The calreticulin (CALR) exon 9 mutations are promising targets for cancer immune therapy.

The calreticulin (CALR) exon 9 mutations are found in ∼30% of patients with essential thrombocythemia and primary myelofibrosis. Recently, we reported spontaneous immune responses against the CALR mutations. Here, we describe that CALR-mutant (CALRmut)-specific T cells are able to specifically recognize CALRmut cells. First, we established a T-cell culture specific for a CALRmut epitope. These specific T cells were able to recognize several epitopes in the CALRmut C terminus. Next, we established a CALRmut-specific CD4+ T-cell clone by limiting dilution. These CD4+ T cells recognized autologous CALRmut monocytes and hematopoietic stem cells, and T-cell recognition of target cells was dependent on the presence of CALR. Furthermore, we showed that the CALRmut response was human leukocyte antigen (HLA)-DR restricted. Finally, we demonstrated that the CALRmut-specific CD4+ T cells, despite their phenotype, were cytotoxic to autologous CALRmut cells, and that the cytotoxicity was mediated by degranulation of the T cells. In conclusion, the CALR exon 9 mutations are targets for specific T cells and thus are promising targets for cancer immune therapy such as peptide vaccination in patients harboring CALR exon 9 mutations.

Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2.

In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-α and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high) , was prospectively analysed in fresh blood, and treatment-associated quantitative and qualitative changes were analysed. By the 4th vaccine, patients showed a marked increase in CD4+ CD25(high) T cell subset from 6% to 22% (P<0.001). At the 6th vaccine, a general decline was observed and a significantly (P=0.01) lower level of CD4+ CD25(high) Treg cells was reached in the group of patients who attained disease stabilization (9.5%) compared to patients with continued progressive disease (14.5%). However, when FoxP3 was employed for retrospective analysis of Tregs on frozen blood, this difference did not reach significance (P=0.09). The vast majority of the Treg produced IL-10 and, to a varying extent, TGF-ß. In addition, sorted CD4+ CD25(high) CD127⁻ Tregs were able to suppress proliferation of peripheral blood mononuclear cells in a dose-dependent manner, thus suggesting a regulatory functionality. These findings emphasize the need for strategies to effectively eliminate Treg cells to optimize the clinical effectiveness of cancer immunotherapy.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays.

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Evidence for involvement of clonally expanded CD8+ T cells in anticancer immune responses in CLL patients following nonmyeloablative conditioning and hematopoietic cell transplantation.

We have analyzed the clonotype composition of CD8+ T cells following nonmyeloablative (NMA) conditioning and hematopoietic cell transplantation (HCT), of patients with chronic lymphocytic leukemia (CLL). Consecutive analyses of blood samples taken up to 2 years following HCT, demonstrated that CD8+ T-cell clonality was highly dynamic in the early phases after HCT, but became more stable after 4-5 months. Moreover, donor lymphocyte infusion (DLI) given for disease progression in one of the patients led to establishment of recurrent as well as new T-cell clonotypes. This coincided with disease remission, strongly suggesting that these T cells were engaged with anti-CLL cytotoxicity. To examine the functional capacity of stable clonally expanded T cells after HCT, CD8+ T cells isolated post-transplant from the recipients were stimulated ex vivo with CLL cells and subsequently analyzed by FACS for surface expression of the marker for cytotoxic activity, CD107a. Stimulation with CLL cells indeed led to surface expression of CD107a, and clonotype analyses of sorted cells demonstrated that CD107a positive T cells were stably expanded following HCT. Our data suggest that clonally expanded CD8+ T-cell clones participate in the ongoing T-cell response against CLL cells following HCT with NMA conditioning.

Longitudinal analysis of MART-1/HLA-A2-reactive T cells over the course of melanoma progression.

An HLA-A2-positive patient with advanced stage IV melanoma was vaccinated with dendritic cells (DCs) pulsed with melanoma antigens, whereby the rapid progression of disease stalled for a period of 10 months. Monitoring of the cellular immune response against one of the vaccinated HLA-A2-restricted epitopes demonstrated both induction and subsequent decline in the number of interferon-gamma (IFN-gamma)-producing MART-1-reactive cells present in the blood. Enumeration of reactive T cells by MART-126-35/HLA-A2 tetramer staining revealed an induction of such cells after three vaccinations and a subsequent decline that most prominent at times of rapid disease progression. However, a substantial number of reactive cells were present even when no MART-1 reactivity was detectable by functional assays. Isolation of such MART-126-35-reactive T cells by means of peptide/HLA-A2-coated magnetic beads demonstrated the persistence of a TCRVbeta14+ T-cell clone in this population over the whole observation period. Intracellular fluorescence-activated cell sorter staining of such TCRVbeta14+ T cells for IFN-gamma and interleukin-2 after maximal stimulation with phorbol 12-myristate 13-acetate/ionomycin revealed an impairment in their capacity to produce cytokines at the end of the observation period. Thus, functional changes of individual T-cell clones, e.g. clonal exhaustion, seem to be responsible for the known discrepancy between functional and phenotype assays for immune monitoring of tumour patients.

Survivin--a universal tumor antigen.

