Differentiation of memory T cells to virus plaque-forming cells and cytotoxic T lymphocytes.

The aims of this study were to define the T-cell subpopulation(s) detected by the virus plaque assay, and particularly to determine whether the virus plaque assay could be used to enumerate cytotoxic T lymphocytes. In addition, studies were undertaken to ascertain whether cell proliferation was required for development of cytotoxic effector function and virus plaque formation by these subpopulations. The results of experiments with a secondary mouse mixed lymphocyte culture (MLC) model indicated that 70 percent of virus plaque-forming cells bore the Ly 1 phenotype and 30 percent the Ly 2,3 phenotype. Three lines of evidence suggested that cytotoxic T lymphocytes (CTL) can be detected by this assay: the fact that some virus plaque-forming cells (V-PFC) bear the same Ly phenotype as CTL; the use of an inhibitor of DNA synthesis indicated that proliferating cells could be eliminated with no effect on V-PFC production and cytotoxic activity of the Ly 2,3 cell population; and that infection of primed lymphocyteswith vesicular stomatitis virus before (MLC) stimulation eliminated cytotoxic activity. In primary MLC, development of V-PFC and CTL was completely abolished by cytosine arabinoside. In contrast, in secondary MLC, some CTL and V- PFC were generated by antigenic stimulation in the absence of proliferation. However, the development of both functions became progressively more susceptible to cytosine arabinoside as the time between primary immunization and in vitro boosting is increased. It is suggested that there may be a considerable disparity between the number of existing effector cells at any given time and the cytotoxic potential, i.e. the number of cells capable of being generated by antigenic stimulation.

Separate cytotoxic T lymphocyte subsets recognize the different H-2 specificities.

Using a monolayer adsorption technique, the fine specificity of cytotoxic effector T lymphocytes (CTL) generated against allogeneic or semi-allogeneic H-2 haplotypes was investigated. The results show that: (a) CTL reacting with the private specificity expressed on an H-2.K molecule can be separated from those reacting with the public specificities expressed on the same molecule and (b) the CTL that recognize cross-reacting H-2 determinants (public specificities) can also be separated into several subpopulations. These data support the hypothesis that an allogeneic stimulation induces a large number of independent T cell clones that react with H-2 determinants.

Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA.

U937 cells lacking mitochondrial DNA (rho [symbol: see text] cells) are auxotrophic for uridine and pyruvate, hypersensitive to hypoglycemic conditions, and resistant to antimycin A-induced apoptosis. In spite of their obvious metabolic defects, rho [symbol: see text] cells possess a normal mitochondrial transmembrane potential, as well as near-normal capacity to generate superoxide anion after menadione treatment. Similarly to rho + controls, rho [symbol: see text] cells undergo apoptosis in response to tumor necrosis factor-alpha plus cycloheximide. Detailed comparison of the apoptotic process in rho + and rho [symbol: see text] cells reveals essentially the same sequence of events. In response to tumor necrosis factor/cycloheximide, cells first lose their mitochondrial transmembrane potential (delta psi m) and then manifest late apoptotic alterations, such as generation of reactive oxygen species and DNA fragmentation. Experiments involving isolated mitochondria from rho + and rho [symbol: see text] cells confirm that rho [symbol: see text] mitochondria can be induced to undergo permeability transition, a process thought to account for the pre-apoptotic delta psi m disruption in cells. Like rho + mitochondria, rho [symbol: see text] mitochondria contain a pre-formed soluble factor that is capable of inducing chromatin condensation in isolated nuclei in vitro. This factor is released from mitochondria upon induction of permeability transition by calcium or the specific ligand of the adenine nucleotide translocator atractyloside. In conclusion, it appears that all structures involved in the maintenance and pre-apoptotic disruption of the delta psi m, as well as a mitochondrial apoptotic factor(s), are present in rho [symbol: see text] cells and thus are controlled by the nuclear rather than by the mitochondrial genome. These findings underline the contribution of mitochondria to the apoptotic process.

Abnormally expanded CD8+/Leu-7+ lymphocytes persisting in long-term bone marrow-transplanted patients are resting pre-cytotoxic T-lymphocytes.

We analyzed the functional status of the small CD8+/Leu-7+ T-lymphocytes that circulate in increased proportions in the blood of many allogeneic bone marrow transplant (BMT) patients. Purified CD8+/Leu-7+ T cells were tested for their effect on T-cell proliferative responses. In contrast to CD8+/Leu-7-T-lymphocytes, such cells behaved as suppressor cells for lectin-induced mitogenic responses of the donor's peripheral blood lymphocytes. However, they did not interfere with the in vitro responsiveness to specific stimuli such as protein purified derivative (PPD) or alloantigens. We demonstrate that CD8+/Leu-7+ T cells are resting pre-cytotoxic T-lymphocytes (CTL) that can be induced by mitogenic lectins to express their cytolytic program in a non-specific, non-major histocompatibility complex-restricted manner against phytohemagglutinin-treated lymphoblasts or K562 target cells. The lectin-triggered cytotoxicity was achieved within a few days, together with limited cell division. Our results suggest that circulating CD8+/Leu-7+ T cells from BMT recipients are in vivo primed CTL awaiting cellular activation.

