Introduction

The study of innate-like T cells, namely invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, is rapidly expanding and gaining interest. Defined by their phenotype, innate-like T cells bear functional T-cell receptor (TCR) rearrangements, yet acquire a memory phenotype during development, and quickly respond to stimulation without undergoing extensive clonal expansion1,2. They are generally thought to recognize conserved antigens3. For these reasons, they are considered as a bridge between adaptive and innate immunity, and are seen as an appealing target for immune therapies. As a prominent member of this group, iNKT cells were initially described as cells with such unconventional phenotype4,5,6,7. Consequently, it was shown that iNKT cells share the same developmental path with conventional αβ T cells until the double-positive (DP) thymocyte stage8. Yet, they follow a different process of thymic selection. Conventional αβ T cells undergo positive and negative selection by thymic epithelial cells (TECs) presenting peptide antigens by classical class I and II major histocompatibility complex (MHC) molecules. In contrast iNKT cells are positively selected on hematopoietic cells (HCs), in particular, another DP thymocyte presenting lipid antigens via the non-classical MHC-like molecule CD1d3,9,10,11. In addition, positive selection of iNKT cells requires a strong TCR signaling, a process known as “agonist selection,” and a secondary co-stimulatory signal provided by homotypic interactions between signaling lymphocyte activation molecule family (SLAMF) receptors12,13. As a result, selected cells upregulate the key transcription factor PLZF, which governs the commitment to the iNKT cell lineage14,15. Subsequently, more innate-like T-cell subsets were described, including PLZF+ γδ T cells and MAIT cells. Similar to iNKT cells, both cell subsets express PLZF and the latter were shown to be positively selected by DP thymocytes16,17. In addition, it was reported that non-classical MHC Ib-restricted cells can be selected by TECs or by HCs. However, they develop an innate-like phenotype only when they are selected on HCs but not on TECs18. Thus, it appears that a common feature of innate-like T cells is being positively selected by thymocytes. Thus, DP–DP interactions are proven to be pivotal for induction of PLZF expression and initiation of an innate-like program in T cells. Yet, the underlining mechanisms governing selection and development of these cells are still elusive.

Strikingly, murine DP thymocytes do not express classical MHC I or MHC II molecules4c). Surprisingly, PIL T cells in T-MHC I mice did not exclusively express CD8, but segregated into CD4 SP, CD8 SP, and DN, similar to those in WT mice, suggesting that PIL T cells in WT mice may be more closely related to MHC I-restricted cells (Fig. 4c). Although we did not observe a severe bias in TCR Vβ chain usage by PIL T cells, there was a noticeable increase in frequency of Vβ3+ and Vβ5.1/Vβ5.2+ cells within the PIL T-cell pool from WT and T-MHC I mice. In addition, a small increase in frequency of Vβ6+ cells was present within the PIL T cells from T-MHC II mice (Fig. 4d).

It was previously shown that thymic iNKT cell subsets display different levels of TCR on their surface, a trait that is more prominent on the BALB/c background33,36. TCR levels are highest on NKT2 cells, lowest on NKT1 cells, and intermediate on NKT17 cells. Moreover, these differences in TCR expression levels, and thus the presumed signaling strength, are reported to be pivotal for iNKT cell subset commitment and differentiation37,38,39. However, surface TCR did not differ significantly between PIL T-cell subsets (Fig. 4e), suggesting that factors other than TCR expression level may dictate subset commitment and differentiation in these cells.

Historical studies in the field of iNKT cell biology used CD44 and NK1.1 as markers to assess iNKT cell maturity and stage of development40. Using these markers, a higher proportion of PIL T cells were found to be CD44low compared to iNKT cells, indicating a less mature stage (Fig. 4f).

Memory phenotype CD8 T-cell development requires CD4 co-receptor engagement in PIL T cells

