Induction Of Cellular Immunity Research Articles

Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia

It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV-16 E7 specific immune responses in patients with different grades of cervical intra-epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T-cell responses were measured by IFN-gamma ELISPOT analysis, after stimulation with recombinant HPV-16 E7 protein. As expected, HPV-16 E7 specific IFN-gamma T cell responses were more frequent in HPV-16 DNA positive patients compared with that in HPV-16 DNA negative patients (39/50 vs. 16/36, (p=0.006, chi2 test). After invasive procedures, a small number of HPV-16 DNA positive CIN patients, but a considerable proportion of HPV-16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV-16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4+ and CD8+ T cells showed E7 specific IFN-gamma production upon in-vitro stimulation. Our study shows that invasive procedures may enhance HPV-specific cell-mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV-specific immune responses is used as intermediate end-point.

DLI: Where are we know?

Allogeneic stem cell transplantation has induced lasting remissions in patients with leukaemia, lymphoma and myeloma otherwise refractory to chemoand radiotherapy. Patients with acute and chronic graft-versus-host disease (GVHD) have a lesser risk or relapse of disease than those without GVHD. Patients given T-cell depleted grafts have a lower risk of GVHD and a higher risk of rejection of the graft and of relapse. The dilemma of either risk of GVHD or leukaemia relapse can be solved in animal models by transfusion of donor lymphocytes at a time when chimerism and tolerance is established. Here the evidence for a graft-versus-leukaemia (GVL) effect is reviewed and ways of improving the GVL effect and overcoming mechanisms of immune escape are discussed. Donor lymphocyte transfusions (DLT) have been introduced in the treatment of leukaemia relapse after allogeneic stem cell transplantation in the late 1980’s and the best results have been observed in patients with chronic myelogenous leukaemia (CML). The first patients are stil alive and in remission. In acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) results were inferior and in acute lymphatic leukaemia (ALL) results were generally poor. In multiple myeloma DLT could induce remissions, but the duration of the responses has generally been limited. There have been reports on the successful treatment of low grade lymphoma, chronic lymphocytic leukaemia and Hodgkin’s disease with DLT, but in general the results are controversial. In most patients of these disease groups conditioning treatment was not myeloablative and patients received grafts of mobilized blood cells containing a high proportion of T-cells. Relapses may have already escaped immune mechanisms of T-cells when they occurred. The hypothesis that dendritic cells of leukaemia origin are formed in CML and AML has been proven by in vitro culture of leukaemia cells in the presence of GM-CSF and interferon-alpha or IL-4. Dendritic cells had the leukaemia karyotype. Consequently treatment of recurrent CML with DLT was combined with GM-CSF and IFN-a in patients with advanced relapse or not responding to DLT alone. Responses were observed in 6 out of 9 patients treated. In AML the combination of low dose cytosine arabinoside (LD-AraC) with mobilized blood cells and GM-CSF followed by DLT in cases without GVHD induced remission of more than 4 and 8 years. The long-term responses were limited to patients that responded to a short course of LD-AraC. The use of GM-CSF is currently studied also in other diseases as multiple myeloma and lymphoma with persistence or relapse. Another possibility to activate T-cells in the GVL reaction is the use of bispecific antibodies. BI20 is a bispecific antibody of rat and mouse origin with one binding site to CD3 and the other to CD20, the Fc part binds to macrophages and dendritic cells inducing phagocytosis of the lymphoma cell. This antibody has been shown to induce CTL against CLL in vitro and depletes leukaemia cells in the blood. However the induction of cellular immunity has not been shown yet in vivo. Finally unlike T cells NK cells are able to kill leukemia and lymphoma cells not expressing HLAantigens. They are inhibited by their own HLAantigens respectively, HLA-associated killer inhibitor receptor ligands. In the HLA-haploidentical situation they are not inhibited, if the recipients leukaemia cells do not express the KIR Ligands of the donor. We have designed a treatment protocol with unmodified mar-

Plasmid-encoded GM-CSF induces priming of the F4(K88)-specific serum IgA response by FaeG DNA vaccination in pigs

We have used FaeG DNA to immunise piglets by the intradermal (ID) and the intramuscular (IM) route in a heterologous prime/boost model. ID immunisation with DNA resulted in a better induction of cellular immunity, whereas only the IM immunisation could prime an F4(K88)-specific serum IgA response. However, ID administration of plasmid-encoded GM-CSF 1 week before the ID immunisation enhanced the F4-specific humoral and cellular immune response and even primed the F4-specific IgA response more efficiently than the IM immunisation did.

