CD39 Is Expressed on Functional Effector and Tissue-resident Memory CD8+ T Cells

Abstract

The ecto-ATPase CD39 is expressed on exhausted CD8+ T cells in chronic viral infection and has been proposed as a marker of tumor-specific CD8+ T cells in cancer, but the role of CD39 in an effector and memory T cell response has not been clearly defined. We report that CD39 is expressed on Ag-specific CD8+ short-lived effector cells, while it's co-ectoenzyme, CD73, is found on memory precursor effector cells (MPECs) in vivo. Inhibition of CD39 enzymatic activity during in vitro T cell priming enhances MPEC differentiation in vivo after transfer and infection. The enriched MPEC phenotype is associated with enhanced tissue resident memory T cell (TRM cell) establishment in the brain and salivary gland following an acute intranasal viral infection, suggesting that CD39 ATPase activity plays a role in memory CD8+ T cell differentiation. We also show that CD39 is expressed on human and murine TRM cells across several nonlymphoid tissues and melanoma, whereas CD73 is expressed on both circulating and resident memory subsets in mice. In contrast to exhausted CD39+ T cells in chronic infection, CD39+ TRM cells are fully functional when stimulated ex vivo with cognate Ag, further expanding the identity of CD39 beyond a T cell exhaustion marker.

Figure 1.. Anti-viral effector T cell response is associated with dynamic modulation of CD39 and CD73.

a. Experimental schematic utilized to track effector virus-specific OT-I T cells in response to an acute VSV_ova infection. b. Representative histogram and combined bar graph depicting CD39 expression on OT-I T cells (CD44+ CD45.1+) and naive CD8+ T cells (day 7, CD44− CD45.1−) on day 7, 10, and 14 post infection in blood. c. Representative histograms and combined bar graph depicting CD73 expression on OT-I T cells (CD44+ CD45.1+) and naive CD8+ T cells (day 7, CD44− CD45.1−) on day 7, 10, and 14 post infection in blood. d. Representative flow plots showing CD39 and CD73 expression on naïve or OT-I T cells in blood, quantified over time in e. Data combined from two experiments. Naive CD8+ and day 7 OT-I n=15, day 10 and day 14 OT-I n=20. b-c. One-way ANOVA with Tukey’s post-test. *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns= not significant. Error bars represent the mean ± SEM.

Figure 2.. CD39 is increased on short-lived effector CD8+ T cells while CD73 is expressed on memory precursors during an acute anti-viral response.

CD45.1+ OT-I were adoptively transferred into naive mice and infected with VSV_ova as in Fig 1a. a. Memory precursor; MPEC (CD127+KLRG1−) and short-lived effector; SLEC (CD127-KLRG1+) OT-I populations on day 7, 10, and 14 post infection in blood. b. Percent MPEC and SLEC of total OT-I T cells in blood over time. c. Representative histogram of CD39 expression on naive CD8+ T cells (CD44− CD45.1−) and day 7 MPEC or SLEC OT-I, quantified over time in d. e. Percent of CD39+/− CD73+/− OT-I T cells within the SLEC compartment. f. Representative histogram of CD73 expression on naive CD8+ T cells (CD44− CD45.1−) and day 7 MPEC or SLEC OT-I, quantified over time in g. h. Percent of CD39+/− CD73+/− OT-I T cells within the MPEC compartment. Vertical lines in c and f indicate gating for CD39+ and CD73+. i. Percent OT-I of CD39+ on day 7, 10 and 14. Data combined from two experiments. Naive CD8+ and day 7 OT-I n=15, day 10 and day 14 OT-I n=20. d (day 7), f (day 7) One way ANOVA with Tukey’s post-test. b, d, f. Unpaired t test. *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns= not significant. Error bars represent the mean ± SEM.

Figure 3.. Inhibiting CD39 during T cell in vitro priming leads to OT-I T cell MPEC skewing and enhanced T_RM establishment.

a. Experimental schematic demonstrating in vitro OT-I T cell priming in the presence or absence (+/−) of POM-1 (50uM), followed by transfer and subsequent infection in vivo. b-c. Representative flow plots showing CD127 and KLRG1 staining in untreated and POM-1 treated OT-I T cells on day 7 (b) and day 14 (c) in blood. d. Frequency of OT-I T cells in blood on day 7 and 14 post infection. e-f. Frequency of MPEC (e) and SLEC (f) populations among OT-I T cells primed +/− POM-1 in blood. g-h. Representative flow plots of CD45.1+ OT-I populations in brain (g) and SG (h) within each experimental group. i. Frequency of total OT-I T cells found in blood, lymph node or spleen 30+ days post infection. j. Frequency of IV-negative OT-I T cells in tissues 30+ days post infection (gated on IV-negative CD45.1 or CD90.1) FRT= female reproductive tract, SG= salivary gland, SI= small intestine, LN= lymph node, ns= not significant. Data in B-F combined from 3 independent experiments. Untreated n=20, POM-1 n=25. Data in G from three experiments (blood n=17 untreated, n=25 POM-1). Data in H from 2-3 experiments (brain and lung from 3 experiments, untreated n= 14, POM-1 n= 15; FRT, SI, Lung, SG from 2 experiments, n=8 per group). Unpaired t test. *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns= not significant. Error bars represent the mean ± SEM.

