Heterogeneity of cytolytic and suppressor clones of alloactivated cells generated from soft agar colonies

Long-term cell cultures (or clones) were developed from soft agar colonies of lymphocytes alloactivated in mixed leukocyte culture reaction (MLR). Two types of colonies were identified: upper colonies that grew on the agar surface, and lower colonies found within the agar layer. Virtually all cytolytic clones originated exclusively from the upper colonies. Two groups of cytolytic clones could be distinguished, one with strong and the other with weak proliferation upon restimulation. Upper clones were capable of inhibiting primary MLR proliferation and this appeared to be related to their cytolytic effect on the stimulator. Many noncytolytic lower clones were found to suppress primary MLR cultures. Considerable heterogeneity was apparent from differences in the magnitude of suppression and the ability of the clones themselves to undergo stimulator-induced proliferation. Kinetic studies of MLR suppression were conducted to further analyze this heterogeneity. Two major kinetic patterns were observed. One showed a biphasic proliferation pattern of the MLR + clone culture. The first peak appeared to reflect an enhanced proliferation of the clone. The second phase seemed to represent diminished proliferation of the MLR responder. This type of suppression may be related to T cell growth factor depletion from MLR by the proliferating clone. The other kinetic pattern showed a consistently low proliferation of the MLR + clone culture throughout the 8-day assay period. Subsequent testing of these suppressor clones in third-party MLR cultures suggested that the specificity of suppression was unrelated to HLA-DR, MB, MT, and SB.     

Protection against tuberculosis by passive transfer with T-cell clones recognizing mycobacterial heat-shock protein 65.

We have previously shown that mice vaccinated by injection with J774 macrophage-like tumour cells that expressed Mycobacterium leprae heat-shock protein (hsp) 65 as a transgene had acquired a remarkably high degree of protection against subsequent challenge with virulent M. tuberculosis. We show here that antigen-specific T cells cloned from spleens of such vaccinated animals can transfer a high level of protection to non-vaccinated recipients. The most efficient cells were of T-cell receptor (TCR) alpha beta+ and CD4- CD8+ type and specifically lysed mycobacteria-infected macrophages. These findings are consistent with the importance for protective immunity of engaging the endogenous antigen-presenting pathway to bias the immune response towards a cytolytic action against a mycobacterial antigen that is expressed at the surface of infected macrophages. TCR gamma delta+ and TCR alpha beta+ cells interacted synergistically.     

Karyotypic analysis of murine cytolytic T-cell lines

G-banding techniques have been used to analyze the karyotypes of four independently derived murine cytolytic T lymphocyte (CTL) lines established in culture 2–4 yr earlier. For two of them, several subclones were included in the analysis. The modal arm number of three of the lines was close to diploid, whereas in the fourth line, both near-diploid and near-tetraploid subclones were found. The most frequent type of rearrangement was the formation of metacentrics by centromeric fusion. A comparison of the abnormalities (markers) occurring in the different CTL lines showed the formation of similar chromosomal rearrangements in independent lines, thus suggesting nonrandom chromosomal changes in these cells. Several of the metacentrics are derived from the fusion of normal murine chromosomes that are apparently prone to form such “markers,” as the occurrence of similar abnormalities has been reported in other established cell lines and tumors. On the other hand, some of the biarmed and telocentric marker chromosomes seem to characteristically occur in CTL but not in other types of cell lines.     

Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones.

