Delayed-type Hypersensitivity Sites Research Articles

Induction of Antigen-Specific T-Cell Responses through Dendritic Cell Vaccination in AML: Results of a Phase I/II Trial and Ex Vivo Enhancement By Checkpoint Blockade

Postremission therapy is critical for successful elimination of minimal residual disease (MRD) in acute myeloid leukemia (AML). Innovative treatment options are needed for patients that are not eligible for allogeneic stem cell transplantation. As the intrinsic immune response against leukemia-associated antigens (LAAs) is generally low, the clinical application of checkpoint inhibitors as monotherapy is less promising in AML compared to other hemato-oncological diseases. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with LAAs is a promising treatment strategy to induce anti-leukemic immune responses.We have conducted a phase I/II proof-of-concept study using monocyte-derived next-generation DCs as postremission therapy of AML patients with a non-favorable risk profile in CR/CRi after intensive induction therapy (NCT01734304). These DCs are generated using a GMP-compliant 3-day protocol including a TLR7/8 agonist, loaded with RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant/surrogate antigen, and are applied intradermally up to 10 times within 26 weeks. The primary endpoint of the trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control.After the safety and toxicity profile was evaluated within phase I (n=6), the patient cohort was expanded to a total of 13 patients. DCs of sufficient number and quality could be generated from leukapheresis in 11/12 cases. DCs exhibited an immune-stimulatory profile based on high costimulatory molecule expression, IL-12p70 secretion, migration towards a chemokine gradient and processing and presentation of antigen. In 9/9 patients that are currently evaluable, we observed delayed-type hypersensitivity (DTH) responses at the vaccination site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. In the peripheral blood, we detected vaccination-specific T cell responses by multimer staining and by ELISPOT analysis: 7/7 patients showed responses to CMVpp65, including both boosting of pre-existing T cells in CMV+ patients and induction of a primary T cell response in CMV- patients. 2/7 patients exhibited responses to PRAME and WT each. 7/10 vaccinated patients are still alive, and 5/10 are in CR, with an observation period of up to 840 days.In vitro, DC-activated T cells showed an upregulation of PD-1 and LAG-3, while the DCs expressed the respective ligands PD-L1 and HLA-DR. Therefore, we studied the capacity of checkpoint blocking antibodies to further enhance T-cell activation by DCs. We found that blockade of PD-1 and particularly of LAG-3 was highly effective in enhancing both IFN-γ secretion and proliferation of T cells. Both pathways seem to target different T-cell subsets, as PD-1 blockade resulted in increased IFN-γ secretion by TN- and TEM-subsets, while blockade of LAG-3 significantly affected TN- and TCM-subsets.We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe, and induces anti-leukemic immune responses in vivo. Our in vitro data supports the hypothesis that T-cell activation by means of the vaccine could be further enhanced by blocking PD-1 and/or LAG-3. DisclosuresSubklewe:AMGEN Research Munich: Research Funding.

Abstract B002: TLR7/8-matured dendritic cells for therapeutic vaccination in AML: Results of a clinical Phase I/II trial

Abstract Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. Using a TLR7/8 agonist, we have developed a GMP-compliant 3-day protocol to differentiate monocytes of intensively pretreated AML patients into highly functional, therapeutic DCs. A phase I/II proof-of-concept study has been initiated using TLR7/8-matured DCs as postremission therapy of AML patients with a non-favorable risk profile in CR or CRi after intensive induction therapy (NCT01734304). DCs have been loaded with in vitro transcribed RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients have been vaccinated intradermally with 5×106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I/II trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. In total, 13 patients have been enrolled into the study. The first 6 patients were analysed in phase I for safety and toxicity of the DC vaccine. No higher grade toxicities were observed during their treatment and hence phase II has been initiated. DCs of sufficient number and quality were generated from leukapheresis in 10/11 cases. DCs exhibited an immune-stimulatory profile based on high surface expression of positive costimulatory molecules, the capacity to secrete IL-12p70, the migration towards a chemokine gradient and processing and presentation of antigen. In 9/9 vaccinated patients, we observed delayed-type hypersensitivity (DTH) responses at the vaccination site, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. TCR repertoire analysis by NGS revealed an enrichment of particular clonotypes at DTH sites. In addition, we detected vaccine-specific T-cell responses by multimer staining and by Interferon-gamma-ELISPOT analysis: 7/7 patients showed responses to CMVpp65 and 2/7 exhibited responses to PRAME and WT1, respectively. In an individual treatment attempt, an enrolled patient with impending relapse was treated with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. Long-term disease control and immunological responses are studied in the ongoing phase II trial. We conclude that vaccination with TLR7/8-matured, LAA-expressing DCs in AML is feasible, safe and induces anti-leukemia-specific immune responses in vivo. Citation Format: Katrin Deiser, Felix S. Lichtenegger, Frauke M. Schnorfeil, Thomas Köhnke, Torben Altmann, Veit Bücklein, Christian Augsberger, Andreas Moosmann, Monika Brüggemann, Mirjam HM Heemskerk, Beate Wagner, Wolfgang Hiddemann, Iris Bigalke, Gunnar Kvalheim, Marion Subklewe. TLR7/8-matured dendritic cells for therapeutic vaccination in AML: Results of a clinical Phase I/II trial [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B002.

