Molecular insights for optimizing T cell receptor specificity against cancer.

Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses.

Photoaffinity labeling of the T cell receptor on cloned cytotoxic T lymphocytes by covalent photoreactive ligand.

The interaction of the T cell antigen receptor with a photoreactive antigenic peptide derivative bound covalently to the H-2Kd (Kd) molecule was studied by photoaffinity labeling on cloned, CD8 positive cytotoxic T lymphocytes. The Kd-restricted Plasmodium berghei circumsporozoite peptide 253-260 (YIPS-AEKI) was conjugated with iodo-4-azidosalicylic acid at the N terminus and with 4-azidobenzoic acid at the T cell receptor residue Lys-259. Cell-associated or soluble Kd molecules were photoaffinity-labeled with the peptide derivative by selective photoactivation of the N-terminal photoreactive group. Incubation of cell-associated or soluble covalent Kd-peptide derivative complexes (ligands) with cytotoxic T lymphocytes that recognized this peptide derivative and activation of the orthogonal photoreactive group resulted in specific photoaffinity labeling of the T cell receptor. The labeling was inhibitable by an anti-Kd antibody and was absent on Kd-restricted cytotoxic T lymphocytes of different specificity. The binding of the soluble ligand reached a maximum after 2-4 min at 37 degrees C, after 30 min at 18 degrees C, and after 3 h at 4 degrees C. In contrast, binding of the cell-associated ligand reached a transient maxima after 50 and 110 min at 37 and 18 degrees C, respectively. The degree of binding at 37 degrees C was approximately 30% lower than that at 18 degrees C. No binding took place at 4 degrees C. Inhibition studies with antibodies and drugs indicated that the binding of the cell-associated, but not the soluble ligand, was highly dependent on T cell-target cell conjugate formation, whereas the binding of the soluble ligand was greatly dependent on CD8.

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection.

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.

Combining MHC tetramer and intracellular cytokine staining for CD8(+) T cells to reveal antigenic epitopes naturally presented on tumor cells.

As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNgamma producing cells with unknown specificities and should be widely applicable.

Homeostasis limits the development of mature CD8+ but not CD4+ thymocytes.

The involvement of a variety of clonal selection processes during the development of T lymphocytes in the thymus has been well established. Less information, however, is available on how homeostatic mechanisms may regulate the generation and maturation of thymocytes. To investigate this question, mixed radiation bone marrow chimeras were established in which wild-type T cell precursors capable of full maturation were diluted with precursors deficient in maturation potential because of targeted mutations of the RAG1 or TCR-alpha genes. In chimeras in which the majority of thymocytes are blocked at the CD4- CD8- CD25+ stage (RAG1 deficient), and only a small proportion of T cell precursors are of wild-type origin, we observed no difference in the maturation of wild-type CD4- CD8- CD25+ cells to the CD4+ CD8+ stage as compared with control chimeras. Therefore, the number of cell divisions occurring during this transition is fixed and not subject to homeostatic regulation. In contrast, in mixed chimeras in which the majority of thymocytes are blocked at the CD4+ CD8+ stage (TCR-alpha deficient), an increased efficiency of development of wild-type mature CD8+ cells was observed. Surprisingly, the rate of generation of mature CD4+ thymocytes was not affected in these chimeras. Thus, the number of selectable CD8 lineage thymocytes apparently saturates the selection mechanism in normal mice while the development of CD4 lineage cells seems to be limited only by the expression of a suitable TCR. These data may open the way to the identification of homeostatic mechanisms regulating thymic output and CD4/CD8 lineage commitment, and the development of means to modulate it.

Thymic lineage commitment rather than selection causes genetic variations in size of CD4 and CD8 compartments.

During their development, immature CD4+ CD8+ thymocytes become committed to either the CD4 or CD8 lineage. Subsequent complete maturation of CD4+ and CD8+ cells requires a molecular match of the expressed coreceptor and the MHC specificity of the TCR. The final size of the mature CD4+ and CD8+ thymic compartments is therefore determined by a combination of lineage commitment and TCR-mediated selection. In humans and mice, the relative size of CD4+ and CD8+ peripheral T cell compartments shows marked genetic variability. We show here that genetic variations in thymic lineage commitment, rather than TCR-mediated selection processes, are responsible for the distinct CD4/CD8 ratios observed in common inbred mouse strains. Genetic variations in the regulation of lineage commitment open new ways to analyze this process and to identify the molecules involved.

Early neutralizing antibody response against mouse mammary tumor virus: critical role of viral infection and superantigen-reactive T cells.

Infectious mouse mammary tumor virus (MMTV) is a retrovirus that expresses a superantigen shortly after infection of B cells. The superantigen first drives the polyclonal activation and proliferation of superantigen-reactive CD4+ T cells, which then induce the infected B cells to proliferate and differentiate. Part of the MMTV-induced B cell response leads to the production of Abs that are specific for the viral envelope protein gp52. Here we show that this Ab response has virus-neutralizing activity and confers protection against superinfection by other MMTV strains in vivo as soon as 4 to 7 days after infection. A protective Ab titer is maintained lifelong. Viral infection as well as the superantigen-induced T-B collaboration are required to generate this rapid and long lasting neutralizing Ab response. Polyclonal or superantigen-independent B cell activation, on the contrary, does not lead to detectable virus neutralization. The early onset of this superantigen-dependent neutralizing response suggests that viral envelope-specific B cells are selectively recruited to form part of the extrafollicular B cell response and are subsequently amplified and maintained by superantigen-reactive Th cells.

