Dysfunctional CD4 T cells in an oncovirus-specific TCR-transgenic in vivo model

Domain: chemistry
Published: 2025-12-18
Editorial outcome: minor_revision

Abstract
T cell exhaustion has been implicated in cancer and infectious diseases. In this study, we report a novel mouse model, “MolT-II”, with T cells expressing a transgenic T cell receptor (TCR) specific for a Moloney virus envelope-derived, MHC class II-presented peptide epitope. Characterization of MolT-II CD4 T cells revealed that they are dysfunctional, showing severely impaired effector functions, reduced proliferation and increased baseline expression of co-inhibitory receptors such as PD-1, LAG-3 and CTLA-4, likely due to chronic exposure to a self-antigen. We further show that epitope-specific peptide vaccination combined with immune checkpoint blockade is able to restore the function of MolT-II CD4 T cells in vivo, associated with enhanced tumor control in mice. The MolT-II mouse strain thus represents an in vivo model for reversible CD4 T cell dysfunction, allowing the study of the role of CD4 T cell regulation in cancer, mechanisms underlying CD4 T cell dysfunction and exhaustion, and novel immunomodulatory therapies aiming to rescue dysfunctional T cells.

Introduction
Prolonged antigen exposure can lead to tolerization and dysfunction of antigen-specific T cells. Such T cell “exhaustion” can occur in the context of chronic infection or growing tumors1–4. While CD8 T cell exhaustion is being studied extensively and many treatments currently in use aim to rehabilitate dysfunctional CD8 T cells, the role of exhaustion or dysfunction of CD4 T cells remains largely understudied, despite their central role in different immune processes5. In cancer, CD4 T cells have been shown to take on a myriad of roles, most important of which is providing help to CD8 T cells for the mounting and maintenance of an effective anti-tumor response6. Further, CD4 T cells can suppress immune responses as regulatory CD4 T cells (Tregs)7,8. Also, CD4 T cells can engage in tumor eradication through direct cytotoxicity9,10 or interaction with other immune cells such as macrophages11–13. Extensive multidimensional analysis of thousands of human solid tumors has shown the presence of both CD4 and CD8 T cells in the tumor microenvironment correlating with immunotherapy efficacy by blocking coinhibitory receptors like PD-1. This indicated dysfunctional states of immune cell clusters in many types of cancer14–16.

T cell dysfunction comes in several flavors which can differ in cause; however, most are characterized by reduced proliferative activity, expression of co-inhibitory markers, downregulation of the TCR signaling machinery and abnormal cytokine expression17–19. In cancer, T cell exhaustion has been extensively researched, especially in relation to immunotherapy, with a primary focus on CD8 T cells. Chronic antigen exposure is thought to be one of the main causes underlying T cell exhaustion3,20,21, and recent studies suggest that dysfunctional states similar to those found in CD8 T cells can also be observed in different CD4 T cell populations, and may indeed be clinically relevant5,22. Several cancer immunotherapies target molecules associated with T cell exhaustion like PD-1, LAG-3 and CTLA-4 in order to reinvigorate T cell function23–25. And while immune checkpoint inhibition is an effective therapeutic option, the high rate of non-responders and the unwanted side effects leave room for improvement2,26,27.

We generated a T cell receptor (TCR)-transgenic mouse model named “MolT-II” (in analogy to the OT-II mouse strain) to investigate tumor-specific CD4 T cell responses to murine leukemia virus (MuLV)-induced cancers. The Friend, Moloney, and Rauscher (“FMR”) virus types are well-characterized exogenous MuLVs that can induce leukemias, lymphomas and sarcomas in mice28. Several virus-derived CD4 and CD8 T cell epitopes have been shown to play critical roles in the immune response against these tumors29,30. Most importantly, previous research has shown that CD4 T cells directed against an I-Ab-presented peptide epitope derived from the Moloney virus envelope protein are by themselves sufficient to protect against virus-induced sarcoma lesions and enhance tumor-specific CD8 T cell responses against MHC class II-negative lymphoma cells29,31.

In this report we describe the TCR-transgenic MolT-II mouse strain, whose CD4 T cells show a dysfunctional phenotype characterized by severely diminished proliferation, lack of cytokine secretion, upregulation of co-inhibitory checkpoint molecules and inadequate response to antigen-specific TCR stimuli. This phenotype appears to be driven by cross-reactivity between the transgenic TCR and the common endogenous murine retrovirus AKV (originally found in AKR mice). Rescue of MolT-II CD4 T cell function can be achieved through antigen-specific TCR stimulation and combined blockade of co-inhibitory checkpoint molecules PD-1, CTLA-4 and LAG-3, and functionally reinvigorated MolT-II CD4 T cells can contribute to tumor control in mice bearing MuLV-induced lymphoma. This new mouse model mirrors the phenotype of exhausted CD4 T cells found in cancer, infections and autoimmune diseases, enabling the study of mechanisms underlying T cell dysfunction and how to reinvigorate T cells.

Results
Reduced CD4 T cell compartment in TCR-transgenic MolT-II mice
The newly generated TCR-transgenic MolT-II mice, expressing the TCRα and β chains derived from a CD4 T cell clone specific for Moloney MuLV env-gp70 peptide sequence 119-137 (envH), bred normally and did not present with any obvious phenotypic aberrations. The majority of peripheral CD4 T cells of MolT-II mice expressed the TCR-transgenic Vβ6 chain, comparable to the expression of the transgenic Vβ5 chain in OT-II mice, another model for transgenic CD4 T cells specific for chicken ovalbumin peptide sequence 323-339 (ovaH) (Fig. 1A). Analysis of the lymphoid system revealed an unusually low frequency of CD4 T cells in the spleens, lymph nodes and blood of MolT-II mice (Fig. 1B). Wild type C57BL/6 mice had a CD8:CD4 T cell ratio with ranges between 0.5:1 and 0.7:1, while the CD8:CD4 ratio in OT-II mice ranged between 0.2:1 and 0.5:1, likely due to positive selection of the TCR-transgenic CD4 T cells in the thymus and lack of negative selection of the ovalbumin-specific TCR. Unexpectedly, in MolT-II mice, we found the CD8:CD4 ratio to be drastically increased to around 1.7:1 in secondary lymphoid organs and blood (Fig. 1C). We further found that the total number of CD3-positive T cells was comparable between the different mouse strains (Fig. 1D), but the number of CD4 T cells in MolT-II spleens was significantly lower than in C57BL/6 and OT-II mice (Fig. 1E). Thus, the CD4 T cells of MolT-II mouse express the transgenic TCR, but the frequency of these CD4 T cells in the periphery is significantly reduced.