Experimental studies have suggested that antigen-specific T lymphocytes are important mediators of resistance to infection with the pathogenic fungus Histoplasma capsulation. To gain a better understanding of the role of T lymphocytes, we developed murine T-cell lines and clones that recognized Histoplasma antigens. These T cells were of the helper/inducer phenotype (Thy-1.2+ Lyt-1+ L3T4+ Lyt-2-) and exerted multiple immunological functions. T-cell lines and 12 clones proliferated vigorously in response to histoplasmin; the T-cell lines and 6 clones also were reactive with heterologous fungal antigens prepared from either Blastomyces dermatitidis or Coccidioides immitis. Recognition of antigen by T cells was H-2 restricted; in the absence of antigen, four clones demonstrated alloreactivity. All T-cell clones conferred local delayed-type hypersensitivity responses when injected with antigen into footpads of mice. Ten of 12 T-cell clones released interleukin-2 after stimulation with antigen, and all clones tested secreted interferon. Moreover, culture supernatants from antigen-stimulated clones armed peritoneal macrophages to inhibit intracellular growth of H. capsulatum yeast cells. All clones assayed exerted nonspecific help. Thus, development of T-cell clones should facilitate analysis of the regulatory properties of Histoplasma-specific T cells.     

Erythrocyte-reactive T-cell lines and clones from normal mice recognize serum-derived antigens.

T-cell clones and cell lines which apparently respond to autologous (syngeneic) erythrocytes have been generated from the spleens of normal mice. The response showed considerable cross-reactivity with red blood cells (RBC) from other species, including chicken, and was 'heteroclitic' in that reactivity against some species of RBC, notably rat and monkey, was greater than to mouse. The clones were Thy-1+ L3T4+ Lyt-2- and recognized antigen in association with I-Ak or I-Ek. At least three specificities were identified on the basis of reactivity to crude lysates of mouse and rat RBC. One of the putative clones, M0.5/1/D2, showed a change in reactivity during culture, proliferating strongly against antigen-presenting cells (APC) without added erythrocytes. Analysis of the clones and lines using an I-Ak I-Ek expressing hybridoma, HB-98, has indicated that only one, M5/1/F5, was likely to be erythrocyte-specific; the remainder were responding to antigens present in foetal calf serum (FCS). The data demonstrate that apparent erythrocyte specificity can be a result of serum components being presented to T lymphocytes via red blood cells.     

Investigation of antigen cross-reactivity of Mycobacterium leprae-reactive murine T-cell lines and clones.

Inguinal lymph node lymphocytes from BALB/c mice immunized intradermally with 10(8) 60Co-irradiated Mycobacterium leprae were cloned by limiting dilution culture. In general, cloned T-cell lines exhibited helper type activity producing interleukin-2, macrophage activation factor and gamma-interferon and lines were further characterized in terms of their cross-reactivities with other species of mycobacteria. M. leprae clones derived after a period of in vitro restimulation were found to cross-react with other species of mycobacteria probably recognizing non-specific or closely related common cell wall associated mycobacterial determinants. On the other hand, lines established by cloning directly from immune mice appeared more M. leprae-specific, exhibiting antigen-dependent lymphokine production and proliferation in vitro.     

Bovine naturally cytolytic cell activation against bovine herpes virus type 1-infected cells does not require late viral glycoproteins.

Freshly isolated or overnight cultured bovine peripheral blood mononuclear (PBM) cells lysed bovine herpes virus 1 (BHV-1)-infected allogeneic and xenogeneic target cells but not non-infected target cells. To determine if late viral genes contribute to target cell lysis, phosphonoacetic acid (PAA), an inhibitor of DNA polymerase activity, was used to block DNA replication that is required for expression of late viral proteins. Both adherent and non-adherent (NA) cell populations mediated lysis against PAA-treated BHV-1-infected target cells in both 4- and 20-hr assays, indicating recognition and killing occurred in the absence of expression of late BHV-1 glycoproteins. Thus recognition of BHV-1 by bovine natural cytolytic effector cells does not require recognition of late BHV-1 glycoproteins for killing virally infected target cells.     