As previously reported, the frequency of CD8 SP T cells was increased in the thymus of T-MHC II mice (Fig. 2b)21,22,31. This was shown to be caused by IL-4 produced by PIL T cells (called T-CD4 cells in that report), which mediates development of memory phenotype CD8 T (TMP) cells through the induction of eomesodermin (Eomes)41. Thus, a large proportion of CD8 SP T cells in T-MHC II mice express Eomes (Fig. 5a) and have a memory phenotype31. To our surprise, there was no increase in Eomes+ CD8 TMP in T-MHC I mice (Fig. 5b). As interaction of the TCR with MHC II can invoke CD4 co-receptor signaling, we reasoned that the induction of IL-4 production by PIL T cells and, consequently, the increase in CD8 TMP cells might be the result of CD4 co-receptor signaling. To test this hypothesis, we crossed T-MHC I mice with CD8.4 transgenic mice. In these mice, the cytoplasmic tail of the endogenous Cd8a gene was substituted with the cytoplasmic tail of CD442. The CD8.4 transgene did not cause an increase in the total number of PIL T cells in T-MHC I mice (Fig. 5c), but it shifted the proportion of PIL T cells in favor of PLZFhi PIL2 cells (Fig. 5d), which produce IL-4. Consistent with this, the number of CD8 TMP was markedly increased in CD8.4 T-MHC I mice (Fig. 5e). A similar trend was seen in the spleen (Supplementary Fig. 7). Thus, the particular co-receptor involved in sensing MHC ligands on DP thymocytes influences effector subset differentiation of PIL T cells and has secondary effects of CD8 TMP development.

Fig. 5: MHC II, but not MHC I, drives type 2 cytokine production in PIL T cells and induces memory phenotype CD8 T (TMP)-cell development.
figure 5

a Representative flow cytometry plots of intracellular staining for Eomes on CD8 SP thymocytes from WT, T-MHC I, and T-MHC II mice. b Number (plotted on the right axis) and frequency (plotted on the left axis) of CD8 TMP cells among CD8 SP cells in the thymus from WT, T-MHC I, and T-MHC II mice, defined by the gating strategy shown in a. c Representative flow cytometry plots of thymocytes comparing PIL T-cell frequency from T-MHC I mice with CD8.4Tg/+ T-MHC I mice. Shown are summary evaluations for PIL T-cell number and frequency (right two panels). d Representative flow cytometry plots and summary evaluation for frequency of PIL2 T-cell subset among thymocytes from T-MHC I and CD8.4Tg/+ T-MHC I mice. Both groups are compared to WT mice. e Exemplarity flow cytometry plots of CD8 TMP cell frequency among CD8 SP cells in the thymus from T-MHC I mice with CD8.4Tg/+ T-MHC I mice (left two panels) and summary evaluation of CD8 TMP cell number (right panel). Each point represents one animal: n = 9 animals per group (WT and T-MHC II groups) in a, b, n = 9 animals (T-MHC I group) in c, d, n = 7 animals (T-MHC I group) in b, e, and n = 4 animals (CD8.4Tg/+ T-MHC I group) in c–e. Data are representative of 7 in a, b and 2 in c–e independent experiments. Unpaired two-tailed Mann–Whitney test was performed in b–e; ns, not significant (p ≥ 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Full size image

PIL T cells compete with iNKT cells for a cellular niche within the thymus

Similar to what have been previously reported with T-MHC II mouse43, we observed a two- to threefold reduction in the number of lipid-specific iNKT cells in mice with increased numbers of PIL T cells due to MHC I or MHC II expression on DP thymocytes (Fig. 2b). Of note, the pooled total cell count of PILs + iNKT cells was not significantly different between WT and T-MHC I mice (data not shown). As both peptide and lipid-specific T cells require SAP signaling from the surface SLAM family co-receptors to develop into PIL T cells or iNKT cells, respectively, this suggests that the two cell types may be in competition with each other for a cellular niche within the thymus. To further test this notion, we examined B6xBALB/c F1 mice, which have a slightly larger (twofold) iNKT cell niche32,44. If a similar niche regulates PIL T cells, then we might expect more PIL T cells in F1 mice. Indeed, both the portion and number of PIL T cells in T-MHC I and T-MHC II mice were higher in animals on the F1 background compared to the B6 background (Fig. 6a, b and Supplementary Fig. 8a, b) inferring an existence of a bigger niche for PIL T-cell development on the F1 background. We observed an inverse relationship between the number of iNKT cells and PIL T cells in both B6 and F1 mice (Fig. 6c and Supplementary Fig. 8c). Taken together, with the expansion of PIL T cells in mice that lack iNKT cells (Fig. 3b), these data strongly suggest that PIL T cells and iNKT cells compete for the same cellular niche. As an aside, the reported skewing of iNKT cells towards the PFZFhi NKT2 subset in F1 mice32 was also observed in PIL T cells (Fig. 6d and Supplementary Fig. 8d). This suggests that the same factors control iNKT and PIL T-cell effector subset skewing in different strains, which are more likely to be environmental cytokines, cell-intrinsic factors, or co-stimulatory molecules rather than specific self-antigens.