Tumor-associated antigens recognized by cellular effectors of the immune system are potential targets for antigen-specific cancer immunotherapy. These antigens are classified as tissue (melanocyte)-specific proteins, cancer-testis antigens (proteins expressed in normal testis and various cancers), tumor-specific peptides derived from mutations in tumor cells, and others. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific cytotoxic T lymphocytes (CTL) responses in vivo. However, most of the peptide epitopes used in these vaccination trials are melanocyte-specific, and these peptides cannot be applied for tumors of non-melanocyte origin. Furthermore, the expression of most tumor antigens is heterogeneous among tumors from different patients and can even vary among metastases obtained from one patient. Immune selection of antigen loss variants may prove to be an additional obstacle for the clinical applicability of most of the known CTL epitopes. Recently, a new tumor antigen, survivin, has been identified on the basis of spontaneous CTL responses in different cancer patients. Survivin is expressed in most human neoplasms, but not in normal, differentiated tissues. Importantly, downregulation or loss of survivin would severely inflict the growth potential of the tumor cell. Since survivin is expressed by a variety of different tumors MHC-restricted survivin epitopes may serve as important and widely applicable targets for anti-cancer immunotherapeutic strategies.

A case of lymphoblastoid natural killer (NK)-cell lymphoma: association with the NK-cell receptor complex CD94/NKG2 and TP53 intragenic deletion.

The clinical, histological, phenotypic and genotypic features of a lymphoblastoid natural killer (NK)-cell lymphoma presenting in the skin in a young caucasian woman are described. The disease behaved aggressively, but long-lasting remission was obtained by combination chemotherapy followed by autologous bone marrow transplantation. The blastoid cells were positive for terminal deoxynucleotidyl transferase, CD34, CD56 and CD4. Furthermore, the NK-cell receptor complex CD94/NKG2 was strongly expressed, as shown by examination with reverse transcription-polymerase chain reaction. The T-cell receptor (TCR)-gamma genes were in germline, and with the exception of CD4 all T-cell antigens were negative, including CD3, TCR-beta, TCR-delta, TIA-1, granzyme B and perforin. Epstein-Barr virus was negative, and no expression was seen of myeloid cell-associated markers. Molecular analysis showed no abnormalities of the CDKN2A (p16), CDKN2B (p15) or TNFRSF6 (Fas) genes. By contrast, a 34-bp deletion in exon 7 of the TP53 (p53) gene was detected. It is suggested that lymphoblastoid NK-cell lymphoma, which is a rare but distinctive disease, originates from NK cell precursors and may be associated with and possibly caused by alterations in the TP53 gene. Experience is too limited to warrant therapeutic suggestions. However, stem cell transplantation may be a useful option in younger patients.

Spontaneous cytotoxic T-cell responses against survivin-derived MHC class I-restricted T-cell epitopes in situ as well as ex vivo in cancer patients.

Recent advances in therapeutic tumor vaccinations necessitate the identification of broadly expressed, immunogenic tumor antigens that are not prone to immune selection. To this end, the human inhibitor of apoptosis, survivin, is a prime candidate because it is expressed in most human neoplasms but not in normal, differentiated tissues. Here, we demonstrate spontaneous cytotoxic T-cell responses against survivin-derived MHC class I-restricted T-cell epitopes in breast cancer, leukemia, and melanoma patients both in situ as well as ex vivo. Moreover, survivin-reactive T cells isolated by magnetic beads coated with MHC/peptide complexes were cytotoxic against HLA-matched tumors of different tissue types. Being a universal tumor antigen, survivin may serve as a widely applicable target for anticancer immunotherapy.

In situ cytokine therapy: redistribution of clonally expanded T cells.

Immunity to tumors relies on recirculating antigen-specific T cells. Whilst induction of antigen-specific T cells by immunotherapy has been convincingly proven, direct evidence for recirculation of such cells is still lacking. Here, employing a recently established in situ immunotherapy model for murine melanoma we directly demonstrate the redistribution of clonally expanded T cells. In this model IL-2 is targeted to the tumor microenvironment by means of specific antibody-IL-2 fusion proteins resulting in the expansion of T cells. The therapeutic effect of the fusion protein is not restricted to tumors expressing the targeted antigen, but extends to antigen negative variants of the tumor if present in the same animal. Analysis of the T cell infiltrate by quantitative reverse transcription-PCR revealed the presence of highly expressed TCR BV regions in both tumor variants. TCR clonotype mapping revealed that the high expressions of these regions were caused by clonal expansions and, notably, that these specific clonotypic TCR transcripts were identical in both tumors. Thus, T cell clones activated locally by targeted IL-2 therapy recirculate and mediate eradication of distant tumor sites not subjected to in situ cytokine therapy.

Targeting of lymphotoxin-alpha to the tumor elicits an efficient immune response associated with induction of peripheral lymphoid-like tissue.