Effect of Skin Barrier Disruption on Immune Responses to Topically Applied Cross-Reacting Material, CRM197, of Diphtheria Toxin

The high accessibility of the skin and the presence of immunocompetent cells in the epidermis makes this surface an attractive route for needle-free administration of vaccines. However, the lining of the skin by the stratum corneum is a major obstacle to vaccine delivery. In this study we examined the effect of skin barrier disruption on the immune responses to the cross-reacting material CRM(197), a nontoxic mutant of diphtheria toxin (DTx) that is considered as a vaccine candidate. Application of CRM(197), together with cholera toxin (CT), onto the tape-stripped skin of mice elicited antibody responses that had anti-DTx neutralizing activity. Vaccine delivery onto mildly ablated skin or intact skin did not elicit any detectable anti-CRM(197) antibodies. Mice immunized with CRM(197) alone onto the tape-stripped skin mounted a vigorous antigen-specific proliferative response. In contrast, the induction of cellular immunity after CRM(197) deposition onto mildly ablated or intact skin was adjuvant dependent. Furthermore, epidermal cells were activated and underwent apoptosis that was more pronounced when the stratum corneum was removed by tape stripping. Overall, these findings highlight the potential for transcutaneous delivery of CRM(197) and establish a correlation between the degree of barrier disruption and levels of antigen-specific immune responses. Moreover, these results provide the first evidence that the development of a transcutaneous immunization strategy for diphtheria, based on simple and practical methods to disrupt the skin barrier, is feasible.

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside.

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

Induction of cellular immunity to varicella-zoster virus glycoproteins tested with pernasal coadministration of Escherichia coli enterotoxin in mice.

A mutant of Escherichia coli enterotoxin promotes the induction of cellular immunity to a live varicella vaccine (the Oka strain) as a mucosal adjuvant in mice. An investigation was carried out to determine which of the purified glycoproteins of the virus among three induced cellular immunity with a single nasal administration. Spleen cells from mice immunized nasally with the vaccine and toxin produced interleukin-2 (IL-2) at the same level on restimulation in vitro with glycoprotein H: glycoprotein L (gH:gL), gB, and gE:gI, but not IL-4. The spleen cells from mice immunized with gH:gL, gB, or gE:gI and toxin produced IL-2 on restimulation with gH:gL, gB, or gE:gI, respectively, and the vaccine, but not IL-4. Immunization with gH:gL and the toxin showed increased thymidine uptake and production of IL-2 and interferon-gamma (IFN-gamma) of the spleen cells, but not IL-4, depending on the dose of gH:gL used for immunization and restimulation in vitro. Purified gE:gI and gB have been reported to be the strongest stimulators of cellular immunity to varicella upon subcutaneous injection and are useful as a subunit vaccine. All the glycoproteins tested are excellent stimulators of cellular immunity to the virus and itself on nasal co-immunization with the toxin.

Induction of HIV-1-specific T-helper responses and type 1 cytokine secretion following therapeutic vaccination of macaques with a recombinant fowlpoxvirus co-expressing interferon-gamma.

Preventive and/or therapeutic vaccines against Human Immunodeficiency Virus (HIV-1) are urgently required. Induction of cellular immunity is favoured since these responses correlate with control of HIV-1. Recombinant fowlpoxvirus (FPV) vaccines encoding both HIV-1 gag/pol and interferon-gamma (FPV gag/pol-IFNgamma) were hypothesised to enhance HIV-specific cellular immunity and were further evaluated in macaques previously infected with HIV-1. A novel assay to detect IFNgamma secretion following HIV antigen stimulation of whole blood was developed to further assess the safety and immunogenicity of the FPV gag/pol-IFNgamma vaccine. Immunisation with FPV gag/pol-IFNgamma safely enhanced HIV-specific IFNgamma secretion following ex vivo stimulation of whole blood, greater than that observed following FPV gag/pol vaccination not co-expressing IFNgamma. Both HIV-specific IFNgamma-spot-forming cells by ELISPOT and CD69 expression by CD4+ lymphocytes were also enhanced following FPV gag/pol-IFNgamma vaccination. Hence, the FPV-HIV vaccine co-expressing IFNgamma stimulated HIV-specific T cell responses in macaques, and should be further evaluated as a therapeutic or preventive HIV vaccine.

Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70

Heat shock proteins 70 (hsp70) derived from tissues and cells can elicit cytotoxic T lymphocyte (CTL) responses against peptides bound to hsp70. However, peptides can markedly differ in their affinity for hsp, and this potentially limits the repertoire of peptides available to induce CTL by the hsp immunization. Hybrid peptides consisting of a high-affinity ligand for the peptide-binding site of hsp70 joined to T cell epitopes by a glycine-serine-glycine linker were constructed. Immunization with hybrid peptides complexed to mouse hsp70 effectively primed specific CTL responses in mice and were more potent than T cell peptide epitopes alone with hsp70. In vivo immunization with hsp70 and hybrid peptides led to rejection of tumors expressing antigen with greater efficacy than immunization with peptide epitope plus hsp70. Induction of CTL responses occurred independently of CD4(+) T cells, suggesting that immunization directly primed antigen-presenting cells to elicit CD8(+) cytotoxic T cell responses without T cell help. Both peptide/hsp70 complexes and mouse hsp70 alone were able to induce cultures of mouse bone marrow-derived dendritic cells (DC) to release cytokines, including DC from endotoxin-resistant C57BL/10Sc mice. Thus, hsp70/hybrid peptide complexes can activate DC for cytokine release, providing a potential adjuvant effect that could bypass T cell help.

Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70.

Heat shock proteins 70 (hsp70) derived from tissues and cells can elicit cytotoxic T lymphocyte (CTL) responses against peptides bound to hsp70. However, peptides can markedly differ in their affinity for hsp, and this potentially limits the repertoire of peptides available to induce CTL by the hsp immunization. Hybrid peptides consisting of a high-affinity ligand for the peptide-binding site of hsp70 joined to T cell epitopes by a glycine-serine-glycine linker were constructed. Immunization with hybrid peptides complexed to mouse hsp70 effectively primed specific CTL responses in mice and were more potent than T cell peptide epitopes alone with hsp70. In vivo immunization with hsp70 and hybrid peptides led to rejection of tumors expressing antigen with greater efficacy than immunization with peptide epitope plus hsp70. Induction of CTL responses occurred independently of CD4(+) T cells, suggesting that immunization directly primed antigen-presenting cells to elicit CD8(+) cytotoxic T cell responses without T cell help. Both peptide/hsp70 complexes and mouse hsp70 alone were able to induce cultures of mouse bone marrow-derived dendritic cells (DC) to release cytokines, including DC from endotoxin-resistant C57BL/10Sc mice. Thus, hsp70/hybrid peptide complexes can activate DC for cytokine release, providing a potential adjuvant effect that could bypass T cell help.

Combination treatment with interleukin-2 and interleukin-12 for murine renal cell carcinoma

