Lack of MHC class I surface expression on neoplastic cells and poor activation of the secretory pathway of cytotoxic cells in oral squamous cell carcinomas.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the secretory pathway of perforin/granzymes to kill their target cells. In contrast to NK cells, CTL responses are MHC class I restricted. In this study we analysed the relative activation of CTL and NK cells in relation with MHC class I expression on oral squamous cell carcinomas (OSCCs). MHC class I expression was investigated in 47 OSCCs by immunohistochemistry using HCA2, HC10 and beta2-m antibodies. The presence of CTLs, NK cells, and its activation, was investigated in 21 of these OSCCs using respectively, CD8, CD57 and GrB7 antibodies. The Q-Prodit measuring system was used for quantification of cytotoxic cells. All OSCCs showed weak or absent staining of beta2-m on the cell surface. The absence of beta2-m was significantly associated with absent expression of MHC class I heavy chain as detected by HC10 antibody (P = 0.004). In tumour infiltrates CTLs always outnumbered NK cells, as reflected by the ratio CD57/CD8 being always inferior to one (mean: 0.19; SD: 0.15). The proportion of activated cytotoxic cells as detected by granzyme B expression was generally low (mean: 8.6%; SD 8.9). A clear correlation between MHC class I expression and the relative proportion of NK cells/CTLs was not found. This study shows that the majority of OSCCs show weak or absent expression of MHC class I molecules on the cell surface, possibly due to alterations in the normal beta2-m pathway. The low proportion of granzyme B-positive CTLs/NK cells indicates that the secretory pathway of cytotoxicity is poor in these patients. The lack of correlation between MHC class I expression and CTL/NK cell activation as detected by granzyme-B expression suggests that, next to poor antigen presentation, also local factors seem to determine the final outcome of the cytotoxic immune response.     

Identification of a lung cancer antigen evading CTL attack due to loss of human leukocyte antigen (HLA) class I expression

The human lung cancer cell line, C831L, lost HLA class I expression due to a mutation of the β2‐microglobulin (β2m) gene, and it may have been the result of immunoediting by CTL cytotoxicity. By restoration of HLA class I expression, we could identify the antigen that may be associated with HLA downregulation. Such an antigen might be a promising target of immunotherapy because it potentially may induce a sufficient immune response to eradicate cancer cells. The CTL clone could be established from lymph node lymphocytes in patient C831 by stimulation with wild‐type β2m‐transduced C831L (C831L‐wβ2m). The CTL clone showed reactivity against C831L‐wβ2m in a HLA‐B*0702‐restricted manner, but not Parental‐C831L or autologous normal cells. The cDNA expression cloning method was used to identify the antigen coding gene recognized by the CTL clone. The cDNA clone exhibited a homology with a part of the mRNA that codes for leucine rich repeat containing eight family member A (LRRC8A). A transfection analysis of minigenes indicated that the antigen peptide was derived from protein translated from the downstream of the registered open reading frame in LRRC8A mRNA. The antigenic 9‐mer peptide (GPRESRPPA) was identified. The present methodology should be useful to find the crucial tumor antigens, which are potentially associated with loss of HLA expression. Furthermore, such an antigen may help in achieving a better understanding of the immunological escape mechanisms and it may also provide a favorable immune response in cancer immunotherapy. (Cancer Sci 2010; 00: 000–000)     

Brefeldin A Blocks the Cytotoxicity of T Cell Receptor α/β and γ/δ Cytotoxic T Lymphocyte Clones Reacting against Human Autologous Cancer Cells

