A virus-specific CD4+ cell-mediated cytolytic activity revealed by CD8+ cell elimination regularly develops in uncloned human antiviral cell lines

Antiviral HLA class II-restricted cytotoxic CD4+ clones have been relatively well characterized in vitro but their significance in the immune response remains unknown. Here anti-influenza A and anti-EBV CD4+ CTL have been studied by using permanent cell lines either untreated or depleted of CD8+ cells. In bulk cultures, HLA class I-restricted anti-viral CD8+ CTL account for all of the detectable killer cell activity, whereas after elimination of CD8+ cells an HLA class II-restricted killer activity mediated by CD4+/2H4-/4B4+ cells was consistently observed. The CD4+ CTL were fully differentiated in all of the cultures tested from the third in vitro passage because they could be demonstrated immediately after elimination of CD8+ cells. These CD4+ killer cells were equivalent to the CD8+ cells in terms of their lytic capacity. The absence of any class II-restricted antiviral activity in bulk cultures seems to be related to the very small numbers of CD4+ cells present in these antiviral cell lines. However, CD4+ cytolytic activity could not be detected during the first two in vitro passages, even when limiting dilution analysis of the CTL precursors were performed, showing that the killer function of Th cells differentiate only after several in vitro stimulations.     

A previously unrecognized large fraction of cytotoxic lymphocyte precursors is present in CD4+ human peripheral blood T cells

We describe a limiting dilution (LD) culture system in which cell sorter-purified CD4+ (and CD8+) peripheral blood T cells are cocultured with irradiated, anti-CD3 mab-producing OKT3 hybridoma cells. Under these conditions, one out of 2-3 CD4+ (and CD8+) T cells is induced to clonal proliferation. In striking contrast to previously described LD culture systems, every growing CD4+ cell clone displayed cytotoxic activity when tested in a lectin-facilitated 51Cr release assay against P815 target cells. This contrasts with the development of cytotoxic CD4+ T cells in alloantigen-stimulated LD cultures, where only one out of 15-20 proliferating CD4+ cells killed P815 in the presence of PHA, and one out of 300-500 proliferating CD4+ cells displayed alloantigen-specific cytotoxic activity. Furthermore, we have established antigen-specific proliferating CD4+ T cell clones which do not exert antigen-specific cytotoxicity but can be cytotoxic when crosslinked to target cells via lectin or monoclonal antibody (anti-CD3, anti-TCR). Our results show that a previously unrecognized large fraction (at least 30-50%) of all peripheral blood CD4+ T cells can give rise to cytotoxic effector cells. The mode of CD4+ T cell activation (OKT3 hybridoma versus alloantigen) thus determines whether the intrinsic cytotoxic capacity of CD4+ T cells is functionally activated or not.     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.     

Human cytotoxic lymphocytes. IV. Frequency and clonal specificity of CD8+CD16-(Leu2+Leu11-) and CD16+CD3-(Leu11+Leu4-) cytotoxic lymphocyte precursors activated by alloantigen or K562 stimulator cells

Cell sorter-purified CD8+CD16- (Leu2+Leu11-) cytotoxic T cell precursors and CD16+CD3-(Leu11+Leu4-) natural killer (NK) cells were cultured under limiting dilution (LD) conditions with allogeneic stimulator cells or with K562 tumor cells in the presence of exogenous interleukin 2. One out of 100-200 alloantigen-stimulated Leu2+ T cells clonally developed into an alloantigen-specific cytotoxic T cell, but only 1 out of 500-3400 of these cells lysed NK-susceptible K562 target cells. In contrast, 1 out of 2-35 alloantigen-stimulated Leu11+ precursor cells developed into an effector cell that lysed K562, but less than 1 out of 500 of these cells lysed allogeneic Con A blast targets. However, clonal activation of Leu11+ precursor cells under LD conditions did not require alloantigenic stimulator cells. Comparable high frequencies (f = 1/3 to 1/28) of anti-K562 cytotoxic lymphocyte precursors were thus measured when Leu11+ precursor cells were cultured on autologous or K562 feeder cells. As shown by a split culture approach, the vast majority of alloantigen-activated Leu2+ effector cells were highly specific for the stimulating alloantigen (i.e., they did not lyse K562), while the majority of Leu11+ microcultures lysed K562 tumor cells but neither autologous nor allogeneic Con A blast targets. On a quantitative basis, these data show that CD8+CD16- T cells and CD16+CD3-NK cells are two mutually exclusive lymphocyte populations which clonally develop into cytotoxic effector cells specific for alloantigen or K562 target cells, respectively.     

