMT.

Human autoreactive (Th0) CD4 + T-cell clones with cytolytic activity recognizing autologous activated T cells as the target

In attempt to obtain a clue to understanding possible physiological roles played by autoreactive T cells, autoreactive T-cell clones originally derived from an allogeneic mixed lymphocyte culture have been analyzed for their target spectrum, lytic function and cytokine profiles. Five CD4 + T-cell clones established from allogeneic MLR, in which the stimulator cells shared certain class II MHC antigens with the responder, turned out to be reactive to autologous PBL. Among these, three clones were cytolytic against autologous B-cell line. These three cytolytic autoreactive clones were shown to be capable of specifically lysing autologous activated T cells expressing class II MHC molecules, raising possibility that such autoreactive clones might play a role in negatively regulating T cell responses. Cytolysis by an autoreactive clone 21C5 was inhibited completely by concanamycin A (CMA) known as a specific inhibitor of perforin, suggesting an involvement of the perforin/granzyme system. T-cell clones derived from the same MLC showed distinct correlation between their specificity and lymphokine profiles. Thus, the three cytolytic autoreactive clones belonged to Th0, whereas the two noncytolytic autoreactive clones belonged to Th2 and three alloreactive CD4 + clones derived from the same culture were of Th1 type.

Reactivity patterns of synovial T-cell lines derived from a patient with rheumatoid arthritis. I. Reactions with defined antigens and auto-antigens suggest the existence of multireactive T-cell clones.

The functional T-cell repertoire in the inflamed joint of a patient with rheumatoid arthritis was analysed at the clonal level. Using limiting dilution techniques and selecting for growth of in vivo activated and/or autoreactive T cells, 149 T-cell lines were established. They were tested in a proliferation assay for reactivity against an autologous Epstein-Barr virus (EBV)-transformed B-cell line and a panel of auto-antigens and foreign antigens. Seventy-five lines (approximately 50%) could be stimulated. Thirty-six lines (approximately 24%) were antigen-reactive. They were stimulated by human collagens type I (15), II (10), IV (7) or V (4), cartilage proteoglycans (4), Mycobacterium tuberculosis (15), the 60 kDa heat-shock protein of M. bovis (13) or tetanus toxoid (10). T-cell lines were either monoreactive (19), bireactive (6), or multireactive (11), i.e. they were stimulated by either one, by two, or by more antigens in the panel. About half of the antigen-reactive lines were at the same time autoreactive towards the autologous B-cell line. These data suggest the existence of multispecific autoreactive T-cell receptors comparable to multireactive or natural autoantibodies and prove the presence of autoantigen-reactive T cells in the inflamed joints of patients with rheumatoid arthritis.

HIV-2-specific cytotoxic T-lymphocyte activity is inversely related to proviral load.

To characterize HIV-specific cytotoxic T-lymphocyte (CTL) activities in HIV-2-infected individuals and to relate these to HIV-2 proviral load. Peripheral blood mononuclear cells were collected from 16 HIV-2-seropositive and four HIV-1/2 dually seropositive subjects. CTL were restimulated with autologous phytohaemagglutinin-stimulated blasts and CTL activities in 'bulk' cultures were evaluated 7 and 14 days later by a standard 51Cr-release assay using autologous B-cell lines infected with recombinant vaccinia expressing HIV-2 Gag, Pol or Nef protein. Proviral load was quantified by polymerase chain reaction (PCR) which used HIV-2 long terminal repeat primers and an external standard control made by an HIV-2CBL-22 chronically infected C8166 cell line. A biotinylated primer was used to capture the 35S dATP-incorporated secondary PCR product in a quantitative radiometric assay. After 14 days of culture CTL responses against Gag or Pol protein were seen in 18 (90.0%) and 14 (70.0%) out of 20 subjects, respectively, whereas a CTL response was noted against Nef protein in five (25.0%) out of 20 subjects. In 14 (70.0%) out of 20 subjects multiple HIV proteins were simultaneously recognized. The sum of specific lysis (%) against HIV-2 Gag, Pol and Nef at 30:1 effector-to-target ratio, or specific lysis of the dominant CTL response, correlated strongly with HIV-2 proviral load expressed as copies per 10(5) CD4+ cells (r = -0.625, P = 0.003 and r = -0.674, P = 0.001, respectively). HIV-2-specific CTL to multiple gene products was demonstrated in most HIV-2-infected individuals. An inverse correlation between the level of CTL activity and proviral load was found, which supports the hypothesis that CTL are important in the control of HIV-2 replication.