MolT-II CD4 T cells show decreased effector functions in response to peptide stimulation
After assessing the diminished cellularity of the CD4 T cell compartment of MolT-II mice, we analyzed the functionality of the CD4 T cells. CD4 T cells from MolT-II and OT-II mice were stimulated by co-culturing splenocytes with envH or ovaH peptide-loaded dendritic cells (DCs) (Figs. 2A, B). While MolT-II CD4 T cells were able to express moderate levels of CD40L, IL-2 and TNF, the magnitude of the response was significantly lower than OT-II CD4 T cells stimulated with their cognate epitope (ovaH), which induced very high expression of CD40L and TNF, as well as increased IL-2 expression.

Further, we tested the proliferative capacities of TCR-transgenic MolT-II and OT-II CD4 T cells after stimulation with envH or ovaH peptide, respectively. To that end, CFSE-labeled splenocytes were incubated with peptide-loaded DCs for 96h, followed by flow cytometric analysis (Fig. 2C). For OT-II CD4 T cells, even low concentrations of ovaH peptide induced strong proliferation, as shown by the high proportion of divided CFSElow CD4 T cells. In contrast, stimulation of MolT-II CD4 T cells with envH peptide induced proliferation only to a low extent, even at high peptide concentrations.

The lack of cytokine expression and reduced proliferative capacity showed that MolT-II CD4 T cells are unable to appropriately respond to stimulation with MHC class II-presented envH peptide. This could be caused by low avidity between MolT-II TCR and the peptide-MHC complex (pMHC) or insufficient interaction time between TCR and pMHC32,33. To assess whether a more potent stimulus would be able to trigger the transgenic MolT-II TCR and lead to an improved cytokine response, C57BL/6, MolT-II and OT-II splenocytes were stimulated with 1 µg/ml each of soluble αCD3 (Signal 1) and soluble αCD28 (Signal 2) (Fig. 2D). Stimulation with these soluble antibodies led to high expression of CD40L, IL-2 and TNF by CD4 T cells from all three mouse strains. In response to the stimulation, MolT-II CD4 T cells produced IL-2 to even higher levels than wild type and OT-II CD4 T cells. These results, indicating regained function by high affinity co-stimulation, show that the low responses observed after envH stimulation do not reflect irreversible dysfunction of MolT-II CD4 T cells.

Expression of co-inhibitory checkpoint molecules is indicative of CD4 T cell dysfunction
Immune regulatory signaling may be involved in the dysfunctional phenotype of CD4 T cells in MolT-II mice. First, we found that MolT-II mice harbor a similar frequency of Tregs (regulatory T cells, FoxP3+CD25+CD4+) compared to wild type mice (Supplementary Fig. 1A). Rather, we found that the number of Tregs in spleens from OT-II mice was lower than in the other two mouse strains.

This suggested that the functional deficits observed in MolT-II CD4 T cells might be due to intrinsic regulatory pathways in the T cells themselves. Flow cytometric analysis of CD4 T cells from naive MolT-II, C57BL/6 and OT-II mice showed that several co-inhibitory immune checkpoint molecules, such as PD-1, LAG-3, CTLA-4, and TIM-3 were upregulated on MolT-II CD4 T cells (Fig. 2E, Supplementary Fig. 2A). We also observed higher expression of co-stimulatory checkpoint molecules ICOS and OX40 on MolT-II CD4 T cells (Supplementary Fig. 2B, C), which has been previously described to occur in exhausted CD4 but not CD8 T cells34.

Stimulation of the cells with αCD3 antibody, without CD28 co-stimulation, using low (200 ng/ml) or high dose conditions (1 µg/ml) further increased the expression of checkpoint molecules, far beyond the expression levels of C57BL/6 and OT-II CD4 T cells (Fig. 2E). On the other hand, stimulation of MolT-II CD4 T cells with peptide envH had no notable effects on the expression of checkpoint molecules (Supplementary Fig. 2D).

Taken together, the high expression of co-inhibitory molecules, lack of cytokine production in response to cognate antigen encounter and limited proliferative capabilities suggest that MolT-II CD4 T cells are in a state of functional exhaustion.

MolT-II CD4 T cells recognize a cross-reactive endogenous peptide ligand
Upon investigation of splenic CD4 T cells we noticed that the MolT-II CD4 T cell population had a significantly smaller proportion of CD62L+CD44- CD4 T cells compared to C57BL/6 and OT-II (Fig. 3A, Supplementary Fig. 3A, B). Furthermore, a larger percentage of CD4 T cells were CD44 positive (CD44+CD62L- and CD44+CD62L+), which is uncharacteristic of naive mice. CD44 is considered a marker for T cell activation, effector and memory T cells and therefore antigen-experience35. The high expression of CD44 might be due to interactions between MolT-II CD4 T cells and an endogenous peptide ligand underlying the exhausted phenotype found in MolT-II T cells. Furthermore, cross-reactivity to an endogenous peptide ligand could lead to selective self-pressure, explaining the relatively low numbers of peripheral CD4 T cells in MolT-II mice. Investigation of thymocytes indicated that negative selection of the TCR-transgenic MolT-II CD4 T cells is not restricted to the periphery. The percentage of CD44-positive, antigen-experienced CD4 T cells in the thymus of MolT-II mice was significantly elevated compared to OT-II and C57BL/6 thymocytes (Supplementary Fig. 3C). Further, MolT-II mice showed a decreased proportion of CD4+CD8+ double-positive thymocytes compared to other mouse strains (Fig. 3B, Supplementary Fig. 3D), resembling findings in other TCR-transgenic mouse strains in which TCR ligands are expressed in the thymus inducing negative selection of self-reactive T cells36,37. As these results suggest the existence of a cross-reactive peptide ligand for the TCR-transgenic MolT-II CD4 T cells, we compared the sequence of the envH epitope to sequences from the mouse genome. This led to the discovery of a highly homologous sequence derived from the envelope protein of the endogenous murine retrovirus AKV (env 138-156, “AKVenv”). The sequences of the endogenous peptide AKVenv and the exogenous Moloney MuLV-derived peptide envH only differ in one amino acid (envH 124, AKVenv 143), where a leucine is replaced by a tyrosine in AKVenv (Fig. 3C). In order to analyze whether this sequence was expressed in the thymus of MolT-II mice, thymic stromal cells were isolated and sorted into different subsets (cortical thymic epithelial cells (cTECs), medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs)) and analyzed by qPCR, using the thymoma cell line EL-4 as a positive control (Fig. 3D). These experiments demonstrate that AKVenv expression in the thymus is largely restricted to mTECs, supporting their role in shaping central tolerance by mediating the deletion of autoreactive CD4+CD8+ thymocytes38. Expression of AKVenv was not detectable in cTECs and only low expression was found in thymic DCs.

