Regulatory T cells constrain the TCR repertoire of antigen‐stimulated conventional CD4 T cells

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non‐self‐antigen, a TCRβ transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T‐cell repertoire was used. The response of EF4.1 mice to an I‐Ab‐associated epitope of the F‐MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg‐depleted EF4.1 mice were immunized, and the extent of the Vα2‐bearing, antigen‐specific TCR repertoire was characterized by high‐throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre‐immune repertoire. Injection of anti‐CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non‐self‐antigens.     

Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.     

TCR‐β repertoire analysis of antigen‐specific single T cells using a high‐density microcavity array

The antigen specificity of cytotoxic T cells, provided by T‐cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single‐cell level is required. In this study, we applied a high‐density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR‐β gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent‐labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T‐cell population. The isolated single cells were then directly utilized for RT‐PCR. By sequencing the amplified PCR products, antigen‐specific TCR‐β repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single‐cell level. This simple, accurate, and cost‐effective technique for single‐cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells. Biotechnol. Bioeng. 2010;106: 311–318. © 2010 Wiley Periodicals, Inc.     

Characterization of age‐associated exhausted CD8+ T cells defined by increased expression of Tim‐3 and PD‐1

Aging is accompanied by altered T‐cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD‐1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T‐cell exhaustion and aging‐associated immune dysfunction. In this study, we demonstrate that T‐cell immunoglobulin mucin domain‐3 (Tim‐3), another exhaustion marker, is up‐regulated on aged T cells, especially CD8⁺ T cells. Tim‐3‐expressing cells also produced PD‐1, but Tim‐3⁺PD‐1⁺ CD8⁺ T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim‐3^?PD‐1⁺ cells. Tim‐3⁺PD‐1⁺ CD8⁺ T cells showed more evident properties associated with exhaustion than Tim‐3^?PD‐1⁺ CD8⁺ T cells: an exhaustion‐related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL‐10 and induced normal CD8⁺ T cells to produce IL‐10, which might contribute to immune dysregulation in aged mice. The generation of Tim‐3‐expressing CD8⁺ T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8⁺ T‐cell population with age‐associated exhaustion was distinguishable by its expression of Tim‐3. These results provide clues for understanding the alterations that occur in T‐cell populations with age and for improving dysfunctions related to the aging of the immune system.     

Hyper‐reactive cloned mice generated by direct nuclear transfer of antigen‐specific CD4+ T cells

T‐cell receptor (TCR)‐transgenic mice have been employed for evaluating antigen‐response mechanisms, but their non‐endogenous TCR might induce immune response differently than the physiologically expressed TCR. Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen‐specific CD4⁺ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre‐rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic‐type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter‐regulated antigen‐specific TCR are a unique animal model with allergic predisposition for investigating CD4⁺ T‐cell‐mediated pathogenesis and cellular commitment in immune diseases.     

A CD8+ T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of peptidomimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of an anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8+ CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization.     

Age-associated parallel increase of Foxp3CD4 regulatory and CD44CD4 memory T cells in SJL/J mice

Effector/memory T cells (Tem) are required to maintain successful immunity, while regulatory T cells (Treg) are required to prevent excessive/uncontrolled inflammation and/or autoimmunity. Although both Tem and Treg cells are increased during aging, the relationship between the increased proportion of Foxp3⁺ Treg cells and CD44⁺ Tem cells with aging is not clearly understood. We found in this report that Foxp3⁺ Treg cells are increased in parallel with CD44⁺ Tem cells in SJL/J mice with aging, and that all Foxp3⁺ Treg cells are of CD44⁺ Tem phenotype, suggesting that the increased Foxp3⁺ Treg cells originated from the expanded pool of CD44⁺ Tem cells with aging. Our in vitro kinetic studies further suggested that Foxp3⁺ Treg cells are converted through the CD44⁺ stage. Furthermore, we observed that although the balance between Foxp3⁺ Treg and CD44⁺Foxp3⁻ Tem cells remained with aging, the aged mice have higher ratios of both Tem and Treg cells vs. naïve T cells resulting in the “shrunken” naïve T cell pools. Our results suggest that an age-associated imbalance of T cell repertoire is a mechanism that contributes to spontaneous occurrence of Hodgkin’s-like lymphoma in aged SJL/J mice.     

