Epitopes in the GPI attachment signal peptide of Trypanosoma cruzi mucin proteins generate robust but delayed and nonprotective CD8⁺ T cell responses

Abstract

Infection with the protozoan parasite Trypanosoma cruzi elicits substantial CD8⁺ T cell responses that disproportionately target epitopes encoded in the large trans-sialidase (TS) gene family. Within the C57BL/6 infection model, a significant proportion (30–40%) of the T. cruzi-specific CD8⁺ T cell response targets two immunodominant TS epitopes, TSKb18 and TSKb20. However, both TS-specific CD8⁺ T cell responses are dispensable for immune control and TS-based vaccines have no demonstrable impact on parasite persistence, a determinant of disease. Besides TS, the specificity and protective capacity of CD8⁺ T cells that mediate immune control of T. cruzi infection is unknown. With the goal of identifying alternative CD8⁺ T cell targets, we designed and screened a representative set of genome-wide, in-silico predicted epitopes. Our screen identified a novel T cell epitope MUCKb25, found within mucin family proteins, the 3^rd most expanded large gene family in T. cruzi. The MUCKb25-specific response was characterized by delayed kinetics, relative to TS-specific responses, and extensive cross-reactivity with a large number of endogenous epitope variants. Similar to TS-specific responses, the MUCKb25 response was dispensable for control of the infection and vaccination to generate MUCK-specific CD8⁺ T cells failed to confer protection. The lack of protection by MUCK vaccination was partly attributed to the fact that MUCKb25-specific T cells exhibit limited recognition of T. cruzi-infected host cells. Overall, these results indicate that the CD8⁺ T cell compartment in many T. cruzi-infected mice is occupied by cells with minimal apparent effector potential.

Introduction

The protozoan parasite Trypanosoma cruzi establishes lifelong infection within human and mammalian hosts, leading to serious cardiac and digestive complications known as Chagas disease [1]. Highly effective immune responses coordinate and maintain control of parasites to very low levels within most infected individuals. However, 30–40% of chronically infected people will eventually progress to symptomatic disease for which treatment options are limited and often fail [2, 3] (reviewed in [4]). The host immune and parasite factors that enable parasite persistence are unclear, though highly relevant if more effective drugs and preventive strategies are to be developed.

Immune control of infection with intracellular pathogens like T. cruzi is dependent on CD8⁺ T cell-mediated cytokine production and/or cytolysis of infected host cells [5, 6]. These potent effector functions are facilitated by T cell receptor (TCR) engagement of pathogen-derived peptide/epitope presented by MHC-I complexes. This highly specific binding interaction leads to the activation and expansion of epitope-specific CD8⁺ T cell responses that coordinate pathogen control. Oftentimes, effective T cell immunity leads to complete pathogen clearance, although some pathogens, including T. cruzi, can delay or even evade immune clearance indefinitely [7, 8].

CD8⁺ T cells are known to mediate and maintain control of T. cruzi throughout infection [9–11], but the antigen specificities of T cells involved in this control are unclear. A significant proportion of CD8⁺ T cells generated within infected mice and humans are specific for epitopes derived from trans-sialidase (TS) proteins [12–21]. Trans-sialidase proteins are encoded by a large, expanded gene family comprised of several thousand highly variable genes, in which there is evidence of frequent recombination and inter/intrastrain diversity [22, 23]. Vaccination strategies eliciting-TS-specific CD8⁺ T cells have been shown to enhance immune control in mice [24–27] but ultimately fail to prevent parasite persistence [21, 28]. During experimental infection of C57BL/6 (H-2^b) mice, the majority (30–40%) of the CD8⁺ T cell response is specific for two immunodominant TS epitopes: TSKb20 (ANYKFTLV) and TSKb18 (ANYDFTLV) [15]. However, neither of these CD8⁺ T cell populations are required for immune control or survival of infection [29, 30], implying a minimal role, if any, for these T cells in control of the infection.

The dispensability of the TSKb18/TSKb20-specific responses suggests that alternative CD8⁺ T cell responses may be mediating immune control of infection. Subdominant CD8⁺ T cell responses have been reported to target H-2^b epitopes within TS [15–18, 21], cruzipain [15], β-galactofuranosyl transferase [15], LYT-1 [31], and flagellar [32, 33] proteins, though the low frequency and variability across individuals complicates efforts to study these responses in depth. In this study, we sought to define and explore the role of other, non-TS-specific CD8⁺ T cells in immune control. In the search for such responses, we discovered a novel epitope MUCKb25 (SAWVCAPL) derived from mucin family proteins, the third most expanded T. cruzi gene family comprised of >500 genes [34]. Similar to TS responses, the MUCKb25 response was found to be non-protective and dispensable, indicating that the majority (50–60%) of the T. cruzi-specific CD8⁺ T cell compartment is comprised of T cells with suboptimal effector capacity. Overall, our findings emphasize the importance in understanding if and how T. cruzi-specific CD8⁺ T cells mediate infection control prior to committing to particular epitope-specific T cells as reliable indicators of anti-parasite immunity or feasible vaccine candidates.

Material and Methods

Mice and parasites

C57BL/6J mice (CD45.2+) and C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J (Nur77-GFP) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The SKH-1 ‘hairless’ mice backcrossed to C57BL/6 were a generous gift from Dr. Lisa DeLouise (University of Rochester). TSKb20/18 Tg mice were maintained from the original generation as previously described [30]. All strains of mice were bred and maintained under specific pathogen-free conditions in the animal facility in Coverdell at the University of Georgia. All mice were euthanized by CO₂.

For T. cruzi infections, 8–12 week old mice were infected via intraperitoneal (ip) injection or footpad (fp) injection of indicated doses of Brazil, CL, Colombiana, or Y strain trypomastigotes derived from passage through Vero cell (ATCC) cultures. Luciferase expressing Colombiana parasites were previously generated by transfection with a pTREX- plasmid containing red-shifted firefly luciferase [35].

Ethics statement

All animal use was performed in accordance with protocol A2015 05–010 and A2018 06–010 approved by the University of Georgia Institutional Animal Care and Use Committee. This protocol adhered to the animal welfare guidelines outlined in Guide for the Care and Use of Laboratory Animals.