Based on these findings, we further analyzed possible cross-reactivity of MolT-II CD4 T cells with the AKVenv epitope. CFSE-labeled MolT-II splenocytes were co-cultured for 96h with DCs loaded with envH, AKVenv, or control peptide ovaH. Results showed that the AKVenv peptide was able to induce proliferation of MolT-II CD4 T cells, similar to, but at a lower level than envH stimulation (Fig. 3E), indicating cross-reactivity of the MolT-II TCR with the endogenous AKVenv peptide ligand. To further assess cross-reactivity of MolT-II cells with the endogenous AKVenv sequence, LPS-matured dendritic cells were loaded with envH, AKVenv, or control peptide ovaH and co-incubated over night with MolT-II splenocytes. Analysis of the MolT-II CD4 T cells by flow cytometry revealed that stimulation with AKVenv resulted in elevated expression of CD40L and TNF compared to the controls (Fig. 3F), but still significantly lower than stimulation with envH. Similarly, AKVenv stimulation also led to increased expression of IL-2, CD25 and, to a lesser degree, CD69 (Supplementary Fig. 3E). Together, these findings indicate that MolT-II CD4 T cells are, albeit weakly, cross-reactive to AKVenv.

Dysfunction of MolT-II CD4 T cells in vivo
Our ex vivo experiments revealed that despite adequate expression of the transgenic TCR Vβ6 chain on MolT-II CD4 T cells, only a minor fraction of cells responded to stimulation with the cognate peptide epitope envH. Based on this, we investigated whether the functionally impaired CD4 T cells could be activated in vivo.

To analyze the in vivo responsiveness of MolT-II CD4 T cells we made use of the dual-color bioluminescence T cell reporter mouse strain “TbiLuc”, which enables in vivo imaging of the localization and activation of T cells39. MolT-II and OT-II mice were crossbred to TbiLuc reporter mice to obtain F1 mice with luciferase-expressing TCR-transgenic CD4 T cells. These CD4 T cells, enriched from the spleens of naive TbiLuc*MolT-II or TbiLuc*OT-II mice, were adoptively transferred to naive C57BL/6 mice to enable accurate bioluminescence imaging and minimize background. After the T cell transfer, the recipient mice were vaccinated twice with envH + CpG (TbiLuc*MolT-II) or ovaH + CpG (TbiLuc*OT-II). Presence of the transferred cells in recipient mice was visualized by a click-beetle luciferase (CBG99), which is constitutively expressed by TbiLuc T cells, and luciferase substrate D-luciferin (Fig. 4A, upper). Upon activation, NFAT (Nuclear Factor of Activated T cells)-driven expression of a firefly luciferase (PpyRE9) enables the imaging of activated T cells with the substrate CycLuc1 (Fig. 4A, lower). Quantification of the average radiance of the CycLuc1 bioluminescence signal showed that vaccination with ovaH + CpG induced significant activation of TbiLuc*OT-II CD4 T cells compared to naive TbiLuc*OT-II CD4 T cells. (Fig. 4B). In contrast, envH + CpG vaccination only slightly increased the activation of TbiLuc*MolT-II CD4 T cells, further showing that MolT-II CD4 T cells are poorly responsive to antigenic stimulation in vivo.

Dysfunctional CD4 T cells can recover function after in vivo vaccination and immune checkpoint blockade
Based on the upregulation of immune checkpoint molecules on MolT-II CD4 T cells we had previously observed (Fig. 2D), we combined peptide vaccination with systemic blockade of the LAG-3, PD-1 and/or CTLA-4 pathways in vivo, aiming to restore T cell function (Fig. 5A). Systemic treatment with αLAG-3, αPD-L1 and αCTLA-4 had no effect on the functional capabilities of MolT-II CD4 T cells (Fig. 5B, C). Vaccination with envH + CpG only slightly increased IL-2 and CD40L expression by MolT-II CD4 T cells. Combining vaccination with αLAG-3 or αPD-L1 had no significant additive effects. CTLA-4 blockade and vaccination could partly restore T cells function, however, simultaneous blockade of LAG-3, PD-L1 and CTLA-4 and vaccination led to a significant increase of IL-2+ and CD40L+ CD4 T cells. In addition, the absolute number of Vβ6+ CD4 T cells in the vaccine-draining lymph node and spleen of MolT-II mice significantly expanded in response to envH + CpG vaccination and triple checkpoint inhibition (Fig. 5D, E). Notably, treatment with triple checkpoint blockade alone had no effects on T cell function in naive MolT-II mice and required antigenic stimulation to take effect. This suggests that the presence of the endogenous peptide ligand AKVenv was not sufficient to overcome the tolerizing environment in vivo that restrains the CD4 T cells in a dysfunctional state.

MolT-II T cells can support tumor control in vivo upon robust vaccination
As we observed that MolT-II CD4 T cells were able to functionally recover in vivo, we evaluated whether MolT-II CD4 T cells were also able to contribute to tumor control. To that end we transferred MolT-II cells into naive C57BL/6 mice. The next day, these recipient mice received a peptide vaccine consisting of envH combined with the dominant MuLV CTL epitope “gagL”, emulsified in incomplete Freund’s adjuvant (IFA). This formulation has shown to be effective in CD8-mediated tumor control in a previous study29. The mice were stratified into four groups receiving either IFA (Control), vaccination (envH + gagL), adoptive transfer of MolT-II cells (MolT-II), or a combination of vaccination and adoptive MolT-II cell transfer (MolT-II + envH + gagL). Two weeks later, the animals were all subcutaneously injected with a lethal dose of RMA tumor cells (Fig. 6A). Treatment with IFA or with MolT-II cells only did not contribute to tumor control (Fig. 6B). Vaccination with envH and gagL delayed the outgrowth of RMA tumors, and protected about 40% of mice. Combining the vaccination with the transfer of MolT-II cells significantly increased the survival of mice to 80% (Figs. 6B, C). These findings indicate that after transfer, MolT-II CD4 T cells can regain functional helper activity when properly activated by robust peptide vaccination, to enhance CD8 T cell-mediated tumor control in vivo.

Methods
Mice
The TCR-transgenic mouse strain MolT-II was generated at the Leiden University Medical Center (‘LUMC’, Leiden, Netherlands) on a C57BL/6 background. MolT-II mice express a TCR recognizing peptide Moloney env 119-137 in association with I-Ab under control of Kb-promotor IgG enhancer element. The transgenic TCR α and β genes were derived from the Moloney env 119-137 specific helper T cell clone 3A1229. DNA sequence determination revealed that the TCR’s variable gene segments were composed of Vα10.2 and Vβ6. The TCR Vα10.2-J-C and TCR Vβ6-D-J-C regions were amplified by PCR from clone 3A12-derived cDNA with specific primers and cloned into the pHSE3’ expression vector78. The TCR α- and β-chain gene constructs were linearized and co-injected directly into the pronuclei of fertilized C57BL/6 oocytes. Transgenic founder mice were identified by PCR using specific primers for the Vα-J-C and Vβ-D-J-C regions, and results were confirmed by Southern blot. Transgenic founder mice were bred with C57BL/6 mice and tail blood of transgenic offspring was analyzed by means of PCR and Vβ6 specific monoclonal antibodies to confirm expression of the transgenic TCR on the surface of T cells.

