P thor Straten

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.

Concurrent disruption of cell cycle associated genes in mantle cell lymphoma : a genotypic and phenotypic study of cyclin D1, p16, p15, p53 and pRb

Mantle cell lymphomas (MCL) are morphologically and immunophenotypically distinctive lymphoid neoplasms characterised by overexpression of cyclin D1. Recent studies have suggested that co-operating aberrations of cell cycle associated genes may provide a growth advantage to a tumour. To address this issue further, we investigated five typical and three aggressive (blastoid) MCL for alterations in the cell cycle regulating genes p15, p16, CDK4, Rb and p53. In 3/3 aggressive cases with cyclin D1 overexpression we found aberration of at least one additional gene. One case showed diminished expression of the retinoblastoma protein (pRb); one case harboured deletion of both p15 and p16; and one case exhibited both deletion of p16 and point mutation of p53. However, we also identified two typical cases which in addition to cyclin D1 overexpression exhibited diminished pRb expression and p15 and p16 hypermethylation, respectively. Our findings confirm and extend other recent investigations and indicate that co-operating genetic alterations of cell cycle-associated genes may contribute to the pathogenesis of MCL.

Clonal T cell responses in tumor infiltrating lymphocytes from both regressive and progressive regions of primary human malignant melanoma.

The T cell receptor (TCR) BV variable (V) gene repertoire of tumor infiltrating lymphocytes (TIL) found in progressive and regressive regions of the same primary human melanomas were characterized by reverse transcription coupled polymerase chain reaction (RT-PCR). After surgery, the tumors were divided into different parts which were judged as regressive or progressive regions by visual inspection. Subsequently this diagnosis was confirmed by histology. From a total of four primary melanomas analyzed, 2 were drawn to be HLA-A2+. Only relatively few BV-gene families were expressed at significant levels in each of the samples. Comparison of the BV-expression in regressive versus progressive regions of the same tumor revealed major differences in all cases examined. Direct sequencing of RT-PCR products indicated that highly expressed BV-gene families were of clonal origin in both the regressive and progressive regions. Together, these data strongly suggest the occurrence of clonal T cell responses in both regressive and progressive areas of the same primary tumor. The differences in expression of certain BV-genes may correlate with the functional activity of certain populations of tumor-infiltrating T cells.

The antiapoptotic member of the Bcl-2 family Mcl-1 is a CTL target in cancer patients

