Inhibitory Receptors Beyond T Cell Exhaustion.

Inhibitory receptors (iRs) are frequently associated with "T cell exhaustion". However, the expression of iRs is also dependent on T cell differentiation and activation. Therapeutic blockade of various iRs, also referred to as "checkpoint blockade", is showing -unprecedented results in the treatment of cancer patients. Consequently, the clinical potential in this field is broad, calling for increased research efforts and rapid refinements in the understanding of iR function. In this review, we provide an overview on the significance of iR expression for the interpretation of T cell functionality. We summarize how iRs have been strongly associated with "T cell exhaustion" and illustrate the parallel evidence on the importance of T cell differentiation and activation for the expression of iRs. The differentiation subsets of CD8 T cells (naïve, effector, and memory cells) show broad and inherent differences in iR expression, while activation leads to strong upregulation of iRs. Therefore, changes in iR expression during an immune response are often concomitant with T cell differentiation and activation. Sustained expression of iRs in chronic infection and in the tumor microenvironment likely reflects a specialized T cell differentiation. In these situations of prolonged antigen exposure and chronic inflammation, T cells are "downtuned" in order to limit tissue damage. Furthermore, we review the novel "checkpoint blockade" treatments and the potential of iRs as biomarkers. Finally, we provide recommendations for the immune monitoring of patients to interpret iR expression data combined with parameters of activation and differentiation of T cells

Large T Antigen-Specific Cytotoxic T Cells Protect Against Dendritic Cell Tumors through Perforin-Mediated Mechanisms Independent of CD4 T Cell Help.

Our newly generated murine tumor dendritic cell (MuTuDC) lines, generated from tumors developing in transgenic mice expressing the simian virus 40 large T antigen (SV40LgT) and GFP under the DC specific promoter CD11c, reproduce the phenotypic and functional properties of splenic wild type CD8α(+) conventional DCs. They have an immature phenotype with low co-stimulation molecule expression (CD40, CD70, CD80, and CD86) that is upregulated after activation with toll-like receptor ligands. We observed that after transfer into syngeneic C57BL/6 mice, MuTuDC lines were quickly rejected. Tumors grew efficiently in large T transgene-tolerant mice. To investigate the immune response toward the large T antigen that leads to rejection of the MuTuDC lines, they were genetically engineered by lentiviral transduction to express luciferase and tested for the induction of DC tumors after adoptive transfer in various gene deficient recipient mice. Here, we document that the MuTuDC line was rejected in C57BL/6 mice by a CD4 T cell help-independent, perforin-mediated CD8 T cell response to the SV40LgT without pre-activation or co-injection of adjuvants. Using depleting anti-CD8β antibodies, we were able to induce efficient tumor growth in C57BL/6 mice. These results are important for researchers who want to use the MuTuDC lines for in vivo studies

Long-lasting stem cell-like memory CD8+ T cells with a naïve-like profile upon yellow fever vaccination.

Efficient and persisting immune memory is essential for long-term protection from infectious and malignant diseases. The yellow fever (YF) vaccine is a live attenuated virus that mediates lifelong protection, with recent studies showing that the CD8(+) T cell response is particularly robust. Yet, limited data exist regarding the long-term CD8(+) T cell response, with no studies beyond 5 years after vaccination. We investigated 41 vaccinees, spanning 0.27 to 35 years after vaccination. YF-specific CD8(+) T cells were readily detected in almost all donors (38 of 41), with frequencies decreasing with time. As previously described, effector cells dominated the response early after vaccination. We detected a population of naïve-like YF-specific CD8(+) T cells that was stably maintained for more than 25 years and was capable of self-renewal ex vivo. In-depth analyses of markers and genome-wide mRNA profiling showed that naïve-like YF-specific CD8(+) T cells in vaccinees (i) were distinct from genuine naïve cells in unvaccinated donors, (ii) resembled the recently described stem cell-like memory subset (Tscm), and (iii) among all differentiated subsets, had profiles closest to naïve cells. Our findings reveal that CD8(+) Tscm are efficiently induced by a vaccine in humans, persist for decades, and preserve a naïveness-like profile. These data support YF vaccination as an optimal mechanistic model for the study of long-lasting memory CD8(+) T cells in humans

T cell receptor alpha variable 12-2 bias in the immunodominant response to Yellow fever virus.

The repertoire of human αβ T-cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8 <sup>+</sup> T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8 <sup>+</sup> T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8 <sup>+</sup> T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for the A2/LLW epitope

Early drop of circulating T cells negatively correlates with the protective immune response to Yellow Fever vaccination.

Lymphocyte recirculation within the human body is essential for efficient pathogen detection and immune responses. So far, immune cell migration has been investigated largely using ovine and murine models, with little evidence in humans. Here, we analyzed peripheral blood of healthy individuals following primary vaccination with the Yellow Fever vaccine YF-17D. We found that the number of leukocytes was transiently and sharply reduced in blood as detected on day 7 after vaccine administration. The T cell drop was restricted to cells expressing the lymph node-homing chemokine receptor CCR7. Interestingly, the vaccine-induced drop positively correlated with the expression of CD69 by the T cells before vaccination. This suggests that CCR7 <sup>+</sup> T cells are being trapped within the lymph nodes through CD69-induced suppression of egress. Strikingly, we further found that the T cell drop negatively correlated with CD8 T cell activation and with production of neutralizing antibodies. In conclusion, early and transient T cell depletion in blood negatively correlated with protective immune response events induced by YF-17D vaccination. Our data highlight baseline CD69 expression and early drop in T cells as potential biomarkers of the Yellow Fever vaccine response

Yellow fever virus vaccination: an emblematic model to elucidate robust human immune responses.