Cell culture

Vero and MC57G fibrosarcoma cells were both purchased from ATCC and cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and with 2% penicillin-streptomycin. Cultures were maintained at 37°C with 5% CO₂ in a humidified incubator and tested periodically for mycoplasma contamination.

Peptides

The genome wide set of H2-Db and H2-Kb binding peptides (8–11mer) were predicted using a combination of NetMHC4.0/NetMHCpan4.0 algorithms [36–38] with input protein sequences retrieved from the TriTrypDB [39] using the reference strain. Any peptides from proteins annotated as trans-sia* were filtered from the list and priority was given to remaining peptides with high genome occurrence (>100 occurrences) and high predicted binding affinity (IC50 <100 nM). Endogenous peptide variants of MUCKb25 were identified through manual alignment using Geneious Prime 2020.1.2 of the C-termini of all mucin proteins identified in the Brazil A4 genome in TriTrypDB. Peptides with predicted IC50 values <200 nM were prioritized. Peptides were synthesized by Synthetic Biomolecules (San Diego, CA) or GenScript (Piscataway, NJ). Lyophilized peptide was dissolved initially in DMSO and used to generate working 1 mM peptide stocks in 1xPBS, stored at −20°C.

ELISPOT

ELISPOT assays to measure IFN-γ secretion were performed as guided by the manufacturer’s instructions (BD Biosciences, San Jose CA). Briefly, single-cell mouse splenocyte suspensions were prepared and plated (2×10⁶ per well) then incubated for 16–20 h with indicated peptide concentrations or 2.5 ug/mL of anti-mouse CD3e antibody (145–2C11 clone, eBioscience) in nitrocellulose-bottomed ELISPOT plates (S2EM004M99, Millipore) coated with capture anti-mouse IFN-γ antibody (BD Biosciences). After overnight stimulation and washing, wells were incubated with detection biotinylated-anti-mouse IFN-γ antibody (BD Biosciences), streptavidin-HRP (BD Biosciences) and AEC substrate (Sigma-Aldrich). Images of developed plates were captured using a CTL Analyzer and spots counted using the Immunospot Software (CTL).

T cell phenotyping

CD8⁺ T cell specificity was determined by staining with MHC class I tetramers: BV421-conjugated TSKb20 (ANYKFTLV/Kb), PE-conjugated TSKb18 (ANYDFTLV/Kb), APC-conjugated MUCKb25 (SAWVCAPL/Kb) synthesized at the Tetramer Core Facility (Emory University, Atlanta, GA) and anti-mouse CD8a antibodies conjugated with APC or PeCy7 (53–6.7, Biolegend). RBCs in single cell suspensions of spleen cells or peripheral blood were lysed in a hypotonic ammonium chloride solution then washed 1x with PAB (1% BSA, 0.02% sodium azide in 1xPBS). Cells were stained for at least 30 min at 4°C and washed in PAB. At least 500,000 events or the maximum allowed in limiting samples were acquired using a CyAn (Beckman Coulter) or Quanteon (Agilent) flow cytometer and analyzed with FlowJo software v10.7.1.

Nur77-GFP T cell activation

In most assays, splenocytes (2×10⁶ cells per well) from naïve or infected Nur77-GFP mice were stimulated directly with indicated peptide concentrations and incubated overnight. For fibroblast peptide presentation, MC57G cells were plated at 150,000 cells/well in 96-well flat bottom plates for at least 1 hour before pre-loading with peptide for 3 hours then splenocytes from infected Nur77-GFP (2×10⁶ cells per well) were added and co-incubated for at least 16 hours.

RMAS MHC-I stabilization assay

TAP-deficient RMAS cells were a kind gift from Dr. Mark Tompkins at University of Georgia. To increase MHC-I surface expression, cells were incubated overnight at room temperature as described previously [40]. Cells were washed, resuspended in reduced serum Opti-MEM I media (Thermo Fisher Scientific), then plated at 500,000 cells per well. Varying concentrations of peptide or no peptide was added to the cells then incubated for 3 h at room temperature then 2 h at 37°C. Cells were washed 1x then stained in PAB with anti-mouse antibodies: H2-Kb-ef450 (AF6-88.5.5.3, eBioscience) and H2-Db-FITC (28-14-8, Thermo Fisher Scientific).

Sequence logo of MUCKb25-epitope containing mucins

Protein sequences containing the MUCKb25 epitope sequence ‘SAWVCAPL’ were identified and extracted from the Brazil A4 genome in the TriTrypDB [41], then aligned using Geneious Prime (version 2020.1.2) to generate a sequence logo.

Dendritic cell vaccinations

Epitope-specific CD8⁺ T cells were generated by dendritic cell vaccination as described previously. Briefly, dendritic cells (DCs) were derived from hematopoietic stem cells of the bone marrow via supplementation with 20 ng/mL recombinant murine GM-CSF (PeproTech) in media. C57BL/6 or ‘hairless’ mice were vaccinated with dendritic cells that were previously activated overnight with 100 ng/mL LPS (Sigma-Aldrich), then washed and loaded with at least 10 uM of peptide in reduced serum media Opti-MEM I for at least 1 h at 37°C. Loaded DCs were washed 1x then resuspended in a final volume of 1xPBS for intravenous injection (iv) of 300,000–500,000 cells per mouse. Mice were boosted 5 days post vaccination with syngeneic splenocytes loaded with at least 10 uM of respective peptides. At least 5–8 days post boost, presence of specific CD8⁺ T cells was confirmed through the staining of lymphocytes prepared from the blood of vaccinated mice.

In vivo imaging and quantification of parasite growth

Mice were infected with Colombiana-luciferase parasites intraperitoneally or at the hind-footpads. For bioluminescent detection, mice were injected intraperitoneally with D-luciferin (150 mg/kg; PerkinElmer) and anesthetized using 2.5% (vol/vol) gaseous isofluorane in oxygen prior to imaging, 13 minutes post-substrate injection on IVIS 100 imager (Xenogen). Quantification of bioluminescence and data analysis was performed using Living Image v4.3 software (Xenogen).

