Cytotoxic T Lymphocytes Recognition Research Articles

Analysis of immune selection as a potential cause for the presence of cleavage site mutation 431V in treatment-naive HIV type-1 isolates

HIV type-1 (HIV-1) protease (PR) and cleavage site (CS) mutations accumulate in protease-inhibitor-resistant isolates. HIV-1 CS mutation 431V is the most frequent treatment-associated CS mutation; however, little is known about its origin in treatment-naive HIV-1 isolates. Recently, it has been shown that the CS mutation 431V is located within the human leukocyte antigen (HLA)-B*13-restricted cytotoxic T-lymphocyte (CTL) epitope RQANFLGKI (RI9). Therefore, we investigated whether the presence of CS mutation 431V might additionally be related to immune escape. CTL recognition of RI9 and of RI9 variants carrying the 431V or the 436R mutation was analysed by ELISPOT in nine HLA-B*13-positive HIV-1-infected patients. Treatment-naive HIV-1-infected patients with primary drug-resistant HIV-1 isolates (n=58) or carrying 431V (n=4) were genotyped for HLA class I alleles. ELISPOT analysis showed different patterns of CTL recognition of RI9. CS mutation 431V could abrogate recognition by RI9-specific CTL in a subgroup of patients. Nevertheless, in our study, the occurrence of 431V in treatment-naive HIV-1 without primary drug resistance could not be explained by HLA-B*13-mediated immune selection. In patients with primary drug-resistant HIV-1 isolates, the frequency of HLA-B*13 was not increased and HLA-B*13 did not correlate with CS mutations 436R or 431V. HIV-1 CS mutation 431V can abrogate CTL recognition, indicating interactions between development of drug resistance and the CTL response. However, we could not find evidence that the presence of 431V in treatment-naive HIV-1 isolates with and without primary drug resistance is related to immune selection by HLA-B*13 or other HLA class I alleles.

Multiple, Non-conserved, Internal Viral Ligands Naturally Presented by HLA-B27 in Human Respiratory Syncytial Virus-infected Cells

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

Abstract 4778: Epigenetic modulation of colorectal cancer cells for cancer-testis antigen-targeted immunotherapy

Abstract Cancer-testis (C-T) antigens represent attractive potential targets for cancer immunotherapy due to their limited expression in normal somatic tissues and their widespread expression in malignant cells of diverse histologies. The utility of C-T antigens for immunotherapy of colorectal cancer (CRC), however, is limited because expression of C-T genes is repressed in most CRC cells by hypermethylation. We investigated whether treatment of CRC lines with the hypomethylating agent 5-aza-2′-deoxycytidine (DAC) could induce C-T gene expression in CRC cells and thereby sensitize them to recognition by CD8+ cytotoxic T lymphocytes (CTL) specific for the highly immunogenic C-T antigen NY-ESO-1. A panel of CRC and primary dermal fibroblast lines was cultured with DAC or medium alone for 48 hours followed by culture with exposure to interferon γ (IFNγ) or medium alone for an additional 48 hours. After the treatment period, cells were harvested for collection of RNA, DNA, and protein; studied with flow cytometry for analysis of MHC class I and II expression; and tested for recognition by HLA-A*0201-restricted CTL specific for NY-ESO-1157–165 in a 51Cr release-cytotoxicity assay. Quantitative RT-PCR analysis of the expression of 20 C-T genes in untreated and DAC-treated cells using a custom PCR array showed that the majority of C-T genes were expressed at low levels in untreated CRC cells but were induced in most CRC cell lines by DAC exposure. Fibroblasts did not show any significant C-T gene expression with or without DAC treatment. Western blot analysis for NY-ESO-1 expression confirmed that DAC induction of NY-ESO-1 transcription was also associated with increased protein expression. DAC-treated HLA-A*0201+ CRC lines were also recognized by CD8+ HLA-A*0201-restricted NY-ESO-1-specific CTL. CTL recognition was enhanced when the cells were treated with IFNγ in combination with DAC, with the most significant effect seen in CRC cells with low baseline expression of MHC class I molecules. These data suggest that epigenetic modulation with DAC or related agents could be used to selectively induce expression of C-T antigens in CRC cells and thereby make them susceptible to C-T antigen-directed immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4778.

