Identification of novel tumor antigens with patient-derived immune-selected antibodies

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions.     

VH4 gene segments dominate the intrathecal humoral immune response in multiple sclerosis

A characteristic feature of the CNS inflammatory response in multiple sclerosis (MS) is the intrathecal synthesis of IgG and the presence of oligoclonal bands. A strong correlation between CD138(+) plasma blast numbers in MS cerebrospinal fluid (CeSF) and intrathecal IgG synthesis suggests that these cells are the major Ab-secreting cell type in MS CeSF. Sequencing of V regions from CD138(+) cells in MS CeSF has revealed somatically mutated and expanded IgG clonotypes consistent with an Ag-targeted response. In the present study, single-cell RT-PCR analysis of CD138(+) cells from 11 MS patients representing differing clinical courses and stages of disease identified expansion of CD138(+) cells with functionally rearranged V(H)4 gene segments as an overriding feature of MS CeSF repertoires. V(H)4 dominance was attributed to the preferential selection of specific V(H)4 genes, particularly gene segment V(H)4-39, which displayed a significant enrichment in CeSF compared with MS peripheral blood B cells. A modest increase in V(H)4 prevalence among MS peripheral blood IgG memory cells was also noted, suggesting that factors shaping the CD138 repertoire in CeSF might also influence the peripheral IgG memory cell pool. These results indicate a highly restricted B cell response in MS. Identifying the targets of CeSF plasma cells may yield insights into disease pathogenesis.     

CD27 is acquired by primed B cells at the centroblast stage and promotes germinal center formation

Studies on human B cells have featured CD27 as a marker and mediator of the B cell response. We have studied CD27 expression and function on B cells in the mouse. We find that B cells acquire CD27 at the centroblast stage and lose it progressively upon further differentiation. It is not a marker for somatically mutated B cells and is present at very low frequency on memory B cells. Enrichment of CD27 among centroblasts and the presence of its ligand CD70 on occasional T and B cells in or near germinal centers (GCs) suggested a role for CD27/CD70 interactions in clonal B cell expansion. Accordingly, GC formation in response to influenza virus infection was delayed in CD27 knockout mice. CD27 deficiency did not affect somatic hypermutation or serum levels of virus-specific IgM, IgG, and IgA attained in primary and recall responses. Adoptive transfer of T and B cells into CD27/CD28(-/-) mice revealed that CD27 promotes GC formation and consequent IgG production by two distinct mechanisms. Stimulation of CD27 on B cells by CD28(+) Th cells accelerates GC formation, most likely by promoting centroblast expansion. In addition, CD27 on T cells can partially substitute for CD28 in supporting GC formation.     

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

Expansions of clonal and oligoclonal T cells in B-cell chronic lymphocytic leukemia are primarily restricted to the CD3(+)CD8(+) T-cell population

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature-appearing clonal B cells exhibiting coexpression of CD5 and CD23. In addition to the accumulation of neoplastic B cells, numerous T-cell abnormalities also occur in B-CLL patients. In this study, the presence, and distribution within the T-cell subsets, of clonal/oligoclonal T cells was studied. Multicolor flow cytometric techniques were employed using combinations of anti-CD3, anti-CD4, and anti-CD8 antibodies coupled with antibodies specific for V(alpha) and V(beta) T-cell receptor (TCR) epitopes. Molecular studies of TCR gene sequences were done to confirm the presence of clonal/oligoclonal T-cell populations. In the flow cytometric studies, examination of V(alpha)/V(beta)expression found evidence of clonal/oligoclonal expansion in 9 of 19 patients studied. In eight of the nine patients, the expansions were restricted to the CD3(+)CD8(+) cell population. Molecular analyses were performed in 16 patients, 12 of whom showed a clonal or oligoclonal pattern. Of the four patients who were negative in the molecular analyses, all demonstrated flow cytometric evidence of clonal/oligoclonal expansions. Thus, when the flow cytometric and molecular analyses were considered together, all 16 patients for whom parallel analyses were done showed evidence of clonal/oligoclonal expansions. These results confirm previous work demonstrating that the majority of B-CLL patients harbor clonal/oligoclonal expansions within the T-cell population. Additionally, based on the relative numbers of cells expressing specific V(alpha) or V(beta)epitopes, these results show that these expansions occur primarily within the CD3(+)CD8(+) T-cell population.     

Single-cell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid

Single-cell RT-PCR was used to sample CD19(+) B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominantly polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for approximately 70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.     