Abstract B146: Next-generation dendritic cell vaccination in postremission therapy of AML: Results of a clinical phase I trial

Abstract Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. We have developed a GMP-compliant 3-day protocol including a TLR7/8 agonist to differentiate monocytes of intensively pretreated AML patients into next-generation DCs. A phase I proof-of-concept study (n=6) was completed using next-generation DCs as postremission therapy of AML patients with a non-favorable genetic risk profile in CR after intensive induction therapy (NCT01734304), and we have already started enrollment into a phase II trial building on these results. DCs are loaded with ivt-RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients are vaccinated intradermally with 5x106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. In total, 10 patients have been enrolled into the phase I/II trial. With two screening failures, DCs of sufficient number and quality were generated from leukapheresis in 7/8 cases. DC analysis demonstrated a positive costimulatory profile, secretion of IL-12p70, migration towards a chemokine gradient, antigen processing and presentation and activation of specific T cells in vitro. 5 patients have completed the vaccination schedule; the 6th patient has received 4/10 vaccinations. We observed delayed-type hypersensitivity (DTH) responses at the vaccination site in 5/5 patients, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. Multimer analysis revealed the induction of antigen-specific T cell responses in 3/3 patients tested. We detected an increase of WT1-specific T cells in one patient and strong inductions of CMVpp65-specific T cells in two CMV-seronegative patients. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. In an individual treatment attempt, we treated one patient with impending relapse with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. From the results of our phase I trial, we conclude that vaccination with next-generation DCs in AML is feasible and safe and induces leukemia-specific immune responses in vivo. The protocol is continued to be studied in an ongoing phase II trial. Citation Format: Felix S. Lichtenegger, Frauke M. Schnorfeil, Thomas Köhnke, Torben Altmann, Veit Bücklein, Andreas Moosmann, Monika Brüggemann, Beate Wagner, Wolfgang Hiddemann, Iris Bigalke, Gunnar Kvalheim, Marion Subklewe. Next-generation dendritic cell vaccination in postremission therapy of AML: Results of a clinical phase I trial. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B146.

Next-Generation Dendritic Cell Vaccination in Postremission Therapy of AML: Results of a Clinical Phase I Trial

Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. We have developed a GMP-compliant 3-day protocol including a TLR7/8 agonist to differentiate monocytes of intensively pretreated AML patients into next-generation DCs.A phase I/II proof-of-concept study has been initiated using next-generation DCs as postremission therapy of AML patients with a non-favorable genetic risk profile in CR after intensive induction therapy (NCT01734304). DCs are loaded with in vitro transcribed RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients are vaccinated intradermally with 5x106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I/II trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control.Based on the safety and toxicity profile of the phase I trial (n=6), phase II has been initiated. In total, 10 patients have been enrolled into the study. DCs of sufficient number and quality were generated from leukapheresis in 8/9 cases. DCs exhibited an immune-stimulatory profile based on high surface expression of positive costimulatory molecules, the capacity to secrete IL-12p70, the migration towards a chemokine gradient and processing and presentation of antigen. 5 patients have completed the vaccination schedule; the 6th and 7th patient have received 7/10 and 4/10 vaccinations, respectively. We observed delayed-type hypersensitivity (DTH) responses at the vaccination site in 6/6 patients, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. Limited by HLA restriction, we have so far analyzed 4 patients by multimer staining. All of them mounted DC vaccination-specific T cell responses: We detected an increase of WT1-specific T cells in one patient and strong expansion/induction of CMVpp65-specific T cells in one CMV-seropositive and two CMV-seronegative patients. In an individual treatment attempt, an enrolled patient with impending relapse was treated with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. Long-term disease control and immunological responses are studied in the ongoing phase II trial.We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe and induces anti-leukemia-specific immune responses in vivo. DisclosuresSubklewe:AMGEN Research (Munich): Research Funding.

