T-cell-receptor-dependent signal intensity dominantly controls CD4(+) T cell polarization In Vivo

Abstract

Polarization of effector CD4(+) T cells can be influenced by both antigen-specific signals and by pathogen- or adjuvant-induced cytokines, with current models attributing a dominant role to the latter. Here we have examined the relationship between these factors in shaping cell-mediated immunity by using intravital imaging of CD4(+) T cell interactions with dendritic cells (DCs) exposed to polarizing adjuvants. These studies revealed a close correspondence between strength of T cell receptor (TCR)-dependent signaling and T helper 1 (Th1) versus Th2 cell fate, with antigen concentration dominating over adjuvant in controlling T cell polarity. Consistent with this finding, at a fixed antigen concentration, adjuvants inducing Th1 cells operated by affecting DC costimulation that amplified TCR signaling. TCR signal strength controlled downstream cytokine receptor expression, linking the two components in a hierarchical fashion. These data reveal how quantitative integration of antigen display and costimulation regulates downstream checkpoints responsible for cytokine-mediated control of effector differentiation.

Figure 1. Exposure of DC to polarizing adjuvants alters the balance of CD4⁺ T cell effector fates in concert with changes in cellular interaction times

Assessment of 5CC7 cell differentiation following priming with (A–C) LPS- vs. papain-treated DC or (F–H) CpG- vs SEA-treated DC at day four post-adoptive transfer. (D & I) Individual cellular interaction times of polyclonal or 5CC7 T cells interacting with adjuvant treated DC, 0–2hr post-transfer as determined following 2P-IVM imaging. (E & J) Mean cellular interaction times for separate 2P-IVM experiments. (A–C & F–H) Data are representative of three experiments (n=4). (D&I) Data are pooled from five or three animals respectively. (E&J) Data represent the mean interaction times from five or three animals respectively. Error bars represent mean +/− s.e.m. * P<0.05, ** P<0.01, *** P<0.001 as determined by (B–E,G,H) 1 way ANOVA with Tukeys post testing or (I,J) Student’s t test.

Figure 2. Exposure of DC to polarizing adjuvants alters Phase-2 and Phase-3 interactions

Distribution of interaction times of polyclonal and 5CC7 cells with pPCC loaded papain- or LPS-treated DC at (A) 12hr post-transfer and (B) 22hr post-transfer. Mean experimental interaction times at (C) 12hr post-transfer and (D) 22hr post-transfer. Mean experimental velocities at (E) 12hr and (F) 22hr post-transfer. (G) Percentage of 5CC7 cell:DC conjugates originally observed still remaining over time. Imaging was conducted for 1hr (A,B,G) Represent pooled data from 4 experiments. (C–F) indicate mean +/− s.e.m of data from 4 experiments. *P<0.05, **P<0.01 and ***P<0.001 as determined by 1 way ANOVA with Tukeys post testing.

Figure 3. Different adjuvant treatments of DCs alter contact duration but not initial contact frequency with CD4⁺ T cells

pPCC loaded papain- and LPS-stimulated DC were alternately labeled and co-transferred into naïve B10.A hosts. Eighteen hrs post transfer 5CC7 cells were adoptively transferred and imaged for 2hr. (A) 5CC7 and DC cell numbers per LN were determined post imaging. (B) Ratio of LPS: papain treated DC per LN. (C) Mean number of CD4⁺ T cell interactions per DC and (D) frequency distribution of interactions. (E) Interaction times of 5CC7 CD4⁺ T cells with DCs. (F) Ratio of 5CC7 cells interacting with LPS-DC: papain-DC after 2 hrs. (A–F) Data are indicative of 5 LN. Means +/− s.e.m. *P<0.05, **P<0.01 and ***P<0.001 as determined by Student’s t test.

Figure 4. Greater Ca²⁺ signaling and synapse size of CD4⁺ T cells interacting with DC exposed to Th1vs. Th2cell promoting adjuvants