We studied the effector mechanism of T cell receptor (TCR) α/β - and γ/δ -type cytotoxic T lymphocyte (CTL) clones that react with human autologous tumor cells. Treatment of tumor cells with a fungal antibacterial reagent, brefeldin A (BFA), resulted in the inhibition of cytotoxicity of an autologous tumor (HST-2)-specific CD8⁺ TCR α/β -type CTL, TcHST-2. Other anti-metabolites such as chloroquine, cycloheximide and colchicine did not affect the cytotoxicity. The cell-surface antigen expression, including MHC class I molecules, was not influenced by BFA treatment. Furthermore, BFA did not influence the cytotoxicity of lymphokine-activated killer cells and natural killer cells. Since BFA blocks the transport of peptides from endoplasmic reticulum to the Golgi apparatus, the above data suggest that BFA could affect washing out of the peptide fragments from the MHC class I groove. Consequently, target tumor cells were protected from killing by CTL, Moreover, we obtained a CD4⁻, 8⁻, TCR γ/δ -type (Vδ1⁺) CTL clone, TcHOT, that reacts against an autologous ovarial carcinoma, HOT. BFA could also inhibit this cytotoxicity, and it is likely that different presenting molecules other than MHC class I proteins participate in the cytotoxicity of this TCR gm/δ - type CTL. These studies suggest that both TCR α/β - and γ/δ -type CTL may require antigenic peptides that are most likely derived from the BFA-sensitive, intracellular endogenous target proteins.     

顺铂对鼻咽癌细胞NKG2D配体表达和NK细胞杀伤活性的增强作用

 目的 研究顺铂(Cisplatin, DDP)作用前后人鼻咽癌细胞CNE2 NKG2D配体和HLA-Ⅰ类分子表达的改变及NK细胞杀伤活性的变化。 方法 MTT法测定DDP对CNE2细胞的50%抑制量（IC₅₀）;以此浓度DDP作用CNE2细胞24h,乳酸脱氢酶释放法检测效靶比20∶1时,NK细胞对DDP作用前后的CNE2细胞的杀伤活性;流式细胞仪检测DDP作用前后的CNE2细胞表面NKG2D配体(MICA/B、ULBP1、ULBP2、 ULBP3)和HLA-Ⅰ类分子表达的变化。 结果 DDP对CNE2细胞的IC₅₀为5mg/L。效靶比20∶1时,NK细胞对5mg/L DDP作用前后的CNE2细胞的杀伤活性分别为 （38.11±1.41）%,（47.71±1.53）%,差异有统计学意义（P＜0.05）,DDP作用后CNE2细胞表面MICA/B、ULBP1、 ULBP3表达显著升高,与作用前相比差异有统计学意义（P＜0.05）。ULBP2、HLA-Ⅰ类分子无明显变化(P＞0.05)。 结论 DDP能提高CNE2细胞NKG2D配体(MICA/B、ULBP1、ULBP3)的表达,从而增强CNE2细胞对NK细胞杀伤的敏感度。     

High-risk human papillomavirus E7 expression reduces cell-surface MHC class I molecules and increases susceptibility to natural killer cells

High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigen-presentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16- and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication.     

NKG2D介导NK 细胞对鼻咽癌细胞杀伤作用的体外研究

 目的 探讨鼻咽癌CNE2细胞表面HLA-Ⅰ类分子表型和NKG2D配体的表达情况，进一步了解其对同种异体NK细胞杀伤活性的影响。方法 流式细胞仪检测NKG2D的配体MICA、MICB、ULBP1、ULBP2、ULBP3在K562、CNE2细胞的表达情况。PCR-SSP法分析CNE2细胞HLA-A、B、Cw分型和NK细胞KIR分型。LDH释放法测定5例健康者NK细胞在不同效靶比时对K562、CNE2细胞的杀伤活性，效靶比20：1时观察抗NKG2D配体的单抗对NK细胞杀伤K562、CNE2细胞活性的影响。结果 CNE2细胞表达MICA、MICB、ULBP2，不表达ULBP1、ULBP3。K562细胞表面表达MICA、MICB、ULBP1、ULBP2、ULBP3。5例健康者NK细胞抑制性KIR与CNE2细胞表面的HLA-Ⅰ类分子之间存在错配。效靶比5：1、10：1、20：1、30：1时NK细胞对K562、CNE2细胞的杀伤活性分别为（29．02±0．45）％、（10．50±2．17）％；（44．43±1．36）％、（27．68±1．47）％；（57．82±1．35）％、（36．99±3．13）％：（71．24±2．36）％、（55．00±2．20）％，在各效靶比时NK细胞对K562细胞的杀伤活性较CNE2细胞明显增强（P=0．000）；在效靶比20：1时anti-MICA、anti-MICB、anti-ULBP1、anti—ULBP2、anti-ULBP3可明显抑制NK细胞对K562细胞的杀伤活性，与阻断前相比有显著性差异（P=0．000）；anti—MICA、anti—MICB、anti—ULBP2可明显抑制NK细胞对CNE2细胞的杀伤活性，与阻断前相比有显著性差异（P〈0．01），但anti—ULBP1、anti—ULBP3不能阻断NK细胞对CNE2细胞的杀伤活性。结论 NKG2D配体影响NK细胞对靶细胞的杀伤活性，提高NKG2D配体的表达有可能提高NK细胞的抗肿瘤活性。     

