Recognition of shared melanoma antigen by HLA-A2-restricted cytolytic T cell clones derived from human tumor-infiltrating lymphocytes

Three melanoma-specific cytotoxic T lymphocytes (CTL) clones were derived from the tumor-infiltrating lymphocyte (TIL) of human melanoma M17, and were used to study the expression of immunogenic melanoma peptides on allogeneic tumors. Antibody inhibition studies showed that two of these TIL clones were restricted by an HLA-A2 molecule which was identified as A2.1 by gene sequencing. The third CTL clone was not restricted by HLA-A2, but by a B or C HLA antigen. HLA-A2-restricted CTL clones M17-1 and M17-2 lysed 5 and 12 out of 15 HLA-A2⁺ allogeneic melanomas, respectively. Since they did not lyse autologous Epstein-Barr virus B cells, HLA-A2.1-transfected P815 cells,13 HLA-A2⁺ non-melanoma tumor cell lines and 10 HLA-A2^? melanomas, these clones appeared specific for melanoma-restricted epitopes presented by the HLA-A2.1 molecule. We then tried to determine why a few HLA-A2⁺ melanomas were refractory to TIL lysis. By using a combination of flow cytometry analysis, partial cloning and sequencing of their HLA-A2 genes, we show that failure to lyse did not result from low expression or polymoprhism of the HLA-A2 molecule, or from deficient expression of the adhesion molecules ICAM-1 and LFA-3 by these melanomas. Taken together, our data confirm at the clonal level the existence of shared melanoma antigens recognized by TIL in the HLA-A2.1 context. They further show that individual peptides derived from these antigens are expressed by a large majority of HLA-A2⁺ melanomas. Identification of such peptides appears crucial for the future of vaccination therapies.     

HLA-A and HLA-DRB1 amino acid polymorphisms are associated with susceptibility and protection to pulmonary tuberculosis in a Greek population

Background

Pulmonary tuberculosis remains the single deadliest infectious disease causing high mortality in humans leading to 1.4 million deaths annually. Inherited genetic factors may explain why some people resist infection more successfully than others.

Methods

The polymorphisms of HLA-class I (-A, -B) and class II (-DRB1, -DQB1) genes have been evaluated using DNA-based typing in a population of 86 non-immunosuppressed, unrelated Greek patients with PTb and 46 healthy unrelated people without a history of PTb, who were all tested purified protein derivative positive (>14 mm).

Results

The HLA-A R¹¹⁴ and HLA-DRβN³⁷ residues are associated with susceptibility. They operate independently from each other and their effect is detected when the population is evaluated for their concurrent presence (A R¹¹⁴ positive or DRβN³⁷ positive or A R¹¹⁴ and DRβN³⁷ positive). Furthermore the HLA-A S⁷⁷ appears to have a protective role, however in the presence of the DRβN³⁷, the A-S⁷⁷ does not exert its protective effect.

Conclusion

The HLA residues A-S⁷⁷, A-R¹¹⁴ and DRβN³⁷ in combination with PTb antigenic elements possibly modulate T-cell responses against MTb that lead to either protection or susceptibility. The HLA-A and -DRB1-dependent T-cell networks may interact among themselves and influence each other resulting in different PTb phenotypes.     

Identification and validation of shrimp-tropomyosin specific CD4 T cell epitopes

Background

Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE.

Objective

We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy.

Method

Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects.

Result

17 epitopes restricted to DRB^∗01:01, DRB1^∗03:01, DRB1^∗04:01, DRB1^∗09:01, DQB1^∗02:01, DQB1^∗03:02 and DQB1^∗05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13.

Conclusion

The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.     

In vitro-expanded CD4CD25Foxp3 regulatory T cells controls corneal allograft rejection

Aims

Natural CD4⁺CD25⁺ regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model.

Methods

Natural CD4⁺CD25⁺ T cells were freshly purified from naïve mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-β1). Suppression ability of iTregs was assayed by co-culturing with CD4⁺CD25⁻ T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling.

Results

Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-β1. Compared with nTregs, iTregs had similar expression of CD62L, and PD- L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4⁺CD25⁻ T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression.

Conclusion

Therapeutic use of in vitro-expanded CD4⁺CD25⁺ T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance.     

B7-H4 overexpression impairs the immune response of T cells in human cervical carcinomas

Objective

To investigate B7-H4 expression and its correlation with the number of infiltrating T lymphocytes and cytokine production by those lymphocytes in human cervical cancer and to determine the effect of recombinant B7-H4 on the active peripheral blood T cells of the patients in vitro.