Toward improved immunocompetence of adoptively transferred CD8+ T cells.

Adoptive transfer of autologous or allogenic T cells to patients is being used with increased frequency as a therapy for infectious diseases and cancer. However, many questions remain with regard to defining optimized procedures for preparation and selection of T cell populations for transfer. In a new study in this issue of the JCI, Gattinoni and colleagues used a TCR transgenic mouse model to examine in vitro-generated tumor antigen-specific CD8+ T cells at various stages of differentiation for their efficacy in adoptive immunotherapy against transplantable melanoma. The results confirm that CD8+ T cells progressively lose immunocompetence with prolonged in vitro cultivation and suggest that effector CD8+ T cells alone may be considerably less potent at protecting hosts with advanced tumors than are less differentiated T cells.

Distinct mechanisms control human naive and antigen-experienced CD8+ T lymphocyte proliferation.

Human Ag-specific CD8(+) T lymphocytes are heterogeneous and include functionally distinct populations. In this study, we report that at least two distinct mechanisms control the expansion of circulating naive, memory, and effector CD8(+) T lymphocytes when exposed to mitogen or Ag stimulation. The first one leads to apoptosis and occurs shortly after in vitro stimulation. Susceptibility to cell death is prominent among primed T cell subsets, and it is inversely correlated with the size of the ex vivo Bcl-2(high) population within these subsets. Importantly, the Bcl-2(high) phenotype is associated to the proportion of responsive CD8(+) T cells, independently of their differentiation stage. The second one depends on the expression of newly synthesized cyclin-dependent kinase inhibitor p16(INK4a) that occurs in a significant fraction of T cells that had been actively cycling, leading to their cell cycle arrest upon stimulation. Strikingly, accumulation of p16(INK4a) protein preferentially occurs in naive as opposed to primed derived T lymphocytes and is not related to apoptosis. Significant levels of p16 are readily detectable in a small number of ex vivo CD8(+) T cells. Our observations reveal that activation-induced p16 expression represents an alternative process to apoptosis, limiting the proliferation potential of activated naive derived T lymphocytes.

Neutrophils contribute to development of a protective immune response during onset of infection with Leishmania donovani.

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4(+) and CD8(+) T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells.

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

IL-10 producing CD8+ T cells in human infection with Leishmania guyanensis.

Cytokines are increasingly recognized as important components of the cellular immune responses to intracellular pathogens. In this study, we analyzed the production of TGF-beta, IL-10 and IFN-gamma by PBMC of unexposed naïve subjects and LCL patients after stimulation with live Leishmania guyanensis (L.g.). We demonstrated that IFN-gamma is produced in controls and LCL patients, IL-10 only in LCL patients and TGF-beta only in naïve subjects. Furthermore, in naive subjects, neutralization of TGF-beta induced IL-10 production. IL-10 produced in naïve subjects when TGF-beta is neutralized or in LCL patients did not modify the IFN-gamma production but inhibit reactive nitrogen species production. Analysis of the phenotype of IL-10 producing cells in naive subjects when TGF-beta is neutralized clearly showed that they are memory CD45RA- CD8+ T cells. In LCL patients, IL-10 producing cells are both CD45RA- CD4 and CD8+ T cells. The role of these IL-10 producing CD8+ T cells in the development of the diseases should be carefully evaluated.

Activation of human melanoma reactive CD8+ T cells by vaccination with an immunogenic peptide analog derived from Melan-A/melanoma antigen recognized by T cells-1.

PURPOSE: As compared with natural tumor peptide sequences, carefully selected analog peptides may be more immunogenic and thus better suited for vaccination. However, T cells in vivo activated by such altered analog peptides may not necessarily be tumor specific because sequence and structure of peptide analogs differ from corresponding natural peptides. EXPERIMENTAL DESIGN: Three melanoma patients were immunized with a Melan-A peptide analog that binds more strongly to HLA-A*0201 and is more immunogenic than the natural sequence. This peptide was injected together with a saponin-based adjuvant, followed by surgical removal of lymph node(s) draining the site of vaccination. RESULTS: Ex vivo analysis of vaccine site draining lymph nodes revealed antigen-specific CD8+ T cells, which had differentiated to memory cells. In vitro, these cells showed accelerated proliferation upon peptide stimulation. Nearly all (16 of 17) of Melan-A-specific CD8+ T-cell clones generated from these lymph nodes efficiently killed melanoma cells. CONCLUSIONS: Patient immunization with the analog peptide leads to in vivo activation of T cells that were specific for the natural tumor antigen, demonstrating the usefulness of the analog peptide for melanoma immunotherapy.

Inactivation of Notch 1 in immature thymocytes does not perturb CD4 or CD8T cell development.

Notch proteins influence cell-fate decisions in many developing systems. Several gain-of-function studies have suggested a critical role for Notch 1 signaling in CD4-CD8 lineage commitment, maturation and survival in the thymus. However, we show here that tissue-specific inactivation of the gene encoding Notch 1 in immature (CD25+CD44-)T cell precursors does not affect subsequent thymocyte development. Neither steady-state numbers nor the rate of production of CD4+ and CD8+ mature thymocytes is perturbed in the absence of Notch 1. In addition, Notch 1-deficient thymocytes are normally sensitive to spontaneous or glucocorticoid-induced apoptosis. In contrast to earlier reports, these data formally exclude an essential role for Notch 1 in CD4-CD8 lineage commitment, maturation or survival.