Figure 4.. CD39 and CD73 are expressed on T_RM.

Memory OT-I T cells were harvested from mice at day 40-50 post infection and CD39/CD73 expression was analyzed across memory subsets. a. Flow plots depicting CD39 and CD73 expression on effector memory (T_EM) and central memory (T_CM) OT-I from the superficial cervical lymph node and IV-negative OT-I across non-lymphoid tissues. b-c. Frequency of CD73+ (b) or CD39+ (c) OT-I T_RM (CD69+/CD103+/−) in the brain, OT-I T_EM (CD62L−), and OT-I T_CM (CD62L+) in the lymph node and naïve (CD44−) CD8+ T cells in spleen. d. Frequency of CD39+/−CD73+/− IV-negative OT-I T_RM in the brain, and OT-I T_EM and T_CM in the lymph node. e. Representative flow plots of CD69 and CD103 residency phenotype across non-lymphoid tissues, quantified in f. g. Frequency of CD39+CD73+ OT-I stratified by CD69 and CD103 expression across tissue. h-i. Cytokine production by OT-I T cells isolated from the brain of OT-I memory mice as described in Figure 1. Cells were stimulated in vitro with viral peptide (SIINFEKL) with or without POM-1, control peptide (gp33), or PMA (positive control). h. Representative flow plots of IFNg and TNFa production by brain OT-I, quantified in i. j-k. Mice were infected intravenously with vaccinia virus, then received an adoptive transfer of 1 x 10⁶ in vitro activated OT-I T cells 30 days later. 7 days after transfer, mice received B16-ova brain tumors and were euthanized at day 16 post tumor challenge. Vaccinia-specific endogenous CD8+ T cells were identified by B8R H2-K^b tetramer. j. MFI of CD39, gated on IV-negative CD39+ OT-I T cells (tumor-specific) or IV-negative B8R+ T cells (virus-specific). Paired data are shown where each line connects datapoints from individual mice. k. MFI of CD39 on CD69+ tumor-specific OT-I T cells, and CD69+ and CD69− memory virus-specific B8R+ T cells. Gated on IV-negative. T_EM= CD44+ CD45.1+ CD69− CD103− CD62L−, T_CM= CD44+ CD45.1+(or Thy1.1+) CD69− CD103− CD62L+, T_RM= IV− CD44+ CD45.1+(or Thy1.1+) CD69+CD103+/−. FRT= female reproductive tract, SG= salivary gland, SI= small intestine, LN= lymph node. a-g. Data combined from 2-3 experiments. n= 10-16 mice. h-i. Data combined from 3 experiments n=3 per group per experiment. j-k. Data combined from two independent experiments, n=17 mice. (b-c, g,i, k) One way ANOVA, (j) unpaired t test. *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns= not significant. Error bars represent the mean ± SEM.

Figure 5.. CD39 is expressed on CD69+ CD103+ CD8+ T cells in human tissue.

a. Percent of CD39+ CD8+ T cells in each tissue, gated on CD45RA− CCR7−. b. Representative flow plots showing CD69 and CD103 expression on CD45RA− CCR7− memory CD8+ T cells in human autopsy tissues quantified in (c). d-g. Representative histograms of CD39 expression on CD8+ T cells. Frequency of CD39+ cells across CD69+/CD103+ and CD69-/CD103− CD8+ T cells, quantified in bar graphs. Graphs connecting individual patient data for cortex (d), meninges (e), lung (f) and skin (g). Data points from 2-3 individual subjects connected via line plot where cortex n=3, meninges n=3, lung n=2, and skin n=3. h. Identification of Epstein Barr Virus (EBV)-specific CD8+ T cells in a patient melanoma tumor by combinatorial HLA-A2 tetramer staining (cells gated on live, CD8+ CD3+ CD45RA− CCR7−). Flow plot depicting CD69 and CD103 expression on EBV-specific cells (right). i. CD39 expression on total CD45RA− CCR7− EBV_GLC-specific CD8+ T cells in blood (blue) compared to EBV-specific CD69+/−CD103+/− T cells in the tumor (purple, red, orange), compared to CD45RA− CCR7− non-viral specific CD8+ T cells in the tumor (green). All samples gated on live, CD8+ CD3+ CD45RA− CCR7−. Numbers in black are percentages, colored numbers inset in peaks are MFI. Melanoma patient sample= 1. d-g. Paired t-test. Error bars represent the mean ± SEM.