Methods

B7-H4 expression was detected in 67 cases of cervical cancer using immunohistochemical staining. Tumor-infiltrating CD8⁺T, CD4⁺T, and FOXP3⁺ (Forkhead Box P3) T lymphocytes and their levels of IFN-γ and TGF-β₁ production were determined by immunofluorescent double-staining. After the peripheral blood T lymphocytes of patients were co-cultured with B7-H4, proliferation, apoptosis, and cell subtypes were analyzed using flow cytometry. Cytokines in the supernatant were detected by ELISA.

Results

B7-H4 was expressed in 46% (31/67) of the cases of cervical cancer. The number of infiltrating CD8⁺T lymphocytes and their IFN-γ production in positive B7-H4 expression cervical cancers was significantly lower than in negative B7-H4 cases (P < 0.01, P < 0.05), but there was no significant difference between cases positive and negative for B7-H4 with respect to infiltrating FOXP3⁺T and CD4⁺T cells or TGF-β1 production. After co-culture with B7-H4 for 48 h, the patients’ activated T lymphocytes were arrested at G1/G2 phase. The Ki67 positive rates of CD4⁺T and CD8⁺T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, and they were lower than in the blank group. The proportion of CD4⁺T and CD8⁺T cells decreased, but CD4⁺T/CD8⁺T and the proportion of CD4⁺CD25⁺Foxp3⁺T cells increased. In addition, concentrations of IL-10 and TGF-β1 in the supernatant of co-cultured T cells increased significantly (P < 0.05, P < 0.05), but that of IFN-γ decreased. B7-H4 had no significant effect on apoptosis of the T cells.

Conclusion

B7-H4 is overexpressed in human cervical cancers, and it is associated with lower numbers of tumor-infiltrating CD8⁺T lymphocytes and therefore less IFN-γ production. In vitro, B7-H4 inhibits the proliferation of CD4⁺T and CD8⁺T but promotes the proliferation of Tregs and the secretion of IL-10 and TGF-β1. B7-H4 plays an important role in depressing the anti-tumor immunity of CD8⁺T cell in microenvironments of cervical cancer.     

Correlation between infiltration of FOXP3 regulatory T cells and expression of B7-H1 in the tumor tissues of gastric cancer

Background

Substantial evidence suggests that the expansion of regulatory T cells (T_regs) plays a pivotal role in immunological evasion of tumors. Recent studies have demonstrated that a majority of tumor cells overexpress B7-H1, and this overexpression is associated with poor disease prognosis. Although an increase of T_regs and B7-H1 has been revealed in several malignancies, their correlation in gastric cancer has not been studied.

Methods

Tumor sections from 111 gastric cancer patients were stained for FOXP3 and B7-H1 by immunohistochemistry. The expression levels of these two molecules were statistically associated with various factors involved in disease progression and prognosis. The correlation between their expression levels was analyzed.

Results

The infiltration of FOXP3⁺ Tregs and expression of B7-H1 were observed in gastric cancer tissues, and there was a highly significant correlation between these two molecules (P < 0.01). The expression of FOXP3⁺ Tregs and B7-H1 was associated with lymph node metastasis and the clinicopathological stage and prognosis of gastric cancer patients. The expression levels of these two determinants in patients with lymph node metastasis and an advanced clinicopathological stage were distinctly higher (P < 0.05). The patients with enhanced expression of FOXP3⁺ Tregs and B7-H1 exhibited a lower overall survival rate and a worse prognosis (P < 0.05).

Conclusions

Increased expression of FOXP3⁺ Tregs and B7-H1 was observed in gastric cancer tissues; the two molecules were closely correlated with each other, suggesting that they might be used as new biomarkers to predict the disease progression and prognosis. Combinatorial immunotherapeutic approaches based on depleting the T_regs and blocking B7-H1 might improve therapeutic efficacy in gastric cancer.     

Expression of IL-7 receptor in human peripheral regulatory T cells

Introduction

Regulatory T cells (Tregs, CD4 + CD25^high Foxp3⁺) play a crucial role in allergy and other inflammatory diseases. However, the isolation of viable Tregs on the basis of intracellular expression of specific Forkhead Box Protein P3 (Foxp3) is difficult. In this study we checked if the expression of IL-7 receptor (CD127) on the Tregs could be a useful marker for isolation of viable Treg Foxp3⁺ cells.