Background. We studied the anti-tumor effectiveness of treatment with interleukin-2 (IL-2) and/or IL-12 for a spontaneously arising murine renal cell carcinoma (RC-2). We also analyzed the immunological effects induced by the treatments. Methods. One week after RC-2 inoculation, treatment with IL-2 (1 × 104 U or 5 × 104 U/mouse, every day for 3 weeks) and/or IL-12 (1 μg/mouse, once a week for 3 weeks) were started. We analyzed the relative mean tumor weight ratio (tumor relative weight/control relative weight [TRW/CRW]), the degree of tumor degeneration (grade), and the survival rate of the mice. We also analyzed the expression of various T-helper (Th)1-derived cytokine mRNAs (interferon-γ, IL-2, tumor necrosis factor-β) and Th2-derived-cytokines mRNAs (ILs-4, 5, 6, 10) in the spleen by reverse transcription-polymerase chain reaction. Results. The combination treatments were effective in terms of the TRW/CRW ratios (4.8% for IL-2 5 × 104 U/mouse plus IL-12 and 8.9% for IL-2 1 × 104 U/mouse plus IL-12) and showed superior survival of mice compared with results for the control mice and those treated with a single agent. However, a small amount of viable tumor cells still remained (grade IIb), indicating the treatment was ineffective. Control spleens expressed Th2-derived cytokine mRNAs predominantly, in accordance with tumor growth. In mice that had combination treatments, the spleen expressed Th1-derived cytokine mRNAs, except for IL-10. Conclusion. We conclude that combination treatment with IL-2 and IL-12 has potent anti-tumor effectiveness, based upon the induction of cellular immunity implying cross-regulation with humoral immunity.

Cutting Edge: CD40 Ligand Is Not Essential for the Development of Cell-Mediated Immunity and Resistance toMycobacterium tuberculosis

AbstractIt has been proposed that the induction of cellular immunity and resistance to intracellular pathogens is dependent upon CD40 ligand (CD40L). In the present study we show that this proposal is not ubiquitously supported. Mice genetically deficient in CD40L (CD40LKO) were resistant to i.v. infection with Mycobacterium tuberculosis when assessed by survival and bacteriologic burden in the spleen, liver, and lungs. Infected CD40LKO mice developed granulomas that lacked epithelioid cells and were less numerous and markedly smaller than those observed in control mice. Upon stimulation with purified protein derivative of M. tuberculosis,CD4+ T cells from infected CD40LKO mice proliferated and produced high levels of IFN-γ but not IL-4. Finally, spleen cells from CD40LKO mice stimulated with M. tuberculosis produced IL-12, TNF, and nitric oxide levels comparable to those produced by control cells. In contrast to original proposals, these data clearly show that protective Th1 immunity can be achieved against intracellular pathogens (e.g., Mycobacterium) independently of CD40L.

Induction of cellular immunity against Mycobacterium tuberculosis

Induction of cellular immunity against Mycobacterium tuberculosis

Induction of cellular immunity against Mycobacterium tuberculosis

Induction of cellular immunity against Mycobacterium tuberculosis

Cellular immuno-competence of infected root canal contents in pathogenesis of periapical lesions

The soluble fractions of infected root canal contents (IRCC) were collected from about 300 human extracted teeth and examined for the presence of mononuclear cell (MNC) chemotaxis and cellular immunocompetence. IRCC showed remarkable chemotactic activity for polymorphonuclear leukocytes but a weak activity for MNC. However, generation of intrinsic MNC chemotaxis and induction of cellular immunity were confirmed in rats given repeated injections of IRCC.

Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA-transfected Epstein-Barr virus-immortalized autologous B cells.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

Interleukin‐12 profoundly up‐regulates the synthesis of antigen‐specific complement‐fixing IgG2a, IgG2b and IgG3 antibody subclasses in vivo

The influence of the cytokine interleukin-12 (IL-12) on humoral immune responses was studied in vivo. CBA/J mice immunized with protein antigens (keyhole limpet hemocyanin, phospholipase A2) adsorbed to aluminum hydroxide (Alum) develop a Th2-like immune response characterized by the production of large amounts of IgG1 as well as some IgE but little IgG2a, IgG2b and IgG3 antibodies. IL-12 is a cytokine that promotes the development and the activation of Th1 cells. Th1 cells are involved in the induction of cellular immunity, which is characterized by low or absent antibody production. On the other hand, some Th1-like immune responses are associated with a strong antibody production of the IgG2a, IgG2b and IgG3 subclasses. Thus, we investigated whether treatment with IL-12 would down-regulate the humoral immune response or stimulate antibody production of the IgG2a, IgG2b and IgG3 subclasses. We observed that: 1) administration of IL-12 to mice together with protein antigens adsorbed to Alum strongly enhanced the humoral immune response by increasing the synthesis of antigen-specific antibodies of the IgG2a, IgG2b and IgG3 subclasses 10- to 1000-fold. The synthesis of IgG1 was not or only slightly (2-5-fold) enhanced, whereas that of the IgE isotype was suppressed. 2) These effects of IL-12 were observed when high (10 micrograms, 100 micrograms) or low doses (0.1 microgram) of antigen were used for immunization. 3) Titration of IL-12 in vitro revealed that IgG2a is strongly up-regulated over a wide dose range of IL-12 (10 to 1000 ng/day). 4) The effects of IL-12 in vivo are at least partially interferon (IFN)-gamma-dependent because an anti-IFN-gamma mAb in combination with IL-12 prevented most of the enhanced IgG2a production. 5) Mice receiving IL-12 showed a strong up-regulation of IFN-gamma but no inhibition of IL-5 synthesis by spleen cells activated ex vivo with antigen. These results suggest that IL-12 is a potent adjuvant for enhancing humoral immunity to protein antigens adsorbed to Alum, primarily by inducing the synthesis of the complement-fixing IgG subclasses 2a, 2b and 3.