Oral Vaccination with AttenuatedSalmonella entericaStrains Encoding T-Cell Epitopes from Tumor Antigen NY-ESO-1 Induces Specific Cytotoxic T-Lymphocyte Responses

Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica serovar Typhimurium SL3261. The T-cell epitopes (NY-ESO-1 p157-165 and p157-167) from NY-ESO-1, which is a promising target antigen in patients for the specific immune recognition of cancer, were incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length of the DNA segment. To improve cytotoxic T-lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) residues. Immunofluorescence microscopy with AgfA-specific antiserum verified the expression of chimeric AgfA, which was also proved by a Congo red binding assay. Oral immunizations of HLA-A*0201 transgenic mice with recombinant SL3261 strains encoding NY-ESO-1 p157-165 or p157-167 induced NY-ESO-1 p157-165-specific CD8(+) T cells, detected by an HLA-A*0201 pentamer, and induced a T-cell response detected by an enzyme-linked immunospot assay. The Salmonella fimbrial display system was efficient at the induction of an antitumor cellular immune response in vivo, providing a new strategy for the development of efficient cancer vaccinations.

Novel Immunogenic HLA-A*0201-restricted Epidermal Growth Factor Receptor-specific T-cell Epitope in Head and Neck Cancer Patients

Therapeutic targeting of the epidermal growth factor receptor (EGFR), which is highly overexpressed and correlated with poor prognosis in colorectal and head and neck squamous cell carcinoma (SCCHN), has shown clinical efficacy using the blocking mAbs, cetuximab or panitumumab, but only in 10% to 20% of patients. Clinical responsiveness is correlated with certain Fcgamma receptor genotypes, suggesting immune activity may contribute to therapeutic efficacy. In addition, cetuximab-resistant tumor cells exhibit ubiquitination and degradation of EGFR, which would increase its processing as a tumor antigen for cytotoxic T lymphocyte (CTL) lysis. Thus, T cell-based immunotherapy might enhance the antitumor efficacy of EGFR-specific mAbs, but CTL epitopes are poorly defined. To permit combinatorial EGFR-targeted immunotherapy, we identified a novel immunogenic wild-type sequence peptide, EGFR853-861 and modified its anchor sequence to enhance HLA-A*0201 binding and stimulation of cross-reactive anti-wild-type EGFR853-861-specific CTL. Cross-reactivity was also observed with HER2861-869. EGFR853-861-specific CTL recognition of SCCHN cells was increased by incubation of tumor cells with cetuximab, which led to EGFR degradation. In addition, EGFR853-861-specific CTLs were elevated in the circulation of SCCHN patients as compared with healthy control peripheral blood mononuclear cells. Thus, a novel, immunogenic EGFR-encoded CTL epitope may be incorporated into vaccines and would be useful for combinatorial immunotherapy with EGFR-specific mAbs in cancer patients.

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti–human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1–specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

Impaired Antigen Presentation in Neoplasia: Basic Mechanisms and Implications for Acute Myeloid Leukemia

During onset, treatment and progression of acute myeloid leukemia (AML), inadequate immune responses against certain myeloid leukemic blasts might be associated with the occurrence of minimal residual disease and subsequent relapse. Several studies on this subject have demonstrated that, in general, solid tumor cells are able to avoid CD8(+) cytotoxic T-cell recognition by downregulating HLA class I-restricted presentation of tumor-associated antigens. In tumor cells that can express HLA class II molecules, such as myeloid leukemic blasts, abnormalities in the processing pathways of endogenous antigens could also result in impaired HLA class II-restricted tumor-associated antigen presentation to CD4(+) T helper cells. More insight into impaired tumor-associated antigen presentation by myeloid leukemic blasts could explain their escape from immune recognition and might be crucial for selecting appropriate strategies to improve whole-cell or dendritic cell-based tumor vaccine efficacy in the treatment of AML patients.