Quantification of parasite burden

T. cruzi parasite equivalents in skeletal muscle were determined by real-time PCR as previously described [42].

Peptide tolerance

SIINFEKL and M12 (SAWVFAPL) peptides were synthesized by Genscript (Piscataway, NJ) and initially reconstituted in DMSO at 100 mg/mL then stored at −20°C. Each peptide was diluted to the desired concentration in 1xPBS for iv injection of 100 uL per mouse. Treated mice received an initial higher dose (600 μg) of peptide one week prior to infection and lower doses (200 μg) at 4 days and 1 day before infection. After infection, mice were given weekly doses (200 μg) until the end of the experiment.

Assessment of T cell recognition of T. cruzi-infected cells

Bone marrow progenitor cells were cultured in complete DMEM supplemented with 10% FBS in the presence of 10 ng/ml of recombinant murine M-CSF (Peprotech) for 6 days to obtain bone marrow derived macrophages (BMDMs). In some experiments, BMDMs were treated with indicated concentrations of recombinant murine IFN-γ (Peprotech) prior to and following infection. For the infection process, T. cruzi trypomastigotes were added to 8 ×10⁴ MC57G fibroblasts or BMDMs, at a parasite to cell ratio of 10:1 or 5:1 respectively, for 3 hours then washed off. Splenocytes or CD8+ T cells isolated using negative selection through magnetic sorting with a CD8a+ T cell isolation kit (Miltenyi) from infected Nur77-GFP mice were co-incubated for the final 16 hours with MC57G or BMDMs that had been infected for various lengths of time with T. cruzi or with corresponding peptides as controls. At the end of the 16 hour co-incubation, CD8⁺ tetramer binding cells were evaluated for Nur77-GFP expression.

Benznidazole preparation and treatment

Benznidazole (BNZ – Elea Phoenix, Buenos Aires, Argentina) was prepared by pulverization of tablets followed by suspension in an aqueous solution of 1% sodium carboxymethylcellulose with 0.1% Tween-80. Indicated doses of BNZ were delivered by oral gavage with each mouse receiving 200 uL of suspension.

Statistical analysis

Data are presented as the mean ± the standard error of mean (SEM). Statistically significant differences between groups were identified by two-way ANOVA multiple comparisons using GraphPad Prism version 9.0.0 software. Only p values less than or equal to 0.05 were considered statistically significant.

Results

Novel mucin epitope elicits strain-dependent immunodominant CD8⁺ T cell responses in T. cruzi-infected mice

To identify potentially protective epitopes outside of the TS family of proteins, we performed a genome-wide search for non-TS epitope sequences (8–11mer) with high occurrence and strong predicted binding affinity to MHC-I proteins H2-Kb/H2-Db (see Materials and Methods), two factors known be important for the generation of CD8⁺ T cell responses [43].

A representative set of 33 peptides was curated (Table I) and each peptide was tested individually for the ability to elicit IFN-γ cytokine production upon direct ex vivo stimulation of splenocytes from mice chronically infected with T. cruzi (Supp Fig 1A). Interestingly, only 1 peptide, MUCKb25 (SAWVCAPLL), from the mucin large gene family, induced IFN-γ production in a significant number of spleen cells, at a level similar to that of TSKb20 (Fig 1A). To confirm that the MUCKb25 peptide is activating CD8⁺ T cells, we stimulated splenocytes from infected Nur77-GFP reporter mice, which have been previously shown to be specific reporters of TCR stimulation and signaling in T cells [44]. Approximately 10% of CD8⁺ T cells from infected mice expressed Nur77-GFP upon stimulation with MUCKb25 peptide (Fig 1B), providing additional evidence that a large MUCKb25-specific CD8⁺ T cell response is generated and maintained during T. cruzi infection.

Table I.

List of genome-wide T. cruzi peptides selected based on occurrence and predicted binding affinity to MHC-I

Peptide sequence	Name	Predicted allele	Gene ID	Predicted IC50 (nM)	Occurrence score	Gene/gene family
RMMCNISVPI	G1	H-2-Db	TcCLB.506217.20	21.9	144	Beta galactofuranosyl glycosyltransferase
RVYGWSGRL	G2	H-2-Kb	TcCLB.506217.20	75.7	181	Beta galactofuranosyl glycosyltransferase
SSYIYYNSWL	G3	H-2-Kb	TcCLB.509949.9	6.7	180	DGF
FSIQNSSWI	G4	H-2-Db	TcCLB.504237.10	7.3	142	DGF
FALLNNTML	G5	H-2-Db	TcCLB.507821.70	7.8	119	DGF
FSVTNVSVV	G6	H-2-Db	TcCLB.508517.9	14.9	1315	DGF
VALPYFLSV	G7	H-2-Kb	TcCLB.510499.10	15.3	1268	DGF
YAVAFYSTV	G8	H-2-Kb	TcCLB.416809.10	29.8	2631	DGF
VQYFCMAAV	G9	H-2-Kb	TcCLB.509427.30	77.5	995	DGF
VAFYSTVSL	G10	H-2-Kb	TcCLB.416809.10	49.4	945	DGF
AMLSNATWV	G11	H-2-Db	TcCLB.503475.10	47.6	860	DGF
HALVNMTNV	G12	H-2-Db	TcCLB.418113.9	15.8	832	DGF
YAILNANYL	G13	H-2-Db	TcCLB.506169.10	2.3	104	Glycine dehydrogenase [decarboxylating]
VGAGYYTAL	G14	H-2-Kb	TcCLB.508009.10	13	144	GP63
VSMLLLLGL	G15	H-2-Kb	TcCLB.504755.10	61.8	105	GP63
SVLAYFTLL	G16	H-2-Kb	TcCLB.510547.46	7	148	Hypothetical protein
VSYALKHPI	G17	H-2-Kb	TcCLB.505935.10	38.7	114	Hypothetical protein
TVALFVASL	G18	H-2-Kb	TcCLB.508293.17	40.2	123	Hypothetical protein
VLLPFTGXL	G19	H-2-Kb	TcCLB.510547.46	63.8	142	Hypothetical protein
FSVGNVTLV	G20	H-2-Db	TcCLB.507691.10	72.9	156	Hypothetical protein
IVFDGVSSL	G21	H-2-Kb	TcCLB.508023.12	98.2	107	Hypothetical protein
YMFLNWHELL	G22	H-2-Db	TcCLB.511183.26	35.1	108	MASP
VSALAYTTL	G23	H-2-Kb	TcCLB.504669.50	30.9	188	Mucin TcMUCI
SALAYTAV	G24	H-2-Kb	TcCLB.511099.23	55	104	Mucin TcMUCI
SSAWVCAPL	G25	H-2-Kb	TcCLB.506909.20	99.9	1987	Mucin TcMUCI
SSYLLYQLL	G26	H-2-Kb	TcCLB.506823.80	5.5	105	RHS
FAFLNGSFV	G27	H-2-Db	TcCLB.507497.20	6.9	167	RHS
VAMVFXGSV	G28	H-2-Kb	TcCLB.506803.30	14	176	RHS
AAYFNGWEEL	G29	H-2-Db	TcCLB.503661.20	66.9	250	RHS
AVSAFLNEDFL	G30	H-2-Db	TcCLB.511549.10	74.5	150	RHS
AGSYLLYQLL	G31	H-2-Kb	TcCLB.470827.10	86.7	463	RHS
FIFRNTVYM	G32	H-2-Db	TcCLB.507153.40	29.8	172	Tryptophanyl-tRNA synthetase
RVPLFVAHL	G33	H-2-Kb	TcCLB.504779.10	22.2	162	UDP-Gal or UDP-GlcNAc-dependent glycosyltransferase

