Identification of human leucocyte antigen (HLA)‐A*0201‐restricted cytotoxic T lymphocyte epitopes derived from HLA‐DOβ as a novel target for multiple myeloma

Despite the recent development of effective therapeutic agents against multiple myeloma (MM), new therapeutic approaches, including immunotherapies, remain to be developed. Here we identified novel human leucocyte antigen (HLA)‐A*0201 (HLA‐A2)‐restricted cytotoxic T lymphocyte (CTL) epitopes from a B cell specific molecule HLA‐DOβ (DOB) as a potential target for MM. By DNA microarray analysis, the HLA‐DOB expression in MM cells was significantly higher than that in normal plasma cells. Twenty‐five peptides were predicted to bind to HLA‐A2 from the amino acid sequence of HLA‐DOB. When screened for the immunogenicity in HLA‐A2‐transgenic mice immunized with HLA‐DOB cDNA, 4 peptides were substantially immunogenic. By mass spectrometry analysis of peptides eluted from HLA‐A2‐immunoprecipitates of MM cell lines, only two epitopes, HLA‐DOB_232–240 (FLLGLIFLL) and HLA‐DOB_185–193 (VMLEMTPEL), were confirmed for their physical presence on cell surface. When healthy donor blood was repeatedly stimulated in vitro with these two peptides and assessed by antigen‐specific γ‐interferon secretion, HLA‐DOB_232–240 was more immunogenic than HLA‐DOB_185–193. Additionally, the HLA‐DOB_232–240‐specific CTLs, but not the HLA‐DOB_185–193‐specific CTLs, displayed an major histocompatibility complex class I‐restricted reactivity against MM cell lines expressing both HLA‐A2 and HLA‐DOB. Taken together, based on the physical presence on tumour cell surface and high immunogenicity, HLA‐DOB_232–240 might be useful for developing a novel immunotherapy against MM.     

A novel multi‐epitope vaccine from MMSA‐1 and DKK1 for multiple myeloma immunotherapy

The identification of novel tumour‐associated antigens is urgently needed to improve the efficacy of immunotherapy for multiple myeloma (MM). In this study, we identified a membrane protein MMSA‐1 (multiple myeloma special antigen‐1) that was specifically expressed in MM and exhibited significantly positive correlation with MM. We then identified HLA‐A*0201‐restricted MMSA‐1 epitopes and tested their cytotoxic T lymphocyte (CTL) response. The MMSA‐1 epitope SLSLLTIYV vaccine was shown to induce an obvious CTL response in vitro. To improve the immunotherapy, we constructed a multi‐epitope peptide vaccine by combining epitopes derived from MMSA‐1 and Dickkopf‐1 (DKK1). The effector T cells induced by multi‐epitope peptide vaccine‐loaded dendritic cells lysed U266 cells more effectively than MMSA‐1/DKK1 single‐epitope vaccine. In myeloma‐bearing severe combined immunodeficient mice, the multi‐epitope vaccine improved the survival rate significantly compared with single‐epitope vaccine. Consistently, multi‐epitope vaccine decreased the tumour volume greatly and alleviated bone destruction. The frequencies of CD4⁺ and CD8⁺ T cells was significantly increased in mouse blood induced by the multi‐epitope vaccine, indicating that it inhibits myeloma growth by changing T cell subsets and alleviating immune paralysis. This study identified a novel peptide from MMSA‐1 and the multi‐epitope vaccine will be used to establish appropriate individualized therapy for MM.     

Identification of HLA-A2 restricted T-cell epitopes within the conserved region of the immunoglobulin G heavy-chain in patients with multiple myeloma