C57BL/6J mice were obtained from Charles River (l’Arbresle, France; Catalog, 632). TCR-transgenic OT-II mice (originally obtained from Charles River, France; Catalog, 643), TbiLuc reporter mice (dual luciferase transgenic mice for bioluminescence imaging of T cells39, TbiLuc*MolT-II and TbiLuc-OT-II mice were bred at the animal facility of the LUMC.

Female and male mice were used in all experiments except for the adoptive transfer, sex was not considered as a relevant biological variable in this study. For adoptive transfer experiments, only female mice were used to avoid adverse immune reactions. All animals were aged 8–12 weeks at the beginning of the experiments, and were euthanized by cervical dislocation. All mouse strains were bred and maintained under specific pathogen-free conditions in IVC cages and were co-housed for the duration of experiments, at the LUMC’s animal facility (20°C–22°C, relative humidity 55%, light cycle with daytime 07:00–18:00h and 30min transition periods before and after). All experiments were approved by the animal experiments ethics committee of Leiden University.

Cell lines and peptides
The D1 cell line is a long term growth factor-dependent immature splenic DC line derived from C57BL/6 (H-2b) mice and was cultured as described elsewhere79. For the current study we used the Moloney env Thelper peptide (EPLTSLTPRCNTAWNRLKL, ‘envH’)80, AKV env peptide (EPLTSYTPRCNTAWNRLKL, ‘AKVenv’)81, ovalbumin-derived Thelper peptide Ova 323-339 (ISQAVHAAHAEINEAGR, ‘ovaH’)82, and the MuLV gag-leader-derived CTL epitope gPr80gag 85-93 (CCLCLTVFL, ‘gagL’)83. Further, for in vivo experiments, we used modified versions of envH and AKVenv in which the cysteines at position 10 have been exchanged with α-aminobutyric acid to prevent oxidation (EPLTSLTPRAbuNTAWNRLKL, EPLTSYTPRAbuNTAWNRLKL). All peptides were synthesized at the LUMC (Leiden, Netherlands) on solid-phase using Fmoc chemistry, purified and characterized by mass spectrometry.

Flow cytometry
Spleens, lymph nodes, blood and thymus were harvested from mice and processed to single cell suspensions. For the identification of surface markers, cells were incubated with fluorochrome-conjugated antibodies in PBA (PBS, 0.5% BSA) for 30 min at 4°C.

For detection of intracellular and intranuclear proteins, cells were fixed, permeabilized and stained using the Cyto-Fast Fix-Perm Buffer Set (Biolegend, USA; Catalog, 426803) or the eBioscience FoxP3/Transcription Factor Staining Buffer Set (Invitrogen, USA; Catalog, 00-5523-00) according to the manufacturer’s recommendations. Samples were acquired on LSR-II (with BD FACSDiva software, BD Bioscience, USA) or Aurora 5L (with SpectroFlo software, Cytek, USA) cytometers, followed by analysis using FlowJo software (FlowJo LLC, USA) and OMIQ (Dotmatics, USA). Details about antibodies used for flow cytometry can be found supplementary table 1. The gating strategy used for identifying different T cell populations is shown in supplementary fig. 4.

CFSE labeling
CFSE labeling of splenocytes was performed as according to kit instructions. Shortly, 25,000 D1 cells were added per well in a 96-well round bottom plate. Antigenic peptides were added to final concentrations of 0.05–5 µM. After incubation at 37°C for 4h, cells were washed twice with PBS to remove all remaining antigens from the supernatant. During the last hour of incubation, splenocytes were collected and washed with pre-warmed PBS/BSA (0.1%). For CFSE-labeling, cells were incubated at 37°C for 10min with 5 µM CFSE (ThermoFisher Scientific, USA; Catalog, C34554) at 10x106 splenocytes/ml in PBS/BSA (0.1%). The labeling reaction was stopped by adding 10-fold volume of complete medium (IMDM, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 25 µM β-Mercaptoethanol). CFSE-labeled cells were spun down and 3x105 splenocytes were added per well to the peptide-loaded D1 cells. After 96h, cells were harvested, washed with PBA, stained and analyzed by flow cytometry.

In vitro and ex vivo (re-)stimulation of T cells
For stimulation of cells with anti-CD3e, 48-well plates were coated with 200 ng/ml or 1000 ng/ml anti-CD3e (clone 145-2C11, BD Biosciences; Catalog, 553057) in PBS for 2h at 37°C. Plates were then washed twice with PBS, 1x106 total splenocytes in complete medium were added per well and incubated for 16h. Cells were then harvested, washed in PBA, stained and analyzed by flow cytometry.

For stimulation of cells with combined anti-CD3e and anti-CD28, 1x106 mouse splenocytes were plated per well in 96-well round bottom plates. A mix of 1 mg/ml each of soluble anti-CD3e (clone 145-2C11, BD Biosciences; Catalog, 553057) and anti-CD28 (clone 37.51, BD Biosciences; Catalog, 553294) was added to the cells. After 1h of incubation, Brefeldin A was added and cells were incubated for another 4–6h.

For stimulation of cells with antigenic peptides, 5x104 D1 cells were seeded per well in 96-well round bottom plates and incubated with 0.05-10 mM peptide or equivalent volumes of the solvent DMSO for 4–5h at 37°C. The plates were then washed twice with PBS to remove any remaining free peptide from the supernatant and 1x106 mouse splenocytes were added per well. After 1h of co-incubation, Brefeldin A was added to prevent cytokine secretion, and the cells were incubated for another 4–6h at 37°C, 5% CO2 before they were washed and stained for flow cytometry. In the case of experiments using mature D1 cells, immature D1 cells were incubated with LPS (4 µg/ml, Sigma-Aldrich, USA) over night at 37°C prior to co-incubation of 2.5x104 mature D1 cells with 7.5x105 MolT-II splenocytes and 5 µM peptide, and treatment with Brefeldin A as described above.

