Martin Prlic

Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response

CD8+ T cells only require a brief stimulation with antigen in vitro to divide and differentiate into effector and memory cells upon transfer in vivo. The efficiency of clonal expansion and the functional characteristics of memory cells derived from briefly stimulated cells are poorly defined. We developed a system that allowed us to examine programming entirely in vivo. This was achieved by rapidly killing peptide-pulsed DCs carrying a diphtheria toxin receptor transgene with timed injections of diphtheria toxin without altering the course of an accompanying infection. The magnitude of clonal expansion, but not the functionality of the effector cells, correlated directly with the duration of antigen exposure. Furthermore, memory T cells were capable of mounting a secondary response, regardless of the length of antigen encounter during the primary response. These results indicate that the duration of initial antigen encounter influences the magnitude of the primary response, but does not program responsiveness during the secondary challenge.

Multiple Choices: Regulation of Memory CD8 T Cell Generation and Homeostasis by Interleukin (IL)-7 and IL-15

The generation of memory T cells in immune responses has been extensively studied over recent years, yet the requirements for production and persistence of a functional memory pool are still unclear. For example, while there is compelling evidence that survival of memory cells does not require TCR–MHC engagements (1, 2), recent evidence indicates that such interactions may be needed to maintain functional activity of these cells (3, 4). Furthermore, there is growing evidence that cytokines play a major role in deciding the fate of CD8 memory cells, although the precise mechanisms by which these effects are mediated remain unknown.

MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data

Single-cell transcriptomics reveals gene expression heterogeneity but suffers from stochastic dropout and characteristic bimodal expression distributions in which expression is either strongly non-zero or non-detectable. We propose a two-part, generalized linear model for such bimodal data that parameterizes both of these features. We argue that the cellular detection rate, the fraction of genes expressed in a cell, should be adjusted for as a source of nuisance variation. Our model provides gene set enrichment analysis tailored to single-cell data. It provides insights into how networks of co-expressed genes evolve across an experimental treatment. MAST is available at https://github.com/RGLab/MAST.

Exploring regulatory mechanisms of CD8+ T cell contraction

A small fraction of the CD8 T cell effector population that responds to an infection progresses through the contraction phase to the memory stage. The factors regulating the extent of contraction are poorly understood. Competition for limited resources has been widely postulated to be the cause of cell death during the contraction phase, but our data show that competition does not affect contraction kinetics. We go on to demonstrate that all effector cells present at the peak of the response have the potential to become bona fide memory cells, thus excluding selection on the basis of functionality. We propose that the fate of a CD8 effector cell is predetermined before the onset of contraction and discuss possible mechanisms of regulation.

Positive selection of mC46-expressing CD4 + T cells and maintenance of virus specific immunity in a primate AIDS model

Despite continued progress in the development of novel antiretroviral therapies, it has become increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV(+) patients. In 2009, an HIV(+) patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5(-/-) donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4(+) T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4(+) T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4(+) T cells, an increase in nonmodified CD4(+) T cells, enhanced cytotoxic T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS.

PKCθ is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice

When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform theta (PKCtheta), a key regulator of TCR signaling. In contrast, PKCtheta was required for alloreactivity and GVHD induction. Furthermore, absence of PKCtheta raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCtheta-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCtheta is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.

Bystander-Activated Memory CD8 T Cells Control Early Pathogen Load in an Innate-like, NKG2D-Dependent Manner

During an infection the antigen-nonspecific memory CD8 T cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T cell receptor (TCR) signals that bystander-activated, cytotoxic CD8 T cells (BA-CTLs) receive in vivo and found evidence of limited TCR signaling. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL-mediated killing led to a significant defect in pathogen clearance. Together, these data suggest an innate role for memory CD8 T cells in the early immune response before the onset of a de novo generated, antigen-specific CD8 T cell response.

Immunology: A metabolic switch to memory

Two therapeutic drugs have been found to enhance memory in immune cells called T cells, apparently by altering cellular metabolism. Are changes in T-cell metabolism the key to generating long-lived immune memory?

Cutting Edge: β-Catenin Is Dispensable for T Cell Effector Differentiation, Memory Formation, and Recall Responses

The molecular mechanisms that regulate mature T cell fate and enable cells to differentiate into memory T cells are largely unknown. Memory T cells share certain key features with stem cells: they both have the ability to self-renew and are long-lived. The Wnt–β-catenin signaling pathway is a key player in regulating stem cell self-renewal and differentiation. We generated a conditional knockout mouse that specifically lacks β-catenin in mature T cells and report in this article that β-catenin is not involved in regulating effector versus memory T cell differentiation. β-catenin–deficient memory T cells were phenotypically and functionally indistinguishable from control cells and made normal recall responses. β-catenin deficiency does not affect T cell migration, T cell function in a model of chronic infection, or lymphopenia-induced proliferation. Together, our data suggest that self-renewal and differentiation are regulated differently in memory T cells compared with epithelial and hematopoietic stem cells.

Dissociating Markers of Senescence and Protective Ability in Memory T Cells

No unique transcription factor or biomarker has been identified to reliably distinguish effector from memory T cells. Instead a set of surface markers including IL-7Rα and KLRG1 is commonly used to predict the potential of CD8 effector T cells to differentiate into memory cells. Similarly, these surface markers together with the tumor necrosis factor family member CD27 are frequently used to predict a memory T cells ability to mount a recall response. Expression of these markers changes every time a memory cell is stimulated and repeated stimulation can lead to T cell senescence and loss of memory T cell responsiveness. This is a concern for prime–boost vaccine strategies which repeatedly stimulate T cells with the aim of increasing memory T cell frequency. The molecular cues that cause senescence are still unknown, but cell division history is likely to play a major role. We sought to dissect the roles of inflammation and cell division history in developing T cell senescence and their impact on the expression pattern of commonly used markers of senescence. We developed a system that allows priming of CD8 T cells with minimal inflammation and without acquisition of maximal effector function, such as granzyme expression, but a cell division history similar to priming with systemic inflammation. Memory cells derived from minimal effector T cells are fully functional upon rechallenge, have full access to non-lymphoid tissue and appear to be less senescent by phenotype upon rechallenge. However, we report here that these currently used biomarkers to measure senescence do not predict proliferative potential or protective ability, but merely reflect initial priming conditions.