Three Protein Cocktails Mediate Delayed-Type Hypersensitivity Responses Indistinguishable from That Elicited by Purified Protein Derivative in the Guinea Pig Model ofMycobacterium tuberculosisInfection

Purified protein derivative (PPD) is a widely used reagent for the diagnosis of Mycobacterium tuberculosis infection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4(+)/CD8(+) T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4(+) and CD8(+) T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis of M. tuberculosis infection.

Induction of delayed-type hypersensitivity and interferon-gamma to Candida albicans and anti-hen-egg white lysozyme antibody as phenotypic markers of enhanced bovine immune response

Delayed-type hypersensitivity (DTH) is a protective localized cell-mediated immune response (CMIR), primarily against intracellular pathogens. DTH is widely used in research to assess immune responsiveness and has been a valuable diagnostic test in commercial settings. In pigs and other species both antibody (AMIR) and CMIR have been considered as reliable phenotypic markers of selection programs for disease resistance. Therefore in cattle, it was also considered important to find antigen/adjuvant combinations capable of inducing AMIR and CMIR without interfering with diagnostic tests. The objectives of the present study were to evaluate the combined use of hen-egg white lysozyme (HEWL) and Candida albicans adjuvanted with Quil A in lactacting Holstein cows for the induction of anti-HEWL antibody, as well as DTH and IFN-γ to C. albicans as phenotypic markers of enhanced immune responsiveness. Thirty one lactating Holstein cows were immunized with HEWL to induce antibody responses and C. albicans to sensitize for DTH. Two test antigens, candin and C. albicans whole cell (CaWC), were used to induce the effector phase of DTH. PBS was used as the negative control. In addition, two different skin sites (neck versus tail) were tested to evaluate differences in skin site responsiveness. C. albicans-induced IFN-γ production, as an indicator of a type 1 response, was evaluated by ELISA. Microscopic evaluation of skin samples at DTH sites was performed in five randomly selected cows and these skin biopsies were scored based on inflammation and cell infiltration. Results demonstrated the presence of classical DTH response to C. albicans, in that DTH responses peaked at 24 h post-intradermal injections and cell infiltration was composed largely of mononuclear leukocytes, typical of DTH skin reactions in cattle. The only difference in test antigens was that DTH to candin showed a higher early response (6 h) than CaWC and a rapid decrease in inflammation from 24 to 48 h. The neck was significantly more sensitive than the tail skin-fold as a DTH test site. IFN-γ was detected on days 14 and 21 post-immunization in plasma from blood incubated with candin. Significant primary and secondary anti-HEWL antibodies were also detected, indicating that this combination of test antigens could be used as phenotypic markers of immune responsiveness in cattle.

Immunohistological analysis of peptide-induced delayed-type hypersensitivity in advanced melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination

In melanoma patients vaccinated with monocyte-derived melanoma peptide-pulsed dendritic cells (DC), the delayed-type hypersensitivity (DTH) reactions have been examined as a surrogate marker to determine if acquired immunity is induced by DC vaccination. To date, however, only limited information has been reported as for histopathological analyses of DTH. To evaluate tumor-specific immunomonitoring histopathologically after DC vaccination in melanoma patients. Seven patients previously vaccinated with monocyte-derived melanoma peptide-pulsed DCs were challenged with recall antigenic peptide injection in the skin of the forearm. Using immunohistochemical techniques, the presence of immune cells and the expression of CD4, CD8, interleukin (IL)-2, IL-4, IL-10, Foxp3, CD1a, CD1d, and interferon (IFN)-gamma was investigated at the site of injection where a DTH reaction developed. Strong DTH reactions from infiltrated erythema to bullae formation were detected in all 7 cases. Biopsies taken from the DTH site revealed heavy infiltration of mononuclear cells and eosinophils in the dermis and subcutaneous tissue. Cells staining positively for CD4, CD8, IL-2, IL-4, Foxp3, CD1d, and IFN-gamma were increased at the site 48h after antigen injection in all cases. Cells positive for IL-10 were never found in any patient. Regulatory T cells appeared 6h after injection and reached their maximum at day 7. The significant induction of CD8(+)T cells as well as both Th1 and Th2-type cells at the site of DTH suggests that effective antigen presentation leading to anti-tumor immune responses has taken place. Inhibitory mechanisms may also develop as the disappearance of the DTH response could be related to an increase in Foxp3+ cells.