CD4(+), HLA class I-restricted, cytolytic T-lymphocyte clone against primary malignant melanoma cells

The involvement of HLA-class I in target cell lysis by CD4(+) cytolytic T cells (CTL) has been a controversial issue. A CTL clone of CD4 phenotype was derived from the peripheral blood lymphocytes of a patient with primary melanoma. The CTL clone stably lysed the autologous primary melanoma cells for approximately 9 months in culture. Both the Valpha2/Vbeta8 T-cell receptor and CD4 were involved in CTL cytotoxicity. Of a large panel of allogeneic primary and metastatic melanoma or colorectal carcinoma cells, autologous and allogeneic Epstein-Barr virus-transformed B cells and autologous fibroblasts, only allogeneic metastatic melanoma cells matched with the autologous tumor cells for HLA-class I (B57[17]) were lysed and induced IFN-gamma secretion by the CTL clone. Lysis of the autologous tumor cells was significantly blocked by monoclonal antibody to HLA-B17. Importantly, allogeneic, HLA-class I- and class II-unmatched melanoma cells were lysed by the CTL only following transfection of the cells with B57[17] cDNA. Our results provide direct evidence for the involvement of both CD4 and HLA-class I in tumor cell lysis by CD4(+) CTL.     

Down-regulation of MHC class I by bovine papillomavirus E5 oncoproteins.

The papillomavirus E5 protein is localized in the endoplasmic reticulum (ER) and Golgi apparatus (GA) of the host cell. Transformed bovine fibroblasts expressing bovine papillomavirus (BPV) E5 are highly vacuolated and have a much enlarged, distorted and fragmented GA. Major histocompatibility complex class I (MHC I) is processed and transported to the cell surface through the GA. Given the cellular localization of E5 in the GA and the morphologically abnormal GA, we investigated the expression of MHC I in cells transformed by E5 from BPV-1 and BPV-4. Two cell lines were used: bovine cells that also express E6, E7 and activated ras, and NIH3T3 cells that express only E5. In addition, PalF cells acutely infected with a recombinant retrovirus expressing E5 were also examined. In contrast to non-transformed normal cells, or transformed cells expressing other papillomavirus proteins, cells expressing E5 do not express MHC I on their surface, but retain it intracellularly, independently of the presence of other viral or cellular oncogenes, or of whether the cells are long-term transformants or acutely infected. We conclude that expression of E5 prevents expression of MHC I to the cell surface and causes its retention within the cell. In addition, lower amounts of total MHC I heavy chain and of heavy chain RNA are detected in E5-transformed cells than in control cells. As surface expression of another glycosylated membrane protein, the transferrin receptor, is not affected, it appears that E5 targets MHC I with at least a degree of specificity. In papillomavirus lesions this effect would have important implications for antigen presentation by, and immunosurveillance of, virally infected cells.     