Autocytotoxic and autosuppressor T-cell lines generated from autologous lymphocyte cultures

Limiting dilution analyses have demonstrated both the generation and suppression of autocytotoxic cells following in vitro stimulation with autologous peripheral blood mononuclear leukocytes (PBL). Therefore, in order to isolate and characterize the autocytotoxic lymphocytes, interleukin 2-dependent cell lines were derived from autologous mixed lymphocyte microcultures. The cell lines were screened for cytolytic activity against autologous phytohemagglutinin-activated lymphoblasts, autologous and allogeneic B-lymphoblastoid cell lines (B-LCL), and the natural killer target K562. Of 189 cell lines analyzed, 26 demonstrated cytotoxicity against autologous target cells. Cell surface phenotyping of all cell lines indicated that they were of T lymphocyte lineage. Two autocytotoxic T-cell clones were subsequently derived in similar fashion. Cell lines were also screened for autoregulatory activity. Two cells lines were identified that inhibited the generation of autocytotoxicity. Neither of the autoregulatory lines was capable of directly lysing an autocytotoxic line, suggesting that these autosuppressor cells exert their inhibitory effect by a mechanism other than direct lysis of the autocytotoxic effector cell. These findings indicate that through the application of limiting dilution analysis and in vitro cell culture techniques, autocytotoxic and autosuppressor lymphocyte populations can be isolated and utilized to analyze the cellular interactions involved in maintaining self-tolerance.     

T-cell immunity against tumors,a delicate balancing act involving dendritic cells

T-cell immunity occurs naturally against tumors induced by viruses and other causes. In the latter case self antigens are increasingly found to be targets of tumor associated CTL. In all categories of tumors the T cell response usually falls short of the maximally possible response. This situation calls for vaccination, primarily in situations of low tumor burden and adoptive transfer with tumor specific T cells in case of higher tumor burden. We recently observed that the outcome of immunization with vaccines containing tumor virus CTL epitopes strongly depends on mode of epitope delivery. Surprisingly, vaccination with MHC class I binding peptides cause CTL tolerance associated with enhanced tumor outgrowth rather than immunity. Such specific CTL tolerance can be induced by a single injection of 1 μg of peptide in adjuvant. However, in vivo presentation of the same peptides on dendritic cells or in viral vector (adenovirus) causes strong antitumor protection. Thus tumors may escape from immune attack by specific tolerance induction. Tumor specificity of autoreactive CTL can be achieved by T cells directed against tumor associated self antigens of limited tissue distribution. Alternatively useful CTL can be directed against strongly overexpressed self antigens, as illustrated in our lab by the successful eradication of tumors overexpressing wild type p53 tumor suppressor protein, by the adoptive transfer of a wild p53-specific CTL clone. Apparently the low expression of p53 in many tissues does not cause the CTL clone to inflict tissue damage, while the p53 overexpressing tumor cells are specifically targeted and eradicated. Recently we showed that CD40 signalling can replace CD4+ T-cells in priming of helper dependent tumor-specific CD8+ responses. Blockade of CD40L results in profound inhibition of CTL priming that is overcome by CD40 signalling. CD40 signalling converted CTL tolerization by a tolerogenic peptide into strong CTL priming. Moreover, supplementation of an already protective tumor-specific peptide vaccine with a CD40 activating antibody endows this vaccine with therapeutic CTL activity in tumor-bearing mice. The CD40–CD40L pair acts as a switch determining CTL priming or tolerance. This supports the clinical use of CD40 stimulating agents as components of anticancer vaccines.     

CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria.

Immunization of BALB/c mice with radiation-attenuated Plasmodium yoelii sporozoites induces cytotoxic T lymphocytes (CTL) specific for an epitope located within the amino acid sequence 277-288 of the P. yoelii circumsporozoite (CS) protein. Several CD8+ CTL clones were derived from the spleen cells of sporozoite-immunized mice, all displaying an apparently identical epitope specificity. All the clones induced high levels of cytolysis in vitro upon exposure to peptide-incubated MHC-compatible target cells. The adoptive transfer of two of these clones conferred complete protection against sporozoite challenge to naive mice. This protection is species and stage specific. Using P. yoelii specific ribosomal RNA probes to monitor the in vivo effects of the CTL clones, we found that their target was the intrahepatocytic stage of the parasite. The protective clones completely inhibited the development of the liver stages of P. yoelii. Some CTL clones were only partially inhibitory in vivo, while others failed completely to alter liver stage development and to confer any detectable degree of protection. The elucidation of the effector mechanism of this CTL mediated protection against rodent malaria should facilitate the design of an effective malaria vaccine. From a broader perspective this model may provide further insight into the mechanism(s) of CTL mediated killing of intracellular non-viral pathogens in general.     

Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients

Background  

Cytomegalovirus (CMV) seronegative recipients (R-) of kidney transplants (KT) from seropositive donors (D+) are at higher risk for CMV replication and ganciclovir(GCV)-resistance than CMV R(+). We hypothesized that low CMV-specific T-cell responses are associated with increased risk of CMV replication in R(+)-patients with D(+) or D(-) donors.     

Identification and characterization of canine dendritic cells generated in vivo.

Emerging evidence suggests that host dendritic cells (DC) initiate and regulate graft-versus-host and graft-versus-tumor reactions after allogeneic hematopoietic cell transplantation (HCT). Even though decades of experimentation in the preclinical canine HCT model have substantially improved our understanding of the biology and safety of HCT in human patients, the in vivo phenotype of potent antigen-presenting cells in dogs is poorly defined. Therefore, peripheral blood leukocytes were obtained from dogs treated with recombinant human Flt3-ligand and phenotypically distinct cell populations, including putative DC, were purified by 4-color flow-cytometry and tested for their stimulatory potential in allogeneic mixed lymphocyte cultures (MLC). Cells characterized by surface expression of CD11c and HLA-DR, and absence of expression of CD14 and DM5, a marker of mature granulocytes, were found to be highly potent stimulators in allogeneic MLC. In contrast, all other immunophenotypically different cell populations tested had either weak or absent allostimulatory potential. Transmission electron microscopy of CD11c+/HLA-DR+/CD14-/DM5- cells revealed the morphology similar to that described for DC in humans and ex vivo-generated canine DC, including long cytoplasmic extensions, discrete lysosomes, and an abundant Golgi apparatus and endoplasmatic reticulum. In summary, CD11c+/HLA-DR+/CD14-/DM5- cells obtained from canine peripheral blood have functional and morphologic characteristics similar to those of human myeloid DC.     

Contact-dependent stimulation of monocytic cells and neutrophils by stimulated human T-cell clones.

By means of direct cell-cell contact, fixed, stimulated T lymphocytes trigger the production of interleukin-1 beta (IL-1 beta) and tumour necrosis factor by monocytes and prime polymorphonuclear leucocytes (PMN) for the respiratory burst induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP). In order to assess whether this activity is displayed by a particular subpopulation of T lymphocytes, 88 T-cell clones (TCC) were generated from healthy human peripheral blood. The clones were stimulated by Phaseolus vulgaris agglutinin (PHA) and phorbol 12-myristate acetate monocytic cell line THP-1. All fixed, stimulated TCC induced IL-1 beta production by THP-1 cells, although to varied degrees. The activity of TCC on THP-1 cells correlated with their activity on PMN (r2 = 0.84, P < 0.001), suggesting that this pro-inflammatory activity of TCC may similarly affect both types of infiltrating cells. The ability of TCC to modulate target cells did not correlate with either their phenotype (CD4 or CD8) or their cytokine production profile (interferon-gamma, IL-4 and IL-6), although it tended to correlate inversely with their capacity to produce IL-6 (P < 0.02). These observations suggest that a large proportion, if not all, of human peripheral blood T lymphocytes may potentially affect monocytes and PMN by direct cell-cell contact. This activity may be relevant to the maintenance of chronic inflammation.     

Characterization of long-term cultured bovine CD4-positive and CD8-positive T-cell lines and clones.