Potential applications of dendritic cells

Dendritic cells (DCs) are the professional antigen‐presenting cells of the immune system. Following infection or inflammation they undergo a complex process of maturation, and migrate to lymph nodes where they present antigens to T cells. Their decisive role in inducing immunity formed the rationale for DC immunotherapy: DCs loaded with tumor antigens are injected into cancer patients to stimulate T cells to eradicate tumors. Effective immune responses and favourable clinical outcomes have indeed been observed, but only in a minority of patients.For effective immunotherapy DCs need to traffic throughout the vascular and lymphatic system to reach the T‐cells located within lymph nodes. Though most immunotherapeutic agents are administered intravenously, DCs are predominantly administered intradermally. We observed that < 5% of intradermally administered mature DCs reach the draining lymph nodes amounting to inefficient homing. Despite this low number we could measure effective immune responses in some patients, but generally this may be too low.We demonstrated that DC maturation is a prerequisite to exert their immunostimulatory capacity in vivo. Immuno‐monitoring revealed a remarkable difference in immune responses. In patients vaccinated with immature DC the KLH responses as well as DTH reactivity against KLH and tumor‐peptides were weak and absent, respectively. In contrast, in patients vaccinated with mature DC a pronounced T cell as well a B cell response (IgG) against KLH were observed. Analysis of the response against the tumor peptides demonstrated little or no reactivity in blood. However, following intradermal administration of a delayed type hypersensitivity (DTH) challenge with gp100‐ and tyrosinase‐peptide loaded DC essentially all patients vaccinated with mature DC showed a positive induration. Moreover, we showed the predictive value of the presence or absence of antigen‐specific T cells in biopsies from DTH sites. In clinically responding patients, T cells specific for the antigen preferentially accumulated in the DTH site in accordance with the applied antigen in the DTH challenge.

Preliminary Analysis of Patients With Progressive Renal Cell Carcinoma Vaccinated With CA9-peptide–Pulsed Mature Dendritic Cells

Carbonic anhydrase-IXG250/MN (CA9) is a renal cell carcinoma (RCC)-associated antigen ubiquitously expressed in the clear-cell subtype of RCC. Two CA9-derived peptides have been identified defining a cytotoxic T-lymphocyte epitope and human leukocyte antigen (HLA)-DR epitope, able to induce T-cell responses in vitro. A phase I clinical trial was performed with CA9-peptide-loaded dendritic cells (DCs) in patients with progressive, cytokine-refractory metastatic RCC to assess the safety, toxicity, and induction of CA9-specific immunity. Patients with objective progressive metastatic RCC received 5 vaccinations of mature DCs pulsed with the CA9-derived peptides and keyhole limpet hemocyanine (KLH). Peripheral blood was collected at regular intervals, delayed-type hypersensitivity (DTH) was tested at baseline and after the last vaccination, and skin biopsies of positive DTH sites were collected for immunomonitoring purposes. Patients were also monitored for clinical responses. No significant toxicity was observed. All patients developed humoral responses against KLH, and demonstrated DTH conversion. Evaluation of biopsy material suggested an increased influx of T-helper cells. In none of the immunomonitoring assays was evidence for the induction of CA9-peptide-specific immunity observed. No clinical responses were observed. The vaccination of DCs pulsed with KLH and 2 CA9-derived peptides was well tolerated. The lack of induction of CA9-peptide-specific immune responses indicates that this particular vaccine regimen is poor in inducing CA9-peptide-specific immune responses.

Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate‐pulsed mature, monocyte‐derived dendritic cells