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An assay which measures the secretion of two lymphokines, granulocyte-macrophage colony-stimulating factor and interleukin 3 (IL-3), by single T cells activated with an anti-TCR antibody, F23.1, was used to analyze the effects of anti-CD4 antibodies on antigen-independent T cell activation. Single cells of a CD4+F23.1+ clone were micromanipulated into wells to which F23.1 had been immobilized, and their lymphokine secretion was measured 24 hr later. The frequency of lymphokine-secreting cells was consistently reduced up to 10-fold in the presence of soluble anti-CD4 antibody (GK1.5) but only up to 2.5-fold by an antibody to the cell adhesion molecule, LFA-1. In both bulk and single-cell cultures, responses to suboptimal concentrations of F23.1 were more susceptible to inhibition by GK1.5 than responses to optimal F23.1. The failure of GK1.5 to inhibit IL-2-stimulated lymphokine synthesis in bulk cultures suggested that CD4 ligation did not deliver a negative signal to the clone. By contrast, when either anti-CD4 or anti-LFA-1 was immobilized on the same surface as F23.1, the frequency of lymphokine-secreting cells could be increased up to 10-fold. It is concluded that anti-CD4 antibodies can act directly on the responding T cell to affect TCR-dependent activation, in the absence of interaction with antigen-presenting cells or any other cell type.     

Transfection of dendritic cells with RNA induces CD4- and CD8-mediated T cell immunity against breast carcinomas and reveals the immunodominance of presented T cell epitopes

Transfection of dendritic cells (DC) with tumor-derived RNA has recently been shown to elicit tumor-specific CTL capable of recognizing and lysing a variety of tumor cells. In our study we analyzed the induction of HLA class I- and II-restricted T cell responses against MCF-7 breast cancer cells. Using this approach we were able to elicit CD4- and CD8-mediated antitumor responses. The CTL specifically lysed MCF-7 cells and DC electroporated with MCF-7 RNA, but spared control cell lines. The specificity of the cytotoxic activity was confirmed in cold target inhibition assays and using mAbs blocking HLA class I molecules. Interestingly, these polyclonal cytotoxic T cells recognized selectively two epitopes derived from the MUC1 and Her-2/neu tumor Ags. The induced Th cells were found to be entirely HLA class II restricted and showed a significant cross-reactivity to a renal cell carcinoma cell line, similar to the results obtained with cytotoxic T cells.     

4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens

In contrast to general findings that mouse and human cytotoxic T lymphocytes (CTL) are restricted in cytotoxic activity by major histocompatibility complex (MHC) class I antigens, we previously found that some herpes simplex virus (HSV) type I-infected cells that shared no HLA class I antigens with the HSV-1-stimulated lymphocytes were lysed. In this study, we addressed the question of the role of HLA antigens in human T cell-mediated lysis of HSV-1-infected cells by generating clones of HSV-1-directed CTL from two HSV-1-seropositive individuals. CTL clones that lysed autologous HSV-1-infected lymphoblastoid cell lines (LCL), but not natural killer-sensitive K562 cells or uninfected or influenza virus-infected LCL, were tested for cytotoxicity against a panel of allogeneic HSV-1-infected LCL. Clone KL-35 from individual KL lysed only HSV-1-infected LCL sharing the HLA class II MB1 antigen with KL. With all four CTL clones isolated from individual PM, only HSV-1-infected LCL sharing DR1 with PM were lysed. Monoclonal antibody s3/4 (directed against MB1 ), but not TS1/16 or B33 .1 (directed against a DR framework determinant), blocked lysis of autologous HSV-1-infected cells by KL-35. In contrast, B33 .1, but not s3/4, blocked lysis of autologous HSV-1-infected cells by the PM CTL clones but not by KL-35. Together, these results indicate that our five human CTL clones which are directed against HSV-1-infected cells, and which are all OKT3+, OKT4+, OKT8-, are restricted in lytic activity by HLA class II MB and DR antigens. These results suggest that the HLA D region-encoded class II antigens may be important in the recognition and destruction of virus-infected cells by human CTL.     