Material and methods

Twenty-five patients sensitized to grass pollen with allergic rhinitis (AR) and ten healthy subjects were included. We compared Foxp3 expression in different CD4⁺ T cell subsets by flow cytometry and we assessed the relationship between the expression of Foxp3 and CD127 within regulatory T cells.

Results

Within the CD⁴+ lymphocytes 3.68 ±2.0% showed expression of Foxp3, 51.82 ±8.03% of CD4⁺CD25^high were Foxp3 positive (Foxp3⁺), whereas 82.12 ±5.4% of CD4⁺CD25^highCD127^low were Foxp3⁺. High intracellular expression of Foxp3 correlated with low superficial CD127 expression (r = 0.42, p = 0.017). There were no significant differences regarding the analysed markers between AR patients and healthy controls.

Conclusions

Regulatory T cells may be purified from the fresh peripheral blood as viable regulatory Foxp3 bright cells using CD4, high expression of CD25 and low expression of CD127 antigen.     

Inflammatory cells in minor salivary glands of patients with chronic hepatitis C: Immunophenotype,pattern of distribution,and comparison with liver samples

Objectives

To characterize the immunophenotype and the distribution of the inflammatory infiltrate (INF) in salivary glands (SG) of patients with chronic hepatitis C, comparing with laboratorial data (genotype, viral load, METAVIR, and HCV RNA in SG), and liver.

Methods

INF was classified as diffuse or focal. Immunohistochemistry for CD3, CD20, CD8, CD4, CD57, CD68, and S100 was performed in 61 SG and 59 livers.

Results

Diffuse INF was more common in SG than in liver. CD3⁺, CD20⁺, and CD8⁺ were the most frequent cells in both tissues, with few CD57⁺, CD68⁺, and S100⁺ cells. CD4⁺ cells were common in liver, but rare in SG. Liver presented higher indexes for all markers, except S100⁺ (p < 0.05). Higher CD3⁺, CD20⁺, and CD8⁺ (p < 0.05) were observed in SG with focal infiltrate than with diffuse infiltrate. In liver, CD20⁺ and CD3⁺ were higher in focal infiltrate, and CD68⁺ in diffuse infiltrate (p < 0.05). Comparisons with laboratorial data did not show statistical significance.

Conclusions

The INF in SG was mainly composed by T and B lymphocytes, mostly cytotoxic T cells. The glandular INF can present differences in composition according to its distribution. A more intense inflammation was observed in liver, but similar cell types were identified in SG, except for CD4⁺.     

Thymopoiesis and regulatory T cells in healthy children and adolescents

OBJECTIVES:

The purpose of this study was to investigate the association between T cell receptor excision circle levels in peripheral blood mononuclear cells and regulatory T cells that co-express CD25 and Foxp3 in healthy children and adolescents of different ages.

MATERIALS AND METHODS:

The quantification of signal-joint T-cell receptor excision circle levels in the genomic DNA of peripheral blood mononuclear cells was performed using real-time quantitative PCR. The analysis of CD4, CD8, CD25, and Foxp3 expression was performed using flow cytometry.

RESULTS:

Ninety-five healthy controls (46 females and 49 males) ranging in age from 1 to 18 years were analyzed. The mean T-cell receptor excision circle count in all individuals was 89.095±36.790 T-cell receptor excision circles per microgram of DNA. There was an inverse correlation between T-cell receptor excision circles counts and age (r = -0.846; p<0.001) as well as between the proportion of CD4⁺CD25⁺Foxp3⁺ T cells and age (r = -0.467; p = 0.04). In addition, we observed a positive correlation between the amount of CD4⁺CD25⁺Foxp3⁺ T cells and the amount of T-cell receptor excision circles per microgram of DNA in individuals of all ages (r = -0.529; p = 0.02).

CONCLUSIONS:

In this study, we observed a decrease in the thymic function with age based on the fact that the level of T-cell receptor excision circles in the peripheral blood positively correlated with the proportion of regulatory T cells in healthy children and adolescents. These findings indicate that although T-cell receptor excision circles and regulatory T cells levels decrease with age, homeostasis of the immune system and relative regulatory T cells population levels are maintained in the peripheral blood.     