We performed a clinical trial to assess the feasibility and efficacy of immunotherapy with peptides, tumor lysate or both peptides and tumor lysate-pulsed mature, monocyte-derived dendritic cells (DC) for advanced malignant melanoma patients that are resistant to conventional therapies. Sixteen patients were enrolled in this trial. All patients received DC vaccines i.d. in the proximal thigh, close to the inguinal lymph nodes, one treatment per week or 2 weeks. Several factors such as clinical findings, computed tomography (CT) images, delayed type hypersensitivity (DTH) response, enzyme-linked immunosorbent spot (ELISPOT) assay, and immunohistochemistry in primary, metastatic lesions and the DTH site were evaluated. Clinical results through DC vaccination were as follows: in 11 evaluable cases, three stable disease, six progression of disease and two disease-free from the time of study entry to the completion of one vaccination course. One patient showed reduction of the tumors in the metastases on chest CT during the first and second course of DC vaccination. Ten out of 14 evaluable cases showed positive DTH responses to more than one treatment with melanoma peptides or tumor lysate. Eight out of 13 evaluable cases showed positive immunological responses to more than one treatment with melanoma peptides or tumor lysate in an ELISPOT assay. As for the experiences with toxicity and adverse reactions, autosensitization dermatitis-like eruptions appeared in five cases during DC vaccination. No severe adverse effects were seen in any of the patients. In our study, the clinical efficacy in prolongation of the patients' survival was confirmed. At the same time, cancer immunoediting of the tumor was also found. It will be necessary to improve the tumor-specificity of this therapeutic approach and to analyze the mechanism(s) of tumor escape from immunosurveillance in melanoma.

Immunomonitoring Tumor-Specific T Cells in Delayed-Type Hypersensitivity Skin Biopsies After Dendritic Cell Vaccination Correlates With Clinical Outcome

Tumor-specific immunomonitoring is essential to evaluate the efficacy of vaccination against cancer. In this study, we investigated the predictive value of the presence or absence of antigen-specific T cells in biopsies from delayed-type hypersensitivity (DTH) sites. In our ongoing clinical trials, HLA-A2.1+ melanoma patients were vaccinated with mature dendritic cells (DC) pulsed with melanoma-associated peptides (gp100 and tyrosinase) and keyhole limpet hemocyanin. After intradermal administration of a DTH challenge with gp100- and tyrosinase peptide-loaded DC, essentially all patients showed a positive induration. In clinically responding patients, T cells specific for the antigen preferentially accumulated in the DTH site, as visualized by in situ tetramer staining. Furthermore, significant numbers of functional gp100 and tyrosinase tetramer-positive T cells could be isolated from these DTH biopsies, in accordance with the applied antigen in the DTH challenge. We observed a direct correlation between the presence of DC vaccine-related T cells in the DTH biopsies of stage IV melanoma patients and a positive clinical outcome (P = .0012). These findings demonstrate the potency of this novel approach in the monitoring of vaccination studies in cancer patients.

CXCR3, IP-10, and Mig are required for CD4 + T cell recruitment during the DTH response to HSV-1 yet are independent of the mechanism for viral clearance

Sensitized CD4 + T cells play an essential role in delayed type hypersensitivity (DTH) elicited by HSV-1 antigen. As activated CD4 + T cells express CXCR3, we investigated whether this chemokine receptor was involved in their recruitment. Antibody blockade of CXCR3 suppressed DTH, whereas ear pinna swelling was not impaired in mice lacking the gene for CCR5, another frequently expressed chemokine receptor. CXCR3 ligands IP-10 and Mig were elevated at the DTH site. Their neutralization significantly reduced DTH ear swelling and CD4 + T cell influx. Furthermore, CXCR3 ligand expression was abrogated and DTH diminished in mice unable to make IFN-γ, a potent inducer of IP-10 and Mig. Interestingly, neutralization of CXCR3 or its ligands did not compromise host resistance to virus replication. Collectively, these results suggest that in the sensitized host, CXCR3, IP-10, and Mig are required for optimal DTH responsiveness but are not essential for containing HSV-1 replication in the ear pinna.

A novel role for neutrophils as a source of T cell-recruiting chemokines IP-10 and Mig during the DTH response to HSV-1 antigen

Analogous to CD4+ T cells, neutrophils are essential participants in delayed-type hypersensitivity (DTH) to Herpes simplex virus type 1 antigen. However, what role they play in this cellular immune response is unclear. The recent recognition that neutrophils are potent producers of chemokines led us to hypothesize that they may help recruit CD4+ effector T cells. In the present study, we show that neutrophil depletion was accompanied by a marked decrease in the numbers of CD4+ and CXC receptor 3+ (CXCR3+)-expressing cells migrating to the DTH site and a sharp drop in the levels of interferon-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig). Purified mouse neutrophils were stimulated directly by IFN-gamma to secrete these chemokines, and neutrophils at the DTH site expressed IP-10. IFN-gamma knockout mice, which manifested depressed ear-swelling following DTH challenge, made little IP-10 and no Mig. Reconstitution of these mice with IFN-gamma induced CXCR3 ligand synthesis. Depletion of neutrophils or CD4+ T cells but not CD8+ T cells markedly reduced IFN-gamma levels, suggesting the former were direct (or indirect) cellular sources of this cytokine. Collectively, our results support the hypothesis that neutrophil production of T cell-recruiting chemokines contributes to the regulation and amplification of the DTH response.