Frequent lack of translation of antigen presentation-associated molecules MHC class I, CD1a and Beta(2)-microglobulin in Reed-Sternberg cells

Epstein-Barr virus (EBV) is present in Reed-Sternberg (RS) cells of a substantial proportion of Hodgkin's lymphoma cases. Most EBV-positive cases are also MHC class I-positive, whereas the majority of EBV-negative cases lack detectable levels of MHC class I expression. Application of the SAGE technique has led to the identification of tags corresponding to MHC class I and beta(2)-microglobulin genes in the EBV- and MHC class I-negative L428 Hodgkin's cell line. Further expression studies indicated that single RS cells that do not express HLA class I also lack beta(2)-microglobulins but frequently contain mRNA coding for these proteins. Another tag was identified corresponding to CD1a, a thymocyte and Langerhans cell antigen structurally related to the MHC class I genes. CD1a expression studies revealed mRNA in all cell lines and in several of the single cells, whereas immunostaining showed a cytoplasmic signal in only 2 of the 4 cell lines and in none of the Hodgkin's lymphoma tissue samples. In conclusion, RS cells frequently lack MHC class I, beta(2)-microglobulin and CD1a protein expression but contain mRNA coding for these proteins in some of the RS cells, suggesting a common mechanism affecting the translation of these antigen presentation-associated molecules.     

E5 protein of human papillomavirus type 16 selectively downregulates surface HLA class I

Papillomaviruses have evolved mechanisms that result in escape from host immune surveillance. The E5 protein is expressed early in papillomavirus infection in the deep layers of the infected epithelium. It is localized to the Golgi apparatus (GA) and endoplasmic reticulum. The E5 protein of bovine papillomavirus (BPV) impairs the synthesis and stability of major histocompatibility (MHC) class I complexes and prevents their transport to the cell surface due to retention in the GA. Here we show that human papillomavirus type 16 (HPV-16) E5 also causes the retention of MHC (HLA) class I complexes in the GA and impedes their transport to the cell surface, which is rescued by treatment with interferon. Unlike BPV E5, HPV-16 E5 does not affect the synthesis of HLA class I heavy chains or the expression of the transporter associated with antigen processing TAP. These results show that downregulation of surface MHC class I molecules is common to both BPV and HPV E5 proteins. Moreover, we determined that HPV-16 E5 downregulates surface expression of HLA-A and HLA-B, which present viral peptides to MHC class I-restricted cytotoxic T lymphocytes (CTLs), but not the natural killer (NK) cell inhibitory ligands HLA-C and HLA-E. Selective downregulation of cell surface HLA class I molecules may allow the virus to establish infection by avoiding immune clearance of virus-infected cells by both CTLs and NK cells.     

Renal cell carcinoma-infiltrating natural killer cells express differential repertoires of activating and inhibitory receptors and are inhibited by specific HLA class I allotypes

Among tumor-infiltrating lymphocytes (TILs) directly isolated from renal cell carcinomas (RCCs), we found substantial numbers of natural killer (NK) cells in most tumor tissues. They could be identified reliably in situ with an antibody directed against the activating receptor (AR) NKp46 that is exclusively expressed by all NK cells. NK-enriched TILs (NK-TILs) showed cytotoxicity against major histocompatibility complex (MHC) class I-negative cell lines. The ability to detect lysis of target cells was dependent on the percentage of NK cells within the TILs, and cytotoxicity was only observed after overnight activation with low-dose interleukin-2 (IL-2). Infiltrating NK cells were found to express various inhibitory receptors (IRs); among these the CD94/NKG2A receptor complex was overrepresented compared to the autologous peripheral blood mononuclear cell (PBMC) population. Other IRs were underrepresented, indicating that NK subpopulations vary in their tumor-infiltrating capacity. IRs expressed by NK-TILs are functional since receptor engagement with MHC class I ligands presented by human leukocyte antigen (HLA)-transfected target cell lines was able to inhibit NK-mediated cytotoxicity. NK-TILs were also able to lyse autologous or allogeneic tumor cell lines in vitro. This activity correlated with low HLA class I surface expression since lysis could be inhibited by interferon (IFN)-gamma-expressing RCC transductants that displayed a higher surface density of HLA class I molecules. Therefore, NK cells infiltrating tumor tissues have an inherent ability to recognize transformed cells, but they require cytokine activation and are sensitive to inhibition by IR ligands.     

Intensity of class I antigen expression on human tumour cell lines and its relevance to the efficiency of non-MHC-restricted killing.