Objective: The aim of this study is the identification of HLA‐A2 restricted T‐cell epitopes in the conserved region of the immunoglobulin‐G‐heavy‐chain (IgG_H) that can be used for immunotherapy in multiple myeloma (MM) patients. Methods: After the IgG_H gene sequence was scanned for HLA‐A2 restricted T‐cell epitopes with a high binding affinity to the MHC‐I‐complex, promising nona‐peptides were synthesized. Peptide specific CD8 ⁺ T‐cells were generated from peripheral blood mononuclear cells (PBMC) of healthy donors (HD) and patients with MM using peptide pulsed dendritic cells (DC) in vitro. The activation and cytotoxicity of CD8 ⁺ T‐cells was analyzed by IFN‐α ELISpot‐assay and ⁵¹Chromium release‐assay. HLA‐A2 restriction was proven by blocking T‐cell activation with anti‐HLA‐A2 antibodies. Results: Two HLA‐A2 restricted T‐cell epitopes–TLVTVSSAS derived from the IgG_H‐framework‐region 4 (FR4) and LMISRTPEV from the constant region (CR)‐induced expansion of specific CD8 ⁺ T‐cells from PBMC in two of three (TLVTVSSAS) and one of three (LMISRTPEV) HD respectively. Specific T‐cells were induced from PBMC in two of six (TLVTVSSAS) and eight of 19 (LMISRTPEV) patients with MM. Specific CD8 ⁺ T‐cells also lysed peptide‐pulsed target cells in ⁵¹Chromium release‐assay. LMISRTPEV specific CD8 ⁺ T‐cells from MM patients lysed specifically the HLA‐A2 ⁺ IgG myeloma cell line XG‐6. Conclusion: We identified two HLA‐A2 restricted T‐cell epitopes–TLVTVSSAS and LMISRTPEV–which can yield an expansion of CD8 ⁺ T‐cells with the ability to kill peptide‐loaded target cells and HLA‐A2 ⁺ IgG⁺ myeloma cells. We conclude that TLVTVSSAS and LMISRTPEV could be T‐cell epitopes for immunotherapy in MM patients.     

Persistent hepatitis C virus (HCV) infection impairs HCV‐specific cytotoxic T cell reactivity through Mcl‐1/Bim imbalance due to CD127 down‐regulation

Summary. In persistent hepatitis C virus (HCV) infection, HCV‐specific cytotoxic T lymphocyte (CTL) reactivity is impaired and this affects HCV control. Interleukin‐7 receptor (CD127) expression on these cells could regulate CTL reactivity through Mcl‐1/Bim balance modulation. Bim is a pro‐apoptotic molecule blocked by the action of Mcl‐1. Mcl‐1/Bim expression and T cell reactivity on HCV‐specific CTLs were compared according to CD127 phenotype. Peripheral blood lymphocytes (PBL) from HLA‐A2⁺ HCV⁺ patients were obtained. HCV‐specific CTLs were visualized by staining PBL with anti‐CD8 and HLA‐A2/peptide pentameric complexes (pentamer). Mcl‐1/Bim/CD127 phenotype of HCV‐specific CTLs was tested by staining detectable CD8⁺/pentamer⁺ cells with anti‐Mcl‐1/Bim/CD127 antibodies. HCV‐specific CTL proliferation ability after specific in vitro challenge was tested in the presence and absence of pancaspase inhibitor z‐VAD‐fmk. All stained cells were analysed by flow cytometry. CD127^low‐expressing HCV‐specific CTLs associated with high HCV viraemia, while CD127^high correlated with undetectable viral loads (P < 0.001). Directly ex vivo, pentamer⁺ cell frequency was similar according to CD127 expression level. Nevertheless, CD127^low pentamer⁺ cell proliferation after specific in vitro challenge was impaired (P < 0.05), although this was corrected by z‐VAD‐fmk treatment (P < 0.05). Mcl‐1 expression was low directly ex vivo (P < 0.01), and Bim was up‐regulated after antigen encounter (P < 0.05) of CD127^low pentamer⁺ cells. The ex vivo difference between Mcl‐1 and Bim expression on pentamer⁺ cells correlated positively with CD127 expression level (P < 0.001) and with pentamer⁺ cell reactivity (P < 0.05). In summary, a low ex vivo Mcl‐1 expression and Bim up‐regulation after antigen encounter are involved in CD127^low HCV‐specific CTL hyporeactivity during chronic infection, but it can be overcome by apoptosis blockade.     