Fig. 6: PIL T cells compete with iNKT cells.
figure 6

a B6 WT, T-MHC I, and T-MHC II mice were crossed to BALB/c mice and F1 generation littermates (labeled as F1, F1 T-MHC I, and F1 T-MHC II) were analyzed by flow cytometry for frequency of iNKT and PIL T cells in the thymus. b Summary evaluation of iNKT and PIL T-cell frequency (left panel) and number (right panel) from WT, T-MHC I, and T-MHC II mice on B6 and F1 background. c Inverse correlation between number of iNKT cells and the number of PIL T cells in the thymus from WT (black dots), T-MHC I (red triangles), and T-MHC II mice (blue inverse triangles) on B6 (left panel) and F1 (right panel) background. d Shown are representative flow cytometry plots comparing iNKT and PIL T-cell subsets from WT, T-MHC I, and T-MHC II mice on B6 (upper row) and F1 (lower row) background. Each point represents one animal: n = 10 animals per group (WT and T-MHC I groups), n = 8 animals (T-MHC II group), n = 9 animals (F1 WT group), n = 6 animals (F1 T-MHC I group), and n = 7 animals (F1 T-MHC II group) in a–d. Data are representative of seven independent experiments. Unpaired two-tailed Mann–Whitney test was performed in b; ns, not significant (p ≥ 0.05), ***p < 0.001, and ****p < 0.0001. R2-values and p-values in c were calculated by fitting nonlinear regression and performing a Goodness-of-Fit test and extra-sum-of-squares F test. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Full size image

Discussion

Here we describe an Nlrc5-transgenic mouse (T-MHC I) allowing cell subset-specific expression of Nlrc5, which is a central regulator of the expression and function of peptide presenting MHC I genes. Consequently, our data show that forced expression of Nlrc5 led to upregulation of MHC I on DP thymocytes. To the best of our knowledge, this is the first transgenic mouse model allowing cell subset-specific MHC I upregulation without altering MHC II regulation. Hence, this mouse line can serve as a powerful tool in future studies where stable MHC I expression is sought. Notably, Nlrc5 expression increased the surface level of some non-classical MHC Ib molecules such as Qa-1 and Qa-2 (peptide-presenting molecules) but did not affect CD1d and MR1 (non-peptide presenting molecules). Therefore, in our mouse model, we assume enhanced self-peptide MHC Ia/Ib presentation by DP thymocytes renders them capable of steering peptide-specific T cells towards an innate-like developmental pathway. Although we do not provide a direct proof for the nature of the antigens that PIL T cells recognize, previous studies showed that DP thymocytes could not select PIL T cells in a foreign antigen-specific TCR transgenic T-MHC II mouse model22. This implies that PIL T-cell selection is self-antigen specific and requires presentation by an opposing DP thymocyte. In addition, a subsequent study showed that PIL T cells from T-MHC II mice have a highly diverse TCR repertoire, implying they may recognize a variety of self-antigens45. Taken together, it is reasonable to consider that PIL T cells could be selected on endogenous peptide self-antigens presented by DP thymocytes, much similar to iNKT cells selected on an endogenous lipid self-antigen presented by DP thymocytes3,9,10,11.

Although DP thymocytes do not normally express classical MHC Ia molecules, a small fraction of PIL T cells were already present in WT mice. Yet, we show that most of these cells are not CD1d or MR1 restricted. Therefore, it remains unclear how these innate-like cells were selected. Interestingly, the small number of PIL T cells present in WT mice more closely resemble PIL T cells from T-MHC I mice than those from T-MHC II mice. This implies that PIL T cells in WT mice might be restricted to MHC Ib molecules, which are found to be still expressed, although at a low level, on DP thymocyte (i.e., H2-M3 and Qa-1). Indeed, it was previously shown that peptide-specific MHC Ib-restricted T cells could be selected on TECs or HCs46. However, those cells acquired an innate-like phenotype only when they are selected on HCs18,46. In addition, this process was shown to be SAP dependent47. Therefore, it is plausible that PIL T cells in WT mice are MHC Ib restricted.