CD8high (CD57+) T cells in normal, healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein-Barr virus-transformed B cell lines.

We have previously identified two subsets of CD8+, CD57+ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8high(CD57+)] and ii) natural killer cells expressing low levels of surface CD8[CD8low(CD57+)]. We investigated the cytotoxic and suppressive function of CD8high(CD57+) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8high(CD57+) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8high(CD57+) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8high(CD57+) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8high(CD57+) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8high(CD57+) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8high(CD57+) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8+ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.

Autologous tumor-specific cytotoxic T-lymphocytes in a child with neuroblastoma

Activation of peripheral blood lymphocytes from a neuroblastoma patient by co-cultivation with autologous neuroblastoma cells in a mixed lymphocyte-tumor cell culture (A-MLTC) resulted in the generation of cytotoxic activity against the autologous neuroblastoma cell line HNB-MS. A-MLTC was set up in the presence of recombinant human interleukin-2 (IL-2). HNB-MS stimulator was treated with recombinant human interferon-γ (IFN-γ) prior to A-MLTC. CTL generated in short-term culture effectively lysed HNE-MS, while they had no effect on an Epstein-Barr virus transformed autologous B-cell line EB-MS. Moreover, CTL lysed 3 different allogeneic neuroblastoma cell lines, but not a rhabdomyosarcoma cell line RBB. Recombinant human tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) enhanced and suppressed CTL generation, respectively, when added to the A-MLTC from the beginning of culture. CD3 + CD4 − CD8 + T cells were the major anti-tumor effectors. Furthermore, 111indium-labeled CTL clearly accumulated in metastatic sites. These results indicate that CTL can be used for adoptive immunotherapy in neuroblastoma. neuroblastoma-specifie CTL / IL-2 receptor

Human cytotoxic T‐cell recognition of a synthetic peptide of myelin basic protein

Previous studies with a panel of myelin basic protein (MBP)-specific human T-cell clones suggested a clustering of epitopes in the middle and at the C terminus of the molecule. The current study demonstrates that 19 of 40 clones recognize a synthetic peptide corresponding to residues 152 to 170 of the human MBP molecule and that 9 clones recognize a synthetic peptide corresponding to residues 86 to 105. Myelin basic protein-specific cytotoxic activity was restricted to the clones that recognized peptide 152-170, and this peptide served as a preferential cytotoxic T-cell target when attached to an autologous B-cell line. The specificity of MBP-directed cytotoxic activity appears to be much more restricted than the specificity demonstrated for proliferative activity.

HLA class II-restricted presentation of cytoplasmic measles virus antigens to cytotoxic T cells.

To analyze the nature of the HLA class II-restricted cytotoxic T-lymphocyte (CTL) response to measles virus, murine fibroblasts were transfected with expressible cDNA clones for human HLA-DR antigen and for measles virus matrix or nucleocapsid proteins. DR-positive murine fibroblasts transfected with measles virus matrix or nucleocapsid genes were lysed by class II-restricted measles virus-specific CTL lines. Lysis was as efficient as with infected autologous B-cell lines, even though the measles virus cytoplasmic proteins were undetectable by antibodies in the transfected target cells. These results demonstrate that cytoplasmic viral antigens can be presented to CTL in the context of HLA class II antigens and that measles virus matrix and nucleocapsid proteins contribute to class II-restricted measles virus-specific CTL responses. These results also show that endogenously synthesized measles virus proteins can be efficiently presented by class II antigens. The implications of these findings for measles virus pathogenesis and for antigen processing are discussed.

Different signals for stimulation of proliferation and lymphokine secretion by a CD3 + WT31 − cloned cytotoxic lymphocyte