Molecular analysis of thymic cells
Isolation of thymic stromal cells was performed as described previously84. Thymic lobes were cleaned of fat and connective tissue, cut in little pieces and stirred for 10 min in 15 ml RPMI 1640 medium at room temperature to release thymocytes. Tissue fragments were then resuspended in 1 ml medium per sample containing 0.2 mg/ml Collagenase IV (Worthington Biochemical, USA), 10 mM HEPES and 2% FCS. The mixture was slowly stirred for 15 min at 30°C, released cells removed and fresh enzyme mixture added for a total of 3 incubations. The remaining fragments were digested with a mixture of Collagenase IV and Neutral Protease (0.2 mg/ml each; Worthington Biochemical, USA), 25 µg/ml DNase I (Roche, Switzerland), 10 mM HEPES and 2% FCS in RPMI 1640. Five incubations for 25 min at 37°C were performed. For DC isolation all cells from the collagenase incubations and the first round of collagenase/neutral protease digestion were pooled, washed and rosettes dissociated by 5 min incubation at 37°C in PBS containing 25 mM EDTA. Cells were stained with anti-CD11c microbeads (Miltenyi Biotec, Germany; Catalog, 130-125-835), run on an AutoMACS (Miltenyi Biotec, Germany) using the “Possel_S” program, blocked with anti-FcR mAb 2.4G2 supernatant including 5% rat serum and stained with anti-CD11c-PE (clone HL3; BD Biosciences, USA; Catalog, 561044), anti-F4/80-FITC (clone CI:A3-1; Bio-Rad, USA; Catalog, MCA497FA) and propidium iodide (PI). Thymic epithelial cells were enriched by pooling cells from collagenase/neutral protease digestion rounds two to five, followed by staining with anti-CD45 microbeads (Miltenyi Biotec, Germany; Catalog, 130-052-301), run on an AutoMACS using the ‘Deplete’ program, blocked with anti-FcR mAb 2.4G2 supernatant and stained with anti-Ly51-FITC (clone 6C3; BD Biosciences, USA; Catalog, 562057), anti-CD80-PE (clone 16-10A1; BD Biosciences, USA; Catalog, 561955), anti-EpCAM-Alexa647 (clone G8.8; kindly provided by Dr. B. Kyewski), anti-CD45-PE/Cy5 (clone 30-F11; BD Biosciences, USA; Catalog, 561870) and PI. Dendritic cells were identified as CD11c+F4/80-. mTECs were identified as CD45-Ly51-EpCAM+ and sorted according to their CD80 expression, as CD80hi or CD80lo, representing the top and bottom 30% of the population. cTECs were defined as CD45-Ly51+EpCAM+. Cell sorting was performed with a FACSAria I cell sorter (Becton Dickinson, USA).

For quantitative PCR analysis cells were purified by cell sorting and EL-4 thymoma cells grown in culture were treated as published, including two separate DNase I incubation steps. Mela (AKVenv) and beta actin transcripts were amplified with Power SYBR Green Master Mix (Applied Biosystems/Thermo Fisher, USA; Catalog, 4368577) on a GeneAmp 7300 system (Applied Biosystems/Thermo Fisher, USA). Primers used for the amplification were kindly provided by Dr. B. Kyewski (produced at the oligonucleotide synthesis facility of the German Cancer Research Center, Germany) and can be found in supplementary table 2.

In vivo T cell transfer. For in vivo bioluminescence imaging of the localization and activation of CD4 T cells, TbiLuc mice were crossed to MolT-II and OT-II mice, to obtain TbiLuc*MolT-II and TbiLuc*OT-II mice. Spleens of TbiLuc*MolT-II and TbiLuc*OT-II mice were collected and processed to single cell suspensions, followed by enrichment of CD4 T cells from the splenocytes using a Mouse CD4 T Lymphocyte Enrichment Set (BD Bioscience, USA; Catalog, 558131). 2x106 enriched CD4 T cells were transferred into C57BL/6 mice through intravenous injection in 200 µl PBS (day 0). The recipient mice were then immunized on day 1 and 11 with 5 nmol peptide envH + 5 nmol CpG (TbiLuc*MolT-II), 5 nmol peptide ovaH + 5 nmol CpG (TbiLuc*OT-II) or left unvaccinated.

In vivo bioluminescence imaging of T cells
Bioluminescence imaging of transferred TbiLuc*MolT-II or TbiLuc*OT-II CD4 T cells occurred under isoflurane anesthesia using an IVIS Spectrum Imager (PerkinElmer, Waltham, MA, USA) with Living Image software (Revvity, USA). Mice were imaged every 2-4 days over a period of 4 weeks. To image the localization of T cells, mice were injected subcutaneously (s.c.) with 150 mg/kg D-luciferin imaged after 10 min at the peak of emission, using the open and 560 nm filters with auto exposure. To image the activation of CD4 T cells, the animals were injected s.c. with 7.6 mg/kg CycLuc1 and imaged after 8 min using the open and 620 nm filters with auto exposure.

Bioluminescence signals were quantified using fixed-sized, fixed-position regions of interest (ROIs) and corrected for background signals with Aura Imaging Software (Spectral Instruments Imaging, USA).

In vivo antibody blocking
MolT-II mice were injected intraperitoneally with 10 mg/kg Rat anti-mouse PD-L1 (MIH5, kindly provided by Dr. R. Arens (LUMC, Leiden, Netherlands)), Rat anti-mouse LAG-3 (clone C9B7W, BioXCell; Catalog, BE0174) and/or Syrian hamster anti-mouse CTLA-4 (clone 9H10, BioXCell; Catalog, BE0131) on days 0, 2 and 4. Control Rat anti-β-galactosidase (clone GL113, kindly provided by Dr. R. Arens) was also injected at a dose of 10 mg/kg. On day 1, mice were immunized with 10 nmol envH peptide mixed with 5 nmol CpG or an equivalent volume of the solvent in PBS. On day 6, spleens and draining lymph nodes were collected and processed into single cell suspensions for intracellular cytokine staining and flow cytometry.

In vivo tumor experiments
Tumor experiments were performed with four groups of C57BL/6 mice. The control group (n=16) was s.c. (subcutaneously) injected with a 1:1 IFA:PBS mixture, the ‘MolT-II’ group (n=8) also received 1:1 IFA:PBS and an intravenous (i.v.) transfer of 1×107 MolT-II cells from the spleens and lymph nodes of naive MolT-II mice. The ‘envH + gagL’ group (n=16) was s.c. vaccinated with 20 nmol gagL peptide and 20 nmol envH peptide emulsified in 1:1 IFA:PBS, the ‘MolT-II + envH + gagL’ group (n=15) received the same vaccination as well as i.v. transfer of 1x107 MolT-II cells. All animals were inoculated with 2000 RMA cells in the right flank, followed by an observation period of 60 days, in which the growth of tumor lesions was monitored. Mice were euthanized by cervical dislocation when tumors exceeded the maximum size allowed by the ethics board of 1000 mm3 or became necrotic. The maximum tumor size was not exceeded. Tumors were measured using calipers and volume was calculated using the equation volume=(length x width x height)/2.

Statistics
Statistical analysis was performed using GraphPad Prism 10.2.3 (GraphPad Software, Boston, MA, USA). Data are shown as mean ± SD or mean ± SEM, depending on the experiment. Differences were considered statistically significant at p<0.05. Multiplicity-adjusted P values are depicted in the figures as follows: *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001.

