Romagnoli et al.

Supplementary Material

Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria

monocytogenes infection

PA Romagnoli1, HH Fu1, Z Qiu2, C Khairallah2, QM Pham1, L Puddington1, KM Khanna1, L Lefrançois1,

and BS Sheridan2

1Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, USA.

2Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook

University, Stony Brook, New York, USA

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Supplementary Figures

Supplementary Figure S1. Localization of LLO-specific CD4 T cells in the intestinal lamina propria and

epithelium after oral infection. 4x104 CD4 T cells from LLO56 TCR transgenic Rag1-/- Thy1.1 mice (CD4 T

cells specific for the LLO190-201 epitope1) were transferred into B6 (Thy1.2) mice 1 day prior to oral infection

with 2x109 cfu InlAM rLm. Sections of small intestine from mice at 9 or 29 dpi were immunostained for the

indicated proteins and with the nuclear counterstain DAPI. White arrows identify LLO56 CD4 T cells in the

epithelium. Scale bars represent 50 m.

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Supplementary Figure S2. Oral infection induces traditional pathogen-specific CD4 T cells in the IEL

compartment. B6 mice were orally infected with InlAM rLm and LLO-specific CD4 T cells were analyzed

for the indicated markers from the IEL compartment (a, b) or spleen, MLN, LP, and IEL (c) compartment

after infection. CD8 and CD8β expression were examined on LLO-specific CD4 T cells from the IEL

at 60 dpi (memory; a) or 7 days after a secondary challenge of mice that were immunized 60 days

previously (recall; b). Representative zebra plots showing CD8 and CD8 expression on LLO-I-Ab+ or

LLO-I-Ab- CD4 T cells are shown below each graph. Numbers within plots are the percentage of cells

within gated quadrants. * p < 0.05, *** p < 0.001 (unpaired Student's t-test). (c) Granzyme B expression

on CD4 T cells from the indicated tissues at 9 dpi. Representative zebra plots gated on CD4 T cells are

shown. Numbers within plots are the percentage of cells within gated quadrants. Graph depicts the mean

± SEM percentage of LLO-specific CD4 T cells that expresses granzyme B in each tissue.

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Supplementary Figure S3. Intestinal CD4 T cells display distinct phenotypic characteristics. B6 mice

were orally infected with InlAM rLm and LLO-specific CD4 T cells were analyzed for the indicated markers

from the spleen, MLN, LP, and IEL after infection. Representative histograms (a,b) or dot plots with

adjunct histograms (c) are gated on LLO-I-Ab+ CD4 T cells at 9 (primary), 60 (memory) dpi or 7 days after

a secondary challenge of mice that were immunized 60 days previously (secondary). (a) The graph

depicts the mean ± SEM of pooled data from 2 independent experiments with 3 – 6 mice total. (c) For

each dot plot, LLO-I-Ab+ CD4 T cells from the LP (dark blue) are overlaid onto LLO-I-Ab+ CD4 T cells from

the spleen (light orange). Numbers within plots are the percentage of cells within gated quadrants and are

color coded to the tissue they derive from.

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Supplementary Figure S4. Cytokine receptor expression on memory CD4 T cells and the impact of IL-

15 deficiency on memory CD4 T cell phenotype. (a) LLO-specific CD4 T cells were analyzed for CD122

(IL-2 receptor ) and CD127 (IL-7 receptor ) expression in the spleen, MLN, LP, and IEL at 60 days

(CD122) or 38 days (CD127) after oral InlAM rLm infection. The blue line depicts a representative

histogram of 3-4 mice from the indicated tissue and is representative of 2 independent experiments. For

CD122 staining, the filled gray histogram depicts CD11ahi CD8 T cells from the spleen as a positive

indicator for staining. For CD127 staining, the filled gray histogram depicts CD8+ IELs as a negative

indicator for staining. (b,c) B6 wild-type (WT) and IL-15 deficient (KO) mice were orally infected with InlAM

rLm and LLO-specific CD4 T cell populations were examined in the spleen (b) and LP (c) 60 days after

infection. Data are representative of 2 independent experiments with 3-5 mice per group.

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Supplementary Figure S5. Early impact of anti-CD4 treatment on protection and cellular responses. (a)

B6 mice that were immunized 88 days previously with 2x109 cfu InlAM rLm-ova were orally infected with

2x1010 cfu InlAM rLm-ova and CD4+ (clone RM4-4) TCR+ T cells and Ova-specific CD8 T cells were

examined from the indicated tissues 2 days later. Mice were treated with 200g anti-CD4 (clone GK1.5)

or isotype control (clone LTF-2) antibodies at days -3, -1, and +1 respective to secondary infection. A bar

graph depicting the mean ± SEM from one experiment with 3-4 mice per group is shown and is

representative of two similar experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, (unpaired

Student's t-test). (b) Naive or B6 mice orally immunized 33 days previously with 2x109 cfu InlAM rLm were

challenged with 2x1010 cfu InlAM rLm via oral infection. Immunized mice were treated with 200g anti-CD4

or isotype control antibodies on days -3, -1, and +1 respective to secondary challenge infection. The

small intestines and MLN were harvested approximately 2 days (40 hours) after challenge infection and

Lm burden was quantified on agar plates supplemented with streptomycin. A bar graph depicting the

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mean ± SEM from one experiment with 6 mice per group is shown and is representative of two similar

experiments. * p < 0.05, ** p < 0.01, (Mann-Whitney test).

Supplemental References

1. Weber, K.S., Li, Q.J., Persaud, S.P., Campbell, J.D., Davis, M.M. & Allen, P.M. Distinct CD4+ helper T cells involved in primary and secondary responses to infection. Proc. Natl. Acad. Sci. USA. 109, 9511-9516 (2012).

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