Although MHC I expression on DP thymocytes led to an expansion of PIL T cells, the magnitude of this expansion was lower than that in the T-MHC II mouse. Further, the increased PIL T-cell frequency in the T-MHC I mouse did not result in a notable expansion of CD8 TMP cells, implying functional difference between PILs selected in T-MHC I and T-MHC II mice. Of note, mature murine iNKT cells do not express CD8αβ. In fact, several studies have previously shown that forced expression of CD8αβ is detrimental for iNKT cell development and leads to profound reduction in iNKT cell numbers7,48. Subsequently, another study showed that the silencing of CD8 expression in develo** iNKT cells is mediated by the transcription factor Th-POK, whose expression is required for functional maturity of iNKT cells49. Therefore, the co-receptor choice seems to govern the functional phenotype of PIL cells as well. As a result, despite being selected on MHC I molecules, PIL cells develo** in T-MHC I mice might be downregulating CD8 expression post selection due to initiation of the innate-like developmental program and expression of Th-POK. In addition, previous reports suggest that TCR signal strength seems to regulate iNKT cell subset commitment and differentiation37,38,50. Stronger TCR signal intensities favor iNKT2 cell development and a lower strength preferentially guides into the iNKT1 cell pathway. Moreover, CD4 co-receptor signaling intensity is higher in comparison to CD851,52,53 and presumably favors PIL2 cell expansion in T-MHC II mice. Therefore, increasing co-receptor signaling intensity by crossing T-MHC I to the CD8.4 mouse42 may have increased the frequency of PIL2 cells and expansion of CD8 TMP cells.

There are several studies suggesting a possible involvement of Nlrc5 expression in pro-inflammatory and type I IFN responses54,55. Although we did not observe any major alterations of conventional T-cell development, forced Nlrc5 expression in our mouse model caused an increase in Treg cell number and frequency. Moreover, here we show that this expansion was mediated in a cell-intrinsic manner by Nlrc5 and it was not because of MHC I expression on neighboring DP thymocytes. Interestingly, Nlrc5 is debatably described as a positive or negative modulator of nuclear factor-κB (NF-κB) signaling56,57,58. NF-κb signaling is also known to be crucial for Treg cell development and function59,60. Taking this into consideration, it is possible that Nlrc5 expression at the DP stage might influence Treg cell selection and development by modulating NF-κB signaling. Further studies will be needed to identify the precise mechanism behind Nlrc5 involvement in Treg cell biology.

Lastly, we provide several lines of evidence supporting the existance of a narrow niche for innate-like T-cell development. First, iNKT cell number and frequency were reduced in both T-MHC I and T-MHC II mice. Second, this inverse correlation between iNKT cell and PIL T-cell numbers was even more profound on a B6xBALB/c F1 background where the niche for iNKT cell development is larger. In addition, CD1d-deficient T-MHC I mice showed an increase in PIL T cells. Innate-like T cells might compete between each other for abundance of cytokines, activation ligands, or could directly inhibit each other’s development and survival via unknown mechanisms. Of note, the local abundance of IL-7, IL-15, and IL-2561,62,63, in the thymus, are crucial factors implicated in iNKT cell development, survival, and terminal subset maturation. Thus, higher PIL T-cell numbers might alter the bioavailability of these cytokines in the thymus. Yet, MAIT cells and γδ NKT cells did not display a reduction trend in the T-MHC I mouse. Therefore, this is suggestive that PIL T cells and iNKT cells probably compete for the same activation ligands, presumably members of the SLAMF receptors.

MHC class I molecules are some of the most widely expressed genes in the body. In contrast to MHC class II molecules, which are expressed primarily by professional antigen presenting cells, MHC class I is generally thought to be expressed by all nucleated cells. Based on this, the absence of MHC I in DP thymocytes is remarkable. The fact that we observed a reduction of lipid-specific iNKT cells when peptide-presenting MHC alleles were forced to be expressed in DP thymocytes suggests an evolutionary rationale for the absence of MHC I in DP thymocytes—to facilitate the development of innate-like T cells that recognize non-peptide ligands. Indeed, iNKT cells do recognize non-peptide ligands such as lipids64. Given that these cells also are semi-invariant means that their rearrangements occur relatively rarely, despite the great numeric abundance of these cells in the body10. Thus, the thymus appears to have a mechanism whereby the development of innate-like T cells from rare TCR gene rearrangements is favored.

Methods

Mice

C57BL/6NCrl (B6) and B6.SJL-PtprcaPepcb/BoyCrCrl (CD45.1) mice were obtained from Charles River (via the National Cancer Institute). BALB/cByJ, B6.129S6-Sh2d1atm1Pls/J (SAP−/−), C57BL/6-Tg(Lck-CIITA)16Spark/J (Plck-CIITA), and B6.129S6-Del(3Cd1d2-Cd1d1)1Sbp/J (CD1d−/−) mice were purchased from the Jackson Laboratories. CD8.4 transgenic mice were kindly provided by Dr. Alfred Singer (NCI/CCR, Bethesda). All animals were maintained under specific pathogen-free conditions at the University of Minnesota. All animals used in this study were 6–10 weeks old at the time of analysis. All experimental procedures were approved by the institutional animal care and use committee at the University of Minnesota (IACUC 1706-34889A and 1709-35136A).