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.     

Maximum immunobioactivity of murine small intestinal intraepithelial lymphocytes resides in a subpopulation of CD43+ T cells

CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs-including some but not all of the TCRalphabeta and TCRgammadelta cells-expressed the CD43 S7(-) reactive determinant. CD43 S7(+) IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-gamma following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7(-) IELs. S7(+) but not S7(-) IELs from the ileum of IL-10(-/-) mice spontaneously produced IFN-gamma. In vivo BrdU uptake by IELs in non-Ag-primed mice was greatest in the S7(+) population, indicating that significantly more S7(+) IELs than S7(-) IELs undergo cell expansion under normal homeostatic conditions. DNA microarray analyses showed that S7(+) IELs expressed higher levels of genes associated with activated T cells, whereas S7(-) IELs expressed genes used in the regulation of NK cells. These findings define two functionally distinct populations of IELs based on CD43 expression independent of TCR class, and they identify a subset of IELs that may serve as a target to better control intestinal inflammation.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.     

Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction

Lymphokine secretion by in vivo-activated T cells was analyzed at the population and single-cell levels in lymphocytes from mice undergoing an acute allogeneic graft-vs-host reaction (GVHR). Three observations were made. First, constitutive lymphokine production by these cells was very low but could be dramatically up-regulated by TCR ligation. Thus, even when harvested at the peak of the GVHR, fewer than 0.1% of lymphocytes secreted detectable granulocyte-macrophage (GM)-CSF, IFN-gamma, or IL-3 in the first 24 h in vitro, and average production of these lymphokines in bulk cultures was less than 10(-5) U/cell. However, when cultured for 24 h with anti-CD3 antibody under conditions which activated less than 0.1% of normal cells, about 30% of GVHR T cells secreted GM-CSF, IFN-gamma, and/or IL-3, and average production levels were increased by 10(3)- to 10(4)-fold. Together with evidence that host alloantigen-induced lymphokine secretion was 10 to 100 times lower than the anti-CD3 response, these data suggest that physiologic lymphokine synthesis by most T cells is low (less than 10(-18) mol of IL-3 per cell) but can be raised above the threshold of detection by TCR cross-linking. Second, individual GVHR lymphocytes varied markedly in their total and relative production of different lymphokines in response to anti-CD3 stimulation, with some cells secreting IL-3 alone, some secreting IL-3 accompanied by other lymphokines (GM-CSF and/or IFN-gamma), and some secreting other lymphokines without detectable IL-3. Finally, both CD4+ and CD8+ T cells from GVHR mice responded to anti-CD3 antibody by secreting IL-3 and other lymphokines: purified CD4+ cells contained an average of 16% and CD8+ cells an average of 10% anti-CD3-inducible lymphokine-secreting cells. By contrast, only 2 to 3% of cells of either subset formed clones in cultures with host allogeneic cells and IL-2, suggesting that clonogenic alloreactive cells were a minority of the T cells activated in the GVHR.     

Analysis of the expression of CD5 by human B cells and correlation with functional activity.

B cells expressing the CD5 marker in the mouse have been suggested to be a separate lineage and a major source of autoantibody production. In man, this relationship is less clear. Studies were therefore undertaken to determine whether human CD5+ B cells represent a distinct lineage of cells that differ in patterns of antibody production from CD5- B cells. In normal B cell populations, CD5 was expressed by a mean of 24.0 +/- 2.8% (n = 10) of CD20+ B cells. Of note, an increased frequency of CD5+ B cells was not found in patients with systemic lupus erythematosus (mean of 17.9 +/- 2.8%, n = 16). Analyzing CD5+ B cells for cell membrane Ig isotype expression demonstrated similar frequencies of IgG and IgA expressing cells as were found on the CD5- B cell population, although the frequency of IgM+ cells was slightly increased. Incubation of CD20+ B cells with phorbol myristate acetate (PMA) for 72 hr increased the frequency of CD5 expressing B cells by more than threefold. CD5 expression was also increased by coculture with anti-CD3-activated T cells and most markedly by simultaneous stimulation with both PMA- and anti-CD3-activated T cells (greater than 50% positive). Analysis of CD5- B cells clearly indicated that stimulation with PMA or anti-CD3-activated T cells induced the majority to become CD5+ transiently. Functional analysis of Ig production by CD5+ and CD5- B cells stimulated with anti-CD3-activated T cells indicated that both populations in normals produced IgM and a variety of autoantibodies in comparable amounts, whereas the CD5- B cells produced greater quantities of IgG. B cells were activated with anti-CD3-stimulated T cells followed by separation into CD5+ and CD5- populations. The largest amount of Ig was produced by CD5- B cells that were induced to express CD5, although all populations produced some Ig. These data suggest that CD5 behaves as an activation marker on human B cells rather than as a marker for a distinct lineage of cells. Moreover, CD5 expression does not appear to identify a population of resting B cells with a greater capacity to produce antibodies to DNA or other autoantibodies.     