Figure 1.

Novel mucin epitope elicits strain-dependent immunodominant CD8⁺ T cell responses in T. cruzi-infected mice

(A) Representative wells of IFN-γ ELISPOT assay with splenocytes from naïve or T. cruzi Brazil strain-infected (61 dpi) C57BL/6 mice re-stimulated for 18 hrs. Bar graph indicates the average number of spots counted per well for each group. (B) Representative flow plots show Nur77-GFP expression and TSKb20 tetramer staining of CD8⁺ T cells from spleens of infected (68 dpi) Nur77-GFP mice after 16 h stimulation with indicated peptides. Summary data of the percentages of CD8⁺ T cells expressing Nur77-GFP are displayed graphically to the right. (C) Line graph shows the %MFI increase in surface expression of H2-Kb in RMAS cells incubated with indicated concentrations of peptide that was calculated based on the MFI of H2-Kb signal detected by antibody staining in peptide-loaded vs unloaded cells. (D) Flow plots show Nur77-GFP expression combined with TSKb20 or MUCKb25 tetramer staining of CD8⁺ cells from spleens of T. cruzi Brazil-strain infected (50 dpi) Nur77-GFP mice incubated for 16 hrs with MC57G fibroblasts pre-loaded with indicated concentrations of peptide for 3 hrs. (E) Dual line graphs show the kinetics of T cell activation (%Nur77-GFP⁺ of CD8⁺ cells) and tetramer binding (% of specific CD8⁺ cells bound to tetramer) of splenocytes from infected (33 dpi) Nur77-GFP mouse upon stimulation with various concentrations of TSKb20 peptide (top) or MUCKb25 peptide (bottom), performed as described in Fig 1D. (F) Representative flow plots of MUCKb25 or TSKb20 tetramer binding CD8⁺ cells in the circulation of C57BL/6 mice 30 dpi with either Brazil, CL, or Y strain parasites. Bar graphs indicate the average percentage of MUCKb25 or TSKb20 tetramer binding of CD8⁺ cells. (G) Line graph shows the kinetics of epitope specific T cell responses during infection with T. cruzi Brazil strain represented as the average % CD8⁺ tetramer⁺ cells detected in the circulation. All data are representative of at least two independent experiments with n=3–10 with the exception of panels F-G, representative of 1 experiment with n=3 and n=10, respectively. Data is represented as mean + SEM. * indicates percentage levels that are significantly different (*P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001) from the SIINFEKL group, unless otherwise stated.

Since the 9mer MUCKb25 peptide contains an 8mer peptide with a higher predicted binding affinity to MHC-I (H2-Kb) (IC50 45.5 nM), we tested and confirmed the minimal epitope to be the 8mer (SAWVCAPL) (Fig 1C) (Supp Fig 1B), hereafter referred to as MUCKb25. MHC-I stabilization assays using RMAS cells indicated that the MUCKb25 peptide is restricted to H2-Kb and binds with a significantly lower affinity compared to strong H2-Kb binders SIINFEKL (from ovalbumin) and TSKb20 (Fig 1C). Further supporting this, MUCKb25-specific CD8⁺ T cells were only able to recognize fibroblasts pulsed with high concentrations (≥ nM) of MUCKb25 peptide, as demonstrated through TCR stimulation via Nur77-GFP expression of MUCKb25 tetramer binding cells (Fig 1D). In comparison, TSKb20-specific CD8⁺ T cells recognized and expressed Nur77-GFP in response to fibroblasts pulsed with peptide concentrations in the pM range, implying that the binding and stability of TSKb20 peptide to MHC-I is greater than MUCKb25, though differences in TCR affinity could also contribute to these differences in T cell recognition. There were also noticeable differences between TSKb20- and MUCKb25-specific T cells in terms of the concentration of peptide that triggered TCR downregulation (Fig 1D–E). TCR downregulation of TSKb20-specific CD8⁺ T cells was detectable in conditions with peptide concentrations as low as 100 pM whereas MUCKb25-specific CD8⁺ T cells required at least 10 nM peptide for TCR downregulation.

Interestingly, the MUCKb25 epitope is located within predicted GPI signal sequences (SS) at the C termini of mucin proteins, downstream of the ω site (Supp Fig 1C), a reported cleavage and GPI anchor addition site in other trypanosomes [45]. This location likely accounts for the high conservation of the epitope sequence in many, otherwise highly variable, mucin sequences across T. cruzi strains irrespective of genetic type (Table II). However, in contrast to the CL and Brazil strains, mice infected with the Y strain of T. cruzi failed to generate detectable MUCKb25-specific responses (Fig 1F), despite having a high number of mucins containing this sequence (Table II). Restimulation of splenocytes from Y strain-infected mice with the MUCKb25 peptide also failed to elicit IFN-γ production (Supp Fig 1D), further demonstrating the lack of a MUCKb25-specific response. Examination of other lab-adapted (Columbiana (TcI) (Supp Fig 1E) and recent field isolates [46] (for which genome sequencing has not been conducted) revealed that this absence of a MUCKb25 response was not unique to the Y strain, as isolate 20392 similarly did not prime a MUCKb25 response (Sup Fig 1F–G).