Immunological responses against an autologous human hepatocellular carcinoma cell line

We performed a detailed analysis of immune responses in a hepatocellular carcinoma (HCC) cell line and effector cells obtained from a patient with HCC. We examined the cytotoxic activity of natural killer (NK) cells, lymphokine-activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) against an autologous tumour cell line (SUHC-1) to investigate the immune mechanism of human lymphocytes against HCC cells. Cytotoxic T lymphocytes were induced by co-culturing of peripheral blood lymphocytes (PBL) and SUHC-1 cells, mixed lymphocyte and tumour cell culture (MLTC). The susceptibility of SUHC-1 to NK and LAK cells was similar to that of other allogeneic cell lines, such as K562, PLC/PRF/5 and Mahlavu. Effector cells induced in the primary MLTC had high cytotoxic acitivity but were not specific for SUHC-1. Cytotoxic T lymphocytes with specific activity against SUHC-1 were induced after PBL were stimulated five times at 7–10 day intervals with SUHC-1 and low-dose recombinant interleukin-2 (rIL-2), suggesting that as the culture progressed, broadly reactive effector cells disappeared and specific effector cells survived. The specific effector cells were identified as CD3⁺/CD4⁺ and CD⁺/CD8⁺ T-lymphocyte subsets. The recognition mechanisms of CD3⁺/CD4⁺ CTL remain unresolved because the cytotoxicities were not inhibited by anti-CD4 and anti-major histocompatibility complex (MHC) class II monoclonal antibodies (MoAb). Treatment of cells with anti-CD3, anti-CD8 and anti-MHC class I MoAb partially inhibited lysis. These results demonstrated that the T-cell receptor (TCR)/CD3 complex appeared to be involved in SUHC-1 specific antigen recognition and antigen recognition of CD3⁺/CD8⁺ CTL was MHC class I restricted.     

Age-matched lymphocyte subpopulation reference values in childhood and adolescence: application of exponential regression analysis

Background: Normal values of lymphocyte subpopulations for healthy children and adults have been published in defined age groups exclusively, which results in difficult data interpretation for patients close to the limit of contiguous age group ranges. In addition, normal values for a number of lymphocyte subpopulations have not been established to date. Objective: The aim of this study was to develop a model which provides continuous age‐dependent reference values. This model was applied for lymphocyte subpopulations such as naïve and memory T cells as well as their activation profile with diagnostic relevance in children and adults. Study design: A total of 100 blood samples, obtained from 80 healthy children and 20 adults were analysed by means of four colour‐flow cytometry. Continuous age‐dependent reference values were computed based on the residual values in an exponential regression model. Results: We calculated a continuous age‐related regression model for both, absolute cell counts and percentages of CD3⁺CD4⁺ T helper (T_H) cells, CD3⁺CD8⁺ cytotoxic T cells, CD56⁺CD3^? natural killer (NK) cells, CD56⁺CD3⁺ T cells, CD3⁺CD4⁺CD45RA⁺ naïve T_H cells, CD3⁺CD4⁺CD45RO⁺ memory T_H cells, CD3⁺CD8⁺CD45RA⁺CD28⁺ naïve cytotoxic T cells, CD3⁺CD8⁺CD45RO⁺ memory cytotoxic T cells, CD3⁺CD8⁺CD69⁺ early activated cytotoxic T cells and CD3⁺CD8⁺HLA‐DR⁺ late activated cytotoxic T cells, respectively, to obtain reference values. Conclusion: Based on an exponential regression model, the obtained reference values reflect the continuous maturation of lymphocyte subsets during childhood.     

Frequency of specific CD8+ T cells for a promiscuous epitope derived from Trypanosoma cruzi KMP‐11 protein in chagasic patients

The K1 peptide is a CD8 ⁺ T cell HLA‐A*0201‐restricted epitope derived from the Trypanosoma cruzi KMP‐11 protein. We have previously shown that this peptide induces IFN‐γ secretion by CD8⁺T cells. The aim of this study was to characterize the frequency of K1‐specific CD8⁺T cells in chagasic patients. Nineteen HLA‐A2⁺individuals were selected from 50 T. cruzi infected patients using flow cytometry and SSP‐PCR assays. Twelve HLA‐A*0201⁺noninfected donors were included as controls. Peripheral blood mononuclear cells were stained with HLA‐A2‐K1 tetramer, showing that 15 of 19 infected patients have K1‐specific CD8⁺T cells (0·09–0·34% frequency) without differences in disease stages or severity. Of note, five of these responders were A*0205, A*0222, A*0226, A*0259 and A*0287 after molecular typing. Thus, a phenotypic and functional comparison of K1‐specific CD8⁺T cells from non‐HLA‐A*0201 and HLA‐A*0201⁺infected patients was performed. The results showed that both non‐HLA‐A*0201 and HLA‐A*0201⁺individuals have a predominant effector memory CD8⁺T cell phenotype (CCR7?, CD62L?). Moreover, CD8⁺T cells from non‐HLA‐A*0201 and HLA‐A*0201⁺individuals expressed IL‐2, IFN‐γ and perforin without any differences. These findings support that K1 peptide is a promiscuous epitope presented by HLA‐A2 supertype molecules and is highly recognized by chagasic patients.     