Pulmonary tuberculosis in BALB/c mice with non-functional IL-4 genes: changes in the inflammatory effects of TNF-alpha and in the regulation of fibrosis.

In BALB/c mice, as in man, progressive pulmonary tuberculosis is accompanied by increasing expression of IL-4. Therefore we have used BALB/c mice with disrupted IL-4 genes (IL-4(-/-)) to investigate the role of IL-4 in pulmonary tuberculosis, with particular emphasis on the toxicity of TNF-alpha and on fibrosis, both of which are neglected aspects of human tuberculosis. Delayed-type hypersensitivity (DTH) sites in IL-4(+/+) mice were sensitive to the toxicity of locally injected TNF-alpha, whereas DTH sites in IL-4(-/-) mice were not. However, intravenous administration of IL-4 to IL-4(-/-) mice restored the sensitivity of the DTH sites to pro-inflammatory effects of TNF-alpha. In late disease, the lungs of IL-4(+/+) mice expressed low IFN-gamma, but high TGF-beta and IL-4, correlating with fibrosis, detected as a high hydroxyproline content. In contrast, TGF-beta peaked 7 days after infection in the lungs of the IL-4(-/-) mice, and then fell to very low levels in the late disease, while IFN-gamma remained high. Accordingly, hydroxyproline content was reduced in infected IL-4(-/-) mice compared to IL-4(+/+) controls. In conclusion, the findings suggest that IL-4 has modestly detrimental effects on the antibacterial efficacy of the Th1 response, and larger effects on the toxicity of TNF-alpha, and on fibrosis.

Stress, leukocyte trafficking, and the augmentation of skin immune function.

Delayed type hypersensitivity (DTH) reactions represent cell-mediated immune responses that exert important immunoprotective (resistance to viruses, bacteria, and fungi) or immunopathologic (allergic or autoimmune hypersensitivity) effects. We have used the skin DTH response as an in vivo model to study neuro-endocrine-immune interactions. We hypothesized that just as an acute stress response prepares the cardiovascular and musculoskeletal systems for fight or flight, it may also prepare the immune system for challenges (e.g., wounding) that may be imposed by a stressor (e.g., an aggressor). Studies showed that acute (2 hours) stress experienced before primary or secondary cutaneous antigen exposure induces significantly enhanced skin DTH. This enhancement involves innate as well as adaptive immune mechanisms. Adrenalectomy eliminates the stress-induced enhancement of DTH. Acute administration of physiological concentrations of corticosterone and/or epinephrine to adrenalectomized animals enhances skin DTH. Compared with those in controls, DTH sites from acutely stressed or hormone-injected animals show significantly greater erythema and induration, numbers of infiltrating leukocytes, and levels of cytokine gene expression. In contrast to acute stress, chronic stress is immunosuppressive. Chronic exposure to corticosterone or acute exposure to dexamethasone significantly suppresses skin DTH. These results suggest that during acute stress, endogenous stress hormones enhance skin immunity by increasing leukocyte trafficking and cytokine gene expression at the site of antigen entry. Elucidation of mechanisms mediating a stress-induced enhancement of skin immune function is important because such immunoenhancement can have protective (wound healing, resistance to infection) or pathological (allergic or autoimmune hypersensitivity) consequences.

CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis

Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mφ) recruitment into M. tuberculosis–infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44–/–) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-γ were reduced in the lungs of infected CD44–/– mice. The production of IFN-γ by splenocytes of CD44–/– mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mφ-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44–/– Mφs. This suggests that CD44 is a receptor on Mφs for binding of M. tuberculosis. CD44–/– mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mφ binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mφ recruitment, and protective immunity against pulmonary tuberculosis.

CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis.

Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mphi) recruitment into M. tuberculosis-infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44(-/-)) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-gamma were reduced in the lungs of infected CD44(-/-) mice. The production of IFN-gamma by splenocytes of CD44(-/-) mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mphi-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44(-/-) Mphis. This suggests that CD44 is a receptor on Mphis for binding of M. tuberculosis. CD44(-/-) mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mphi binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mphi recruitment, and protective immunity against pulmonary tuberculosis.