CD3 + WT31 − T cells were sorted from peripheral blood of a normal healthy donor by a FACS IV and cloned by limiting dilution in the presence of a phorbol ester (tetradecanoyl phorbol acetate, TPA), calcium ionophore (ionomycin, Io), interleukin-2 (IL-2), allogeneic cells, and phytohemagglutinin (PHA). One of the derived clones, 290-2, was investigated in detail. 290-2 mediated strong natural killer (NK) but not lymphokine-activated killer (LAK) activity. It proliferated in the presence of IL-2 but not IL-4. It carried the surface phenotype CD3 + WT31 − CD4 weak+ CD8 −, CD16 −, and Leu 19 +. Expression of CD4 was heterogeneous within the clone, since two of three subclones were also CD4 weak+ but one was CD4 −. NK activity was blocked by monoclonal antibody (moAb) to CD11a (LFA1), but not by monoclonal antibody (moAb) to either CD3 or CD4. Northern blotting revealed T-cell receptor (TCR-γ) but not α- or full-length β-chain mRNA. 290-2 proliferated autonomously when stimulated with a combination of TPA + Io, with PHA or CD3 moAb and autologous B-cell lines (B-LCL) (and this was inhibited by an anti-IL-2 receptor moAb), but not to allogeneic B-LCL or any of the other stimulating agents alone. Unexpectedly, the TPA + Io stimulus which resulted in maximal proliferative responses did not trigger interferon-γ or granulocyte/macrophage colony-stimulating factor production, although both lymphokines were secreted in the presence of B-LCL + TPA + Io. Proliferative responses were not enhanced by the presence of B-LCL. Thus, activation signals sufficient for autocrine proliferative responses were insufficient for secretion of other lymphokines. Such clones will provide valuable reagents for investigating the biology of the TCR-γ + T cell.

Antigen presentation to HLA class II-restricted measles virus-specific T-cell clones can occur in the absence of the invariant chain.

A human fibroblast line expressing HLA-DR1 antigen on its surface was generated by transfection with DR alpha and DR beta cDNAs. Expression of the invariant chain gene was not detectable in the transfected fibroblasts and was not induced by infection with measles virus. Lysis of measles virus-infected cells occurred with DR1- but not with DR4-restricted measles virus-specific cytotoxic T-lymphocyte (CTL) clones and was inhibited by a monoclonal antibody specific for DR antigen. Therefore, the invariant chain is not required for DR-restricted presentation of measles virus antigens by this fibroblast line. Transfected fibroblasts were lysed as efficiently as an autologous B-cell line even though they expressed much less surface DR antigen. Lysis of both the transfected fibroblasts and the B-cell line was insensitive to treatment with chloroquine. These results demonstrate that expression of a DR alpha beta heterodimer at the surface of this fibroblast line is necessary and sufficient for presentation of measles virus antigens to specific CTL clones.

Generation and specificity of a heteroantiserum to human peripheral blood monocytes

An antiserum to human monocytes was raised in a rabbit by immunization with adherent mononuclear cells. After absorption with AB erythrocytes and an autologous B-cell line, the specificity of the antiserum was studied by complement-mediated lysis and direct fluorescence on a Fluorescence-Activated Cell Sorter (FACS). No reactivity was found with fresh B and T lymphocytes or T- and B-cell lines. More than 75% of adherent mononuclear cells were reactive with the antiserum. A mean of 26% of normal donors' peripheral blood mononuclear cells were labeled by direct immunofluorescence. Most of the large cells defined by light scatter profile on the FACS were stained by the antiserum but a fraction of cells of a smaller size was also positive. Morphologic and functional studies confirmed that the antiserum was specific for monocytes.

Ia determinants on stimulated human T lymphocytes. Occurrence on mitogen- and antigen-activated T cells.

Human T-cell blasts were generated by stimulation with mitogens and antigens. A proportion of these blasts expressed Ia antigens detectable by immunofluorescence with both allo- and hetero-antiserums. The maximal expression of Ia antigens was delayed and usually occurred after the peak of blastogenesis. Among the three mitogens used, pokeweed mitogen (PWM) was most effective in giving a high percentage and intense Ia staining of T-cell blasts. Phytohemagglutinin and concanavalin A blasts gave weaker and lower percentages of Ia staining. Activation by alloantigens and soluble antigens such as tetanus toxoid and purified protein derivative resulted in Ia expression on T cells comparable to PWM stimulation. Depletion of Ia+ cells from freshly isolated T cells with anti-Ia and complement decreased subsequent Ia expression, suggesting that a proportion of Ia+ blasts were derived from Ia-bearing peripheral blood T cells. When the specificities of the Ia antigens on T-cell blasts were examined with alloantiserums, it was evident that the T blasts expressed similar HLA-DR determinants to those on B cells from the same donor; occasional minor differences between stimulated T cells and autologous B-cell lines or fresh B cells were encountered.