Table II.

List of identified endogenous MUCK peptide variants and their presence within mucin proteins across T. cruzi strains

Peptide information				# of mucin proteins containing peptide sequence within each T. cruzi strain
Sequence	Peptide name	Predicted IC50 (nM)	Response	Brazil A4 (TcI)	CL Brener Esmeraldo-like (TcVI)	CL Brener non-Esmeraldo-like (TcVI)	Y C6 (TcII)
SAWVCAPL	MUCKb25	45.5	+	223	206	204	415
SAWMCAPL	M1	31.8	+	8	3	1	8
SAWLCAPL	M2	36.8	+	1	0	2	0
SAWVCARL	M3	42.2	+	1	0	1	0
SVWVCAPL	M4	50.2	+	8	1	2	1
SAWVCSPL	M5	58	+	2	6	1	6
SAWVCASL	M6	62.6	+	11	1	11	4
SAWVCTPL	M7	71.1	+	2	3	0	5
SAWVCATL	M8	74.8	+	2	4	7	5
SAWACALL	M9	94.1	+	2	0	0	0
SAWVSCLL	M10	119.6	-	5	1	2	1
SAWVCVPL	M11	125.3	+	8	4	0	1
SAWVFAPL	M12	4.3	+	11	7	3	15
SAWVYAPL	M13	4.6	+	1	0	0	0
SAWRVAPL	M14	28.2	+	1	0	0	0
SAWACAPL	M15	49.8	+	10	11	6	8
SAWVLPPL	M16	52.9	+	1	0	0	0
SAWVRASL	M17	57.2	+	2	0	0	0
SALVCAPL	M18	72.3	+	1	2	0	2
STWMCVPL	M19	80.4	+	1	0	0	0
SAWMCVPL	M20	83.4	+	1	0	0	0
SAWVCALL	M21	84.4	+	1	2	0	4
FAWVCAPL	M22	123.7	+	3	7	3	12
FTWVCAPL	M23	133.9	+	3	0	0	0
AAWVCALL	M24	168.6	+	1	0	0	0

As previously reported, the immunodominant TSKb18 and TSKb20-specific CD8⁺ T cell responses were detectable beginning at 9–10 days post-infection (dpi), peaking at ~20 dpi before contracting to a low but detectable population in the chronic phase (Fig 1G) [15]. However, MUCKb25-specific CD8⁺ T cells were not detected until 15–16 dpi then plateaued at approximately 26 dpi (while the TS-specific responses were contracting) and maintained this plateau until >60 days post-infection. The kinetics of the MUCKb25-specific response were not impacted by the presence of the TS-specific T cells, as mice that lack both TSKb18 and TSKb20-specific CD8⁺ T cells, due to expression of these epitopes in a transgene (TS18/TS20 Tg [30]), showed the same pattern of response as WT mice (Supp Fig 1H). Collectively, these results demonstrate that a conserved mucin peptide within a GPI SS primes a delayed CD8⁺ T cell response that varies in presence and magnitude depending on the infecting parasite strain.

MUCKb25-specific CD8⁺ T cells cross-react with MUCK epitope variants

Although the MUCKb25 sequence is by far the predominant sequence in this region of the mucin GPI SS in all T. cruzi strains examined, a number of variants of this sequence are also present at low frequency (Table II). Given the relatively low affinity of MUCKb25 for H2-Kb, we considered that one or more of these variants could be responsible for driving this robust response. To explore this, a set of 24 of these peptide variants was assayed for the ability to induce IFN-γ production by splenocytes from chronically infected mice. The majority of variants (21/24) activated T cells from chronically infected mice, although to varying extents, with several variants (M4, M11–14) eliciting recall IFN-γ responses (Fig 2A) and Nur77-GFP expression in CD8⁺ T cells (Supp Fig 2A) at a magnitude similar to that of MUCKb25. Staining of the splenocytes from infected Nur77-GFP mice with the MUCKb25 tetramer confirmed that the MUCKb25-specific CD8⁺ T cells were cross-reactive with these variants (Fig 2B). The variant peptides elicited a variety of response patterns (Fig 2B–D) (Supp Fig 2B) but notably several variants (M4, M12, M13) strongly activated Nur77-GFP expression (Fig 2B–C) in all MUCKb25-specific CD8⁺ T cells with substantial TCR downregulation (Fig 2D).

Figure 2.

MUCKb25-specific CD8⁺ T cells recognize MUCK epitope variants

(A) Bar graph representing the average number of spots per well in an IFN-γ ELISPOT assay using splenocytes from naïve or Brazil strain infected C57BL/6 mice (98 dpi) restimulated with the indicated peptides for 18 hrs. Groups marked as significant were statistically different from media group. (B) Selected flow plots show the gating strategy used to define the %MUCKb25 tetramer⁺ (black) and %Nur77-GFP⁺ (grey) of CD8⁺ cells upon re-stimulation of splenocytes from infected Nur77-GFP mice with each variant peptide for 16h. (C) Bar graph shows the change in the % Nur77-GFP⁺ cells of CD8⁺ cells (with media set to 0), performed as described in B. (D) Bar graph depicts the % of CD8+ cells that remain bound to MUCKb25 tetramer upon stimulation with peptide, performed as described in B. (E) Line graph shows the %MFI increase in surface expression of H2-Kb in RMAS cells calculated based on the MFI of H2-Kb signal detected by antibody staining in peptide-loaded vs unloaded cells. Groups marked as significant were statistically different from SIINFEKL group. (F) Flow plots depict Nur77-GFP expression combined with MUCKb25 tetramer binding of CD8⁺ cells from spleens of infected (82 dpi) Nur77-GFP mice incubated for 16 hrs with MC57G fibroblasts pre-loaded with indicated concentrations of peptide for 3 hrs. All data are representative of at least two independent experiments with n=3. Data is represented as mean + SEM. * indicates percentage levels that are significantly different (*P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001) between groups.