A modified tetrazolium reduction assay (MTT) was used to assess the relation between HLA class I antigen expression on tumour cells and their susceptibility as a target for non-MHC restricted LAK/NK cytotoxicity using interleukin-2 activated peripheral blood mononuclear cells (MNC) from normal individuals. At 20/1 effector/target ratio this ranged from no killing to 77%. The efficiency of killing was dependent on duration of effector cell culture with IL-2, peaking at day 10 and declining thereafter. This killing could be enhanced by addition of other cytokines including interferons alpha, beta and gamma. Study of a panel of 15 tumour cell lines using a single effector showed that there was no statistically significant inverse correlation (using Spearman rank test) between the degree of tumour class I expression and LAK/NK killing at 20/1 (r = 0.23 P = 0.39) and 10/1 (r = 0.30, P = 0.27) and at 5/1 E/T ratio r = 0.47, P = 0.08) respectively. Lack of inverse correlation between these two parameters came from study of one bladder tumour line (FEN), whose absent class I antigens had been corrected by transfection with beta 2 microglobulin gene. At high E/T ratio (20/1) there was an increase in the susceptibility of target cells to lysis (36% parent cell, 45% transfected cell), whilst at lower E/T ratios (1/1) there was significantly more killing of the non-transfected cells (10% vs 31%). The addition of anti-class I antibody W6/32 increased killing by 18% but this was non-specific as the same increase occurred with a class II antibody. These data suggest that overall there was not an inverse correlation between class I expression and LAK/NK killing at high E/T ratios, whilst at low (5/1 or lower) E/T ratios this correlation nearly reached statistical significance suggesting that the conflicting literature reports may be due to a threshold levels of effector cells above which the masking effects of MHC antigens disappears.     

The level of MHC class I expression on murine adenocarcinoma can change the antitumor effector mechanism of immunocytokine therapy

The huKS1/4-IL2 fusion protein, directed against the human epithelial cell adhesion molecule (huEpCAM) has been shown to induce a strong CD8+ T-cell-dependent, natural killer (NK) cell-independent, antitumor response in mice bearing the huEp-CAM-transfected CT26 colon cancer CT26-EpCAM. Here we investigate the effectiveness of huKS1/4-IL2 against CT26-Ep21.6, a subclone of CT26-EpCAM, expressing low levels of MHC class I. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays in the presence of huKS1/4-IL2 demonstrate that murine NK cells from spleen and blood can kill CT26-Ep21.6 significantly better than they kill CT26-EpCAM. NK-mediated ADCC of CT26-EpCAM can be enhanced by blocking the murine NK cell-inhibitory receptor, Ly-49C. A potent in vivo antitumor effect was observed when BALB/c mice bearing experimental metastases of CT26-Ep21.6 were treated with huKS1/4-IL2. The depletion of NK cells during huKS1/4-IL2 treatment significantly reduced the antitumor effect against CT26-Ep21.6. Together our in vitro and in vivo data in the huEp-CAM-transfected CT26 models indicate that the amount of MHC class I expressed on the tumor target cell plays a critical role in the in vivo antitumor mechanism of huKS1/4-IL2 immunotherapy. A low MHC class I level favors NK cells as effectors, whereas a high level of MHC class I favors T cells as effectors. Given the heterogeneity of MHC class I expression seen in human tumors and the prevailing T-cell suppression in many cancer patients, the observation that huKS1/4-IL2 has the potential to effectively activate an NK cell-based antitumor response may be of potential clinical relevance.     