HER2 Dendritic Cell Vaccines

HER2/ neu, a tumor antigen overexpressed by a third of breast cancers, is a potential target for vaccine therapies. A particularly potent immunization strategy to induce T-cell responses against tumor antigens is to use dendritic cells (DCs) loaded with the tumor antigen. We performed two small studies to test the safety, feasibility, and immunologic and clinical responses to immunizations with in vitro—generated DCs loaded with either a human leukocyte antigen A2—restricted peptide fragment of the extracellular domain of the tumor antigen HER2 (E75) or a HER2 intracellular domain (ICD) protein in patients with high-risk resected breast cancer or metastatic cancers expressing HER2. There were no toxicities due to the immunizations in any of the patients. In the study of DCs loaded with the E75 peptide, 1 of 6 patients with metastatic HER2-expressing malignancies who completed all immunizations had stable disease for 6 months; the remainder of the patients had progressive disease. Delayed-type hypersensitivity (DTH) reactivity (2-3 mm of induration) at E75-loaded DC injection sites was observed in 2 of 5 patients evaluated but was similar at the unloaded DC injection sites. In 2 patients, the DTH sites underwent biopsy and a perivascular infiltrate of CD4 and CD8 cells was demonstrated, which was greater in the E75-loaded DC injection sites than in the unloaded DC sites. In the pilot study of ICD-loaded DC in patients with high-risk resected breast cancer, all 3 patients enrolled had no evidence of recurrence at a follow-up of up to 2.5 years. Intracellular domain—specific T-cell responses were detected directly from the peripheral blood by enzyme-linked immunospot and proliferation assay in 2 patients. We conclude that it is feasible and safe to generate and administer HER2-loaded DCs to patients with advanced HER2/ neu-expressing malignancies and high-risk breast cancer. The magnitude of the immune responses generated is fairly modest, and more potent DC loading and maturation strategies will be necessary to optimize these vaccines.

Stress-induced augmentation of immune function—The role of stress hormones, leukocyte trafficking, and cytokines

Delayed-type hypersensitivity (DTH) reactions represent cell-mediated immune responses that exert important immunoprotective (resistance to viruses, bacteria, and fungi) or immunopathological (allergic or autoimmune hypersensitivity) effects. We initially utilized the skin DTH response as an experimental in vivo model to study neuro–endocrine-immune interactions in rodents. We hypothesized that just as an acute stress response prepares the cardiovascular and musculoskeletal systems for fight or flight, it may also prepare the immune system for challenges which may be imposed by a stressor. The skin DTH model allowed us to examine the effects of stress at the time of primary and secondary exposure to antigen. Studies showed that acute (2 h) stress experienced before primary or secondary antigen exposure induces a significant enhancement of skin DTH. Importantly, this enhancement involved innate as well as adaptive immune mechanisms. Adrenalectomy eliminated the stress-induced enhancement of DTH. Acute administration of physiological (stress) concentrations of corticosterone and/or epinephrine to adrenalectomized animals enhanced skin DTH. Compared with controls, DTH sites from acutely stressed or hormone-injected animals showed significantly greater erythema and induration, numbers of infiltrating leukocytes, and levels of cytokine gene expression. In contrast to acute stress, chronic stress was immunosuppressive. Chronic exposure to corticosterone, or acute exposure to dexamethasone significantly suppressed skin DTH. These results suggest that during acute stress, endogenous stress hormones enhance skin immunity by increasing leukocyte trafficking and cytokine gene expression at the site of antigen entry. While these results are discussed from a mechanistic and clinical relevance perspective, it is acknowledged that much work remains to be done to elucidate the precise mechanisms mediating these bi-directional effects of stress and stress hormones and their clinical ramifications.

Addition of an IL-15 mutant/FCγ2A antagonist protein protects islet allografts from rejection overriding costimulation blockade