A malaria vaccine strategy based on the induction of cellular immunity

In this article, Michael Good offers an alternative to the current emphasis on developing subunit, humoral-response-inducing malaria vaccines. He develops a three-pronged proposal comprising (1) a greater emphasis on cellular immunity, (2) analysis of the role of the spleen in protection, and (3) the development of an attenuated vaccine.

Progress Toward a Malaria Vaccine

In initial human trials, synthetic vaccines have induced humoral immunity sufficient to prevent clinical infection in some cases and delay it in others. Progress in induction of cellular immunity is also noteworthy with identification of determinants recognized by T cells. Antigenic variation and consequent blunting of immunogenicity may not be as troublesome as feared.

Adjuvanticity of dimethyl dioctadecyl ammonium bromide in guinea pigs. I. Skin test reactions.

The effect of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the induction of cellular immunity in guinea pigs was studied. DDA, a surface-active lipid, was mixed with the antigen bovine serum albumin (BSA) or with a conjugate of BSA and dinitrophenol (DNP22--BSA) and injected into the footpads of guinea pigs. At varying intervals skin tests were performed to test the immediate and delayed hypersensitivity (DH) reactions. Optimal DH reactions to BSA were observed from 3 to 6 weeks after immunization with BSA in DDA. The hapten-specific response to DNP22--BSA had an optimum at 3 weeks and was highly specific for the homologous antigen. Histological examinations of skin test sites confirmed that the reaction was rather of the tuberculin type than of the cutaneous basophil hypersensitivity type. When guinea pigs were immunized with DNP22--BSA in Freund's complete adjuvant (FCA) a long-lasting DH to both carrier and hapten groups developed but the DH was always complicated by an Arthus reaction due to antibodies to the DNP hapten. In conclusion, DDA is superior to FCA as adjuvant for the induction of a state of pure DH in guinea pigs.

A Pilot Study Concerning the Early Immunosuppressive Effects of Rifampin in Man

The early effects of rifampin-isoniazid therapy on humoral and cell-mediated immunity were studied in six patients with pulmonary tuberculosis and nine normal control subjects. Humoral antibody responses were tested using two different DNA viral bacteriophages. T2 immunization was performed before and Φ X 174 was administered two weeks after therapy was started. Neutralizing titers for both bacteriophages were measured after incubation of the serum with 2-mercaptoethanol (2-ME), which distinguishes immunoglobulin G antibodies from other immunoglobulins. The total antibody responses to both bacteriophages were higher in the patients, but not statistically different from the control subjects. The 2-ME resistant Φ X 174 antibody titer, which measures mainly immunoglobulin G antibodies, was significantly depressed at 14 and 28 days. Six weeks of rifampin-isoniazid therapy produced no noticeable impairment of cellular immunity when measured by the responses either to antigens, to which pre-existing cellular immunity existed, or by induction of cellular immunity with 1-chloro-2, 4-dinitrobenzene. The data suggest that two weeks of rifampin therapy in the usual recommended dosage is associated with some partial suppression in immunoglobulin G synthesis. From all evidence, it appears that this mild depression is not of clinical significance.