Conserved HIV‐1 Epitopes Continuously Elicit Subdominant Cytotoxic T‐Lymphocyte Responses

The epitope specificities and antiviral activities of class I HLA-restricted CD8(+) T cells, especially those induced during human immunodeficiency virus type 1 (HIV-1) primary infection, are important considerations in designing HIV-1 vaccines. Conserved epitopes may be more commonly and persistently recognized than variable epitopes, as they may be more likely to be present in infecting viruses. However, some studies have shown preferential or similar targeting of variable versus conserved epitopes during primary infection. We analyzed cytotoxic T-lymphocyte (CTL) responses toward predefined conserved and variable epitopes in 45 subjects during primary (n = 34) and/or chronic infection (n = 16). Conserved and variable CTL epitopes were recognized with similar probabilities, whereas conserved epitopes generally elicited subdominant responses during both primary and chronic infection. During primary infection, CTL responses against Gag versus responses against Env and variable epitopes tended to be associated with lower and higher viral loads, respectively. During chronic infection, Env-specific responses tended to be associated with lower CD4(+) cell counts. Subdominant CTL recognition of conserved HIV-1 epitopes commonly occurs from the primary through chronic stages of HIV-1 infection. These findings underscore the challenge in designing T cell-based vaccines that can induce immunodominant CTL responses to conserved HIV-1 regions.

HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphocyte Recognition

HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphocyte Recognition

Immune Response and Possible Causes of CD4+T-cell Depletion in Human Immunodeficiency Virus (HIV) - 1 Infection

In this review, immune response and possible causes of depletion of CD4 + T-cell counts in patients with human immunodeficiency (HIV)-1 infection, have been documented. HIV has been recognized as a global problem; however, the developing countries are the most affected by epidemic diseases. Countries in the sub-Saharan Africa seem to bear the bulk of the HIV burden among the developing countries with about 24.7 million (� 63%) of all people living with HIV globally in 2006. The major factor obstructing progress towards an effective vaccine to prevent or modulate HIV - 1 in- fection is that the critical features needed for a protective immune response are not fully understood. Although, it has been found that potent neutralizing antibodies can protect against experimentally acquired HIV infection in animal models, they are scarcely generated in vivo in the infected person and neutralization resistant viral variants have been monitored to develop rapidly in chronic infection. It is generally believed that cellular immune responses, particularly specific cytotoxic T lymphocytes (CTL), are significant in the host response to HIV - 1 infection. Scientists have observed that CTL develop very early in acute HIV - 1 infection, coincident with a rapid fall in plasma vireamia, whereas in chronic infection their levels are inversely related to viral load. However the potent HIV-specific CTL response ultimately fails to control HIV replication. This could be as a consequence of the emergence of viral variants that escape CTL recognition or impairment of CTL function.

Reciprocal recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope by CD8+ T cells and NK cells

The HIV-1 Nef protein selectively downregulates human leukocyte antigen (HLA)-A and HLA-B but not HLA-C molecules on the surface of infected cells. This allows HIV-infected cells to evade recognition by most cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We investigated the recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope SFNCGGEFF (SF9) and its variant SFNCGGEFL (SL9) by T cells and NK receptors. Recognition of HIV-1 gp120 peptides (SF9 and SL9) by T-cell clones was measured by staining with HLA-Cw4-peptide tetrameric complexes and cytolytic assays using target cell pulsed with either peptides. KIR2DL1 binding to these two peptides was measured using surface plasmon resonance and tetramer staining of an NK cell line. : CTLs could recognize SF9 better than the variant SL9, as shown by both tetramer staining and cytolytic assays. Intriguingly, an HLA-Cw4 tetramer folded with the 'escape' variant SL9 could bind to KIR2DL1 on NK cell lines with higher affinity than HLA-Cw4-SF9. The binding of KIR2DL1 to its ligand results in inhibition of NK cell function. Our results indicate that the HIV-1 gp120 variant peptide SL9 could potentially escape both from NK cell and CTL recognition by increasing its affinity for KIR2DL1 binding. These data suggest that HIV-1 can acquire mutations that are capable of escaping from both CTL and NK cell recognition, a phenomenon we have termed 'double escape'.

Predicting the Impact of Blocking Human Immunodeficiency Virus Type 1 Nef In Vivo