Human growth hormone gene transfer into tumor cells may improve cancer chemotherapy.

Chemotherapy remains the main tool for the treatment of cancers, but is often hampered by tumor cell resistance. In this context, the transfer of genes able to accentuate the effect of anticancer drugs may constitute a useful approach, as exemplified by inactivation of nuclear factor (NF)-kappa B via direct transfer of a gene encoding a negative dominant of its natural inhibitor I kappa B, leading to improved response to cancer chemotherapy. Following our previous report that transfection of human growth hormone (hGH) gene into human monocytic cell lines may also inactivate NF-kappa B in another situation, we decided to test the consequences of hGH gene transfer on cancer treatments. We demonstrated that hGH-transfected human myeloid leukemia U937 cells were sensitized to an apoptotic signal mediated by the anticancer drugs. In parallel, we found that, by inhibiting degradation of I kappa B, hGH gene transfer diminished NF-kappa B entry into the nuclei of U937 cells exposed to daunorubicin. Finally, we report that hGH-transfected tumor cells engrafted in nude mice responded in vivo to chemotherapy with nontoxic doses of daunorubicin whereas, under the same conditions, control tumor cells remained insensitive. Overall, this study therefore suggests that hGH gene transfer may offer new therapeutic prospects in cancer therapy.

Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro.

Specific gene transfer into targeted tumor cells remains a critical issue for the development of systemic gene therapy protocols. With this end in view, we have tested the possibility of selectively directing genes to tumor cells through the recognition of tumor-associated antigens (TAA). This was approached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conjugated to a plasmid DNA conveying a reporter activity. This mAb directed to a TAA that is present on 95% of primary RCCs and on 60% of metastatic human RCCs was extensively characterized, including during clinical trials. Epifluorescence microscopy analysis indicated that upon specific binding to G250 TAA, G250 mAb alone or conjugated to plasmid DNA was internalized by an active endocytic process and colocalized with the transferrin concentrated in the late recycling perinuclear compartment. We also observed that both unconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for > or = 20 hours and were not rapidly translocated to lysosomes or recycled to the plasma membrane. In contrast, unconjugated plasmid DNA was not internalized. After transfection of G250 TAA-positive RCC lines with G250 mAb conjugated to a plasmid cDNA encoding mouse interleukin-2, a significant and sustained production of mouse interleukin-2 protein was detected from days 5-15 and was abrogated by inhibiting the internalization process. Altogether, our data showed that endocytosis of G250 TAA should be the basis of gene transfer to RCC, suggesting that targeting of TAA capable of internalization may be the basis of new approaches for designing alternative cancer gene therapy procedures.

EBV cell-lines (LCL) and E- cells as stimulator cells for limiting dilution analysis (LDA) of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). Comparison of their stimulator capacity and ability to generate specific alloreactivity.

The choice of stimulator cells is crucial in determining the frequency of alloreactive cells by limiting dilution analysis (LDA). In humans, E- cells or EBV-lymphoblastoid cell lines (LCL) are available for this purpose. The E- cells have been mostly used until now, but they appear to stimulate much less than LCL in other models. Consequently, we undertook a systematic comparison of the efficiency of both types of cell for the LDA of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). We show that LCL are stronger stimulator cells both for IL-2 secretion and for cytotoxicity induction. However, high levels of non-allogeneic reactivity were also induced. Therefore, to measure the frequency of specific alloreactive IL-2-SC and CTLp, T cells were depleted of such irrelevant reactive cells by a suicide technique: culture with autologous LCL in the presence of BUDR and use of the remaining live cells as responding effectors in LDA. Using this methodology, no non-allogeneic reactive T cells remain in the responding cells: after restimulation by autologous LCL, no IL-2-SC could be seen and no cytotoxic activity could be observed against autologous, irrelevant or LAK sensitive targets. In contrast, the number of IL-2-SC and CTLp against allogeneic targets was preserved. Therefore with this methodology, the strong stimulator capacity of LCL could be used whereas non-specific activation could be avoided in order to assess the frequency of allo-specific IL-2-SC and CTLp.

Long-term persistence of transferred PPD-reactive T cells after allogeneic bone marrow transplantation.