Some of these same variants (e.g. M4, M12, and M13) were predicted to bind to H2-Kb with high affinity (Table II) and direct testing of this capacity using the RMA-S MHC-I binding assay revealed that, indeed, variants M12 and M13 stabilized H2-Kb to a similar extent as the known strong H2-Kb binders SIINFEKL and TSKb20, while variant M4 and MUCKb25 showed significantly less stabilization overall (Fig 2E). Comparison of T cell responsiveness to fibroblasts pulsed with M12 or MUCKb25 peptides demonstrated that the M12 variant triggered T cell activation (Nur77-GFP) and TCR downregulation (reduced tetramer binding) in the pM range compared to MUCKb25 which only operated in the nM-μM range (Fig 2F and Supp Fig 2C). Thus, a number of MUCKb25 variants present at low frequency in the mucin gene family are better MHC-I binders and trigger specific CD8⁺ T cell recognition at low, physiological concentrations.

Mucin-specific CD8⁺ T cells have minimal impact on infection outcomes

To assess the immune protective capacity of mucin-specific CD8⁺ T cells, we first attempted without success to generate a high frequency of MUCKb25-specific T cells via vaccination with peptide-loaded bone marrow-derived dendritic cells (BMDCs) (Supp Fig 3A). However, the M12 variant induced frequencies of MUCKb25 tetramer-binding T cells that were intermediate between the TSKb20 and OVA SIINFEKL peptides (Fig 3A). Groups of mice were vaccinated with peptide-loaded (SIINFEKL, TSKb20, or M12) BMDCs then boosted with peptide-loaded splenocytes and rested for 6 weeks prior to challenge in the footpads with luciferase-expressing T. cruzi (Fig 3B). Chronically infected mice were re-infected alongside these groups to put into direct context the level of protection that is achieved in mice with on-going T. cruzi-specific effector/memory responses compared to that of vaccinated mice with single epitope-specific CD8⁺ T cell responses.

Figure 3.

Mucin-specific CD8⁺ T cells generated via vaccination minimally participate in early immune control and fail to impact parasite persistence

(A) Bar graphs show the % of CD8⁺ T cells binding a specific tetramer found within the circulation of vaccinated mice 8 days (left) and 38 days (right) after the boost (dpb). (B) Timeline of vaccination strategy and parasite challenge in mice. C57BL/6 mice were vaccinated with peptide-loaded dendritic cells derived from progenitors in the bone marrow injected intravenously (iv) and boosted in the same route 5 days later with congenic splenocytes loaded with respective peptide. Six weeks after the boost, mice were challenged in each footpad with 25,000 Colombiana-luciferase expressing parasites and imaged for luciferase expression every 2–4 days for 18 days. Mice with existing chronic infection with Brazil-strain parasites served as a positive control for resistance to re-infection. (C-D) Line graphs indicates the % of CD8⁺ T cells binding TSKb20 tetramer or MUCKb25 tetramer within the circulation of mice during challenge infection. (E) Representative images show luciferase expression in mouse footpads of each group after challenge infection, including previously naïve, unvaccinated mice (PBS) and chronically infected mice (re-infected) in comparison to vaccinated groups SIINFEKL, TSKb20, and M12. (F) Line graph depicts the overall kinetics of luciferase expression within the footpads of each group after challenge infection. (G) Scatter plot shows the quantification of parasite load by qPCR of the skeletal muscle of challenged mice at 106 dpi. The dotted line represents the limit of detection for qPCR. All data are representative of at least two independent experiments with n=5–6. Data is represented as mean + SEM. * indicates percentage levels that are significantly different (*P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001) or not (ns) between groups.

In the case of both TSKb20 and MUCKb25, the vaccine-induced CD8⁺ T cell responses to these epitopes were boosted by the challenge infection (Fig 3C–D). However, in contrast to the rapid and marked control of the footpad infection in mice with an ongoing chronic infection, the M12 and TSKb20-vaccinated mice demonstrated minimal (but statistically significant) reductions in parasite load only at 10 dpi, as compared to the negative control SIINFEKL-vaccinated mice (Fig 3E–F). Additionally, there was no long-term impact of vaccination on parasite load and persistence (Fig 3G).

To determine if MUCKb25-specific T cells generated during the course of infection impacted infection control, we tolerized mice with weekly high doses of peptides M12 or SIINFEKL [29] prior to and following infection with luciferase-expressing T. cruzi (Fig 4A). The absence of epitope-specific CD8⁺ T cells was confirmed in M12-tolerized mice by lack of MUCKb25 tetramer staining of cells in the circulation at 29 dpi (Fig 4B). Importantly, there was no significant impact of the tolerization protocol on TSKb20-specific CD8⁺ T cells as well as the percentage of overall CD8⁺ cells (Fig 4C). Systemic monitoring of parasite load demonstrated a transient reduction in parasite load at 30 dpi in the M12-tolerized group but an otherwise unimpacted parasite load profile in the tolerized mice (Fig 4D). Collectively, these results demonstrate that pre-primed TSKb20 and MUCKb25-specific CD8⁺ T cells minimally impact both early and long-term control of T. cruzi infection and that MUCKb25-specific CD8⁺ T cells are dispensable for overall control of T. cruzi infection.

Figure 4.

Lack of MUCK-specific CD8⁺ T cells does not impact overall control of T. cruzi infection

(A) Protocol used to induce epitope-specific tolerance using high dose peptide administration. Mice were injected (iv) with an initial high dose (600 ug) of peptide then lower doses (200 ug) at days 4 and 1 prior to infection with 100,000 Colombiana-luciferase expressing parasites (ip). After infection, mice were injected weekly with maintenance doses (200 ug) and monitored for parasite load based on luciferase expression at 5 day intervals. (B) Representative flow plots of MUCKb25 or TSKb20 tetramer binding of CD8⁺ cells from the circulation of each group of tolerized mice at 29 dpi after infection. Bar graph indicates summary of percentage of tetramer⁺ cells of CD8⁺ cells. (C) Bar graph indicates summary of percentage of CD8⁺ cells in the circulation of tolerized groups of mice at 29 dpi. (D) Representative images (left) of luciferase expression seen in infected mice. Line graph (right) shows the control of parasite load based on total flux (p/s) of luciferase signal in the whole body of infected mice. All data are representative of at least two independent experiments with n=5–6. Data is represented as mean + SEM. * indicates percentage levels that are significantly different (** P≤0.01, **** P≤0.0001) between groups.