Long-term cultured CD4+ or CD8+ bovine T-cell lines and clones were established. The CD8+ T-cell line and clones had a strong lectin-dependent cytotoxicity, whereas the CD4+ T-cell line did not. Both phenotype cell lines grew in an interleukin-2 (IL-2)-dependent manner and expressed 50,000-55,000 MW and 65,000-75,000 MW proteins associated with a putative IL-2 receptor (IL-2R), as demonstrated by the cross-linking of radioiodinated recombinant human IL-2 (rhIL-2). Additional molecules of 13,000 and 27,000 MW were also observed on CD8+ T cells. The binding of rhIL-2 was blocked by crude bovine IL-2, and Scatchard plot analysis of the binding data showed that both phenotype cells expressed two different affinity IL-2R that had equilibrium dissociation constants of 12-20 pm (3000-6000 sites/cell) and 146-490 pM (16,000-25,000 sites/well). Only IL-2 stimulated DNA synthesis in these cell lines, whereas mitochondrial enzymes activity, protein synthesis and protein secretion were enhanced by IL-2, mitogens and phorbol myristate acetate. The supernatant from mitogen-stimulated CD4+ cells was unable to enhance the DNA synthesis of either the CD4+ or CD8+ lines, whereas both freshly prepared Con A blasts and anti-immunoglobulin-treated bovine B cells showed elevated DNA synthesis under the same conditions.     

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.     

Artificially generated dendritic cells misdirect antiviral immune responses

Dendritic cells (DCs) are critical to the outcome of many viral infections. Questions still remain as to the relevance of artificially generated DCs in models of in vivo immune responses. We compared different DC generation pathways, in terms of phenotypic expression, cytokine production, apoptosis, and T cell proliferation, following viral infection. Direct viral infection of monocytes or monocytes cultured with supernatants from virally infected lung epithelial cells (A549 DCs) induce distinct DC subsets compared with viral infection of artificially generated IL-4 DCs and IFN-DCs. These virally infected DC subsets stimulated different cytokine secretion profiles and displayed contrasting sensitivities to viral-induced apoptosis. It is most interesting that we observed marked differences in the proliferation of purified CD3+ T cells from the virally infected DC subsets. In conclusion, artificially generated DCs skew immune responses to viral infections, and direct viral infection of monocytes and DCs, generated from monocytes cultured with supernatants from infected epithelial cells, appears to be a more relevant pathway of producing DCs, which mimic those generated in vivo.     

Follicular dendritic cells: beyond the necessity of T-cell help.

Follicular dendritic cells (FDCs) are potent accessory cells for B cells, but the molecular basis of their activity is not understood. Several important molecules involved in FDC-B-cell interactions are indicated by blocking the ligands and receptors on FDCs and/or B cells. The engagement of CD21 in the B-cell coreceptor complex by complement-derived CD21 ligand on FDCs delivers a crucial signal that dramatically augments the stimulation delivered by the binding of antigen to the B-cell receptor (BCR). The engagement of Fc gamma receptor IIB (FcgammaRIIB) by the Ig crystallizable fragment (Fc) in antigen-antibody complexes held on FDCs decreases the activation of immunoreceptor tyrosine-based inhibition motifs (ITIMs), mediated by the crosslinking of BCR and FcgammaRIIB. Thus, FDCs minimize a negative B-cell signal. In short, these ligand-receptor interactions help to signal to B cells and meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help.     

Functional differences and complementation between dendritic cells and macrophages in T-cell activation.