IL-15, a powerful chemoattractant and growth factor for antigen-activated T cells and NK cells, contributes to lymphocyte homeostasis and selectively induces the activation and proliferation of memory CD8+ T cells.1,2 Analysis of the phenotype of IL-15−/− mice3 and IL-15Rα−/− mice4 demonstrate that IL-15 is a critical factor for innate and adaptive immune responses. IL-15 is produced by the monocyte/macrophage lineage, by epithelial and endothelial cells but not by T-cells. The IL-15 receptor (IL-15R) expressed upon T cells is composed of three chains. Two are constitutively expressed and shared with the IL-2 receptor (IL-2R), the IL-2Rβ and IL-2Rγc chains, and a unique IL-15Rα chain is expressed upon activation of, but not during resting of mononuclear leukocytes.5,6 Because IL-15 and IL-2 share two receptor signaling components (IL-2/IL-15Rβγc), they display similar biological properties in vitro. Nevertheless, there are differential patterns of IL-15/IL-15Rα expression and in vivo studies have revealed major nonredundant roles for these two T-cell growth factors (TCGFs). In organ transplantation, T cells play a critical role in initiating the immune response leading ultimately to the effector mechanisms mediating allograft rejection. An analysis of biopsies taken from rejecting human renal allografts has revealed that intragraft gene expression of IL-15 is a far more consistent marker of rejection than IL-2 gene expression.7 Moreover, IL-2 expression is not a prerequisite for allograft rejection as IL-2 knockout (KO) mice promptly reject islet and cardiac allografts.7,8 Immunohistology in the IL-2 KO recipients reveals infiltration of rejecting grafts by CD4 and CD8 T cells as well as robust intragraft expression of the IL-4, IL-7, and IL-15 as well as granzyme B genes.8 Since costimulation blockade drastically decreases post-transplant expression of IL-2,9 the clonal proliferation of alloreactive T cells in hosts treated with costimulation blockade may be explained by the action of a non-T-cell-derived cytokine, such as IL-15. Although costimulatory blockade of CD28/CTLA4/B7 or CD40/CD40L interactions reduces immune activation and provides a powerful inhibition of alloimmune responses in both rodents and primates,9–11 in some circumstances it failed to induce permanent engraftment. Several reports suggest that CD8+ T cells are responsible for costimulation blockade-resistant rejection in various models, such as intestinal and skin allograft models.12,13 For example, adjunctive treatment with anti-CD8 mAb or other agents to deplete CD8+ T cells, are required to induce tolerance in these models.14 The resistance of CD8+ T cells to costimulation blockade may be explained by a poor effect of CTLA4Ig and αCD154 Abs to regulate activation and proliferation of CD8+ compared to CD4+ T cells.14,15 Recently, Smith et al16 have reported the prolonged survival of heart allografts in a treatment protocol consisting of soluble IL-15 receptor- α chain proteins and nondepleting anti-CD4 mAb. Thus the expression of IL-15 may be linked to CD8-dependent costimulation-resistant rejection. As costimulation blockade resistant rejection is mediated by CD8+ T cells and as IL-15 is essential for the activation and proliferation of CD8+ T cells, we hypothesized that targeting the IL-15/IL-15R system in conjunction with costimulation blockade would provide a new perspective for inducing allograft tolerance. The development of agents targeting the receptor and signaling elements of IL-15 may provide new perspectives for treatment of diseases associated with the IL-15/IL-15R pathway. The idea of creating an IL-15 antagonist was based on the homology of glutamine residues within the C-terminus of the four helix structures shared by IL-2 and IL-15, and on the report that a C-terminal glutamine in IL-2 is crucial for IL-2 binding to IL-2Rγ chain.17 In our laboratory a novel IL-15 mutant/Fcγ2a protein was genetically constructed, linked to murine Fcγ2a and expressed in mammalian cells. The IL-15 mutant was constructed by replacing the codons for the C-terminal glutamine amino acid residues with codons for aspartic acid (ie, Q101 and Q108).18 The mutant IL-15 moiety is a high-affinity IL-15R site-specific antagonist. The Fc portion of this fusion protein confers longevity and is designed to support cytocidal activities against IL-15R α+ cells insofar as the Fc-related sequences of mouse IgG2a isotype that support complement activation and activation of FcR+ phagocytes are present.18 Moreover, this IL-15 mutant/Fcγ2a protein competitively inhibits IL-15-triggered cell proliferation and does not activate the STAT-signaling pathway. Because the receptor site-specific antagonist IL-15 mutant/Fcγ2a protein had a prolonged half-life in vivo and the potential for destruction of IL-15R + leukocytes, the immunosuppressive effects of this agent were examined in mice with induced delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA), a murine model for RA. In the DTH model, treatment with IL-15 mutant/Fcγ2a protein attenuated DTH responses and mononuclear leukocyte infiltration within the DTH sites.18 In the CIA model, treatment with IL-15 mutant/Fcγ2a protein markedly decreased the incidence and severity of arthritis in mice (S. Ferrari-Lacraz, inpreparation). As this unique IL-15 mutant/Fcγ2a protein displays promising effects on T-cell-derived pathogenesis, we wanted to determine its role and relative merits as a new therapeutic strategy in transplantation.