Novel epitope evoking CD138 antigen-specific cytotoxic T lymphocytes targeting multiple myeloma and other plasma cell disorders

The development of an immunotherapeutic strategy targeting CD138 antigen could potentially represent a new treatment option for multiple myeloma (MM). This study evaluated the immune function of CD138 peptide-specific cytotoxic T lymphocytes (CTL), generated ex vivo using an HLA-A2-specific CD138 epitope against MM cells. A novel immunogenic HLA-A2-specific CD138(260-268) (GLVGLIFAV) peptide was identified from the full-length protein sequence of the CD138 antigen, which induced CTL specific to primary CD138(+) MM cells. The peptide-induced CD138-CTL contained a high percentage of CD8(+) activated/memory T cells with a low percentage of CD4(+) T cell and naive CD8(+) T cell subsets. The CTL displayed HLA-A2-restricted and CD138 antigen-specific cytotoxicity against MM cell lines. In addition, CD138-CTL demonstrated increased degranulation, proliferation and γ-interferon secretion to HLA-A2(+) /CD138(+) myeloma cells, but not HLA-A2(-) /CD138(+) or HLA-A2(+) /CD138(-) cells. The immune functional properties of the CD138-CTL were also demonstrated using primary HLA-A2(+) /CD138(+) cells isolated from myeloma patients. In conclusion, a novel immunogenic CD138(260-268) (GLVGLIFAV) peptide can induce antigen-specific CTL, which might be useful for the treatment of MM patients with peptide-based vaccine or cellular immunotherapy strategies.     

Naïve subset develops the most important alloreactive response among human CD4+ T lymphocytes in Human Leukocyte Antigen‐identical related setting

In longitudinal clinical studies, receiving a high percentage of allogeneic donor‐derived CD4⁺CCR7⁺ T cells, which include naïve and central memory subsets have been correlated with increased incidence and severity of acute GVHD. Whether naïve and central memory CD4⁺ T‐cell subsets contribute more or equally to alloimmune responses are still unclear in human. The aim of this study was to investigate in vitro the alloreactive response of purified naïve, central memory, and effector memory CD4⁺ T‐cell subsets in HLA identical setting. By coculturing monocyte‐derived dendritic cells and purified CD4⁺ T‐cell subsets, from healthy HLA‐identical male and female sibling pairs, we found that naïve CD4⁺CCR7⁺CD45RA⁺ T cells developed the highest proliferative response upon stimulation by minor histocompatibility antigens and were progressively driven to produce high levels of interferon‐γ, tumor necrosis factor, and interleukin‐6. Comparatively, the central memory CD4⁺CCR7⁺CD45RA^neg subset proliferated to a lower extent and produced very low amounts of pro‐inflammatory cytokines while the CCR7^neg effector memory CD4⁺ subset was unresponsive. This study demonstrates the superior capacity of naïve CD4⁺ T cells to mount a primary alloreactive response as compared to central memory T cells. Their proliferative response associated with a pro‐inflammatory differentiation makes them potentially acute GVHD inducers. These in vitro results in line with what we have observed in clinical studies and may also lend support to approaches of partial selective T‐cell depletion for GVHD prevention.     

Generation of cellular immune responses to HCV NS5 protein through in vivo activation of dendritic cells

Summary. Chronic hepatitis C (HCV) infection is a substantial medical problem that leads to progressive liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The aim of this study was to achieve sustained cellular immune responses in vivo to a HCV nonstructural protein using dendritic cell (DC)‐based immunization approach. We targeted the HCV NS5 protein to DCs in vivo by injecting microparticles loaded with this antigen. The DC population was expanded in BALB/C mice (H‐2^d) by hydrodynamic injection of a plasmid pUMVC3‐hFLex expressing the secreted portion of the human Fms‐like tyrosine kinase receptor‐3 ligand (hFlt3). Mice were subsequently injected with microparticles coated with HCV NS5 protein via the tail vein. Cellular immune responses were determined with respect to secretion of INFγ and IL2 by CD4⁺ cells and cytotoxic T‐lymphocyte (CTL) assays in vitro; inhibition of tumour cell growth was employed for the assessment of CD8⁺ generated activity in vivo. We found that Flt3L treatment expanded the DC population in the spleen to 43%, and such cells displayed a striking upregulation of CD86 as well as CD80 and CD40 co‐stimulating molecules. Viral antigen‐specific T_H1 cytokine secretion by splenocytes was generated, and CTL activity against syngeneic NS5 expressing myeloma target cells was observed. In addition, these cells inhibited tumour growth indicating that NS5‐specific robust CTL activity was operative in vivo. Thus, the capability of activating DCs in vivo using the methods described is valuable as a therapeutic vaccine strategy for chronic HCV infection.     