Study approval
All animal experiments were performed in accordance with the guidelines of the Dutch Animal Ethics Committee and the recommendations set by the LUMC and by the Dutch Experiments on Animals Act. All animal experience were approved by the Animal Welfare Body of LUMC (permit AVD11600202013796).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Spitzer et al describes novel CD4 TCR transgenic mouse reactive against a peptide derived from the Maloney virus envelope protein presented in the context of I-Ab. The mouse was generated with the goal of studying CD4 responses to Maloney virus-induced tumors but, unexpectedly, the TCR cross-reacted to a protein derived from an endogenous retrovirus genetic sequence that resulted in deletion in the thymus and dysfunction of T cells that escape thymic selection. The investigators compare these T cells to T cells from wild-type B6 mice and TCR transgenic T cells from OT-II mice which react against a peptide from ovalbumin that is not expressed in the thymus or periphery. The authors show that the dysfunction of the MolT-II T cells is reversible and use the mouse to explore mechanism of, and approaches to, overcome the exhaustion. As pointed out by the authors, CD4 T cell dysfunction, while less-well described than CD8 T cell dysfunction has been described. However, this is first description of a TCR transgenic mouse that models CD4 dysfunction.\nComments:\n1) Figure 2D. Do the Molt-II cells upregulate checkpoint molecules upon peptide stimulation?\n2) I assume Figure 3A is gated on all splenic T cells. What do the VB6 gated (TCR transgenic) T cells look like?\n3) Figure 3E and F. Cross-reactivity is shown using proliferation only. Addition data such as cytokine expression would enable better assessment of degree of functional crossreactivity.\n4) The delta in naïve vs vaccinated CD40L expression 4A for the OT-II is not very impressive. Other analyses (including phenotype without ex-vivo restimulation) would better support robustness of the vaccine.\n5) A major gap in the studies is evidence for in vivo functionally – that is control of tumors for example. This could easily be assessed using tumors that express both ovalbumin and Moloney envelope. This would markedly improve the robustness of the data supporting both dysfunction and reversal of dysfunction.\n6) Where is the ERV-derived peptide (AKVenv) expressed? Presumably in the thymus but where in the periphery?", "reviewer_2": "Spitzer et al. describe the generation and characterization of a novel TCR-transgenic mouse model, MolT-II, which expresses a CD4 T cell receptor specific for a peptide derived from the Moloney murine leukemia virus envelope protein. The authors demonstrate that MolT-II CD4 T cells exhibit a dysfunctional, exhaustion-like phenotype, which is likely due to chronic exposure to a cross-reactive endogenous retroviral peptide (AKVenv). They further show that this dysfunctional state can be partially reversed in vivo through peptide vaccination in combination with triple immune checkpoint blockade. This model presents a unique and valuable tool to investigate CD4 T cell dysfunction and reinvigoration, particularly in settings of chronic antigen exposure.\nThe manuscript is clearly written and structured in a way that makes it easy to follow the scientific rationale and findings. Overall, this work could make a meaningful contribution to the fields of T cell biology and immunotherapy. Addressing the following points could further strengthen the study and enhance its impact on the field.\nMajor points:\n1. The claim that AKVenv is the driving endogenous ligand for thymic deletion and peripheral dysfunction is central to the manuscript. However, direct evidence for AKVenv expression in the thymus or peripheral APCs is lacking. Inclusion of RT-qPCR or RNA in situ hybridization to detect AKV transcripts in thymic epithelial cells or APCs would considerably strengthen the conclusion that MolT-II CD4 T cells are tolerized/exhausted due to chronic exposure to an endogenous antigens.\n2. Given that 50 nM peptide appears to saturate proliferation in both OT-II and MolT-II T cells, CFSE assays may not be sensitive enough to resolve differences in TCR avidity. While CFSE-based proliferation to AKVenv peptide was observed compared to unstimulated control, more direct evidence of TCR recognition, such as MHC class II tetramer binding, early activation markers (e.g., CD69, CD25), or peptide titration with functional readouts, would strengthen the claim of cross-reactivity to the endogenous AKVenv peptide.\n3. While the MolT-II CD4 T cells display features of dysfunction such as impaired proliferation and elevated inhibitory receptors, it remains unclear whether these cells mirror classical exhaustion, tolerance, or an intermediate phenotype. Comparative transcriptomic or phenotypic profiling, particularly CD4 T cells from chronic viral or tumor models, would clarify the relationship. The authors used ICOS and OX40 expression as markers of exhausted CD4 T cells (ref. 37), but it would strengthen the interpretation to include functional comparisons with known exhausted CD4 T cell populations.\n4. The rescue of function via triple blockade is an important finding. It would be valuable to assess changes in key transcription factors associated with T cell exhaustion and reinvigoration (e.g., TOX and TCF1), providing insight into whether MolT-II CD4 T cells undergo molecular reprogramming toward a less dysfunctional state.\n5. While the authors provide compelling evidence that MolT-II CD4 T cells can be reinvigorated in vivo through combined checkpoint blockade and antigenic stimulation, the functional consequences of this reinvigoration remain unclear. The restoration of proliferation and cytokine production offers important mechanistic insights. However, assessing whether reinvigorated CD4 T cells support downstream immune responses, such as enhancing CD8 T cell function or controlling tumor growth, would further strengthen the translational relevance of this model, especially in contexts where CD4 help is critical.\nAlthough such experiments may be beyond the current scope and present experimental challenges, addressing this point would significantly enhance the impact and applicability of the model for future studies on CD4 T cell–mediated immune modulation in chronic antigenic settings like cancer or persistent infection.\nMinor points and suggestions:\n1. The authors should spell out \"MolT-II\", “AKV”, and “TbiLuc” early in the manuscript for reader clarity.\n2. Figures 1D and 1E should be appropriately called out.\n3. In Figure 2A, adding quantitative graphs with statistics would improve clarity. Also, including an irrelevant control peptide would better control for background responses."}, "author_response": "Response to Referees\nWe thank both reviewers for their constructive criticism and comments and we answered all questions in a point-by-point reply below.\nReviewer #1\n1) Figure 2D; Do the MolT-II cells upregulate checkpoint molecules upon peptide stimulation?\nWe agree this is an important issue which we indeed did not address properly in the previous version. To answer this, we reexamined our data from experiments in which we exposed MolT-II cells to peptide stimulation. Coculture of MolT-II cells with peptide-loaded D1 cells does not lead to any significant changes in the expression of checkpoint molecules by CD4 T cells compared to no stimulation. This is consistent with our other observations showing that peptide stimulation does not induce any significant functional responses in MolT-II CD4 T cells. The corresponding data has been added to the manuscript in the form of supplementary figure 2D, and is referenced in the results section (lines 155-156).