DC were loaded with pPCC and treated with either (A–F) LPS or papain or (G–J) CpG or SEA and transferred into naïve B10.A hosts. Eighteen hrs post transfer 5CC7 cells loaded CMTPX and Fluor-4 were adoptively transferred and imaged for 2hr. (A) Representative micrograph of 5CC7 cell tracking during Ca²⁺ flux analysis by 2P-IVM. (B) 5CC7 cell tracks were analyzed post-2P-IVM and normalized Ca²⁺ flux (blue) and instantaneous velocity (red) were used to characterize interactions. (C,D,G&H) Individual 5CC7 cell Ca²⁺ flux tracings for cells interacting with adjuvant treated DC are shown for durations of T cell:DC contacts classified as involving no interaction (blue), brief interactions (green), or stable interactions (red). (E&I) Mean Ca²⁺ flux and (F&J) integrated Ca²⁺ flux areas were calculated for individual cell tracks. (K) Distribution of 5CC7 cell:DC interaction interface sizes and (L) experimental mean interaction interface sizes. (E,F&K) five, (I&J) three pooled experiments, (L) means from five separate experiments. Mean +/− s.e.m. * P<0.05, ** P<0.01, *** P<0.001 as determined by 1 way ANOVA with Tukeys post testing.

Figure 5. The magnitude of antigen-dependent signal strength dominates over qualitative effects of adjuvants on DC

(A) Assessment of 5CC7 cell differentiation following priming with LPS- or papain-treated DC loaded with 10μM-0.01μM pPCC, at day four post-adoptive transfer. (B) Quantification of percent of 5CC7 cells expressing IL-4 or IFNγ, (C) Number of 5CC7 cells expressing IL-4 or IFNγ and (D) ratio of %IFNγ: %IL-4 producers. (E) Cellular interaction times 0–1hr post-transfer of 5CC7 cells with Lo-LPS- or Hi-papain-treated DC. (F) Mean interaction times for individual experiments. (G) Integrated Ca²⁺ flux areas were calculated for individual cellular tracks. (H) Mean Integrated Ca²⁺ flux areas for individual experiments. (A–D) are representative of four experiments (n=4). (E&G) Data are pooled from four experiments. (F&H) Data are representative of four experiments. Mean +/− SEM. *P<0.05, **P<0.01 and ***P<0.001. 1 way ANOVA with Tukeys post testing.

Figure 6. T cell interpretation of antigen display is affected by CD80 co-stimulation

pPCC loaded LPS- or papain-treated DC were isolated from dLN at 24hr post-transfer and analyzed. (A) MHCII, (B) CD80 and (C) PCC peptide presentation reported as MFI post-staining. (D) Percent of 5CC7 cells producing IL-4 and (E) IFNγ at day four post-adoptive transfer, following priming with LPS- or papain-treated DC, +/− blocking with anti-CD80. (F) Cellular interaction times 0–2hr post-transfer of 5CC7 cells with LPS or papain treated. (H) Mean interaction times for individual 2P-IVM experiments. (H) Mean Ca²⁺ flux values and (I) integrated Ca²⁺ flux areas were calculated for individual cellular tracks. (A–D) are representative of two experiments (n=4). (E&F) Data are representative of three experiments (n=4). (G,I&J) Data are pooled from three experiments. (H) Data are representative of three experiments. Error bars indicate mean +/− s.e.m. *P<0.05, **P<0.01 and ***P<0.001, as determined by (A–E,H&I) 1 way ANOVA with Tukeys post testing or (F, G)Student’s t test.

Figure 7. Signal strength determines the ability of CD4⁺ T cells to respond to polarizing cytokines

5CC7 cells were activated in vitro using P13.9 artificial antigen presenting cells pre-incubated with the concentrations of pPCC indicated. Twenty-four hr post-activation IL-12Rβ2 expression by the T cells was determined by flow cytometry. (A) Representative plots of IL-12Rβ2 expression by stimulated (black lines) or naïve (grey lines) 5CC7cells, (B) Percent of of activated (CD69⁺) and non-activated (CD69⁻) 5CC7 cells expressing IL-12Rβ2, (C) comparison of IL-12Rβ2 MFI for activated vs. non-activated 5CC7 cells. WT 5CC7 or 5CC7 Il4^−/−Ifng^−/− (2ko) cells were stimulated in vitro with P13.9 cells pre-incubated with either 0.01μM or 10μM pPCC and (D) IL-12Rβ2 expression determined. 5CC7 cells were activated in vivo with LPS or papain treated DC and compared to control 5CC7 cells from non-dLN. Ex vivo expression of (E) IL-12Rβ2 and (F & G) pSTAT4 by CD69⁺ 5CC7 CD4⁺ T cells was then determined at 24hr post transfer. Data in (A–C) are representative of two experiments (n=4), (D, E, F & G) are representative of three experiments (n=4), means are plotted +/− s.e.m. *P<0.05, **P<0.01 and ***P<0.001 as determined by 1 way ANOVA with Tukeys post testing.