Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing

Snyder, Jennifer E.; Bowers, William J.; Livingstone, Alexandra; Lee, F Eun-Hyung; Federoff, Howard J.; Mosmann, Tim R.

doi:10.1038/nm821

Cited by 87 publications

(64 citation statements)

References 19 publications

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“…Thus, both populations expand significantly and thereby reach a frequency allowing a more effective interaction either directly or through Ag-presenting cell intermediates. It appears that at this stage, the suppression of cytolytic activity is more effective than the suppression of cytokine production or proliferation, i.e., cytotoxicity appears most sensitive to suppression, a notion consistent with observations by others that these effector functions can be regulated independently (27,(34)(35)(36)(37), perhaps because they require different intensity of TCR signaling (38). Also, the cytokine requirement for proliferation of primary and memory CD8 cells may differ, and, hence, Treg may be more efficient in suppressing the proliferation of memory than of primary CD8 T cells.…”

Section: Discussionsupporting

confidence: 86%

Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-β signalsin vivo

Chen

Pittet

Gorelik

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

723

549

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Cancer patients can harbor significant numbers of CD8 and CD4 T cells with specificities to tumor antigens (Ags). Yet, in most cases, such T cells fail to eradicate the tumor in vivo. Here, we investigated the interference of Ag-specific CD4 ؉ CD25 ؉ regulatory T cells (Treg) with the tumor-specific CD8 T cell immune response in vivo, by monitoring the homing, expansion, and effector function of both subsets in draining and nondraining lymph nodes. The results show that CD8 cells expand to the same extent and produce similar levels of IFN-␥ in the presence or absence of Ag-specific Treg. Nevertheless, these Treg abrogate CD8 T cell-mediated tumor rejection by specifically suppressing the cytotoxicity of expanded CD8 cells. The molecular mechanism of suppression involves TGF-␤ because expression of a dominant-negative TGF-␤ receptor by tumor-specific CD8 cells renders them resistant to suppression and is associated with tumor rejection and unimpaired cytotoxicity. (5-7). Treg also can control the magnitude of recall CD8 T cell responses in different settings that include viral (8, 9) and bacterial (10) infections as well as allograft transplantation in vivo (11,12). A role in Treg function has been attributed to IL-2, which stimulates Treg and in turn may inhibit division of memory CD8 T cells (13,14). However, these studies did not trace Ag-specific Treg in draining lymph nodes (LN), and it is not clear to what extent the different outcomes reflect accumulation of Treg by specific homing and local expansion. In fact, most studies were conducted with polyclonal Treg of unknown specificity where such questions cannot be addressed, and, therefore, the mode of inhibition cannot be correlated with specific Treg accumulation. Analysis of tumor-bearing patients suggests that suppression of CD8 T cell cytotoxicity by Treg may be causally related to tumor progression, because tumor-specific CD8 cells and tumor-specific CD4 Treg frequently accumulate in tumors from melanoma patients, and tumor-specific CD8 cells fail to exert cytotoxic T lymphocyte effector function (15)(16)(17).This study investigates how Treg suppress primary CD8 T cell immune responses directed against tumor cells expressing influenza hemagglutinin (HA) as a surrogate tumor-specific Ag. Naïve CD8 and regulatory CD4 T cells with transgenic receptors specific for distinct peptides of HA were used to allow us to follow the fate of these cells in tumor draining LN. The results show that Treg interfere with CD8 T cell-mediated tumor rejection relatively early during the immune response, and the mechanism by which Treg suppress naïve CD8 cells differs from the ones that have been reported for memory CD8 cells (10)(11)(12)14). Treg influenced neither the kinetics of proliferation nor the commitment of recently activated CD8 cells to produce inflammatory cytokines. Nevertheless, CD8 cells failed to undergo normal functional maturation in the presence of Treg as evidenced by the fact that their cytotoxic potential to destroy specific targets in vivo was abolis...

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Section: Discussionsupporting

confidence: 86%

Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-β signalsin vivo

Chen

Pittet

Gorelik

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

723

549

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“…Effector CD8 T cells (45) and cloned CTL lines (10) have cytolytic activity but are unable to produce IL-2 upon restimulation. Snyder et al have reported the generation of CD8 effector cells capable of secreting IFN-␥, but defective for cytolytic activity (46); however, their individual cell-based assay was not designed to detect effectors of the opposite phenotype. Two groups have independently described CD8 T cell populations capable of cytolytic function, but not IFN-␥ secretion (47,48).…”

Section: Discussionmentioning

confidence: 99%

IL-21 Promotes Differentiation of Naive CD8 T Cells to a Unique Effector Phenotype

Casey

Mescher

2007

The Journal of Immunology

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IL-21, the most recently described member of the common γ-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-α have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-γ or IL-4 production and can partially block IL-12 induction of IFN-γ production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44high, PD-1low, CD25low, CD134low, and CD137low. Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-γ.

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“…Our findings in this study further support a model in which calcium signals can play a possible quantitative role in this functional separation (for example, by setting different thresholds for cytotoxic and cytokine programs). We further hypothesize that the relationships between calcium signals and effector functions observed in NK cells could be extrapolatable to T cells and may help explain the dichotomy observed between cytotoxic activity and cytokine production reported for CD8 T cells in several studies (9,(44)(45)(46) (47), kinase activities (48), or transcriptional factors (49) using fusion proteins or reporter mouse (50)] should also allow better resolving the contributions of the dynamics and magnitudes of a wide variety of molecules to functional diversification at the single-cell level. Establishment of this new tool now opens the door to pursue these and many other previously intractable hypotheses on immune cell communication, activation, and function at the single-cell level.…”

Section: Discussionmentioning

confidence: 74%

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

Dura

Servos

Barry

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

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Resolving how the early signaling events initiated by cell-cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell-cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting singlecell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell.microfluidics | cell pairing | single-cell analysis | cell-cell interactions | multiparameter assay I nitiation and progression of immune responses require direct cell-cell interactions. Molecular interactions at the contact interface mediate cellular cross-talk and trigger a series of wellorchestrated downstream signaling events. The magnitude and dynamics of these signals promote and coordinate immune cell activation, and underlie a broad array of functional outcomes, such as target cell elimination, secretory activity, and proliferation (1, 2). Understanding how these early signaling events are transduced into functional responses demands correlated measurements at different stages as these responses unfold.Typically, these relationships are probed by first activating cells in bulk cocultures, often mixing cell populations and initiating contacts via a brief centrifugal cosedimentation, and then piecing together measurements from independent assays performed at different time points. This approach may determine broad outcomes of intercellular interactions, but it suffers from three major limitations. First, individual assay measurements are averaged over many different combinations of interactions due to the uncontrolled and indefinite nature of cell-cell interactions in bulk cocultures. For example, varying times of initiation and duration of contacts (1, 3), number of interacting partners (4), and differences in antigen presenting cells (5) can all modulate immune cell responses. Unpredictable variation is therefore introduced into measured responses, masking the true intrinsic cellular heterogeneity and blurring the correlations. Second, conventional as...

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Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing

Cited by 87 publications

References 19 publications

Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-β signalsin vivo

Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-β signalsin vivo

IL-21 Promotes Differentiation of Naive CD8 T Cells to a Unique Effector Phenotype

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

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