Fabrice Lemaître

Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice.

CTLs have the potential to attack tumors, and adoptive transfer of CTLs can lead to tumor regression in mouse models and human clinical settings. However, the dynamics of tumor cell elimination during efficient T cell therapy is unknown, and it is unclear whether CTLs act directly by destroying tumor cells or indirectly by initiating the recruitment of innate immune cells that mediate tumor damage. To address these questions, we report real-time imaging of tumor cell apoptosis in vivo using intravital 2-photon microscopy and a Förster resonance energy transfer-based (FRET-based) reporter of caspase 3 activity. In a mouse model of solid tumor, we found that tumor regression after transfer of in vitro-activated CTLs occurred primarily through the direct action of CTLs on each individual tumor cell, with a minimal bystander effect. Surprisingly, the killing of 1 target cell by an individual CTL took an extended period of time, 6 hours on average, which suggested that the slow rate of killing intrinsically limits the efficiency of antitumor T cell responses. The ability to visualize when, where, and how tumor cells are killed in vivo offers new perspectives for understanding how immune effectors survey cancer cells and how local tumor microenvironments may subvert immune responses.

CD8 Expression Allows T Cell Signaling by Monomeric Peptide-MHC Complexes

Physiologically, TCR signaling is unlikely to result from the cross-linking of TCR-CD3 complexes, given the low density of specific peptide-MHC complexes on antigen-presenting cells. We therefore have tested directly an alternative model for antigen recognition. We show that monomers of soluble peptide-MHC trigger Ca2+ responses in CD8alphabeta+ T cells. This response is not observed in CD8- T cells and when either the CD8:MHC or CD8:Lck interactions are prevented. This demonstrates that an intact CD8 coreceptor is necessary for effective TCR signaling in response to monomeric peptide-MHC molecules. We propose that this heterodimerization of TCR and CD8 by peptide-MHC corresponds to the physiological event normally involved during antigen-specific signal transduction.

Enumeration of human antigen-specific naive CD8 T cells reveals conserved precursor frequencies

The number of antigen-specific naive CD8(+) T cells is believed to be important in the shaping of adaptive immune responses, and is predictive for the magnitude of priming responses in mouse models. Because of extremely low precursor frequencies, knowledge about these cells comes from indirect techniques and estimations. Here, we present a strategy based on the combination of tetramer staining, magnetic-bead enrichment, and multiparametric cytometry, which permitted direct detection and analysis of CD8(+) T cells reactive for 6 different naive epitopes (MART-1(26-35), HIV-1 Gag p17(77-85), hepatitis C virus [HCV] NS3(1406-1415), HCV Core(132-140), NY-ESO-1(157-165), and cytomegalovirus [CMV] pp65(495-503)). Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6 x 10(-6) to 1.3 x 10(-4)), but conserved frequencies among humans. Development of a procedure for direct assessment of T-cell precursor frequency in humans has important implications, with particular relevance to vaccine development and monitoring of tumor and self-reactive T cells.

Preexisting BCG-Specific T Cells Improve Intravesical Immunotherapy for Bladder Cancer