Generation of Nlrc5-stopflox transgenic mouse

The Nlrc5-stopflox transgenic mice were generated by the insertion of a CAG-LoxP-STOP-LoxP-Nlrc5-WPRE-pA construct into mouse Rosa26 locus through the CRISPR/Cas9 system with C57BL/6J embryo. The generated mice were screened and further verified for the correct insertion by sequencing and Southern blotting. The generation of Nlrc5-stopflox mice was conducted by Biocytogen.

BM chimeras

BM cells were isolated from the femur and tibia of B6 CD45.1/2+, B6 CD45.2+, and T-MHC I CD45.2+ donor mice. Following isolation, cells were counted and washed twice in cold phosphate-buffered saline (PBS). B6 CD45.1/2+ cells (5 × 105) mixed with 4.5 × 106 T-MHC I CD45.2+ cells were mixed and transferred intravenously into lethally irradiated (900 rads) CD45.1 recipient mice. The control group received 5 × 105 B6 CD45.1/2+ cells mixed with 4.5 × 106 B6 CD45.2+ cells. Mice were killed 8 weeks post reconstitution when the spleen and thymi were collected for flow cytometry analysis.

Flow cytometry

Organs were collected and single-cell suspensions were prepared on ice in fluorescence-activated cell sorting buffer (PBS/3% fetal calf serum). All surface stainings were performed for 30 min on ice. Intracellular detection of cytokines and transcription factors were done using the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences, TNB-0607-KIT), following the protocol provided by the manufacturer. Antibodies used (clone name, dilution factor) were as follows: anti-mouse CD4 BUV395 (GK1.5, 1 : 400), anti-mouse CD8a BUV737 (53-6.7, 1 : 400), anti-mouse Qa-1(b) PE (6A8.6F10.1A6, 1 : 100), anti-mouse CD1d PE (1B1, 1 : 100), anti-mouse PLZF AF647 (R17-809, 1 : 200), anti-mouse CD45.2 BUV737 (104, 1 : 200), anti-mouse RORγt BV786 (Q31-378, 1 : 400), anti-mouse/rat CD44 BV510 (IM7, 1 : 200), anti-mouse IL-4 APC (11B11, 1 : 100), anti-mouse Vβ2 TCR FITC (B20.6, 1 : 100), anti-mouse Vβ3 TCR FITC (KJ25, 1 : 100), anti-mouse Vβ4 TCR FITC (KT4, 1 : 100), anti-mouse Vβ5.1/Vβ5.2 TCR FITC (MR9-4, 1 : 100), anti-mouse Vβ6 TCR FITC (RR4-7, 1 : 100), anti-mouse Vβ7 TCR FITC (TR310, 1 : 100), anti-mouse Vβ8.3 TCR FITC (1B3.3, 1 :100), anti-mouse Vβ10b TCR FITC (B21.5, 1:100), anti-mouse Vβ11 TCR FITC (RR3-15 1 : 100), anti-mouse Vβ12 TCR FITC (MR11-1, 1 : 100), anti-mouse Vβ13 TCR FITC (MR12-3, 1 : 100), and anti-mouse Vβ14 TCR FITC (14-2, 1 : 100) all from BD Bioscience; anti-human HLA-A,B,C PE (W6/32, 1 : 200), anti-mouse I-A/I-E BV510 (M5/114.15.2), anti-mouse H-2Ld/H-2Db PE (28-14-8, 1 : 100), anti-mouse H-2Kb PE (AF6-88.5, 1 : 100), anti-mouse Qa-2 FITC (695H1-9-9, 1 : 100), anti-human/mouse/rat MR1 APC (26.5, 1 : 100), anti-mouse TCRb BV421 (H57-597, 1 : 100), anti-mouse TCR γ/δ PE-Cy7 (GL3, 1 : 100), anti-mouse CD45.1 BV510 (A20, 1 : 200), anti-mouse NK1.1 FITC (PK136, 1 : 100), anti-mouse CD138 PE (281-2, 1 : 200), anti-mouse CD122 PE (TM-β1, 1 : 100), anti-mouse CXCR3 FITC (CXCR3-173, 1 : 100), anti-mouse CD196 (CCR6, 1 : 100) PE (29-2L17), anti-mouse CD25 PE (PC61, 1 : 100), and anti-mouse IL-17A PE (TC11-18H10.1, 1 : 100) all from BioLegend; and anti-mouse/rat Foxp3 PE (FJK-16s, 1 : 200), anti-mouse PLZF AF488 (Mags.21F7), anti-mouse CD69 PE (H1.2F3, 1 : 200), anti-mouse CD19 PerCP-Cy5.5 (eBio1D3, 1 : 200), anti-mouse CD279 (PD-1) PE (J43, 1 : 200), anti-mouse IFNγ PE (XMG1.2, 1 : 100), and anti-mouse Eomes AF488 (Dan11mag, 1 : 100) all from eBioscience. CD1d tertramer loaded with PBS57 (analog of a-galactosylceramide) and MR1 tetramer loaded with 5-OP-RU were provided by the tetramer facility of the US National Institutes of Health. Flow cytometric analysis was performed on LSR Fortessa (BD Biosciences) using FACSDiva software (version 6.1.3, BD Biosciences) and data analysis was done using FlowJo software (Treestar).