Autoreactive and immunoregulatory T-cell subsets in insulindependent diabetes mellitus

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is a T-cell mediated autoimmune disease. Several subsets of T-cells, in particular CD4⁺ and in vivo activate CD45RA⁺RO⁺ T-cells, have been shown to be increased at disease onset. The functional implications of these relative increases in CD4 T-cells were investigated. Methods. Subsets of T-cells were sorted on the basis of their activation status (CD45RA⁺ naïve cells, CD45RA⁺RO⁺ recently activated cells and CD45RO⁺ memory cells) and stimulated with autoantigens or recall antigen in vitro. Results. Proliferative responses to tetanus toxoid were primarily or exclusively observed in resting memory T-cells (CD45RO⁺). Autoimmune T-cell responses were, however, primarily measured in activated T-cells (CD45RA⁺RO⁺) in newly diagnosed Type I diabetic patients, whereas those with longer disease duration reacted to autoantigens with memory T-cells (CD45RO⁺) (p < 0.004). Interestingly, in non-diabetic control subjects not responding to autoantigens in the regular assay, considerable autoreactive T-cell responses were detectable after sorting in the CD45RO⁺ or CD45RA⁺RO⁺ lymphocyte subsets. Remixing these subsets showed that these autoimmune responses in activated cells could be down-modulated by CD45RA⁺ lymphocytes, whereas resting memory cells appeared unaffected by the suppressive CD45RA subset. Conclusion/interpretation. These results show that autoimmune T-cell responses can be linked to particular subsets which differ depending on clinical status. Furthermore, the CD45RA T-cell subset harbours lymphocytes potentially capable of suppressing autoimmune T-cell responses. The changes in responsiveness to exogenous insulin may help to unravel the mechanism by which isohormonal therapy could prevent the onset of Type I diabetes. [Diabetologia (1999) 42: 443–449]     

Myeloma cells act as tolerogenic antigen‐presenting cells and induce regulatory T cells in vitro

Regulatory T cells (Tregs) are essential for maintenance of self‐tolerance; however, tumor cells can exploit the tolerance to escape the immune system. We investigated the Tregs frequency in patients with multiple myeloma (MM) and in those with monoclonal gammopathy of undetermined significance (MGUS), and found that CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ Tregs were significantly increased in patients with MM and correlated with the active phase. Both Tregs subsets were expanded in cocultures of CD3⁺ lymphocytes and fresh CD138⁺ MM plasma cells or RPMI8226 and U266 cell lines and functioned as natural (n) and inducible (i) Tregs insofar as they inhibited the proliferation of stimulated CD3 lymphocytes via contact‐dependent and contact‐independent pathways. Induction of Tregs by MM plasma cells required a contact‐dependent pathway, implying antigen recognition by T cells. MM plasma cells acted as immature and tolerogenic antigen‐presenting cells (APCs), in that they displayed low CD80/CD86 expression associated with a phagocytic activity. By acting as immature APCs, MM plasma cells plausibly expand (n)Tregs and (i)Tregs both through conversion of CD3⁺FoxP3^? into CD3⁺FoxP3⁺ T cells and proliferation of CD3⁺FoxP3⁺ T cells, which may suppress the anti‐MM immune response.     