Human immunodeficiency virus type 1 (HIV-1) Nef is a multifunctional protein that confers an ability to evade killing by cytotoxic T lymphocytes (CTLs) as well as other advantages to the virus in vivo. Here we exploited mathematical modeling and related statistical methods to estimate the impact of Nef activity on viral replication in vivo in relation to CTLs. Our results indicate that downregulation of major histocompatibility complex class I (MHC-I) A and B by wild-type Nef confers an advantage to the virus of about 82% in decreased CTL killing efficiency on average, meaning that abolishing the MHC-I downregulation function of Nef would increase killing by more than fivefold. We incorporated this estimate, as well as prior estimates of replicative enhancement by Nef, into a previously published model of HIV-1 and CTLs in vivo (W. D. Wick, O. O. Yang, L. Corey, and S. G. Self, J. Virol. 79:13579-13586, 2005), generalized to permit CTL recognition of multiple epitopes. A sequence database analysis revealed that 92.9% of HIV-1 epitopes are A or B restricted, and a previous study found an average of about 19 epitopes recognized (M. M. Addo et al., J. Virol. 77:2081-2092, 2003). We combined these estimates in the model in order to predict the impact of inhibiting Nef function in the general (chronically infected) population by a drug. The predicted impact on viral load ranged from negligible to 2.4 orders of magnitude, depending on the effects of the drug and the CTL dynamical scenario assumed. We conclude that inhibiting Nef could make a substantial reduction in disease burden, lengthening the time before the necessity of undertaking combination therapy with other antiretroviral drugs.

Modulation of Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus Proteins

Mesenchymal stem cells (MSC) are promising candidates for cell replacement therapy since they have the ability to differentiate, under appropriate conditions, into a broad range of specialized cell types. Furthermore, MSC have low inherent immunogenicity, immunomodulatory properties, and preferentially home to/engraft damaged tissues. However, they are not invisible to the immune system, and upon allogeneic transplantation, MSC can elicit an immune response, that results in activation of the recipient's cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells and hence rejection of the transplanted cells. Human cytomegalovirus (HCMV), a virus ubiquitously present in humans, has developed several strategies to evade CTL and NK cell recognition. HCMV avoids CTL attack by producing proteins coded for by the unique short region (US) of the genome that downregulate MHC-I surface expression. Moreover, expression of HCMV UL18 protein, which is an MHC-I homolog or decoy, renders CMV-infected cells resistant to NK lysis. Thus, we hypothesized that simultaneous expression of HCMV US protein US2, US3, US6, or US11 with UL18 would render MSC less susceptible to CTL and NK cell recognition and killing. To this end, we started by transducing MSC with the retroviral vectors encoding: US2; US3; US6; or US11, and tested the effect of each of these proteins on expression of MHC-I and CD59, a complement membrane attack complex inhibitor whose expression has been shown to prevent destruction of transplanted cells. MSC expressing US3, US2, US11 and US6 showed substantial MHC-I downregulation of 69%, 74%, 78 % and 95% respectively, when compared to untransduced MSC. Moreover, MSC expressing either US6 or US2 proteins displayed a significant up-regulation of CD59, with US6 causing a 2.5 and US2 a 3.9 fold increase, when compared to untransduced MSC (Mean Fluorescence Intensity: 869.8; 1331.19; 344.99, respectively). Each of the US transduced MSC were then tested for their capability to activate MHC-I mismatched CTL proliferation in both an allogeneic and a xenogeneic setting, using MSC transduced with an empty retrovirus as a control. MSC expressing US2 or US6 reduced CTL proliferation by the same degree, whether CTL were mismatched human- or sheep-derived. Using MSC-US2 we obtained a reduction in CTL proliferation by 18% and 23% in human-or sheep-CTL, respectively, while using MSC-US6 resulted in a reduction in human and sheep CTL proliferation of 60% and 53%. Expression of US11 on MSC was more efficient at abrogating the sheep CTL proliferation than that of mismatched human CTL (reduction in proliferation by 53% vs 31%, respectively). Finally, expression of US3 on MSC only reduced proliferation of human CTL by 21.5%, but had no effect on sheep CTL. In conclusion, engineering MSC to over-express US6 seems to be the most effective way to enable MSC to evade the host immune system, resulting in efficient downregulation of MHC-I surface expression, significant reduction in CTL proliferation, and a reduced formation of the membrane attack complex at the end of the complement cascade, which will reduce destruction of MSC by this system. In vivo studies using a large animal sheep model are underway to demonstrate whether the results obtained in vitro using US6 HCMV protein expression will translate into improved engraftment in vivo.