T cells play an important role in protective immunity against tuberculosis. As patients are not reimmunized with BCG after BMT, the question arises as to whether PPD-specific memory T cells are transferred from the marrow donor to the recipient and persist in the long-term. We studied long-term survivors of non-T cell-depleted allogeneic bone marrow transplantation for in vitro PPD-induced proliferative responses (n = 14), and delayed-type hypersensitivity after intradermal injection of tuberculin (n = 20). We also studied 7 patients who received T cell-depleted bone marrow. Proliferative responses in the first group were low, but were increased by concentrating CD4+ T cells, the major responding cells in this system. In contrast, PBL from patients who received T cell-depleted marrow remained unresponsive to PPD, although they responded normally to CMV antigens. Of the 15 healthy patients in the first group who underwent tuberculin skin tests, 13 had positive reactions, while only two failed to react. (Five patients of the first group were suffering from chronic GVHD and 3 of them were negative.) All the patients in the second group had negative delayed-hypersensitivity responses. The difference between the two groups of patients was highly significant (P < 0.003). These results show that transferred PPD-specific T cells persist in long-term survivors of non-T cell-depleted BMT, even in the absence of reimmunization.

A possible role for specific "anergy" in immunologic hyporeactivity to donor stimulation in human kidney allograft recipients.

The low immunological reactivity toward donor cells usually observed in transplant recipients has been linked to clonal deletion or suppression of alloreactive cells. However, the anergy of donor-specific reactive cells is another possibility not extensively tested until now in humans. In this case, donor-specific reactive cells would be present and eventually be activated without becoming effector cells (i.e., without secreting IL-2 or becoming cytotoxic) after donor-specific stimulation. We studied 8 patients under low-dose immunosuppressive drugs who displayed hyporeactivity toward donor stimulation. IL-2 production, proliferative response, and cytotoxic activity toward donor cell stimulation was decreased (respectively 22, 53, and 19% of response toward third-party stimulation). In order to evidence anergy, we studied two activation markers (cell size increase and expression of IL-2 receptor [CD25]) in allografted recipient T cells after autologous (background), donor (experimental), and third-party cell stimulation (positive control). We showed that the percentage of CD25+ cells and the cell size increase were similar after donor or third-party cell stimulation and clearly above the background as early as days 1-2 after the beginning of the mixed lymphocyte culture. Moreover, CD4+ and CD8+ cells similarly expressed CD25 after donor or third-party stimulation. Thus, donor-specific reactive cells not only were present but could be activated without becoming effector cells. These data suggest that anergy may be an important phenomenon in allograft tolerance.

Decreased lymphokine-activated killer cells in kidney transplant recipients. Correlation with a diminished number of CD3-/NKH1+ cells.

The activity of lymphokine-activated killer cells, measured either by a clonal or polyclonal technique, was assessed in 30 kidney transplant recipients (TX), in 13 hemodialyzed patients (HD-CRI), and in 18 normal (N) controls. A highly significant decrease of the LAK activity in TX in comparison with HD-CRI or N (P = 0.0001) was observed. Moreover, the percentage of CD3-/NKH1+ cells was decreased in TX in comparison with N (P = 0.01). LAK activity was strongly correlated (r = 0.72; P = 0.0001) with the percentage of CD3-/NKH1+ cells and not with that of double-positive CD3+/NKH1+ cells. Multivariate analysis showed that the sole independent variable that determined the LAK activity was the percentage of CD3-/NKH1+ cells: the pathological status (TX, HD-CRI, and N) variable was statistically not significant. On the other hand, two T cell-specific functions (IL-2 secretion and specific cytotoxic activity) were, on the whole, preserved in TX. Altogether, these results suggest that TX are LAK deficient predominantly because they have a decreased number of CD3-/NKH1+ cells. The normality of T cell functions suggests that the high rate of malignancies seen in TX is related to this LAK deficiency. Moreover, our study indicates that, in vivo, CD3+ cells do not significantly contribute to the LAK precursors.

A versatile ELISA-PCR assay for mRNA quantitation from a few cells.

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step: < 0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.

Pattern of cytokine expression in circulation CD57+ T cells from long-term renal allograft recipients.

We made a quantitative analysis of the lymphokine mRNA and of proteins produced by CD57+ and CD57- circulating T cells isolated from long-term kidney-transplanted patients with expanded CD4+/CD57+ and CD8+/CD57+ T cells, and from normal individuals. We concentrated on IL-2 and IFN-gamma, which define a Th1-like type of lymphokine production, and on IL-4 which defines a Th-2-like type. We also analysed the production of IL-10 which is endowed with inhibitory effects on IL-2 and IFN-gamma synthesis, and of TNF-alpha, a pleiotropic inflammatory cytokine. On ionomycin + PMA stimulation, which reveals the intrinsic potential of lymphokine production by T cells, the CD57+ T cell subsets from all individuals produced high amounts of IFN-gamma and TNF-alpha mRNA and protein. They also produced IL-2, but to a much lesser extend than their CD57- counterparts, and little IL-4 and IL-10. They were no more capable of producing IL-2 when stimulated through the CD3/TCR in the presence of monocytes, yet still synthesized IFN-gamma. Our data suggest that the in vivo expansion of CD57+ T cells in stable allograft renal recipients might correspond to Th1 energized cells which on triggering of cell surface receptors hardly secrete lymphokines involved in cell cycle progression, but can still exert some effector functions, including IFN-gamma secretion.

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.