MUCKb25 epitope availability limited during in vitro infection of host cells

The relative lack of impact of MUCKb25-specific T cells on infection control as well as the location of the MUCKb25 epitope within the GPI SS of immature mucin proteins prompts questions concerning the accessibility for T cell recognition of this epitope in infected host cells, as these SS regions are likely not part of the mature mucin proteins. To assess the recognition of MUCKb25 epitopes by infected host cells, splenocytes from infected mice were exposed to fibroblasts infected with T. cruzi for various periods and epitope recognition was assessed by Nur77-GFP expression by tetramer-binding T cells. Similar to overall CD8⁺ T cells (Supp Fig 4A–B), TSKb20-specific T cells were activated as early as 16 hours after host cell infection with the peak of recognition occurring at 96 hours (Fig 5A–B), a timepoint when the intracellular parasite replication cycle is being completed and parasites (along with presumably cytoplasmic debris from infected cells) are being released. In contrast, MUCKb25-specific T cells demonstrated very little or no recognition at all timepoints except 96 hours, implying that the MUCKb25 epitope is not available for direct presentation by infected cells.

Figure 5.

MUCKb25 epitope availability limited during in vitro infection of host cells

(A) Representative flow plots show the Nur77-GFP expression of either TSKb20 or MUCKb25-tetramer binding CD8⁺ cells of splenocytes from a chronically infected Nur77-GFP mouse restimulated with murine fibroblasts infected with T. cruzi Brazil strain parasites for the indicated lengths of time. Restimulation with respective peptide served as controls. (B) Line graph shows the average kinetics of TSKb20 or MUCKb25 T cell recognition, performed as described in A. (C) Line graphs show the kinetics of TSKb20 (left) and MUCKb25 (right) T cell responses represented as % tetramer⁺ of CD8⁺ cells upon treatment with a single oral dose (dosage as indicated) of benznidazole (BNZ) at 10 dpi. (D) Example flow plots show T cell recognition measured by Nur77-GFP expression of TSKb20 or MUCKb25 tetramer binding CD8⁺ cells from splenocytes of infected Nur77-GFP mouse incubated with BMDMs that were pre-treated overnight with indicated concentrations of recombinant IFN-g before infection with Brazil strain parasites (MOI: 5), then left for 8 h prior to a 16 h incubation with splenocytes from a Nur77-GFP infected mouse. (E) Bar graphs show summary of data described in D. All data are representative of 2 independent experiments with at least n=3, except C which is representative of 1 experiment with n=5–6 and D-E, representative of 1 experiment. Data is represented as mean + SEM. * indicates percentage levels that are significantly different (*P≤0.05, *** P≤0.001, **** P≤0.0001) between groups.

As overall CD8⁺ T cell recognition of T. cruzi-infected fibroblasts was relatively low, we next assessed the recognition of infected bone marrow-derived macrophages (BMDMs). TSKb20-specific T cells and overall CD8⁺ T cells demonstrated a similar kinetic of recognition of infected BMDMs, compared to fibroblasts, with a higher level of recognition at both early (16–24 hours) and late timepoints (72 hours) (Supp Fig 4C), though the level of T cell recognition remained relatively low. MUCKb25-specific T cells, however, demonstrated recognition only at later timepoints (≥64 hours), similar to the recognition of fibroblasts, supporting that the availability of the MUCKb25 epitope is restricted and not directly presented by infected cells.

The absence of a MUCKb25 response during Y strain infection is likely due to a lack of availability of the MUCKb25 epitope for MHC-I presentation. Indeed, MUCKb25-specific T cells did not recognize Y-strain infected fibroblasts at any of the timepoints assayed (Supp Fig 4D), including 96 hours, confirming that the MUCKb25 epitope is likely not presented by MHC-I during infection. Interestingly, Y-strain infected fibroblasts were poorly, if at all, recognized by either TSKb20-specific T cells or overall CD8⁺ T cells (Supp Fig 4E) prior to 72 hours, unlike Brazil strain-infected cells (Supp Fig 4D–E), highlighting distinct, strain-dependent differences in T cell recognition of T. cruzi-infected host cells.

Due to its location within GPI SS and lack of presentation during direct infection, we hypothesized that the MUCKb25 epitope may be predominantly internal to the parasite and only made available to the immune system upon parasite death. To explore if enhanced parasite death might potentiate the MUCKb25-specific T cell response we treated groups of infected mice at 10 dpi with various doses of the anti-trypanosomal compound benznidazole (BNZ) [42]. However, the timing of MUCKb25 T cell priming was unaffected by BNZ treatment, even at suboptimal doses, and instead, all treated groups had a significantly reduced MUCKb25-specific CD8⁺ T cell response by 17 dpi (Fig 5C). In contrast, the TSKb20-specific response, which is initiated prior to day 10, was largely unaffected by the BNZ treatment. Additionally, we pre-treated in vitro cultures of BMDMs with IFN-γ prior to infection to induce enhanced parasite killing, as described previously [47, 48], and found MUCKb25 T cell recognition of the infected BMDMs also to be unchanged (Fig 5D–E). Thus, facilitated destruction of T. cruzi failed to enhance MUCKb25 T cell responsiveness.

Discussion

CD8⁺ T cells mediate immune control of T. cruzi, yet the antigen specificity of the T cells involved in this control remains elusive. The dispensability of immunodominant TS-specific responses in C57BL/6 mice [29, 30] implies that T cells of other, undefined specificities may be critical to immune control. As such, the primary goal of this study was to identify alternative targets of T. cruzi-specific CD8⁺ T cells and to assess their role in immune control of infection.

