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. 2023 Jun 15;210(12):1950-1961.
doi: 10.4049/jimmunol.2200337.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated

Michael C Jones  1 , Catherine Castonguay  1 , Padma P Nanaware  1 , Grant C Weaver  1 , Brian Stadinski  1 , Olivia A Kugler-Umana  1 , Eric S Huseby  1 , Lawrence J Stern  1 , Karl Kai McKinstry  2 , Tara M Strutt  2 , Priyadharshini Devarajan  1 , Susan L Swain  1
Affiliations

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated

Michael C Jones et al. J Immunol. .

Abstract

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

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Figures

Figure 1:
Figure 1:. Model to evaluate peptide avidity.
(A-C) Naïve FluNP.Thy1.1+/− cells were transferred to B6 hosts, then infected with PR8. (A) Lungs were collected at 4, 6, 8, 12, 21 and 63 dpi and numbers of donor FluNP and responding host CD4+, CD44hi cells were determined by FACS. (B) Day 8 Effector phenotype and function. Expression of markers associated with subsets of CD4 effectors were analyzed at 8 dpi in lung and spleen: Th1 (T-Bet+, IL-2+, IFNγ+, TNFα+), Triple positive (IL-2+, IFNγ+, TNFα+), Th17 (IL-17+), Treg (FoxP3+), lung ThCTL (NKG2A/C/E) and spleen TFH (CXCR5+, BCL-6+). (C) Memory CD4 subsets were analyzed at 21 dpi: lung TRM (CD69+), spleen TEM (CD127+ CD44+ CD62L-) and spleen TCM (CD127+ CD44+ CD62L+). (D-H) Characterization of NP peptide panel. (D) Peptide name, aa sequence (P1=Y, mutations in red) and the I-Ab binding affinities of the NP311–325 length variants and mutants were experimentally determined to identify the I-Ab binding frame. The reciprocal of the IC50 is shown. (E) Maximal response for a given TCR concentration was plotted for each peptide-MHC complex and nonlinear fits were generated using the equation Y=Bmax*X/(KD + X). The fit was constrained to share a consistent Bmax and yield a KD value of <500. The resulting KD values are shown in μM with 95% confidence intervals. (F-H) Naïve FluNP CD4 T cells were co-cultured with BMDC pulsed with each of the NP peptides for 2d in vitro. Induction of markers functionally associated with TCR signal strength was measured. (F) CD69; (G) CD25; and (H) Nur77. Top histograms display level of marker expression following stimulation with Ag/APC pulsed at 10−4 M. Bottom displays dose response curve to a broad range of peptide concentrations used to pulse APC. The rank of peptide functional avidity is shown on right. Statistical evaluations: (A) Days 4, 6, 8, 21, 63 Pooled data, n = 9–10, two experiments. Day 12 one experiment n = 5. Mean +/− SEM. (B) Pooled data, n = 11, two experiments, mean +/− SEM. (C) Pooled data, n = 7, two experiments, mean +/− SEM. (D) Pooled data, n=3–4, two experiments. (E-G) Representative data, n = 6, two experiments. (F-H) Pooled data, n = 6, two experiments, mean +/− SEM (% of FluNP). Statistical significance determined by two-tailed independent t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Figure 2:
Figure 2:. Peptide avidity during the primary effector phase dictates the size of the memory population.
(A) Experimental design: comparison of PR8 infection to high peptide/APC memory generation. Naïve FluNP.Thy1.1+/− cells were transferred to B6 hosts, then infected with PR8. At 6 dpi, FluNP effector cells were isolated from the 1st hosts and co-transferred with either peptide Ag/APC into uninfected 2nd hosts or without APC into day 6 PR8 infection matched hosts (infected 6d previously). Fifteen days later (21 dpi) 2nd hosts were sacrificed, and donor FluNP cells were analyzed by FACS. (B) FluNP cell numbers were enumerated by FACS in the dLN, lung and spleen at 21 dpi. (C) Experimental design: Impact of peptide avidity on memory generation. 1.5×106 6d FluNP effectors were co-transferred with Ag/APC into uninfected 2nd hosts. The panel of different peptides was used to pulse groups of activated BMDC yielding peptide/APC with different avidity for the TCR of the FluNP T cells. (D) Representative FACS plots showing donor FluNP memory cells by CD44 and CD90.1 expression in the spleen at 21 dpi. (E) Number of FluNP memory cells detected at 21 dpi in the dLN, lung and spleen of second hosts. Statistical significance determined by two-tailed independent t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Spleen comparisons: highs vs. unpulsed, highs vs. lows, NPT vs. A7 / highs vs. K5; mids vs. unpulsed, K5 vs. Q2; Q2 vs. unpulsed / Q7 vs. unpulsed. dLN comparisons: NPT vs. unpulsed / NP311–325 vs. unpulsed, NPT vs. lows / NP311–325 vs. lows, NPT vs. mids / NP311–325 vs. mids; A7 vs. unpulsed / K5 vs. unpulsed, A7 vs. lows / K5 vs. lows; Q2 vs. unpulsed / Q7 vs. unpulsed. Lung comparisons: NPT vs. NP311–325; NPT vs. unpulsed / NP311–325 vs. unpulsed, NPT vs. lows / NP311–325 vs. lows, NPT vs. mids / NP311–325 vs. mids; mids vs. unpulsed, mids vs. Q2 / mids vs. Q7; Q2 vs. unpulsed / Q7 vs. unpulsed. (F) FluNP cell number fold change relative to unpulsed (left), low peptide-pulsed (right) and mid peptide (below) in dLN, lung and spleen 21 dpi, x-axis lists fold change denominator. (B) Pooled data, n = 10, three experiments, mean +/− SEM. (D) Representative data, n = 8–15, four experiments. (E-F) Pooled data, n = 8–15, four experiments, mean +/− SEM.