α‐Galactosylceramide activates antitumor immunity against liver tumor

Aim: α‐Galactosylceramide (α‐GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. We investigated the detailed process of activating liver dendritic cells (DC) and immune cells after α‐GalCer treatment in the mouse liver tumor model. Methods: BALB/c mice bearing CMS4 liver tumor (p53 peptide‐expressing tumor) were treated by α‐GalCer. We evaluated the activation of liver DC and immune cells after α‐GalCer treatment. Interferon (IFN)‐γ enzyme‐linked immunosorbent spot (ELISPOT) assay was performed to detect p53 peptide‐specific cytotoxic T lymphocytes (CTL). To assess the impact of systemic acquired immunity by α‐GalCer treatment, 28 days after liver tumor treatment, CMS4 cells or Colon26 cells were re‐challenged s.c. Results: The liver weights of α‐GalCer‐treated mice were significantly lighter than those of vehicle‐treated mice. Depletion experiments revealed that natural killer (NK) cells were essential for the antitumor effect of α‐GalCer. α‐GalCer treatment significantly increased the population of DC and NK cells in the liver. The expressions of co‐stimulatory molecules on liver DC significantly increased with the peak at 1 day after α‐GalCer administration. IFN‐γ ELISPOT assay demonstrated that p53 peptide‐specific CTL was generated efficiently in α‐GalCer‐treated mice. ⁵¹Cr‐release assay revealed that CD8⁺, not CD4⁺, CTL against CMS4 cells were generated in α‐GalCer‐treated mice. The mice that had been protected from CMS4 liver tumor by α‐GalCer injection became resistant against s.c. CMS4 re‐challenge, but not against Colon26 re‐challenge. Conclusion: These results demonstrated the therapeutic potential of α‐GalCer against liver cancer through activating liver DC and immune cells in the liver.     

Individual myeloma‐specific T‐cell clones eliminate tumour cells and correlate with clinical outcomes in patients with multiple myeloma

Despite novel treatment strategies, multiple myeloma (MM) remains an incurable disease with low immunogenicity and multiple immune defects. We developed an ex vivo strategy for inducing myeloma‐specific cytotoxic T lymphocytes (CTLs) and demonstrate the possibility of identification and long‐term in vivo monitoring of individual myeloma‐specific T‐cell clones using the most sensitive clonotypic assay that is able to detect low frequencies of T‐cell clones (1 clonotypic cell in 10⁶ cells). Ten patients with MM were examined for the presence of tumour‐reactive T cells using dendritic cells loaded with autologous tumour cells. All patients had detectable myeloma‐reactive T cells in vitro. Expanded myeloma‐reactive T cells demonstrated specific cytotoxic effects against autologous tumour cells in vitro (median 39·6% at an effector:target ratio of 40:1). The clonality of myeloma‐specific T cells was studied with a clonotypic assay, which demonstrated both oligoclonal and monoclonal populations of myeloma‐specific T cells. CD8⁺ CTLs were the most immunodominant myeloma‐specific T‐cell clones and clinical responses were closely associated with the in vivo expansion and long‐term persistence of individual CD8⁺ T‐cell clones, usually at very low frequencies (10^?3–10^?6). We conclude that the clonotypic assay is the most sensitive tool for immunomonitoring of low‐frequency T cells.     

Identification of an immunogenic DKK1 long peptide for immunotherapy of human multiple myeloma

Dickkopf-1 (DKK1), broadly expressed by tumor cells from human multiple myeloma (MM) and other cancers but absent from most normal tissues, may be an ideal target for immunotherapy. Our previous studies have shown that DKK1 (peptide)-specific cytotoxic T lymphocytes can effectively lyse primary MM cells in vitro. To develop DKK1-based vaccines that can be easily and inexpensively made and used by all patients, we identified a DKK1 long peptide (LP), DKK1_3-76-LP, that contains 74 amino acids and epitopes that can potentially bind to all major MHC class I and II molecules. Using HLA-A*0201- and HLA-DR*4-transgenic mouse models, we found that DKK1-specific CD4⁺ and CD8⁺ T-cell responses, detected by DKK1 short peptide (P20 and P66v)-HLA-A*0201 tetramer staining and cytotoxic assay for CD8⁺ T cells or by carboxyfluorescein diacetate succinimidyl ester (CSFE) dilution and IFN-g secretion for CD4⁺ T cells, respectively, can be induced in vivo by immunizing mice with the DKK1_3-76-LP. In addition, DKK1_3-76-LP also induced anti-DKK1 humoral immunity in the transgenic mice and the DKK1 antibodies were functional. Finally, DKK1_3-76-LP stimulated human blood T cells ex vivo to generate DKK1-specific CD4⁺ and CD8⁺ T-cell responses from 8 out of 10 MM patients with different MHC backgrounds. The generated DKK1-specific CD8⁺ cells efficiently lysed autologous MM cells from these patients. Thus, these results confirm the immunogenicity of the DKK1_3-76-LP in eliciting DKK1-specific CD4⁺ and CD8⁺ T-cell responses in vitro and in vivo, and suggest that the DKK1_3-76-LP can be used for immunotherapy of MM and other cancers.     