A recombinant antibody-lymphotoxin-alpha fusion protein induced an adaptive immune response protecting mice from melanoma. Importantly, this fusion protein elicited the formation of a lymphoid-like tissue in the tumor microenvironment containing L-selectin+ T cells and MHC class II+ antigen-presenting cells, as well as B and T cell aggregates. Furthermore, PNAd+/TCA4+ high endothelial venules were observed within the tumor, suggesting entry channels for naive T cell infiltrates. Over the course of therapy, a marked clonal expansion of certain TCR specificities occurred among tumor-infiltrating lymphocytes that displayed reactivity against melanoma cells and the TRP-2(180-188) peptide. Consequently, naive T cells may have been recruited to as well as primed and expanded in the lymphoid-like tissue induced by the lymphotoxin-alpha fusion protein at the tumor site.

Oligoclonal T-cell receptor usage of melanocyte differentiation antigen-reactive T cells in stage IV melanoma patients.

Ex vivo ELISPOT analysis of peripheral blood lymphocytes obtained from stage IV melanoma patients demonstrated reactivity against peptides derived from MART-1 and gp100. However, the number of reactive T cells was < 1% that of total lymphocytes as detected by flow cytometry using tetrameric MHC/peptide complexes. Despite this low frequency, we were able to directly isolate these populations ex vivo by means of magnetic beads coated with MHC/peptide complexes and to subject these cells to T-cell receptor clonotype mapping. This analysis revealed that the MART-1/A*0201- and gp100/A*0201-reactive T-cell populations are composed of oligoclonal T cells that engage several T-cell receptor beta chain families. Longitudinal studies using this approach may result in a better correlation between T-cell reactivity and the course of neoplastic disease.

Comparative delineation of T cell clonotypes in coexisting syngeneic B16 melanoma.

B16 is a murine melanoma of C57B1/6 origin, which rapidly develops as a tumor when inoculated into syngeneic immunocompetent hosts. Nevertheless, B16 tumors are considered to be immunogenic since tumor regression can be induced by means of immunotherapeutic intervention. Furthermore, B16 melanoma cells express several melanoma-associated antigens that may serve as targets for autologous T cells. To study the in vivo T cell response against B16, with particular emphasis on diversity and systemic involvement, we examined the spectra of T cell clonotypes in coexisting B16 melanoma lesions in C57B1/6 mice. Three tumors from each animal (n = 8) were examined for the presence of clonotypic T cells using the highly sensitive T cell receptor (TCR) clonotype mapping technology. Systematic analysis of the TCRB variable regions 1-16 revealed from 19 to more than 30 clonotypic TCR transcripts in each tumor. To study intra- and inter-individual variations in the T cell response further, more than 600 clono-typic TCR transcripts were compared for sequence identity. Overall, approximately 2% of the T cell clonotypes were detected in more than one tumor from the same animal. Furthermore, none of the detected clonotypes was present in more than one animal, arguing against recurrent or "public" T cell responses against B16 melanoma. Our data strongly suggest that anti-melanoma T cell responses in this murine model encompass mainly localized T cells, and that systemic involvement is limited.

Tumor infiltrating lymphocytes in melanoma comprise high numbers of T-cell clonotypes that are lost during in vitro culture.

Melanoma is generally accepted as being an antigenic tumor capable of eliciting T-cell responses that, however, in most cases are inadequate to control tumor growth. Tumor-infiltrating lymphocytes (TIL) in melanoma lesions comprise clonotypic T cells, indicating the in situ recognition of melanoma-associated peptide epitopes. Cultured TIL have been studied in order to unveil characteristics of TIL and the interactions of TIL and melanoma cells. Whether in vitro cultured TIL mirrors the in situ situation has, however, been questioned. In the present study we have taken advantage of T-cell receptor clonotype mapping methodology to conduct a full and detailed analysis of the T-cell clonotypes in melanoma lesions and in corresponding lines of TIL established in vitro. All melanoma lesions and the corresponding TIL cultures comprised high numbers of T-cell clonotypes, typically in the range of 40 to more than 60. The subsequent comparison of T-cell clonotypes present in the original lesions and in the corresponding T-cell lines established in vitro demonstrated that a very limited number of the T-cell clonotypes established in vitro are identical to the T-cell clonotypes expanded in situ. These results demonstrate that in situ T-cell clonotypes in melanoma are not readily expanded in vitro and that the majority of T-cell clonotypes present in cultured TIL are not present in situ.

Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein.

Mutations in the tumour suppressor gene p53 are among the most frequent genetic alterations in human malignancies, often associated with an accumulation of the p53 protein in the cytoplasm. We have generated a number of cytotoxic T lymphocyte (CTL) clones that specifically recognize the HLA-A*0201 p53 wild type peptide RMPEAAPPV [65-73], designated R9V, by the in vitro stimulation of CD8 enriched peripheral blood lymphocytes from a healthy HLA-A*0201 donor using peptide loaded autologous dendritic cells. A total of 22 CTL clones were generated from the same bulk culture and demonstrated to carry identical T-cell receptors. The CTL clone, 2D9, was shown to specifically lyse the HLA-A*0201+ squamous carcinoma cell line SCC9 and the breast cancer cell line MDA-MB-468. Our data demonstrate that human peripheral blood lymphocytes from normal healthy individuals comprise T cells capable of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate target antigens for the immunotherapeutic treatment of cancer.