By preventing infectious diseases, vaccines contribute substantially to public health. Besides, they offer great opportunities to investigate human immune responses. This is particularly true for live-attenuated virus vaccines which cause resolving acute infections and induce robust immunity. The fact that one can precisely schedule the time-point of vaccination enables complete characterization of the immune response over time, short-term and over many years. The live-attenuated Yellow Fever virus vaccine strain YF-17D was developed in the 1930's and gave rise to the 17D-204 and 17DD vaccine sub-strains, administered to over 600 million individuals worldwide. YF vaccination causes a systemic viral infection, which induces neutralizing antibodies that last for a lifetime. It also induces a strong T cell response resembling the ones of acute infections, in contrast to most other vaccines. In spite of its use since 1937, learning how YF vaccination stimulates such strong and persistent immune responses has gained substantial knowledge only in the last decades. Here we summarize the current state of knowledge on the immune response to YF vaccination, and discuss its contribution as a human model to address complex questions on optimal immune responses

A stepwise protocol to coat aAPC beads prevents out-competition of anti-CD3 mAb and consequent experimental artefacts.

Artificial antigen-presenting cells (aAPC) are widely used for both clinical and basic research applications, as cell-based or bead-based scaffolds, combining immune synapse components of interest. Adequate and controlled preparation of aAPCs is crucial for subsequent immunoassays. We reveal that certain proteins such as activatory anti-CD3 antibody can be out-competed by other proteins (e.g. inhibitory receptor ligands such as PDL1:Fc) during the coating of aAPC beads, under the usually performed coating procedures. This may be misleading, as we found that decreased CD8 T cell activity was not due to inhibitory receptor triggering but rather because of unexpectedly low anti-CD3 antibody density on the beads upon co-incubation with inhibitory receptor ligands. We propose an optimized protocol, and emphasize the need to quality-control the coating of proteins on aAPC beads prior to their use in immunoassays

The three main stumbling blocks for anticancer T cells.

Memory and effector T cells have the potential to counteract cancer progression, but often fail to control the disease, essentially because of three main stumbling blocks. First, clonal deletion leads to relatively low numbers or low-to-intermediate T cell receptor (TCR) affinity of self/tumor-specific T cells. Second, the poor innate immune stimulation by solid tumors is responsible for inefficient priming and boosting. Third, T cells are suppressed in the tumor microenvironment by inhibitory signals from other immune cells, stroma and tumor cells, which induces T cell exhaustion, as demonstrated in metastases of melanoma patients. State-of-the-art adoptive cell transfer and active immunotherapy can partially overcome the three stumbling blocks. The reversibility of T cell exhaustion and novel molecular insights provide the basis for further improvements of clinical immunotherapy

LAG-3 and PD-1+LAG-3 inhibition promote anti-tumor immune responses in human autologous melanoma/T cell co-cultures.

Despite the success of immunotherapy using checkpoint blockade, many patients with solid tumors remain refractory to these treatments. In human cancer, the experimental options to investigate the specific effects of antibodies blocking inhibitory receptors are limited and it is still unclear which cell types are involved. We addressed the question whether the direct interaction between T cells and tumor cells can be enforced through blocking a set of inhibitory receptors including PD-1, TIM-3, BTLA and LAG-3, blocked either individually or in dual combinations with the anti-PD-1 antibody, and to determine the condition that induces maximal T cell function preventing tumor cell proliferation. Using short-term Melan-A-specific or autologous re-stimulations, checkpoint blockade did not consistently increase cytokine production by tumor-derived expanded T cells. We next set up a 5-day co-culture assay with autologous melanoma cell lines and expanded tumor infiltrating T cells, originating from tumor specimens obtained from 6 different patients. Amongst all combos tested, we observed that blockade of LAG-3 alone, and more strongly when combined with PD-1 blockade, enforced T cell responses and tumor cell growth control. The combination of anti-LAG-3 plus anti-PD-1 acted through CD8 T cells and led to increased IFNγ production and cytotoxic capacity. Our results show that LAG-3 and PD-1 are regulating the direct interaction between tumor cells and autologous T cells, suggesting that therapy effects may be promoted by enhanced access of the corresponding blocking reagents to the tumor microenvironment

Shared acute phase traits in effector and memory human CD8 T cells.

CD8 T cells have multiple functional properties that mediate acute phase and long-term immune protection. Several effector and memory CD8 T cell subsets have been described with diverse functionalities and marker profiles. In contrast to the many comprehensive mouse studies, most human studies lack samples from the acute infection phase, a major reason why current knowledge of human T cell subsets and differentiation remains incomplete, particularly with regard to the T cell heterogeneity early during the immune response. Here we analysed the human CD8 T cell response to yellow fever vaccination as the best-known model to study the human immune response to acute viral infection. We performed flow cytometry on 21 markers conventionally used in mice and in humans to describe differentiation, activation, cycling, and so-called effector functions. We found clearly distinct 'acute traits' at the peak of the response that are shared amongst all non-naïve antigen-specific subsets, including memory-differentiated cells. These acute traits were low BCL-2 and high KI67, CD38, HLA-DR, as well as increased Granzyme B and Perforin, previously attributed only to effector cells at the peak of the response. Furthermore, analysis of chromatin accessibility at the single cell level revealed that memory- and effector-differentiated cells clustered together specifically in the acute phase. Altogether, we demonstrate 'acute traits' across differentiation subsets, and point out the need to discriminate the differentiation states when studying human CD8 T cells that undergo an acute response