Figure 3:
Figure 3:. Peptide avidity determines survival but not proliferation of donor cells 3 days post transfer.
(A) Experimental Design. Naïve FluNP CD4 T cells were transferred to B6 hosts, infected with PR8. At 6 dpi, FluNP effector cells were isolated from the 1st host, labeled with cell trace violet (CTV) and transferred to 2nd hosts given 106 APC, pulsed or not, with high, mid or low peptides. At 3 days post transfer (dpt), 2nd hosts were sacrificed and FluNP (CD4+ CD90.1+) cells were analyzed. (B) Representative FACS plots of FluNP CTV dilution in spleen 3 dpt. Gated on live singlets, CD4+, CD90.1+ cells. (C) Percent survival of FluNP effectors measured by live/dead and caspase 3/7 FACS (double-negative) in spleen 3 dpt. (D) Number of donor FluNP cells recovered 3 dpt in the dLN, lung and spleen. (E) IL-2 receptor subunit expression: IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rγ (CD132) expression was determined by FACS analysis of the 6 dpi effectors (left) and 3 dpt donor cells (right). (F) FluNP 6 dpi effectors were restimulated for 6 hours with indicated NP peptides at 10 μM and IL-2 expression determined by FACS. (G) FluNP effectors were co-cultured with NP peptide-pulsed APC for 48 hr and IL-2 production in the supernatant was determined by ELISA. (H) The area under the curve was calculated to quantitate impact on IL-2 production. (B) Representative data, n = 10, two experiments. (C-E) Pooled data, n = 10, two experiments, mean +/− SEM. (F) Pooled data, n = 10, two experiments, mean +/− SEM. (G-H) Pooled data, n = 3–6, two experiments, mean +/− SEM.
Figure 4:
Figure 4:. IL-2c Treatment when Ag/APC are low affinity and CD25 Expression on APC Enhance Memory Generation.
(A-D) IL-2 complex treatment (IL-2c tx) rescues low stimulated FluNP memory cells. (A) Experimental design. 6 dpi FluNP effectors were co-transferred with NPT (high) or Q2 (low) peptide-pulsed WT BMDC and mice received PBS or IL-2c tx i.p for 3 days post transfer. Number of memory FluNP cells determined by FACS analysis in (B) spleen, (C) dLN and (D) lung. (E-F) CD25 Expression on APC in vivo. Mice were infected or not with PR8 influenza and sacrificed 4, 6 and 8 dpi. (E) CD25 expression was measured by FACS staining on I-Ab+, CD11c+ cells in the dLN of infected (black line, no fill) or uninfected (gray) mice. (F) Kinetics of CD25+ I-Ab+, CD11c+ cells in the dLN, lung, and spleen of infected mice at 4, 6 and 8 dpi. (G-J) Impact of CD25 deletion in APC on memory generation. (G) Experimental Design. 6 dpi FluNP effectors were co-transferred with peptide-pulsed WT or CD25KO BMDC to uninfected 2nd hosts. 2nd hosts were sacrificed 15 dpt (21 dpi), and donor memory cells were analyzed by FACS. Number of memory FluNP cells determined by FACS analysis in (H) spleen, (I) dLN and (J) lung. (B-D) Pooled data, n = 7–8, two experiments, mean +/− SEM. (E) Representative data, n = 10, two experiments. (F) Pooled data, n = 9–10, two experiments, mean +/− SEM. (H-J) Pooled data, n = 6–9, two experiments, mean +/− SEM. Statistical significance determined by two-tailed independent t test (*p<0.05, **p<0.01).
Figure 5:
Figure 5:. Increased peptide avidity at the effector checkpoint promotes a more protective population of memory cells.
(A) Experimental design: FluNP effector cells (6 dpi) were co-transferred with peptide-pulsed APC into uninfected 2nd hosts and rested for 15 d (21 dpi). At 21 dpi, 2nd hosts were challenged with PR8. (B) Weight loss was determined following 2LD50 PR8 challenge. Statistical significance determined by two-tailed independent t test (*p<0.05). (C) Survival was measured following 4LD50 PR8 challenge. Statistical significance determined by log-rank (Mantel-Cox) test (*p<0.05). (B) Pooled data, n= 10–15, two experiments, mean +/− SEM. (C) Pooled data, n= 8, two experiments. (E-G) Pooled data, n= 8–9, two experiments, mean +/− SEM.
Figure 6:
Figure 6:. Peptide avidity during the secondary effector checkpoint regulates the size of the secondary memory population.
(A) Experimental design. Primary 6 dpi FluNP effectors were generated and 1.5×106 co-transferred to 2nd hosts with 106 NPT (high) peptide-pulsed BMDCs. At the memory stage, 21 dpi for the donor cells, 2nd hosts were infected with PR8 influenza (0.3 LD50) and secondary 6 dpi effectors were isolated via CD90.1 MACS and co-transferred to uninfected 3rd hosts. Fifteen days later 3rd hosts were sacrificed, and donor secondary memory cells were analyzed by FACS. (B) Number of secondary memory donor FluNP cells determined FACS analysis in the spleen, dLN and lung. Spleen and dLN pooled data, n = 6–8, two experiments, mean +/− SEM. Lung representative data n = 6–8, two experiments, mean +/− SEM. Statistical significance determined by two-tailed independent t test (*p<0.05, **p<0.01).
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