Preexisting immunity to BCG improves treatment response to intravesical BCG therapy for bladder cancer in mouse models and humans. Bugs as Drugs Although still in its infancy for the treatment of many tumor types, immunotherapy has been and remains the standard of care for patients with bladder cancer. The concept behind this success is repeated instillations of bacillus Calmette-Guérin (BCG; the same bacteria used as a vaccine for Mycobacterium tuberculosis) in the bladder, inducing an immune response that fights the cancer. The clinical response rate in bladder cancer patients is 50 to 70%, higher than any other immunotherapy but still leaving room for improvement. Biot et al. provide new insight into the mechanisms behind BCG’s success in the bladder and suggest that parenteral BCG exposure may boost the success rate for bladder cancer treatment. Using a mouse model of bladder cancer, the authors show that a single instillation of BCG induced an immune response, but repetitive instillations were required for robust T cell trafficking to the bladder. Previous parenteral exposure to BCG overcame this requirement in the mice, inducing an enhanced innate immune response, accelerating T cell trafficking to the bladder, and improving the immune response to the tumor. Furthermore, bladder cancer patients who had preexisting immunity to BCG (secondary to their childhood M. tuberculosis vaccination) showed improved recurrence-free survival after standard BCG therapy relative to those patients without preexisting immunity. Thus, immunizing BCG-naïve cancer patients before therapy may provide a simple strategy that could improve therapeutic response. Therapeutic intravesical instillation of bacillus Calmette-Guérin (BCG) is effective at triggering inflammation and eliciting successful tumor immunity in patients with non–muscle invasive bladder cancer, with 50 to 70% clinical response. Therapeutic success relies on repeated instillations of live BCG administered as adjuvant therapy shortly after tumor resection; however, the precise mechanisms remain unclear. Using an experimental model, we demonstrate that after a single instillation, BCG could disseminate to bladder draining lymph nodes and prime interferon-γ–producing T cells. Nonetheless, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. Parenteral exposure to BCG before instillation overcame this requirement; after the first intravesical instillation, BCG triggered a more robust acute inflammatory process and accelerated T cell entry into the bladder, as compared to the standard protocol. Moreover, parenteral exposure to BCG before intravesical treatment of an orthotopic tumor markedly improved response to therapy. Indeed, patients with sustained preexisting immunity to BCG showed a significant improvement in recurrence-free survival. Together, these data suggest that monitoring patients’ response to purified protein derivative, and, in their absence, boosting BCG responses by parenteral exposure before intravesical treatment initiation, may be a safe and effective means of improving intravesical BCG-induced clinical responses.

Intravital Imaging Reveals Distinct Dynamics for Natural Killer and CD8+ T Cells during Tumor Regression

Recognition of NKG2D ligands by natural killer (NK) cells plays an important role during antitumoral responses. To address how NKG2D engagement affects intratumoral NK cell dynamics, we performed intravital microscopy in a Rae-1β-expressing solid tumor. This NKG2D ligand drove NK cell accumulation, activation, and motility within the tumor. NK cells established mainly dynamic contacts with their targets during tumor regression. In sharp contrast, cytotoxic T lymphocytes (CTLs) formed stable contacts in tumors expressing their cognate antigen. Similar behaviors were observed during effector functions in lymph nodes. In vitro, contacts between NK cells and their targets were cytotoxic but did not elicit sustained calcium influx nor adhesion, whereas CTL contact stability was critically dependent on extracellular calcium entry. Altogether, our results offer mechanistic insight into how NK cells and CTLs can exert cytotoxic activity with remarkably different contact dynamics.

Regulatory T Cells Increase the Avidity of Primary CD8+ T Cell Responses and Promote Memory

Treg-ulating Immune Responses There are many checks and balances to keep the immune system from running amok. One of the most critical is a specialized population of T cells, called regulatory T cells (Tregs). In their absence, a lethal autoimmune disease develops in both humans and mice. Although Tregs are well known for their suppression of autoimmune responses, how they modulate responses to infectious agents is less well understood. Using inducible deletion of Tregs in mice, Pace et al. (p. 532) showed that Tregs are important for shaping the avidity of CD8+ T cell responses. In the absence of Tregs, CD8+ T cell responses were of lower avidity, and the CD8+ T cells were more responsive to lower-affinity antigens. When Tregs were absent, stable interactions between T cell and antigen-presenting cells were increased as a result of higher amounts of chemokine expression in the lymph nodes. Treg depletion also resulted in a lower-avidity CD8+ T cell response to infection with the bacterial pathogen Listeria monocytogenes. Regulatory T cells enhance the avidity of T cells, thereby promoting responses to foreign antigens instead of self-antigens. Although regulatory T cells (Tregs) are known to suppress self-reactive autoimmune responses, their role during T cell responses to nonself antigens is not well understood. We show that Tregs play a critical role during the priming of immune responses in mice. Treg depletion induced the activation and expansion of a population of low-avidity CD8+ T cells because of overproduction of CCL-3/4/5 chemokines, which stabilized the interactions between antigen-presenting dendritic cells and low-avidity T cells. In the absence of Tregs, the avidity of the primary immune response was impaired, which resulted in reduced memory to Listeria monocytogenes. These results suggest that Tregs are important regulators of the homeostasis of CD8+ T cell priming and play a critical role in the induction of high-avidity primary responses and effective memory.