Feline herpesvirus‐1 down‐regulates MHC class I expression in an homologous cell system

Cytotoxic T lymphocytes (CTLs) are an essential component of the immune defense against many virus infections. CTLs recognize viral peptides in the context of the major histocompatibility complex (MHC) class I molecules on the surface of infected cells. Many viruses have evolved mechanisms to interfere with MHC class I expression as a means of evading the host immune response. In the present research we have studied the effect of in vitro Feline Herpesvirus 1 (FeHV-1) infection on MHC class I expression. The results of this study demonstrate that FeHV-1 down regulates surface expression of MHC class I molecules on infected cells, presumably to evade cytotoxic T-cell recognition and, perhaps, attenuate induction of immunity. Sensitivity to UV irradiation and insensitivity to a viral DNA synthesis inhibitor, like phosphonacetic acid, revealed that immediate early or early viral gene(s) are responsible. Use of the protein translation inhibitor cycloheximide confirmed that an early gene is primarily responsible.

Functional Consequences of Human Immunodeficiency Virus Escape from an HLA-B*13-Restricted CD8 + T-Cell Epitope in p1 Gag Protein

The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI(429-437) (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.

Role of Cytotoxic T-Lymphocyte-Mediated Immune Selection in a Dominant Human Leukocyte Antigen-B8-Restricted Cytotoxic T-Lymphocyte Epitope in Nef

To study the role of cytotoxic T-lymphocyte (CTL) escape for disease progression in HIV-1 infection, we analyzed the CTL response to the dominant human leukocyte antigen (HLA)-B8-restricted CTL epitope FLKEKGGL (FL8) in HIV-1 Nef. HIV-1 nef genes derived from 56 patients were analyzed by polymerase chain reaction (PCR)-based sequencing. T-cell responses against FL8 and mutated FL8 variants were detected by gamma-interferon (gamma-IFN) enzyme linked immunospot (ELISPOT) assay. The longitudinal analysis of an HIV-1-infected patient with good control of HIV-1 viremia for several years demonstrated an association of rising viremia with the emergence of CTL escape mutations within the HLA-B8-restricted Nef-specific CTL epitopes FLKEKGGL and WPAIRERM. Analysis of nef genes in 56 HIV-1-infected patients demonstrated a significant correlation between the occurrence of mutations in the FL8 epitope and the presence of HLA-B8. The mutations within the FL8 epitope could decrease CTL recognition; however, there was strong variation regarding the recognition of viral variants between individual donors. The presence of FL8 mutations was associated with lower CD4 cell counts and higher viral loads. Our data demonstrate a strong CTL selection pressure on the immunodominant HLA-B8-restricted CTL epitope FL8 in HIV-1 Nef. The association of FL8 mutations with lower CD4 cell counts indicates an important role of CTL escape mutations for disease progression.

HIV-1/AIDS vaccine development: are we in the darkness before the dawn?

HIV-1/AIDS vaccine development: are we in the darkness before the dawn?

DDX3Y encodes a class I MHC–restricted H-Y antigen that is expressed in leukemic stem cells

AbstractThe Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8+ cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705–restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)–identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705+ patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8+ T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.

The Mamu B ⁎17-restricted SIV Nef IW9 to TW9 mutation abrogates correct epitope processing and presentation without loss of replicative fitness

CD8 + cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A ⁎01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B ⁎17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4 +T cells infected with an engineered SIV mac239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4 +T cells, the TW9 mutant virus failed to induce IFN-γ expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.

STAT3 and immune escape in head and neck squamous cell carcinoma

One strategy of immune escape involves downregulation of the antigen processing machinery (APM), enabling tumor cell evasion by cytotoxic T lymphocytes (CTL), however the mechanism responsible for this has not been determined. Previous work in our and other laboratories has shown that cell lines and tissues derived from HNSCC downregulate APM components such as transporter associated with antigen processing (TAP)‐1 and TAP2. Treatment with IFN‐γ upregulates APM components, and restores the ability of tumor antigen‐specific CTL to recognize and lyse these tumor cell lines. HNSCC tumors overexpress signal transducer and activator of transcription (STAT)‐3, a transcription factor activated by interleukin‐6 (IL‐6). Pre‐treatment of HNSCC cell lines with IL‐6 abrogates IFN‐γ mediated STAT1 activation, suggesting that STAT3 negatively regulates STAT1 activation. Thus, we hypothesized that overexpression of STAT3 in HNSCC may contribute to downregulation of APM components and CTL recognition by decreasing STAT1 activity, thereby contributing to immune escape. Preliminary studies indicate that IL‐6 pre‐treatment can decrease IFN‐γ mediated upregulation of APM components at both the transcriptional and translational level. Investigation is underway to elucidate the interaction of STAT1 and STAT3, and their potential contribution to APM component mediated tumor immune escape from CTL recognition.