Functional differences and cell collaboration between murine lymphoid dendritic cells (DC) and macrophages (M phi) in antigen presentation for T-cell activation were analysed with splenic DC and M phi, culture-derived bone-marrow (BM)-M phi, and DC-like and M phi-like cell lines. DC were the best stimulators of allogeneic mixed leucocyte reaction (MLR), but splenic M phi and small activated BM-M phi were almost as effective. In contrast to MLR stimulation, small activated BM-M phi were the most effective antigen-presenting cells (APC) for the presentation of whole Corynebacterium parvum (CP) organisms, possibly by virtue of their phagocytic and lysosomal functions, which could be particularly important for processing particulate antigens. Large activated BM-M phi were ineffective in stimulating MLR and CP-specific T-cell proliferation. The functional differences between BM-M phi subsets could not be explained by failure to express surface Ia or to take up antigen. Non-phagocytic APC, such as DC and the DC-like line P388AD.4, had low presenting activity for CP and were much less effective at presenting glutaraldehyde-fixed CP than M phi. This suggests that DC are dependent on the shedding of soluble antigen (reduced by glutaraldehyde fixation) from the bacteria, and they may also be less efficient than M phi at processing the fixed bacteria. The Ia- M phi-like line. P388D1, was devoid of APC activity, but could greatly enhance P388AD.4-induced T-cell proliferation to whole bacterial organisms. Similarly, co-culture of splenic DC and M phi produced very pronounced synergistic effects in proliferative responses to CP and keyhole limpet haemocyanin. The function of M phi n this partnership was sensitive to chloroquine and could not be replaced by M phi culture fluids or recombinant interleukin-1. Thus, M phi may contribute processed antigen in a form more suitable for presentation by DC. These results provide a rationale for the functional dichotomy between DC and M phi.     

An early antigen-presenting cell defect in HIV-1-infected patients correlates with CD4 dependency in human T-cell clones.

We have used a defined panel of nine HIV peptide-specific T-cell clones (TLC) generated from a healthy volunteer to evaluate the antigen-presenting cell (APC) function of human immunodeficiency virus-1 (HIV- 1)-infected patients. Peripheral blood mononuclear cells (PBMC) from HLA-matched seropositive and uninfected volunteers were compared for their capacity to present peptide to TLC specific for the V3 loop of HIV- 1 envelope glycoprotein gp120, influenza haemagglutinin or the mycobacterial 19,000 MW antigen APC from uninfected volunteers (HIV- APC) invariably presented peptides to all TLC with comparable efficiency. In contrast using APC from HIV- 1-infected subjects (HIV+ APC) three patterns of responsiveness were observed. The first group of TLC was not stimulated by HIV+ APC even early in infection. The second responded to all APC comparably. The third and intermediate group, responded to APC from some clinically asymptomatic, but not acquired immune deficiency syndrome (AIDS), patients. The two additional TLC, derived from other donors and with specificity for non-HIV peptides, showed similar variation in response to HIV+ APC. The different patterns of response to HIV APC did not correlate with the fine specificity or cytokine phenotypes of the TLC. Neither was the defect due to decreased levels of expression of APC molecules involved in delivering the first or second signal required for T-cell activation APC mixing experiments showed no evidence of APC-derived inhibitory factor. Furthermore, the defect was independent of T cells or their products and was equally expressed in monocytes and dendritic cells. Instead, responsiveness was inversely related to the degree of CD4 dependency suggesting that the underlying mechanism was a CD4 APC-associated gp120 interaction. The early appearance of this defect in HIV- 1 infection co-incident with the loss of recall responses is consistent with a role for APC dysfunction in pathogenesis.     

Lymphokine secretion and cytotoxic activity of human CD4+ T-cell clones against Bordetella pertussis.

Human CD4+ T-cell clones specific for pertussis toxin and other Bordetella pertussis antigens have been tested for their cytotoxic activity, lymphokine production, and capacity to induce immunoglobulin synthesis. Clones specific for the S1 subunit of pertussis toxin were cytotoxic for autologous Epstein-Barr virus-transformed B cells, which had been pulsed with the native antigen, the recombinant S1 subunit of pertussis toxin, or synthetic peptides derived from the S1 amino acid sequence. The killing of antigen-pulsed target cells was class II restricted. All of the T-cell clones produced mostly interleukin-2 and gamma interferon and assisted allogeneic B cells in the production of immunoglobulins M and G but not immunoglobulin E. The potential in vivo role of the cytotoxic activity of these clones is discussed.