Analysis of intracellular cytokines

Total thymocytes were plated at a density of 1 × 106 ml−1 and incubated at 37 °C for 4 h in the presence of Cell Stimulation Cocktail (eBioscience, 00-4975-93) and Protein Transport Inhibitor Cocktail (eBioscience, 00-4980-93) in RPMI 1640/10% fetal bovine serum followed by cytokine detection by intracellular staining.

Enrichment of MAIT cells

Single-cell suspensions were prepared from the thymus or spleen and incubated with PE-MR1–5-OP-RU tetramer for 30 min at ambient temperature. Following incubation, anti-PE microbeads (Miltenyi) were used for immunomagnetic enrichment following the manufacturer’s instructions.

Immunofluorescence

Thymi were fixed with 4% paraformaldehyde overnight, transferred to 30% sucrose solution for 24 h, and snap frozen in optimum cutting temperature compound. Sections (7 μm) were blocked with 5% bovine serum albumin and Fc block (anti-CD16/CD32; 2.4G2, Tonbo Biosciences) in dilution 1 : 100 for 1 h at room temperature (RT) prior to staining. The sections were incubated with Rabbit-anti-b5t in dilution 1 : 200 (MBL International) and fluorescein-labeled Ulex europaeus agglutinin I (UEA-I) (Vector Laboratories) at 4 °C overnight, followed by Goat-anti-Rabbit-AF555 in dilution 1 : 500 for 1 h at RT (Thermo Fisher Scientific). Sections were next stained with 4′,6-diamidino-2-phenylindole and mounted using ProLong antifade mounting medium (Life Technologies). Images were acquired using a Leica DM6000B epifluorescent microscope.

Cloning mNlrc5 and lentiviral transduction experiments

The full-length CDS of murine Nlrc5 was cloned through PCR using primers Nlrc5-Frw: 5′-GATTCTAGAGCCACCATGGACGCTGAGAGCATCC-3′ and Nlrc5-Rev: 5′-GCAATCGATTTAATTAATCAAAGAGTCTGCTGGTCAGTG-3′ with cDNA from B6 CD8 T-cell splenocytes. Nlrc5 cDNA was further subcloned into lentiviral expression vector lentiCas9-EGFP and further verified by DNA sequencing. Nlrc5 expression lentivirus (lentiNlrc5-EGFP) was generated as previously described65. As a control, an empty construct (lenti-EGFP) was generated encoding EGFP (enhanced green fluorescent protein) as a marker for transduction efficacy. Viral supernatants were used to transduce HEK-293T cell. HEK-293T cells were analyzed for MHC I expression by flow cytometry 48 h post transduction.

Statistics and reproducibility

Statistical analysis was performed using GraphPad Prism v7 and v8 Software. An unpaired, two-tailed Mann–Whitney U-test was done when group sample size n was ≤4 in one or both of the groups, which were compared. For groups with sample size n > 4, a Kolmogorov–Smirnov test was used to check for the distribution normality of the data points in each of the groups to be compared. When both groups showed a normal distribution, then an unpaired two-tailed Student’s t-tests was performed. When one or both of the compared groups showed non-normal distribution, then an unpaired two-tailed Mann–Whitney U-test was used as indicated in the figure legends. Samples are shown with medians with error bars showing the SD. p-values of <0.05 (*), <0.01 (**), <0.001 (***), or <0.0001 (****) indicated significant differences between groups. All attempts at replicating experiments were successful.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.