A study of lymphocyte subsets in human normal tonsil and lymph node

A series of T and B lymphocyte specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulation in frozen and paraffin tissue sections of human normal tonsil and lymph node by means of immunocytochemical technique. In the paracortical and interfollicular area of tonsil and lymph node, most lymphocytes reacted with Leu 1, Leu 3 a, Leu 4 and OKT4. The numbers of Leu 2 a and OKT8 positive cells were rare in tissue. These cells were not only limited in paracortical area, they also appeared in considerable numbers in medullary cords of lymph nodes. Leu 2 a and OKT 8 positive cells decreased with prominent follicular hyperplasia of tonsils. In addition, substantial leu 3 a and Leu 4 cells were found in the germinal centers. This finding supports the importance of these lymphocyte subsets in regulation of human immune response. In the mantle zone of secondary follicles, the majority of lymphocytes were positive for OKB 2 and BA 1, whereas, the IgM positive cells were predominately observed in the cytoplasma and extracellular substance of B lymphocytes in the germinal centers, but the lymphocytes bearing sIgM were rarely observed. In the mantle zone, the IgM were frequently found on the surface of membrane of small lymphocytes, however, the staining intensity was much than that in the germinal centers.     

Alphavirus-induced encephalomyelitis: antibody-secreting cells and viral clearance from the nervous system

Sindbis virus (SINV) infection of the central nervous system (CNS) provides a model for understanding the role of the immune response in recovery from alphavirus infection of neurons. Virus clearance occurred in three phases: clearance of infectious virus (days 3 to 7), clearance of viral RNA (days 8 to 60), and maintenance of low levels of viral RNA (>day 60). The antiviral immune response was initiated in the cervical lymph nodes with rapid extrafollicular production of plasmablasts secreting IgM, followed by germinal center production of IgG-secreting and memory B cells. The earliest inflammatory cells to enter the brain were CD8(+) T cells, followed by CD4(+) T cells and CD19(+) B cells. During the clearance of infectious virus, effector lymphocytes in the CNS were primarily CD8(+) T cells and IgM antibody-secreting cells (ASCs). During the clearance of viral RNA, there were more CD4(+) than CD8(+) T cells, and B cells included IgG and IgA ASCs. At late times after infection, ASCs in the CNS were primarily CD19(+) CD38(+) CD138(-) Blimp-1(+) plasmablasts, with few fully differentiated CD38(-) CD138(+) Blimp-1(+) plasma cells. CD19(+) CD38(+) surface Ig(+) memory B cells were also present. The level of antibody to SINV increased in the brain over time, and the proportion of SINV-specific ASCs increased from 15% of total ASCs at day 14 to 90% at 4 to 6 months, suggesting specific retention in the CNS during viral RNA persistence. B cells in the CNS continued to differentiate, as evidenced by accumulation of IgA ASCs not present in peripheral lymphoid tissue and downregulation of major histocompatibility complex (MHC) class II expression on plasmablasts. However, there was no evidence of germinal center activity or IgG avidity maturation within the CNS.     

Development and maintenance of a B220- memory B cell compartment

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.     

Memory B cells express a phenotype consistent with migratory competence after secondary but not short-term primary immunization

The cell surface phenotype of dinitrophenol (DNP)-specific memory B cells, defined by their capacity to transfer IgG responses into syngeneic irradiated recipients, was assessed using two markers of relevance to lymphocyte migratory properties: (i) peanut agglutinin, which binds to terminal galactosyl residues expressed at high levels by several nonmigrating lymphocyte subsets and, among lymph node B cells, is highly specific for germinal center cells; and (ii) MEL-14, a monoclonal antibody specific for lymphocyte surface receptors required for migration from the blood into peripheral lymph nodes. At various times after primary or secondary immunization with DNP-keyhole limpet hemocyananin (KLH), lymph node B cells were separated by fluorescence-activated cell sorting on the basis of staining with PNA and/or MEL-14, and the presence of B-memory cells in each fraction was assessed by adoptive transfer with antigen (DNP-KLH) and helper T cells. One week after immunization, most of the memory sorted in the PNAhi population, confirming a previous report by R. F. Coico, B. S. Bhogal, and G. J. Thorbecke (J. Immunol. 131, 2254, 1983) that early memory B cells or their precursors are contained within the germinal center cell population, a population which is known to be MEL-14- and migratory-incompetent. Six weeks after primary stimulation, however, the bulk of memory cells, unlike germinal center cells, were MEL-14hi. After secondary immunization, memory was still predominantly MEL-14+ and PNAlo, although in some experiments adoptive responses were transferred by all sorted fractions. The results are consistent with the hypothesis that antigen-specific B cells initially undergo local (sessile) differentiation and proliferation in germinal centers, where they develop the capacity for adoptive transfer of antigen-specific secondary responses, but that with continued development their long-lived memory-containing progeny express a phenotype permitting their reentry into the recirculating lymphocyte pool.     