HIV Controller CD4+ T Cells Respond to Minimal Amounts of Gag Antigen Due to High TCR Avidity

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment. Emerging evidence indicates that HIV control is mediated through very active cellular immune responses, though how such responses can persist over time without immune exhaustion is not yet understood. To investigate the nature of memory CD4+ T cells responsible for long-term anti-HIV responses, we characterized the growth kinetics, Vβ repertoire, and avidity for antigen of patient-derived primary CD4+ T cell lines. Specific cell lines were obtained at a high rate for both HIV controllers (16/17) and efficiently treated patients (19/20) in response to the immunodominant Gag293 peptide. However, lines from controllers showed faster growth kinetics than those of treated patients. After normalizing for growth rates, IFN-γ responses directed against the immunodominant Gag293 peptide showed higher functional avidity in HIV controllers, indicating differentiation into highly efficient effector cells. In contrast, responses to Gag161, Gag263, or CMV peptides did not differ between groups. Gag293-specific CD4+ T cells were characterized by a diverse Vβ repertoire, suggesting that multiple clones contributed to the high avidity CD4+ T cell population in controllers. The high functional avidity of the Gag293-specific response could be explained by a high avidity interaction between the TCR and the peptide-MHC complex, as demonstrated by MHC class II tetramer binding. Thus, HIV controllers harbor a pool of memory CD4+ T cells with the intrinsic ability to recognize minimal amounts of Gag antigen, which may explain how they maintain an active antiviral response in the face of very low viremia.

Subcellular dynamics of T cell immunological synapses and kinapses in lymph nodes

In vitro studies have revealed that T cell activation occurs during the formation of either dynamic or stable interactions with antigen-presenting cells (APC), and the respective cell junctions have been referred to as immunological kinapses and synapses. However, the relevance and molecular dynamics of kinapses and synapses remain to be established in vivo. Using two-photon imaging, we tracked the distribution of LAT-EGFP molecules during antigen recognition by activated CD4+ T cells in lymph nodes. At steady state, LAT-EGFP molecules were preferentially found at the uropod of rapidly migrating T cells. In contrast to naïve T cells that fully stopped upon systemic antigen delivery, recently activated T cells decelerated and formed kinapses, characterized by continuous extension of membrane protrusions and by the absence of persistent LAT-EGFP clustering. On the other hand, activated CD4+ T cells formed stable immunological synapses with antigen-loaded B cells and displayed sustained accumulation of LAT-EGFP fluorescence at the contact zone. Our results show that the state of T cell activation and the type of APC largely influence T cell–APC contact dynamics in lymph nodes. Furthermore, we provide a dynamic look at immunological kinapses and synapses in lymph nodes and suggest the existence of distinct patterns of LAT redistribution during antigen recognition.

T cell adhesion lowers the threshold for antigen detection

Antigen detection and initiation of TCR signaling only occur, under physiological conditions, when T cells are adherent, and not in suspension. We show here that T cell adhesion causes an increase in the Ca2+ content of intracellular stores and of the amount of phosphatidylinositol 4,5‐bisphosphate in the plasma membrane, and enhances TCR‐induced Ca2+ signaling. This priming can be observed in freshly isolated T cells, in activated T cells, and in some T cell lines. Stimulation of T cells by specific monomeric MHC‐peptide complexes only triggers Ca2+ responses after T cell adhesion. This solves a controversial issue concerning the minimum valency of activatory TCR ligands. Adhesion‐induced T cell priming not only occurs upon binding to artificial substrates such as immobilized ligands, but also upon interaction with dendritic cells. Thus, this phenomenon is likely to contribute to the high sensitivity of antigen detection by T cells in secondary lymphoid organs.

HIV Controllers Maintain a Population of Highly Efficient Th1 Effector Cells in Contrast to Patients Treated in the Long Term

ABSTRACT HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4+ T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin+/CD107a+ expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4+ T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4+ T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.