Mucosal T‐cell responses to HIV: responding at the front lines

Mucosal surfaces of the body serve as the major portal of entry for human immunodeficiency virus (HIV). These tissues also house a majority of the body’s lymphocytes, including the CD4⁺ T cells that are the major cellular target for HIV infection. Mucosal surfaces are defended by innate and adaptive immune mechanisms, including secreted antibodies and CD8⁺ cytotoxic T cells (CTL). CTL in mucosal lymphoid tissues may serve to limit viral replication, decreasing the host’s viral burden as well as reducing the likelihood of sexual transmission to a naïve host. This review summarizes recent literature on HIV‐specific T‐cell responses in mucosal tissues, with an emphasis on the gastrointestinal tract.     

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer

Generation of tumor-antigen specific CD4⁺ T-helper (T_H) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4⁺ T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T_H lines. We isolated phenotypically defined CD4⁺ T-cell subpopulations from circulating lymphocytes of DR52b⁺ healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO_119-143, autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO_119-143 tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer⁺ cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer⁺ cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T_H lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer⁺ T_H lines from conventional CD4⁺CD25⁻ naïve and central memory populations, but not from effector memory populations or CD4⁺CD25⁺ Treg. In vitro primed T_H lines recognized ESO with affinities comparable to ESO-tetramer⁺ cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T_H lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.     

Human heat shock protein‐specific cytotoxic T lymphocytes display potent antitumour immunity in multiple myeloma

Tumour cell–derived heat shock proteins (HSPs) are used as vaccines for immunotherapy of cancer patients. However, it is proposed that the peptide chaperoned on HSPs, not HSPs themselves, elicited a potent immune response. Given that HSPs are highly expressed by most myeloma cells and vital to myeloma cell survival, we reasoned that HSPs themselves might be an ideal myeloma antigen. In the present study, we explored the feasibility of targeting HSPs themselves for treating multiple myeloma. We identified and chose HLA‐A*0201‐binding peptides from human HSPB1 (HSP27) and HSP90AA1 (HSP90), and confirmed their immunogenicity in HLA‐A*0201 transgenic mice. Dendritic cells pulsed with HSPB1 and HSP90AA1 peptides were used to stimulate peripheral blood mononuclear cells from healthy volunteers and myeloma patients to generate HSP peptide‐specific cytotoxic T lymphocytes (CTLs). HSP peptide‐specific CTLs efficiently lysed HLA‐A*0201⁺ myeloma cells (established cell lines and primary plasma cells) but not HLA‐A*0201^? myeloma cells in vitro, indicating that myeloma cells naturally express HSP peptides in the context of major histocompatibility complex class I molecules. More importantly, HSP peptide‐specific CTLs effectively reduced tumour burden in the xenograft mouse model of myeloma. Our study clearly demonstrated that HSPs might be novel tumour antigens for immunotherapy of myeloma.     

T cell cytotoxicity of autologous and allogeneic lymphocytes in a patient with Crohn's disease

We report a 27-year-old male with Crohn's disease (CD) of the small and large intestine, whose peripheral blood lymphocytes (PBL) showed increased cell-mediated cytotoxicity (CTL). Autologous and allogeneic effector cells from PBL and intestinal lymph nodes (LN) were isolated on a Ficoll-Hypaque gradient. Colonic cells were prepared as the target and were incubated for 6 h with effector cells, after being labeled with Na₂ ⁵¹CrO₄. The CTL activity [effector/ target (E/T) ratio, 100:1] of PBL for autologous targets was increased by 38% compared with that in normal subjects (<10%), while that shown by LN was not increased (14%). The CTL activity of allogeneic PBL prepared from three of four other CD patients was also increased. Anti-major histocompatibility (MHC) class I and II and CD4 and CD8 monoclonal antibodies (50μg/ml) significantly inhibited CTL, activity. Complement-mediated depletion of CD2⁺ cells significantly reduced CTL activity. These results suggest that MHC-restricted CTL may play a role in mucosal damage in some patients with Crohn's disease.