Vaccination provides superior in vivo recall capacity of SARS-CoV-2-specific memory CD8 T cells

1. Download : Download high-res image (187KB)
2. Download : Download full-size image

     

Molecular properties of gp100‐reactive T‐cell receptors drive the cytokine profile and antitumor efficacy of transgenic host T cells

To study the contribution of T‐cell receptors (TCR) to resulting T‐cell responses, we studied three different human αβ TCRs, reactive to the same gp100‐derived peptide presented in the context of HLA‐A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen‐induced IFN‐γ responses, which correlated with TCR affinity for peptide–MHC in the order T4H2 > R6C12 > SILv44. However, SILv44 elicited superior IL‐17A release. Importantly, in vivo, SILv44‐transgenic T cells mediated superior antitumor responses to 888‐A2 + human melanoma tumor cells upon adoptive transfer into tumor‐challenged mice while maintaining IL‐17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T‐cell receptor in defining host T‐cell physiology than traditionally assumed, while parameters beyond IFN‐γ secretion and TCR affinity ultimately determine the reactivity of tumor‐reactive T cells.     

T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%–8% of T cell from healthy individuals expressed the V19 TCR. One BMT patient exhibited V19 expression on more than 60% of peripheral T cells, while additional patients expressed V19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8⁺ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V repertoire of CD4⁺ and CD8⁺ T cell populations. We found that biased V expression characterized both CD4⁺ and CD8⁺ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8⁺ T cells alone may not fully correct repertoire abnormalities following BMT.     

Human CD8+ T cells transduced with an additional receptor bispecific for both Mycobacterium tuberculosis and HIV‐1 recognize both epitopes

Tuberculosis (TB) and human immunodeficiency virus type 1 (HIV‐1) infection are closely intertwined, with one‐quarter of TB/HIV coinfected deaths among people died of TB. Effector CD8⁺ T cells play a crucial role in the control of Mycobacterium tuberculosis (MTB) and HIV‐1 infection in coinfected patients. Adoptive transfer of a multitude of effector CD8⁺ T cells is an appealing strategy to impose improved anti‐MTB/HIV‐1 activity onto coinfected individuals. Due to extensive existence of heterologous immunity, that is, T cells cross‐reactive with peptides encoded by related or even very dissimilar pathogens, it is reasonable to find a single T cell receptor (TCR) recognizing both MTB and HIV‐1 antigenic peptides. In this study, a single TCR specific for both MTB Ag85B_199‐207 peptide and HIV‐1 Env_120‐128 peptide was screened out from peripheral blood mononuclear cells of a HLA‐A*0201⁺ healthy individual using complementarity determining region 3 spectratype analysis and transferred to primary CD8⁺ T cells using a recombinant retroviral vector. The bispecificity of the TCR gene‐modified CD8⁺ T cells was demonstrated by elevated secretion of interferon‐γ, tumour necrosis factor‐α, granzyme B and specific cytolytic activity after antigen presentation of either Ag85B_199‐207 or Env_120‐128 by autologous dendritic cells. To the best of our knowledge, this study is the first report proposing to produce responses against two dissimilar antigenic peptides of MTB and HIV‐1 simultaneously by transfecting CD8⁺ T cells with a single TCR. Taken together, T cells transduced with the additional bispecific TCR might be a useful strategy in immunotherapy for MTB/HIV‐1 coinfected individuals.     

Asymmetric cell division safeguards memory CD8 T cell development

1. Download : Download high-res image (134KB)
2. Download : Download full-size image

     

Development of CD8+ T cells expressing two distinct receptors specific for MTB and HIV‐1 peptides

The immune response in individuals co‐infected with Mycobacterium tuberculosis (MTB) and the human immunodeficiency virus (MTB/HIV) gradually deteriorates, particularly in the cellular compartment. Adoptive transfer of functional effector T cells can confer protective immunity to immunodeficient MTB/HIV co‐infected recipients. However, few such effector T cells exist in vivo, and their isolation and amplification to sufficient numbers is difficult. Therefore, enhancing immune responses against both pathogens is critical for treating MTB/HIV co‐infected patients. One approach is adoptive transfer of T cell receptor (TCR) gene‐modified T cells for the treatment of MTB/HIV co‐infections because lymphocyte numbers and their functional avidity is significantly increased by TCR gene transfer. To generate bispecific CD8⁺ T cells, MTB Ag85B_199–207 peptide‐specific TCRs (MTB/TCR) and HIV‐1 Env_120–128 peptide‐specific TCRs (HIV/TCR) were isolated and introduced into CD8⁺ T cells simultaneously using a retroviral vector. To avoid mispairing among exogenous and endogenous TCRs, and to improve the function and stability of the introduced TCRs, several strategies were employed, including introducing mutations in the MTB/TCR constant (C) regions, substituting part of the HIV/TCR C regions with CD3ζ, and linking gene segments with three different 2A peptides. Results presented in this report suggest that the engineered T cells possessed peptide‐specific specificity resulting in cytokine production and cytotoxic activity. This is the first report describing the generation of engineered T cells specific for two different pathogens and provides new insights into TCR gene therapy for the treatment of immunocompromised MTB/HIV co‐infected patients.