The identification of antigens from Mtb that stimulate CD4+
T cells has been hampered in the past by the use of techniques that relied on non–T cell components of the immune response for their identification. For example, the majority of Mtb antigens characterized thus far were originally identified based on their reactivity with antibodies, or as a consequence of their abundance and subsequent ease for purification 789
. The potential problem with these approaches is that antigens that stimulate potent T cell responses are not necessarily the same as those that stimulate strong antibody responses, nor are they necessarily the most abundant antigens. Indeed, of five Mtb antigens that we recently isolated using human serum from patients with TB, only one induced strong T cell responses in PPD+
. The technique we describe in this paper minimizes this problem because it uses T cells to directly identify antigens that are expressed as recombinant antigens in E
Previous studies have demonstrated that antigenic proteins expressed by recombinant E
can be presented by class II MHC molecules to antigen-specific T cells 19
. Indeed, Shastri and colleagues 2021
demonstrated that this property of E. coli–
expressed antigens could be applied to identify two previously unknown antigens from Listeria monocytogenes
using murine CD4+
T cells. This technique relied on the fusion of the antigen-specific T cells with a hybridoma partner containing a reporter gene (lacZ), and on screening for T cell activation at the single cell level. The technique we describe in this paper is much simpler and is amenable to high throughput screening, as it can be used with either T cell clones or lines, and does not require the generation of either hybridomas or a single cell assay. By taking advantage of the fact that blood-derived DCs are both phagocytic and are extremely potent APCs 17
, we were able to use an antigen-reactive T cell line to detect a previously identified Mtb antigen (Mtb39 ), as well as a family of previously unknown Mtb antigens (the Mtb9.9 family). In addition, we have used T cells from a TB-immune human donor, which presumably has relevance for the development of human vaccines.
Several antigens derived from Mtb have been reported to stimulate T cell responses from Mtb-infected humans and mice. In particular, the antigen 85B has been reported to be an immunodominant T cell antigen 22
. Here, we show that upon preliminary analysis, rMtb9.9A appears to be a more potent T cell antigen than antigen r85B. Of the 12 PPD+
donors tested, 10 made a significant proliferative response to rMtb9.9A (83%) compared with 8 who responded to r85B (67%). In particular, the magnitude of the responses to rMtb9.9A was stronger than to r85B, such that the mean SI with rMtb9.9A from PPD+
donors was 39.0 compared with 15.3 with r85B. Finally, rMtb9.9A induced strong IFN-γ responses in PPD+
but not PPD−
donors, further implicating Mtb9.9A as a candidate for inducing protective immune responses to Mtb.
The use of APCs infected with live Mtb to generate T cell lines ensures that the antigens that are detected are available to the immune response during infection with Mtb. Interestingly, Mtb9.9 was found to be primarily located in Mtb lysate, although low levels of protein were detected in CFPs by Western blotting. However, the quantity of Mtb9.9 in CFPs was sufficient to stimulate T cell responses, as the donor 201 line that reacted with Mtb9.9C was generated using CFPs as antigen. This highlights the sensitivity of the expression cloning technique, as it was able to identify an antigen present at low levels within a complex mixture of proteins. Indeed, rMtb9.9A is able to stimulate T cell responses at extremely low protein concentrations, with as little as 10 ng/ml resulting in optimal recall responses ( B), and as little as 100 pg/ml able to stimulate responses above background (data not shown).
Several groups have reported a heterogeneous response to Mtb antigens among healthy PPD+
individuals and patients with TB 1023
. Interestingly, Boesen et al. 24
described preferential recognition of low-mass CFPs in the majority of patients with minimal TB. This fraction contains several proteins, including 6-kD early secretory antigenic target, Mtb8.4, and Mtb9.9A. Although the Mtb9.9 family of proteins is of low mass, they were found to contain at least five distinct T cell epitopes ( and ). In addition, the donors used in this study represented a variety of ethnic backgrounds and HLA types. This may be important in the design of a subunit vaccine for Mtb, where T cell responses to antigens presented by a large number of MHC alleles would be required to vaccinate the majority of humans. In addition, most PPD+
donors (>80%) made a T cell response to rMtb9.9A, again suggesting that immunization of an outbred human population could potentially induce responses in the vast majority of individuals. Indeed, in preliminary studies, immunization of inbred mice and outbred guinea pigs with rMtb9.9A induced strong antigen-specific T cell responses (data not shown).
Response of PBMCs from four PPD+ donors to rMtb9.9A and overlapping synthetic peptides derived from Mtb9.9A, Mtb9.9B, and Mtb9.9C sequences (peptides derived from Mtb9.9A denoted by number only). Peptides and rMtb9.9A were used at 10 μg/ml.
Despite the very high amino acid homology among the members of the Mtb9.9 family, we observed heterogeneity in the T cell response to each of the antigens. For example, T cells from donor 201 were found to react only with peptides from Mtb9.9C and not Mtb9.9A or Mtb9.9B. In addition, PBMCs from donor 7 only responded to peptide 6 from Mtb9.9A and Mtb9.9C but not from Mtb9.9B. Whether this represents a means for Mtb to evade the immune system by antigenic drift remains to be investigated.
In conclusion, we have developed a very simple and sensitive expression cloning strategy for identifying antigens that are recognized by CD4+ T cells. Such a strategy is likely to be applicable to the identification of CD4+ T cell antigens from other infectious disease microorganisms, as well as antigens from other sources.