We recently identified spontaneous T-cell responses against Bcl2-derived peptides in patients suffering from cancers of different origin. Myeloid cell factor-1 (Mcl-1) is a death-inhibiting member of the Bcl-2 family that is expressed in early monocyte differentiation and can promote viability on transfection into immature myeloid cells. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization of myeloid cells. Elevated levels of Mcl-1 have been reported for a number of human cancers including prostate cancers, pancreatic cancers, melanoma, breast cancers, ovarian cancer patients, and cervical cancer, as well as Bcell chronic lymphocytic leukemia (B-CLL) and in AML and ALL upon relapse. In B-CLL patients, higher levels of Mcl-1 are strongly correlated with failure to achieve complete remission after single-agent therapy. In multiple myeloma, Mcl-1 plays an important role in the survival of malignant cells. In this regard, it has been demonstrated that mice expressing an mcl-1 transgene under the control of its own promoter develop Bcell neoplasias with high frequency, ranging from follicular lymphoma to diffuse large-cell lymphoma. Thus, the overexpression of Mcl-1 in many cancers of different origin and the fact that immune escape by downregulation or loss of expression of this protein would impair sustained tumor cell growth highlight Mcl-1 as a very attractive target for immunotherapy of cancer. To investigate whether Mcl-1 is a natural target for T cells in cancer patients, we examined the protein sequence for the most probable HLA-A2 nonaand decamer peptide epitopes, using the main HLA-A3-specific anchor residues. Subsequently, we synthesized six Mcl-1-deduced peptides (Mcl-1185–194 (YLREQATGAK), Mcl-1293–302 (SITDVLVRTK), Mcl-1267–276 (LISFGAFVAK), Mcl-195–103 (RLLFFAPTR), Mcl-1300–308 (RTKRDWLVK), Mcl-1236–244 (DIKNEDDVK)) and scrutinized PBL from HLAA3þ cancer patients of different origin for reactivity against these peptides, taking advantage of the ELISPOT assay. This method has previously been shown to be highly efficient for the identification of tumor-specific CTL in cancer patients. Indeed, strong and frequent CTL responses were detected against two Mcl-1-derived peptides in cancer patients of different origin as exemplified in Figure 1a: an HLA-A3þ breast cancer patient hosting a response against Mcl-195–103 and an HLA-A3þ pancreatic cancer patient hosting a response against Mcl-1300–308 (Figure 1a). Overall, five out of six examined HLAA3þ breast cancer patients hosted an immune response against one of these two Mcl-1 peptides. Thus, five breast cancer patients hosted a response against Mcl-195–103 (responders are defined as the average number of antigen-specific cells7>2 standard deviation 425/10 cells), and three patients hosted a response against Mcl-1300–308 (Figure 1b). Additionally, two out of two examined HLA-A3þ pancreatic cancer patients hosted an immune response against the Mcl-195–103 peptide, whereas one of these also reacted against Mcl-1300–308. Additionally, we examined the PBL from six patients suffering from B-CLL and identified a response against Mcl-195–103 in two of these patients. As a control, PBL from 10 healthy HLA-A3þ individuals were examined. Importantly, no responses were detected against either the Mcl-195–103 or the Mcl-1300–308 peptide in any of the healthy donors (Figure 1b). Similarly, no responses could be detected against any of the additional four Mcl-1-derived peptides in any of the cancer patients or healthy controls (data not shown). In a recent study, we demonstrated CTL reactivity against the Bcl-2 protein in cancer patients. Together with the data presented here, this strongly suggests that cellular immune responses against antiapoptosis members of the Bcl-2 family represent a general phenomenon in cancer, and consequently that this group of proteins represents attractive universal target proteins for anticancer immunotherapy. The crucial role of Bcl-2 family of proteins, like Bcl-2 and Mcl-1, in cancer cell survival, implies that these proteins are highly relevant targets also for other therapeutic strategies in cancer treatment, for example, antisense oligonucleotides or small molecule inhibitors. However, our reports are the first to describe that the cellular immune system reacts against a member of this protein family, and that anticancer immunotherapy directed against this group of proteins, consequently, might be a novel effective way of attacking cancer. Furthermore, since this group of proteins is correlated with drug resistance, the combination of cytotoxic chemotherapy with immunotherapeutic targeting of Bcl-2 family of proteins might improve the efficacy of both treatments. In an attempt to maximize the impact of immunotherapy on disease progression, it is important to consider the expression profile and prognostic significance of the relevant target, for example, Mcl-1 and/or Bcl-2 in the particular disease, or disease stage. Thus, in some diseases like ovarian cancer, expression of Mcl-1, but not Bcl-2, is associated with advanced stage and poor survival for which reason Mcl-1 might be the prime antigen, whereas in diseases such as CLL, where Bcl-2 and Mcl-1 are cooverexpressed, simultaneous targeting of both proteins may represent a more effective strategy than targeting either molecule alone.

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.

Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

Abstract Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens  –  present also in normal melanocytes  –  and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells.

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 CD25high, 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+CD25high 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+CD25high 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‐β. In addition, sorted CD4+CD25highCD127− 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.

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‐γ (IFN‐γ)‐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 TCRVβ14+ T‐cell clone in this population over the whole observation period. Intracellular fluorescence‐activated cell sorter staining of such TCRVβ14+ T cells for IFN‐γ 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.

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.

Identification of an HLA-A1 restricted CTL epitope from Mcl-1

The lymphochip: a specialized cDNA microarray for the genomicscale analysis of gene expression in normal and malignant lymphocytes. Cold Spring Harb Symp Quant Biol 1999; 64: 71–78. 9 Nacheva E, Dyer MJ, Metivier C, Jadayel D, Stranks G, Morilla R et al. B-cell non-Hodgkin’s lymphoma cell line (Karpas 1106) with complex translocation involving 18q21.3 but lacking BCL2 rearrangement and expression. Blood 1994; 84: 3422–3428. 10 Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE et al. SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 2001; 28: 29–35.