Platelet-derived MHC class I confers a pseudonormal phenotype to cancer cells that subverts the antitumor reactivity of natural killer immune cells

Natural killer (NK) cells are cytotoxic lymphocytes that play an important role in tumor immunosurveillance, preferentially eliminating targets with low or absent expression of MHC class I and stress-induced expression of ligands for activating NK receptors. Platelets promote metastasis by protecting disseminating tumor cells from NK cell immunosurveillance, but the underlying mechanisms are not well understood. In this study, we show that tumor cells rapidly get coated in the presence of platelets in vitro, and circulating tumor cells of cancer patients display coexpression of platelet markers. Flow cytometry, immunofluorescent staining, confocal microscopy, and analyses on an ultrastructural level using immunoelectron microscopy revealed that such coating may cause transfer of MHC class I onto the tumor cell surface resulting in high-level expression of platelet-derived normal MHC class I. The resulting "phenotype of false pretenses" disrupts recognition of tumor cell missing self, thereby impairing cytotoxicity and IFN-γ production by NK cells. Thus, our data indicate that platelets, by conferring an unsuspicious "pseudonormal" phenotype, may enable a molecular mimicry that allows metastasizing tumor cells to downregulate MHC class I, to escape T-cell-mediated immunity without inducing susceptibility to NK cell reactivity.     

Differential modulation of natural killer cell cytotoxicity by 17β-estradiol and prolactin through the NKG2D/NKG2DL axis in cervical cancer cells

Natural killer (NK) cells play a crucial role in cervical cancer (CC). As estrogens and prolactin (PRL) have been reported to be involved in CC, the present study attempted to elucidate the effects of both hormones on NK cells in CC. For this purpose, NKL cells, as well as CC-derived cell lines (HeLa, SiHa and C33A) and non-tumorigenic keratinocytes (HaCaT cells) were stimulated with 17β-estradiol (E2; 10 nM), PRL (200 ng/ml), or both (E2 and PRL) for 48 h. The expression of hormone receptors (estrogen receptor α and β, G protein-coupled estrogen receptor 1 and PRL receptor) and NK cell activating receptors [natural killer group 2D (NKG2D), natural cytotoxicity triggering receptor 3, natural cytotoxicity triggering receptor 2 and natural cytotoxicity triggering receptor 1] were measured using western blot analysis and flow cytometry, respectively. In the HeLa, SiHa, C33A and HaCaT cells stimulated with the hormones, the expression of NKG2D ligands [MHC class I polypeptide-related sequence A/B (MICA/B)] on the membrane and the soluble form of MICA was evaluated using flow cytometry and ELISA. Cytotoxicity assay was performed using GFP-transfected K562 cells as target cells. E2 reduced NKL cell-mediated cytotoxicity, while PRL exerted the opposite effect. NKL cells expressed different hormone receptor forms, of which PRL only induced a decrease in NKG2D expression compared to the untreated control NKL cells. PRL increased MICA/B expression in HeLa cells and E2 and PRL reversed this effect. However, in SiHa cells, the concurrent incubation with the two hormones decreased MICA/B expression. E2 and PRL, either alone or in combination, decreased soluble MICA secretion in all CC cell lines, while E2 solely increased soluble MICA secretion in SiHa cells. On the whole, the present study provides evidence that E2 and PRL mediate the mechanisms through which NK and CC cells mediate a cytotoxic response and these have an antagonistic effect on NK cell-mediated cytotoxicity.     

Recognition of breast cancer-associated peptides by tumor-reactive, HLA-class I restricted allogeneic cytotoxic T lymphocytes

Strategies to identify tumor-associated antigens rely on the paradigm that tumor-associated peptides presented in the context of HLA-class I are recognized by the cellular immune system. Approaches to isolate tumor-specific cytotoxic T lymphocytes (CTL) from tumor-infiltrating lymphocytes are difficult because long-term growth of the CTL requires autologous tumor cells and lymphocytes (PBL) as feeder cells. In this study, a CTL line (BL.HBL-100 CTL) was generated from PBL from a normal healthy donor by stimulating with irradiated, HLA-class I partially matched breast cancer cell line HBL-100. Activated T lymphocytes generated expressed TCR alpha/beta+ with a predominant CD8+ population after 12 stimulations (98.54% CD8+ vs. 0.18% CD4+). These CTL lysed HLA-A1+, but not HLA-A1-, breast cancer cell lines. Moreover, HLA-A1+, non breast cancer cell lines were not recognized. The lytic activity of BL.HBL-100 CTL against HLA-A1+ breast cancer cell lines was blocked by monoclonal antibodies (MAbs) to HLA-class I and CD8, but not by anti-HLA-class II and CD4. Recognition of HLA-A1+ breast cancer cells by the CTL was dependent on peptides associated with HLA-class I since the lysis was inhibited by acid elution of HLA bound peptides. HBL-100 tumors were grown in severe combined immunodeficient (SCID) mice. Immunohistochemical staining of the HBL-100 tissue harvested from SCID mice demonstrated human breast cancer cells. HLA-class I molecules were affinity purified from the HBL-100 harvested from the SCID mice; class I bound peptides were eluted and separated by RP-HPLC. Pooled HPLC peptide fractions were tested for reconstituting antigenic epitopes recognized by the BL.HBL-100 CTL and found to reside within fraction 40. Our results show that a tumor reactive, HLA-class I restricted CTL was produced by stimulating normal PBL against an HLA-class I matched breast cancer cell line. We also provide evidence for a breast cancer-associated, HLA-class I bound peptide antigen(s) that reconstitutes the antigenic epitope(s) recognized by these CTL.     