\n2) I assume Figure 3A is gated on all splenic T cells. What do the VB6 gated (TCR transgenic) T cells look like?\nFigure 3A does indeed show the expression of CD44 and CD62L on all splenic CD4 T cells, in order to allow for comparison between the TCR-transgenic strains MolT-II (Vβ6) and OT-II (Vβ5) with wildtype C57BL/6 mice. Comparing the expression of CD44 and CD62L on CD4 T cells expressing the transgenic TCR to the expression by the overall population yields a similar pattern, as around 90% ±5% of all T cells carry the transgenic TCR in both mouse models (MolT-II and OT-II; see figure 1A).The figure below shows the direct comparison between total and TCR-transgenic CD4 T cells of MolT-II (Vβ6) and OT-II (Vβ5) for these two markers.\n3) Figure 3E and F; Cross-reactivity is shown using proliferation only. Addition data such as cytokine expression would enable better assessment of degree of functional cross-reactivity.\nWe fully agree with this assessment and accordingly added additional data comparing the expression of cytokines and activation markers by MolT-II CD4 T cells after stimulation with envH and AKVenv. To be precise, the histograms showing CFSE proliferation has been removed, the original figure 3F showing a bar graph depicting CFSE-staining based proliferation is now referenced as figure 3E and we added a new figure 3F, showing a bar graph of the frequency of CD40L+ TNFα+ CD4 T cells after peptide stimulation. Further, we added supplementary figure 3D, depicting the effects of stimulation with envH and AKVenv on the expression of IL-2, CD25 and CD69 by MolT-II CD4 T cells. These data all show that MolT-II CD4 T cells cross-react with AKVenv, although the response is lower compared to stimulation with the cognate epitope envH. We adapted the text of the results section (lines 198-206) to reflect the newly added data.\n4) The delta in naïve vs vaccinated CD40L expression 4A for the OT-II is not very impressive. Other analyses (including phenotype without ex-vivo restimulation) would better support robustness of the vaccine.\nWe agree that the data in former figure 4A do not show the robustness of the vaccine. This is however clearly illustrated in the luciferase reporter transfer experiment (former figure 4C now 4B) where in vivo activation is induced in OT-II cells and to a lesser extent in MolT-II cells. In addition, we have used this transfer-vaccination setting in a new tumor experiment (figure 6) which shows effectiveness of the vaccine (see below).\n5) A major gap in the studies is evidence for in vivo functionally – that is control of tumors for example. This could easily be assessed using tumors that express both ovalbumin and Moloney envelope. This would markedly improve the robustness of the data supporting both dysfunction and reversal of dysfunction.\nAs noted by the reviewer, adding further studies demonstrating the dysfunctional state of MolT-II cells, the reversal of dysfunction and the functional capabilities of reinvigorated MolT-II cells (e.g. in the form of tumor control) would greatly add to the content of this manuscript. We have new data showing that MolT-II cells can contribute to the control of envH-expressing RMA tumors in a cell transfer setting combined with robust vaccination, which we have now incorporated in the newly added figure 6 of our manuscript. The results section (lines 250-266) as well as the discussion (lines 384-401) were adjusted to include the data of this experiment. A transfer of MolT-II cells combined with vaccination can firmly improve the survival of RMA tumors in C57BL/6 mice compared to vaccination alone, whereas only transferring MolT-II cells does not improve tumor control, reflecting the sustained dysfunctional state of the cells without additional intervention.\nOur future aims are to further explore ways to reinvigorate MolT-II cells to gain tumor-specific CD4 immunity and understand mechanisms of combined immunotherapeutic interventions of cancer in vivo.\n6) Where is the ERV-derived peptide expressed? Presumably in the thymus but where in the periphery?\nThe AKVenv peptide, which is derived from the murine endogenous retrovirus AKV, is indeed expressed by thymic stromal cells, especially in mTECs who play and important role for the establishment of central tolerance, and, to a lesser degree, by thymic DCs. We added qPCR data to reflect this in figure 3D and in the text (lines 185-192; 331-332). Regarding the expression of AKVenv in the periphery, it is well established that murine ERV sequences identical to AKVenv are commonly expressed in the lymphoid system and have been found in expression data from peripheral tissues (1, 2), and especially are upregulated in cancerous tissues (3, 4).\nReviewer #2\n1) The claim that AKVenv is the driving endogenous ligand for thymic deletion and peripheral dysfunction is central to the manuscript. However, direct evidence for AKVenv expression in the thymus or peripheral APCs is lacking. Inclusion of RT-qPCR or RNA in situ hybridization to detect AKV transcripts in thymic epithelial cells or APCs would considerably strengthen the conclusion that MolT-II CD4 T cells are tolerized/exhausted due to chronic exposure to an endogenous antigens.\nAs requested, we explored the expression of the AKVenv sequence in the thymus and found that AKVenv is indeed expressed by cells in the thymus, specifically by medullary thymic epithelial cells and to a lesser extent by dendritic cells. These results have been included in the manuscript in figure 3D, as well as in the results text (lines 185-192) and in the discussion (lines 331-332).\nRegarding the expression of AKVenv in the periphery, it is well established that murine ERV sequences identical to AKVenv are commonly expressed in the lymphoid system and have been found in expression data from peripheral tissues (1, 2), and especially are upregulated in cancerous tissues (3, 4).\n2) Given that 50 nM peptide appears to saturate proliferation in both OT-II and MolT-II T cells, CFSE assays may not be sensitive enough to resolve differences in TCR avidity. While CFSE-based proliferation to AKVenv peptide was observed compared to unstimulated control, more direct evidence of TCR recognition, such as MHC class II tetramer binding, early activation markers (e.g., CD69, CD25), or peptide titration with functional readouts, would strengthen the claim of cross-reactivity to the endogenous AKVenv peptide.\nWe agree with the reviewer’s assessment that further proof of cross-reactivity to AKVenv is required. While we unfortunately do not have an adequate MHC II tetramer at this time, we now included data showing the expression of cytokines (figure 3F, supplementary figure 3D) and activation markers (supplementary figure 3D) by MolT-II cells after stimulation with envH and AKVenv. These data show that while AKVenv induces lower responses compared to envH, these responses are still significantly higher than those induced by stimulation with an irrelevant peptide (ovaH). We adapted the text accordingly (lines 198-206).\n3) While the MolT-II CD4 T cells display features of dysfunction such as impaired proliferation and elevated inhibitory receptors, it remains unclear whether these cells mirror classical exhaustion, tolerance, or an intermediate phenotype. Comparative transcriptomic or phenotypic profiling, particularly CD4 T cells from chronic viral or tumor models, would clarify the relationship. The authors used ICOS and OX40 expression as markers of exhausted CD4 T cells (ref. 37), but it would strengthen the interpretation to include functional comparisons with known exhausted CD4 T cell populations.