Foxp3+ follicular regulatory T cells control the germinal center response

Follicular helper (T(FH)) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T(FH) numbers maintains self tolerance. We describe a population of Foxp3(+)Blimp-1(+)CD4(+) T cells constituting 10-25% of the CXCR5(high)PD-1(high)CD4(+) T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T(FR)) cells share phenotypic characteristics with T(FH) and conventional Foxp3(+) regulatory T (T(reg)) cells yet are distinct from both. Similar to T(FH) cells, T(FR) cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T(FR) cells originate from thymic-derived Foxp3(+) precursors, not naive or T(FH) cells. T(FR) cells are suppressive in vitro and limit T(FH) cell and germinal center B cell numbers in vivo. In the absence of T(FR) cells, an outgrowth of non-antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T(FH) differentiation pathway is co-opted by T(reg) cells to control the germinal center response.     

Inhibition of CD73 improves B cell-mediated anti-tumor immunity in a mouse model of melanoma

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8(+) T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20(+) B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti-IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8(+) T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.     

Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses

Peyer's patches are known as mucosal inductive sites for humoral and cellular immune responses in the gastrointestinal tract. In contrast, functionally equivalent structures in the respiratory tract remain elusive. It has been suggested that nasal-associated lymphoid tissue (NALT) might serve as a mucosal inductive site in the upper respiratory tract. However, typical signs of mucosal inductive sites like development of germinal center reactions after Ag stimulation and isotype switching of naive B cells to IgA production have not been directly demonstrated. Moreover, it is not known whether CTL can be generated in NALT. To address these issues, NALT was structurally and functionally analyzed using a model of intranasal infection of C3H mice with reovirus. FACS and histological analyses revealed development of germinal centers in NALT in parallel with generation and expansion of IgA(+) and IgG2a(+) B cells after intranasal reovirus infection. Reovirus-specific IgA was produced in both the upper respiratory and the gastrointestinal tract, whereas production of reovirus-specific IgG2a was restricted to NALT, submandibular, and mesenteric lymph nodes. Moreover, virus-specific CTL were detected in NALT. Limiting dilution analysis showed a 5- to 6-fold higher precursor CTL frequency in NALT compared with a cervical lymph node. Together these data provide direct evidence that NALT is a mucosal inductive site for humoral and cellular immune responses in the upper respiratory tract.     

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.     

Immunoglobulin genes expressed by B-lymphocytes infiltrating cervical carcinomas show evidence of antigen-driven selection

Lymphocyte infiltration is often present in cervical cancer lesions, possibly reflecting an ongoing (but ineffective) immune response to the tumour. B-lymphocytes are the predominant lymphocyte infiltrate in pre-malignant cervical lesions, where they are thought to comprise the host immune response to active human papillomavirus (HPV) infection. Although B cells are less frequently detected in cervical tumours, a high proportion of terminally differentiated plasma cells expressing tumour-specific immunoglobulin (Ig) remain. The antigen specificity and functional significance of the antibody response to cervical tumours is unknown. As part of a study to characterise the antibodies expressed by the tumour-infiltrating B cells (TIL-B) in cervical tumours using antibody phage display, we examined expressed Ig gene sequences to determine if there was molecular evidence of a selective response to antigenic changes in the transformed epithelial cells. We found that biased variable region gene usage by the B cells and the rate of somatic hypermutation in the rearranged Ig heavy chain variable regions (VH) both indicated antigenic selection of the B cells. We also found evidence of affinity maturation, as indicated by the detection of antibodies of the IgG1, IgG2 and IgA isotypes, and possible clonal selection of the Ig receptors. These data support the notion that B-lymphocytes and plasma cells infiltrating cervical carcinomas are the result of an antigen-induced response to HPV infection or transformation.     

Antigen binding of human IgG Fabs mediate ERK-associated proliferation of human breast cancer cells

Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.     

人体正常扁桃体和淋巴结T、B淋巴细胞亚群的研究

本文用了一系列T、B淋巴细胞单抗,用免疫细胞化学技术观察了人淋巴结和扁桃体内T,B淋巴细胞及其亚群。T细胞及其亚群主要位于滤泡间的副皮质。此外在淋巴结髓质也有一定数量的T细胞亚群的分布;次级滤泡生发中心也常出现辅助T或Leu 4阳性T细胞。B淋巴细胞及其亚群多集中在初级和次级滤泡,如OKB_2和BA 1阳性细胞多集中于次级滤泡的帽状区,而IgM主要位于次级滤泡的生发中心。LN-2抗体选择性地与生发中心??和帽状区的B细胞的核膜起反应,是研究B细胞表型的一种重要试剂。     

Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.     

Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel,class II HLA-restricted melanoma antigen

Recent studies increasingly point to a pivotal role of CD4(+) T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4(+) T cells recognizing the autologous tumor cells in vitro. In fact, the CD4(+) T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase kappa was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4(+) T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient's peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4(+) T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.