Cytotoxic T cells isolated from healthy donors and cancer patients kill TGFβ-expressing cancer cells in a TGFβ-dependent manner

Transforming growth factor-beta (TGFβ) is a highly potent immunosuppressive cytokine. Although TGFβ is a tumor suppressor in early/premalignant cancer lesions, the cytokine has several tumor-promoting effects in advanced cancer; abrogation of the antitumor immune response is one of the most important tumor-promoting effects. As several immunoregulatory mechanisms have recently been shown to be targets of specific T cells, we hypothesized that TGFβ is targeted by naturally occurring specific T cells and thus could be a potential target for immunomodulatory cancer vaccination. Hence, we tested healthy donor and cancer patient T cells for spontaneous T-cell responses specifically targeting 38 20-mer epitopes derived from TGFβ1. We identified numerous CD4⁺ and CD8⁺ T-cell responses against several epitopes in TGFβ. Additionally, several ex vivo responses were identified. By enriching specific T cells from different donors, we produced highly specific cultures specific to several TGFβ-derived epitopes. Cytotoxic CD8⁺ T-cell clones specific for both a 20-mer epitope and a 9-mer HLA-A2 restricted killed epitope peptide were pulsed in HLA-A2⁺ target cells and killed the HLA-A2⁺ cancer cell lines THP-1 and UKE-1. Additionally, stimulation of THP-1 cancer cells with cytokines that increased TGFβ expression increased the fraction of killed cells. In conclusion, we have shown that healthy donors and cancer patients harbor CD4⁺ and CD8⁺ T cells specific for TGFβ-derived epitopes and that cytotoxic T cells with specificity toward TGFβ-derived epitopes are able to recognize and kill cancer cell lines in a TGFβ-dependent manner.     

M1- and M2-macrophage polarization in rat liver cirrhosis induced by thioacetamide (TAA), focusing on Iba1 and galectin-3

Introduction

Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1⁺ (Iba1⁺) and galectin-3⁺ (Gal-3⁺) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1⁺ and Gal-3⁺ macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1⁺ and Gal-3⁺ macrophages in chemical-induced liver cirrhosis.

Materials and methods

Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100 mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs.

Results

Activation of myofibroblasts was most pronounced in livers at week 15. CD68⁺ (M1), CD204⁺ (M2), Iba1⁺ and Gal-3⁺ macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1β, IL-6, and TNF-α) and M2-(TGF-β1, IL-4, and IL-10) related factors. Iba1⁺ and Gal-3⁺ macrophages showed both M1- and M2-immunophenotypes. CD163⁺ macrophages showed a persistent increase, consistent with TGF-β1 upregulation. MHC class II⁺ macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis.

Conclusion

Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1⁺ and Gal-3⁺ macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.     

Distribution of Killer cell immunoglobulin like receptor genes in end stage renal disease among North Indian population

Introduction

NK cell function is regulated by cell surface inhibitory and activating receptors including the C-type lectin receptors and Killer Immunoglobulin-like receptors (KIR). The effect of immune modulating cytokines produced by NK cells in the pathogenesis of end stage renal disease (ESRD) remained intriguing. In this regard the present study assesses the combinatorial association of KIR gene content and KIR receptor–HLA ligand in the North Indian ESRD patients.

Material and methods

KIR gene polymorphism as a susceptible marker in ESRD among 512 patients and 512 ethnically matched controls was analyzed. PCR-SSP based genotyping for KIR gene content and HLA-A, B, C typing was carried out.

Results

Significant difference in frequencies of KIR2DS1–HLA-C2 (p ? 0.0001, OR = 1.98, CI = 1.50–2.61), KIR2DS2–HLAC1 (p ? 0.0001, OR = 1.87, CI = 1.42–2.46), KIR3DS1–HLA-Bw4 (p = 0.0038, OR = 1.46, CI = 1.13–1.88) combinations for ESRD was found. In the combinatorial analysis Bw4⁺/3DL1⁻/3DS1⁺ (p ? 0.0001, OR = 4.90, CI = 2.75–8.71) and C1⁺/2DL2⁻/2DL3⁻/2DS2⁺/2DS3⁺ (p = 0.0037, OR = 2.50, CI = 1.35–4.63) showed risk association. KIR3DS1 was observed to be susceptible for all four primary kidney disease groups.

Conclusion

NK cell de-regulation due to HLA ligand binding KIR receptors may be involved in the patho-physiology of ESRD. Upon analyzing the data in this context it was found that C2/C2 donor might improve the clinical outcome of patients having C2 ligands.     

Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8+ T cell responses in HLA-A transgenic mice

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8⁺ T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8⁺ T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8⁺ T cell responses in COVID-19 patients.     

Airway constriction in asthma during sustained emotional stimulation with films

Background

Individuals with asthma have been shown to respond to unpleasant stimuli with bronchoconstriction, but little is known about the time course of responding during sustained emotional stimulation and whether it varies with patients’ experience.