Here, we have identified a novel mucin-specific CD8⁺ T cell response through the screening of a representative set of epitope sequences occurring with high frequency (e.g. multiple genes) in the T. cruzi genome. Like the previously described TS-specific CD8⁺ T cells [15], these MUCKb25-specific CD8⁺ T cells occur at a very high frequency in this model, although with distinct kinetics relative to the TS-specific CD8⁺ T cells. The immunodominance of these three (MUCKb25, TSKb18, and TSKb20) epitope-specific T cell responses clearly indicates that epitopes from large gene family proteins are the major targets of the T. cruzi-specific CD8⁺ T cells induced during infection, accounting for 50–60% of all CD8⁺ T cells in these mice. The dominant focus of CD8⁺ T cell immunity on these epitopes is perhaps not surprising, given that these epitopes and their variants are present in the several hundred (mucin) and several thousand (TS) proteins encoded by these families of genes dispersed throughout the T. cruzi genome. What is surprising is that, despite the combined space in the T cell compartment occupied by TS- and mucin-specific T cells, the abolition of these responses has essentially no impact on long-term parasite control (herein and [29, 30]). Furthermore, vaccination to induce these mucin-specific T cells failed to boost resistance to infection in naïve mice. Thus, in this infection, substantial resources appear to be committed to expanding and maintaining, as a majority of the CD8 T cell compartment, pathogen-specific T cells with seemingly limited anti-parasite activity.

One reason for the apparent irrelevance of these TS- and mucin-specific T cell populations to overall parasite control, despite their high frequency, is that the epitopes they recognize are relatively poorly presented on infected cells. For the MUCKb25 epitope, its location within GPI signal sequences of mucin proteins would make it challenging for the epitope to reach the class I MHC presentation pathway in infected cells. Although several studies have reported signal peptides as important targets of CD8⁺ T cell responses against viral and tumor antigens [49, 50], these are systems where such peptides are present in the host cell cytoplasm and thus directly exposed to the MHC-I processing pathway. But in non-viral intracellular pathogens like T. cruzi, GPI signal sequences are cleaved from the mature protein, and presumably stay within these pathogens and are degraded therein. A number of other T cell epitopes within signal sequences have been identified both within the GPI-SS of TS proteins ASP-1 and TSA-1 in T. cruzi [19] and the signal sequences of the Plasmodium falciparum proteins EXP-1 and LSA-1 [51], though these epitopes all elicit modest responses in infected subjects. These reports support that signal sequences are indeed a bona fide source of T cell epitopes, though the mechanism of presentation is unclear. In contrast, the epitopes from mature TS proteins, like TSKb20, are detectably, yet poorly presented by infected host cells. Indeed, our results using sensitive assessment of T cell activation in the Nur77-GFP reporter system (Fig 5A), demonstrate that a low percentage of CD8⁺ T cells present in chronically infected mice can recognize T. cruzi-infected fibroblasts in vitro, particularly during the period that amastigote numbers are rapidly increasing (between >24 and <96 hrs). Collectively, these results suggest that the predominant CD8⁺ T cell populations in T. cruzi-infected hosts are likely induced by cross-presentation by professional APCs, as is the case for most pathogen-specific CD8⁺ T cells [52] [53], and are inefficient in the direct recognition of infected host cells.

Two other characteristics of the MUCKb25-specific T cells distinguish them from the previously described TS-specific T cell population: a delayed generation and the extended kinetics, and a parasite strain variation in their induction. In contrast to the GPI SS localization of the MUCKb25 epitopes, TS epitopes are part of the mature TS surface proteins abundantly expressed on trypomastigotes and amastigotes of T. cruzi, which are released from live parasites via multiple pathways [54–56]. MUCKb25 may only be made available for T cell recognition when parasites containing immature mucins (pre-SS-cleavage) and/or the cleaved mucin GPI SS are processed by APCs. Thus, the delayed generation of the MUCKb25 response may have to await the induction of T and B cell responses capable of killing parasites and/or making them accessible to efficient cross-presentation (due to antibody-mediated uptake in APCs, for example). We failed to enhance the MUCKb25-specific responses by enhancing parasite killing via trypanocidal drugs, among other methods, but this result may not be surprising since this process may not enhance antigen presentation and likely reduces the overall parasite numbers in these hosts.

With the high occurrence of genes encoding the MUCKb25 epitope in T. cruzi, it is surprising that several parasite isolates fail to elicit a MUCKb25-specific CD8⁺ T cell response. Though varying in magnitude, TSKb20-specific CD8⁺ T cell responses have been shown to be elicited during infections with Y strain as well as other strains and isolates from naturally infected macaques [15, 46]. The lack of a MUCKb25 response cannot be due to a reduced presence of the MUCKb25 epitope, as the sequence is found at an even higher frequency in the Y strain as compared to other strains (Table II). There may be strain-dependent differences in the expression of epitope-containing mucins, though if that were the case, we might expect the MUCKb25 response to be reduced, not entirely absent in certain strains. The complete lack of a MUCKb25 response does suggest that the accessibility of the epitope may differ during infection with particular strains/isolates and this may be related to its location within mucins, and, for example, a differential fate for cleaved signal peptides in various isolates.

Overall, the findings presented herein highlight that the targets of highly protective CD8⁺ T cell immunity differ from the major targets that are naturally selected by the T. cruzi-specific CD8⁺ T cell response, although we have had limited success in identifying such highly protective targets using multiple approaches [15, 30] (Bunkofske, in preparation). Alternatively, the apparent limited recognition of infected host cells by T cells of any specificity (Fig 5) may potentially indicate that T cells capable of directly recognizing T. cruzi-infected host cells do not exist or are so inefficiently generated during infection as to be undetectable. Instead, a few immunodominant CD8⁺ T cell responses targeting large gene families, likely facilitated by the high abundance of these proteins and epitope variants in the MHC-I pathway, dominate the T cell repertoire and contribute to the insufficient pattern of immune control, with only rare instances of sterile cures, that characterizes T. cruzi infection in all hosts.

Key points:

• Mucin-derived epitope (MUCKb25) elicits immunodominant T cell response

• MUCKb25 T cell response is nonprotective and not required for parasite control

• Detection of T. cruzi-infected cells by MUCKb25 T cells is limited