Comparative analysis of CD4-mediated down-regulation of T cell adhesion to B cells by flow cytometry and fluorescence microscopy

We have previously shown by means of fluorescence microscopy that antigen-independent adhesion of resting CD4 T cells to EBV-transformed B cells can be down-regulated by ligand interaction with CD4. In this study we used flow cytometry analysis of conjugate formation to confirm these findings. No conjugates between resting CD4 + T cells and B cells were initially detected in the latter method, because flow velocity in the flow chamber induces hydrodynamic elongation forces which disrupt low-affinity conjugates. After forcing cell conjugation by low-speed centrifugation of T and B cells, conjugates became detectable although in smaller numbers than in fluorescence microscopy. "Forced" cell conjugates had similar characteristics to their unforced counterparts, i.e., 37 degrees C temperature dependency, mediation by LFA-1/ICAM-1 and CD2/LFA-3 pathways, and transiency. The latter characteristic was at least partly mediated by CD4/HLA class II interaction, since adhesion of CD4 + T cells to HLA class II-B cells was more stable. In addition, adhesion was inhibited by anti-CD4 antibodies but not by an HLA DR-derived peptide known to inhibit unforced CD4 + T cell adhesion to B cells. This blocking effect was partially reproduced by reducing the centrifugation time prior to the adhesion assay. These results show that a) CD4-mediated down-regulation of T cell adhesion can be observed by means of two different techniques, and b) analysis of cell-cell adhesion after increasing centrifugation times (and possibly speeds) is a simple way of measuring adhesion forces semiquantitatively.     

Functional CD8<Superscript>+</Superscript> T cells infiltrate into nonsmall cell lung carcinoma

Infiltration of CD3(+)CD8(+) cytotoxic T cells was analyzed by multiparameter confocal laser microscopy in a panel of 16 randomly selected stage I nonsmall cell lung carcinomas. T-cell infiltration was observed in the stroma (range 57-2,093 T cells/mm(2)) but also in the tumor epithelium (range 21-892 T cells/mm(2)) and showed wide variation between individual tumors. Interestingly, a significantly higher percentage of CD3(+)CD8(+) T cells was detected in the tumor epithelium compared to the stroma illustrating that cytotoxic T cells may preferentially migrate into tumor epithelium. Aberrant HLA class I antigen expression was observed in 69% of the nonsmall-cell lung carcinoma (NSCLC) tumors. One tumor of a squamous cell lung carcinoma patient with the highest number of tumor infiltrating CD3(+) and CD3(+)CD8(+) cells was studied in detail and the majority (90%) of these cells were shown to be functionally activated granzyme B-positive cytotoxic T cells. DNA oligotyping of a lung carcinoma cell line established from this tumor revealed loss of one HLA haplotype corresponding with a translocation involving chromosome 6, as observed by COBRA-FISH. HLA class I-restricted tumor specific T cells could be isolated from PBMC. One further characterized cytotoxic CD8(+) T cell clone, that released TNF-alpha, IFN-gamma, and granzyme B upon co-incubation with the autologous tumor cells, was shown to be restricted by the remaining HLA-A11 allele, which was also shown to be expressed in the tumor tissue. Our data indicate that, despite HLA-haplotype loss a vigorous antitumor immune response mediated by CD8(+ )T-cells can be present in NSCLC offering possibilities for specific immunotherapy.     

CD4 and CD7 molecules as targets for drug delivery from antibody bearing liposomes.

We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation of d-[3H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4-targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.