Objective

To examine the time course of oscillatory resistance (R_os) during emotionally evocative films in 15 asthma patients and 14 healthy controls.

Methods

Participants viewed unpleasant, surgery, and neutral films, each ranging 3–5 min in duration. R_os and the respiratory pattern (respiration rate, tidal volume, minute ventilation) were monitored continuously. Following each film, participants rated their affective response and symptoms. The time course of R_os during films was explored using multilevel modeling.

Results

Compared to neutral film sequences, unpleasant films (including those with surgery scenes) elicited a uniform pattern of initial increases in R_os with peaks within the first 1–2 min, followed by a gradual decline. Increases were more pronounced in asthma and during surgery films. Including additional respiratory parameters as time-varying covariates did not affect the temporal course of R_os change. The rate of decline in R_os (after the initial increase) was less in participants who experienced greater arousal and in patients who reported more shortness of breath. Patients more susceptible to psychological triggers in daily life showed slower rates of decline in R_os.

Conclusion

The temporal course of bronchoconstriction to unpleasant stimulation is highly uniform in asthma, with strong constriction in early stages of stimulation. More sustained constriction in emotion-induced asthma could be a risk factor for developing asthma exacerbation in daily life.     

Peripheral blood natural killer cells activation status determined by CD69 upregulation predicts implantation outcome in IVF

Problem

NK lymphocytes play critical yet poorly defined role in implantation and during development in early pregnancy.

Methods of study

Recently, we showed that the proportion of NK that expressed CD69⁺ after incubation with K562 (CD69^stim) cells reflected the NK population excitation potential. In the present study, we investigate the significance of NK activation levels in predicting embryo implantation.

Results

A qualitative analysis of values distribution in two groups showed that 25/33 (75.8%) women who became pregnant had CD69^stim levels that were >30 but <60% (conditionally normal zone). In contrast, CD69^stim levels in patients who failed to become pregnant were either elevated, as in 10/51 (19.6%), or reduced, as in 20/51 (39.2%) of the patients. Accentuated CD69^stim levels were predictive for implantation failure, extremely significant for decreased (OR 6.9, p = 0.0004) and not quite significant for increased CD69^stim levels (OR 3.9, p = 0.062). Accordingly, conditionally normal CD69^stim levels were favourable for implantation (OR 4.46, p = 0.0032).

Conclusion

We confirm that actual peripheral blood natural killer cells activation status have an influence on embryo implantation. We showed that exactly normal NK cell activity predicting successful implantation.     

Identification of HLA-A24-restricted CD8(+) cytotoxic T-cell epitopes derived from mammaglobin-A, a human breast cancer-associated antigen

Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8⁺ cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8⁺ CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8⁺ CTL lines lysed the HLA-A24⁺/Mam-A⁺ stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24^-/Mam-A⁺ and HLA-A24⁺/Mam-A^- breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8⁺ CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.     

Peptide-pulsed dendritic cells induce tumoricidal cytotoxic T lymphocytes from healthy donors against stably HLA-A*0201-binding peptides from the Melan-A/MART-1 self antigen

The melanoma antigen Melan-A/MART-1 was screened for the presence of potential HLA-A*0201-binding cytotoxic T lymphocytes (CTL) epitopes. The immunodominant nonamer epitope AAGIGILTV demonstrated weak binding to T2 but a significant half-life of binding to HLA-A*0201 in contrast to the decamer EAAGIGILTV. In addition to the immunodominant CTL epitope, we describe two peptides, GILTVILGV and ALMDKSLHV, that display stable binding to HLA-A*0201. Using cultured autologous dendritic cells pulsed with these peptides, CTL lines were induced from peripheral blood lymphocytes that displayed reactivity with HLA-A2⁺, Melan-A/MART-1⁺ melanoma cells. CTL reactivity against the immunodominant epitope could be induced with the nonamer epitope alone, but not with the decamer variant. CTL clones generated from an (EAAGIGILTV + AAGIGILTV)-induced CTL line recognize the appropriate melanoma cells and normal melanocytes. Upon further characterization of one of these CTL clones, it was found to be of surprisingly high affinity considering that it is directed against a self antigen. This study demonstrates that immunogenic peptides can be selected based on stability (half-life) of peptide/HLA binding. In addition, cultured DC were found to efficiently induce CTL responses in vitro against such selected peptides, and some of these CTL were capable of recognizing endogenously processed antigen.