\nWe agree with this suggestion to do such comparison. CD4 T cell exhaustion remains understudied, and different phenotypes and subsets are described. However, the phenotype of MolT-II CD4 T cells is similar to signatures of exhausted CD4 T cells described in both murine and human tumors (5-8), as well as in chronic infection models (9) and partially matches signatures described in autoimmune diseases (10). For example, a study investigating T cell exhaustion in a murine glioblastoma model describes exhausted CD4 T cells as co-expressing PD-1, CTLA-4, LAG-3, TIM-3, ICOS and OX40, just as MolT-II CD4 T cells do (8). Similarly, other studies show that exhausted CD4 T cells from tumor patients respond poorly to stimulation and co-express different combinations of co-inhibitory and co-stimulatory checkpoints, again with PD-1, CLTA-4, LAG-3, TIM-3, ICOS and OX40 among them (5-7). The signature we find in MolT-II CD4 T cells also resembles one described for CD4 T cells exhausted by chronic LCMV infection, in which the cells show are only capable of very limited cytokine production and express PD-1, CLTA-4, TIM-3, ICOS and OX40 (9). We also compared the phenotype of MolT-II cells with large transcriptomics datasets describing immune cells and immune cell subsets in cancer patients, such as EcoTyper (11),but found no exact matches. We adapted the discussion (lines 305-313) to better reflect how the phenotype we describe in the MolT-II mice fits into what is currently known about CD4 T cell exhaustion.\n4) The rescue of function via triple blockade is an important finding. It would be valuable to assess changes in key transcription factors associated with T cell exhaustion and reinvigoration (e.g., TOX and TCF1), providing insight into whether MolT-II CD4 T cells undergo molecular reprogramming toward a less dysfunctional state.\nWe agree that studies further exploring the reinvigoration of CD4 T cells through vaccination and checkpoint blockade is crucial, and are planning to explore the molecular state of MolT-II cells more in depth in the future. This is an extensive study and we feel that this is outside of the scope of the current report.\n5) While the authors provide compelling evidence that MolT-II CD4 T cells can be reinvigorated in vivo through combined checkpoint blockade and antigenic stimulation, the functional consequences of this reinvigoration remain unclear. The restoration of proliferation and cytokine production offers important mechanistic insights. However, assessing whether reinvigorated CD4 T cells support downstream immune responses, such as enhancing CD8 T cell function or controlling tumor growth, would further strengthen the translational relevance of this model, especially in contexts where CD4 help is critical.\nAlthough such experiments may be beyond the current scope and present experimental challenges, addressing this point would significantly enhance the impact and applicability of the model for future studies on CD4 T cell–mediated immune modulation in chronic antigenic settings like cancer or persistent infection.\nWe agree and while in-depth studies assessing the functional capabilities of reinvigorated MolT-II CD4 T cells in vivo are indeed beyond the scope of this study, we were able to evaluate the capabilities of MolT-II CD4 T cells in supporting anti-tumor immune responses. In the newly added figure 6 we show that MolT-II cells combined with robust tumor-specific vaccination improve the survival of RMA tumors (expressing envH). We added text explaining the experiment in the results section (lines 250-266) and in the discussion (lines 384-401).\nMinor points and suggestions\n1. The authors should spell out \"MolT-II\", “AKV”, and “TbiLuc” early in the manuscript for reader clarity.\nWe agree and have spelled out and explained the above names in the manuscript in the appropriate locations. The MolT-II mouse was named in analogy to the well-known OT-II mouse model, as it is a TCR transgenic mouse strain with CD4 T cells recognizing a Moloney MuLV-derived, MHC class II-presented peptide (“envH” = envelope help). The TbiLuc mouse is a dual-color bioluminescence reporter mouse which enables the in vivo imaging of T cells. The AKV murine leukemia virus is an endogenous retrovirus, originally found in AKR mice, from which it derives its name.\n2. Figures 1D and 1E should be appropriately called out.\nWe want to thank the reviewer for pointing this out, the text (lines 103-104) has been adjusted to appropriately refer to the figures.\n3. In Figure 2A, adding quantitative graphs with statistics would improve clarity. Also, including an irrelevant control peptide would better control for background responses.\nWe agree with the reviewer that a quantitative graph illustrating the differences in cytokine expression between MolT-II and OT-II CD4 T cells would provide more clarity. Therefore, we have added a graph showing this data in figure 2B, which also includes the irrelevant peptide controls (stimulation of MolT-II with ovaH and of OT-II with envH).\nReferences\n1. Ikeda H, et al. Virological properties and nucleotide sequences of Cas-E-type endogenous ecotropic murine leukemia viruses in South Asian wild mice, Mus musculus castaneus. J Virol.2001;75(11):5049–58.\n2. James E, et al. Differential suppression of tumor-specific CD8+ T cells by regulatory T cells. J Immunol.2010;185(9):5048–55.\n3. Baylac-Kalabokias H, et al. A new leukemogenic retrovirus isolated from tumor cells derived from a radio-induced lymphoma of C57BL/6 mice: analysis of the env and LTR sequences. Virus Res.1991;18(2-3):117–34.\n4. Wedekind MF, et al. Endogenous retrovirus envelope as a tumor-associated immunotherapeutic target in murine osteosarcoma. iScience.2021;24(7):102759.\n5. Chi H, et al. T-cell exhaustion signatures characterize the immune landscape and predict HCC prognosis via integrating single-cell RNA-seq and bulk RNA-sequencing. Front Immunol.2023;14:1137025.\n6. Tietscher S, et al. A comprehensive single-cell map of T cell exhaustion-associated immune environments in human breast cancer. Nat Commun.2023;14(1):98.\n7. Ozkazanc D, et al. Functional exhaustion of CD4(+) T cells induced by co-stimulatory signals from myeloid leukaemia cells. Immunology.2016;149(4):460–71.\n8. Khan SM, et al. Impact of CD4 T cells on intratumoral CD8 T-cell exhaustion and responsiveness to PD-1 blockade therapy in mouse brain tumors. J Immunother Cancer.2022;10(12).\n9. Crawford A, et al. Molecular and transcriptional basis of CD4(+) T cell dysfunction during chronic infection. Immunity.2014;40(2):289–302.\n10. Saggau C, et al. Autoantigen-specific CD4(+) T cells acquire an exhausted phenotype and persist in human antigen-specific autoimmune diseases. Immunity. 2024;57(10):2416–32 e8.\n11. Luca BA, et al. Atlas of clinically distinct cell states and ecosystems across human solid tumors. Cell.2021;184(21):5482–96 e28."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately responded to reviewer comments including the addition of new data that address overlaping comments from both reviewers- related to central tolerance and in vivo function of reinvigorated T cells.", "reviewer_2": "The authors have appropriately addressed all the reviewer's comments and revised the manuscript accordingly. I have no further concerns."}, "author_response": ""}]

Decision letter