Anna Senik

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.

DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

In vivo-targeted gene delivery using antibody-based nonviral vector.

Tissue-specific gene transfer remains one of the main challenges to deliver genes into designated and/or disseminated cells. We have previously shown successful gene transfer with a nonviral gene delivery system based on the simple chemical conjugation of plasmid DNA with antibody. However, this approach was hampered by low efficiency due to the poor translocation rate of DNA to the nucleus. To improve this approach, we have modified our vector by introducing noncovalent binding between the antibody and DNA, allowing the possibility to introduce different important molecules. The noncovalent association was achieved with neutravidin and biotinylated components: (1) biotinylated antibodies; (2) a biotinylated hemagglutinin fusogenic peptide of influenza virus to favor endosomal escape; and (3) biotinylated histone H1 to compact, protect, and associate DNA to the complex. We report here that this delivery system can be internalized by tumor cells targeted by a specific monoclonal antibody, permits the protection of the transfected DNA, and allows its subsequent transfer into the nucleus after escape from the endosomal compartment. We also demonstrate that, in vitro, gene transfer with this vector showed much higher reporter activity in cells (15 vs. 0.5%) and a stronger production of murine interleukin 2 as compared with our previous vector. In vivo, a single intravenous injection of the vector containing an antibody directed to the G250 renal cell carcinoma-associated antigen led to beta-galactosidase expression in engrafted tumor bearing G250 but not in G250-negative tumor or in other tissues. Altogether, these results indicate that our antibody-based vector is suitable to promote gene delivery in vitro and in vivo in tumor cells.

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.

Induction of cytolytic function in resting peripheral blood CD8+/Leu-7+ T cells through IL2/p 75 IL2-receptor interaction: a study in the allogeneic human bone marrow transplantation model.

CD8+/Leu-7+ T cells which circulate in increased proportions in the blood of long-term surviving BMT patients are for the most part high-density resting lymphocytes lacking IL2R-alpha (p55) expression. We show that they can be induced by IL2 to manifest cytolytic function after 24-48 hr stimulation by using rather high concentrations of IL2 (at least 50 U/ml). This function was much more readily induced in high-density CD8+/Leu-7+ T cells than in high-density CD8+/Leu-7+ T cells and occurred in the presence of minimal cell proliferation. Other cytokines involved in primary CTL differentiation (IFN-gamma, IL4 and IL6) were without effect suggesting that CD8+/Leu-7+ T cells are, in the BMT model, in vivo preactivated CTL ready to differentiate into cytolytic effectors under the sole IL2 stimulus. TU27 Mab directed at IL2R-beta (p75) subunit almost completely prevented IL2-induced cytolytic function of CD8+ T cells while 33B3.1 Mab directed at IL2R-alpha (p55) subunit was ineffective, suggesting that the signal for this function has its origin in IL2R-beta chains constitutively expressed by these cells.

EQUIVALENT DECREASE OF H‐2Kk AND H‐2Dk EXPRESSION AFTER VACCINIA VIRUS INFECTION

Expression of H‐2Kk and H‐2Dk molecules was studied by indirect immunofluorescence on vaccinia virus‐infected Ls cells (derived from the L929 cell line), using D‐23b and ASA‐21 monospecific alloantisera, respectively directed against the H‐2.23 and H‐2.32 private specificities of the H‐2k haplotype. Our study demonstrates that the increase in vaccinia virus‐induced antigens on the membrane of infected cells (as a function of the multiplicity of infection) is associated with a concomitant decrease in the expression of both H‐2Kk and H‐2Dk serologically defined private specificities. Absorption experiments of D‐23b and ASA‐21 sera, using infected or uninfected Ls cells, also indicate that after virus infection, H‐2Kk and H‐2Dk private specificities are equally altered in their serological expression. We finally show that this alteration consists in a mere quantitative decrease of H‐2 molecules, since the cytolytic capacity of a rabbit anti‐H‐2 serum, specifically reacting against the constant part of the heavy chains of H‐2 molecules, was significantly more absorbed by uninfected cells than by infected cells. We conclude that no preferential decrease in the expression of H‐2 Kk and H‐2 Dk molecules is induced during cell infection by vaccinia virus.

PUBLIC H‐2 SPECIFICITIES ARE TARGET DETERMINANTS FOR ALLOREACTIVE CYTOTOXIC T LYMPHOCYTES

The specificity of he corss‐killing exerted by cytotoxic T lymphocytes (CTLs) generated against H‐2 region products was investigated in a 51Cr release assay on a panel of target cells from a number of different H‐2 haplotypes.

The anti-caspase inhibitor Q-VD-OPH prevents AIDS disease progression in SIV-infected rhesus macaques

Apoptosis has been proposed as a key mechanism responsible for CD4+ T cell depletion and immune dysfunction during HIV infection. We demonstrated that Q-VD-OPH, a caspase inhibitor, inhibits spontaneous and activation-induced death of T cells from SIV-infected rhesus macaques (RMs). When administered during the acute phase of infection, Q-VD-OPH was associated with (a) reduced levels of T cell death, (b) preservation of CD4+/CD8+ T cell ratio in lymphoid organs and in the gut, (c) maintenance of memory CD4+ T cells, and (d) increased specific CD4+ T cell response associated with the expression of cytotoxic molecules. Although therapy was limited to the acute phase of infection, Q-VD-OPH–treated RMs showed lower levels of both viral load and cell-associated SIV DNA as compared with control SIV-infected RMs throughout the chronic phase of infection, and prevented the development of AIDS. Overall, our data demonstrate that Q-VD-OPH injection in SIV-infected RMs may represent an adjunctive therapeutic agent to control HIV infection and delaying disease progression to AIDS.

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.