Human CD1c-autoreactive T cells recognise Mycobacterium tuberculosis–infected antigen-presenting cells and display cytotoxic effector programmes

Reviewer #1 (Public review):

Summary:

T cells that recognize lipids - CD1c - are frequent in circulation; however, their role in infection is unclear. This study aims to understand how Mtb infection can shape the responses of CD1c-specific T cells. CD1c is expressed in MTB granuloma, but in lower amounts than in nearby inflamed tissue. Mtb infection downregulates the expression of CD1c on monocyte-derived DCs. Single-cell RNA sequencing revealed the cytotoxic program inherent to the lipid-CD1c-specific T cells. Using an in vitro APC system where CD1c expression remains intact upon Mtb infection, the authors suggest that these T cells react better to Mtb-infected than uninfected Cd1c-expressing APC and reduce Mtb burden in infected cells. Therefore, Cd1c downregulation could be an immune evasion strategy used by Mtb.

Strengths:

This study asks an important question. The single-cell transcription analysis suggests the inherent cytotoxic program of lipid-CD1c cells and provides insights into their phenotypic and potential functional profiles. Function experiments suggest that these autoreactive T cells can react to Mtb infection, adding to the paradigm of infection control by these non-conventional T cell populations.

Weaknesses:

The study lacks sufficient rigor; conclusions may be strengthened with the incorporation of more controls, and some deeper characterization of the THP1 system and the CD1c-specific T cells isolated from blood. Crucial conclusions are drawn from the cell mixing experiments involving the engineered THP-1 system and CD1c-lipid-specific T cells from blood. These cells need more in-depth characterization. The expression of MHC-I/II is clearly reduced in THP1-CD1c cells. However, it is important to ensure that it is completely abolished, since a residual expression can skew the result with activation of conventional T cells in the blood or low levels of conventional T cells that may be present in the CD1c-tetra/multimer sorted T cells. CD1c-tetra/multimer sorting should include more markers than used in this study.

Figure 2: The immunohistochemistry appears to be shown only for one biopsy; it may be worth quantifying the immunohistochemistry of all five. The expression of CD1 molecules goes up during the differentiation of MoDC. And Mtb infection prevents or dampens the upregulation. Does Mtb infection downregulate the CD1 expression of mature DCs? Can the effect of Mtb on the expression of CD1a,b,c molecules be investigated using CD1c-expressing DCs from blood? What could be the reason THP-1 cells do not downregulate CD1 molecules upon Mtb infection, and how about the expression of CD1a and b?

Figure 3: (F) What does the X-axis read for the no infection group? The value for MOI = 0 should be incorporated for the infected T cell group.

Figure 4: In the lysis assay, THP1-CD1c cells (uninfected and infected) incubated alone should be incorporated.

Figure 5: A quantitative brief on the single cell TCR sequencing - including how many T cells were sequenced and the frequency of different clone including EM1 and EM2 - should be shown.

Reviewer #2 (Public review):

Summary:

The study by Milton et al titled "Human CD1c-autoreactive T cells recognise Mycobacterium tuberculosis-infected antigen-presenting cells and display cytotoxic effector programmes" characterises CD1c-restricted autoreactive T cells and their potential role in controlling Mtb infection. The authors develop a well-controlled system to assay for the functioning/activation of autoreactive T cells. They report the presence of CD1c-restricted autoreactive T cells in the circulating blood of healthy donors. They show that these T cells respond to CD1c and get activated even in the absence of any exogenous antigen. They next show that CD1c, along with CD1a and b, are typically downregulated on APCs during Mtb infection. These autoreactive T cells are cytotoxic, indicating they respond to Mtb treatment and/or to changes in the T cell ratio. The autoreactive T cells could effectively lyse Mtb-infected or PAMP-stimulated CD1c+APCs. Next, using TCR sequencing, they show that T cell responses were mediated by specific TCR clones with common sequence features. They show that these autoreactive T cells could curtail Mtb growth as measured by luminescence. Finally, using scRNAseq, they selectively identify the CD1c-reactive T cell pool and detect enrichment of typical effector memory CD4 and CD8 cells expressing cytolytic markers such as Granzyme, granulolysin, etc. The lung biopsy staining, along with the other data presented here, suggests that while CD1c-restricted T cells could have potential anti-bacterial roles, Mtb downregulation effectively shuts down this mechanism for TB control.

Strengths:

The study is designed well and has developed many exciting tools to generate specific information.

Weaknesses:

The study has weaknesses in two important parameters - novelty and relevance in controlling TB. Further, the results could be better presented and discussed to allow easy understanding of the experimental design. For example, at several places, UV-killed or live Mtb were used. What is the rationale behind that? Why use irradiated THP1-CD1c cells for activating T cells?

While functional assays identified only CD4+ cells as CD1c-restricted, scRNAseq shows that both CD4+ and CD8+ cells exhibit this phenotype. Identifying the specific lipid antigen presented by CD1c could add greater value to the study.

Since autoreactivity was independent of exogenous antigen, the cytotoxic activity should also be independent of exogeneous antigens? What additional signal a THP1-CD1c cells treated with UV-killed Mtb express that is absent from the untreated cells?

The relative Mtb growth assay is confusing. CD1c cells with Mtb infection triggers massive lytic response, as shown in Figure 4. Under similar conditions, in Figure 6, the authors report a significant decline in Mtb growth in these cells. The problem is that with the kind of lytic response observed, a lot more Mtb could be present extracellularly and would evade killing. How do we reconcile the two observations?

Reviewer #3 (Public review):

Summary:

Despite the rising global prevalence of TB, the role of non-classical T-cell pathways in host immunity remains unclear. The present study by Milton et al. is a novel contribution to the field of unconventional T-cell immunity in Mtb infection. The study addresses the role of CD1c-autoreactive T-cells and demonstrates that upon Mtb infection, these cells are significantly activated, resulting in increased cytokine production and cytotoxicity, and a reduction in the bacterial burden, specifically against Mtb-infected CD1c+ APCs (antigen-presenting cells). This defines their role as a plausible candidate for lipid-directed immunity in TB, complementary to MHC-restricted responses.

Strengths:

The manuscript is well written, and the novelty, impact, and limitations of this study are precisely highlighted by the authors.

Weaknesses:

The authors mention that they did not identify any specific lipids presented by CD1c on Mtb-infected APCs, making it unclear whether they are of host or bacterial origin. This leaves a gap in understanding why the response is enhanced in Mtb-infected cells, whether it is through altered self-presentation of lipids arising from Mtb-induced changes, infection-induced stress signals, or Mtb lipids, or through CD1c-dependent co-stimulation/infection signals. Direct lipid identification via lipidomics/MS of CD1c-bound lipids from Mtb-infected APCs would clarify whether the enhancement arises from altered self-lipids or subtle Mtb lipids.