Selective responsiveness to common gamma chain cytokines in peripheral blood-derived cytotoxic T lymphocytes induced by Melan-A/MART-1(27-35)targeted active specific immunotherapy

Tumours can be recognised by CTL and NK cells. CTL recognition depends on expression of MHC Class I loaded with peptides from tumour antigens. In contrast, loss of MHC Class I results in NK activation. In our study a large set of samples from patients with primary operable invasive breast cancer was evaluated for the expression of MHC Class I heavy and light by immunohistochemical staining of 439 breast carcinomas in a tissue microarray. Forty-seven percent (206 of 439) of breast carcinomas were considered negative for HLA Class I heavy chain (HC10), whereas lack of anti-beta2m-antibody staining was observed in 39% (167 of 424) of tumours, with only 3% of the beta2m-negative tumours expressing detectable HLA Class I heavy chain. Correlation with patient outcome showed direct relationship between patient survival and HLA-negative phenotype (log rank = 0.004). A positive relationship was found between the intensity of expression of MHC Class I light and heavy chains expression and histological grade of invasive tumour (p < 0.001) and Nottingham Prognostic Index (p < 0.001). To investigate whether HLA Class I heavy and light chains expression had independent prognostic significance, Cox multivariate regression analysis, including the parameters of tumour size, lymph node stage, grade and intensity of HC10 and anti-beta2m staining, was carried out. In our analysis, lymph node stage (p < 0.001), tumour grade (p = 0.005) and intensity of MHC Class I light and heavy chains expression were shown to be independent prognostic factors predictive of overall survival (p-values HC10 = 0.047 and beta2m = 0.018).     

Split anergized natural killer cells halt inflammation by inducing stem cell differentiation,resistance to NK cell cytotoxicity and prevention of cytokine and chemokine secretion

The mechanism of suppression of NK cytotoxicity in cancer patients is not clearly established. In this paper we provide evidence that anergized NK cells induce differentiation of healthy Dental Pulp Stem Cells (DPSCs) or transformed Oral Squamous Cancer Stem Cells (OSCSCs) resulting in cell growth inhibition, resistance to NK cell-mediated cytotoxicity and prevention of inflammatory mediators secretion. Induction of cytotoxicity resistance in differentiated cells correlated with increased CD54 and MHC class I surface expression and mediated by the combination of IFN-γ and TNF-α since antibodies to both, but not each cytokine alone, was able to inhibit resistance. In contrast, inhibition of cytokine and chemokine release was mediated by IFN-γ since the addition of anti-IFN-γ antibody, and not anti-TNF-α, restored secretion of inflammatory mediators in NK cell cultures with differentiated DPSCs and OSCSCs. There was a gradual and time dependent decrease in MHC class I and CD54 expression which correlated with the restoration of NK cell cytotoxicity, augmentation of cytokine secretion and increased cell growth from days 0–12 post NK removal. Continuous presence of NK cells is required for the maintenance of cell differentiation since the removal of NK cell-mediated function reverses the phenotype and function of differentiated cells to their stem-like cells.