The Role of Naive T Cell Precursor Frequency and Recruitment in Dictating Immune Response Magnitude

It is well established that the magnitude of the primary T cell response is influenced by the amount and duration of presentation of the relevant peptide:MHC (p:MHC) ligands by APCs in secondary lymphoid organs (1–4). Peptides that are derived from abundant proteins or that are processed efficiently or bind strongly or stably to MHC are more likely to be displayed in larger amounts and for longer periods of time on APCs than are peptides that lack these properties (5, 6). Abundantly presented p:MHC complexes will then trigger more intense TCR signaling in cognate T cells than will less abundant complexes, which will promote effector and memory cell formation. However, it is also possible that responses to certain p:MHC ligands are strong because the cognate naive T cell population is larger than average.

The latter possibility has been difficult to test because the frequency of T cells specific for individual p:MHC ligands is so low (7). This infrequency is a direct consequence of the vast number of αβ TCRs that can be produced by random joining of Tcra and Tcrb V(D)J segments and nontemplated N-region additions (8). Thus, within the vast pool of TCRs displayed by individual cells in the naive T cell repertoire, only a few are likely by chance to have high affinity for any individual p:MHC ligand. We now know that the frequency of such cells is, at most, ∼100 cells/million naive T cells (Tables I, II) (9). This low frequency, together with the stringent activation requirements of naive T cells, explains why conventional 96-well plate proliferation-assays containing ∼10⁶ T cells/well are incapable of detecting p:MHC-specific T cell populations in individuals who were not previously immunized (10).

Sallusto and colleagues (11) recently devised a clever “T cell library” approach to solve this problem. These investigators cultured 384,000 naive human CD4⁺ T cells in 192 wells at 2000 cells/well with the PHA mitogen, allogeneic blood cells, and IL-2. These conditions led to 1000-fold expansion of all of the naive cells in each well and converted them into hardy effector cells that could be restimulated with a foreign Ag plus autologous blood cells as APCs. If 1 of the 192 original wells contained cells that proliferated in response to the foreign Ag, then it could be deduced that the frequency of naive T cells specific for peptide:MHC class II (p:MHCII) ligands derived from the Ag was 1/384,000. Using this approach, it was determined that naive CD4⁺ T cells specific for p:MHCII ligands derived from keyhole limpet hemocyanin exist at frequencies of 10–70 cells/million naive CD4⁺ T cells and for Bacillus anthracis-protective Ag at 10–26 cells/million. If there are 10 p:MHCII epitopes derived from keyhole limpet hemocyanin, and each epitope is recognized by a naive population of the same size, then each naive population would exist at a frequency of one to seven cells/million naive CD4⁺ T cells. An advantage of this technique is that it does not require knowledge of the subject’s MHC molecules and yields a total frequency that is the sum of the frequencies of the populations specific for all of the relevant p:MHCII epitopes from the protein. A disadvantage of the technique is that it does not reveal the frequency of T cells specific for individual p:MHCII epitopes.

Recently, however, the combined use of fluorochrome-labeled p:MHC tetramers and magnetic particle-based cell enrichment has solved this problem. Fluorochrome-labeled p:MHC tetramers bind to the TCRs on specific T cells, marking them for detection (12). However, the difficulty has been the limited capacity of flow cytometers to analyze only ∼10⁶ cells at a time, while the rare naive T cells of interest are mixed in with ∼2 × 10⁸ total nucleated cells from the secondary lymphoid organs of a mouse or 100 ml of human peripheral blood. This problem was solved by staining all of the cells in the secondary lymphoid organs with a p:MHC tetramer and then with magnetic beads coupled to Abs specific for the fluorochrome component of the tetramer (13–22). The sample could then be passed over a magnetic column to capture all of the tetramer-bound cells plus ∼10⁶ contaminants. Therefore, the total number of cells in this bound fraction was small enough that it could be analyzed in its entirety by flow cytometry. The tetramer-bound cells could be distinguished from contaminants by staining with a mixture of fluorochrome-labeled Abs specific for T cell- and non-T cell-specific surface proteins.

This approach was used to identify naive CD4⁺ T cells specific for different foreign p:MHCII ligands in C57BL/6 (B6) mice (7, 23–27). The tetramers used in these studies contained the I-A^b MHC class II (MHCII) molecule bound to the 2W variant of peptide 52–68 from the I-E MHCII α-chain (28), peptide 427–441 from the FliC protein of Salmonella typhimurium (29), peptide 329–337 from chicken OVA (30), or peptide 190–201 from the listeriolysin O protein of Listeria monocytogenes (31). The 2W:I-A^b–binding and listeriolysin O peptide 190–201:I-A^b–binding CD4⁺ T cell populations consisted of ∼260 cells/mouse (7, 23–25, 27) and 80 cells/mouse (25), respectively, whereas the FliC:I-A^b–binding (7, 23, 24, 26) and OVA:I-A^b–binding (23) populations contained ∼20 and 40 cells/mouse. Assuming that mice contain 2.5 × 10⁷ CD4⁺ T cells with a naive phenotype (9), the frequencies of naive T cells specific for these individual p:MHCII complexes ranged from 0.8–10 cells/million naive CD4⁺ T cells.

Recently, Kwok et al. (32) used p:MHCII tetramer-based cell enrichment to enumerate human naive CD4⁺ T cells specific for three B. anthracis-protective Ag peptide:HLA-DR1 epitopes in unvaccinated individuals. The frequencies of these naive populations were 0.2, 2, and 10 cells/million naive CD4⁺ T cells (Table II). This range of frequencies is remarkably similar to the 0.8–10 cells/million range reported for mouse T cells specific for individual p:MHCII epitopes determined by the same method (Tables I, II). This congruence is surprising, because the TCR diversity of humans has been estimated to be 10 times greater than that of mice (33, 34).

The fact that Kwok et al. (32) and Geiger et al. (11) both studied B. anthracis-protective Ag allows for a comparison between the p:MHCII tetramer-based cell enrichment and T cell library methods. The sum of the frequencies of the three peptide:HLA-DR1–specific naive populations measured by p:MHCII tetramer-based cell enrichment was ∼12 cells/million naive CD4⁺ cells. The collective frequency of T cells specific for all of the p:MHCII epitopes in the protein measured by the T cell library approach was 10–26 cells/million. The similarity of the frequencies derived from p:MHCII tetramer staining and the T cell library approach that relies on Ag-driven T cell proliferation suggests that the former method may not greatly underestimate the number of T cells that are capable of responding to Ag, as recently suggested (35).

Peptide:MHC class I (p:MHCI) tetramer-based cell enrichment has also been performed by several laboratories to enumerate the preimmune frequencies for many foreign p:MHCI-specific CD8⁺ T cell populations (Tables I, II). The number of naive phenotype CD8⁺ T cells in B6 mice ranged from 15 lymphocytic choriomeningitis virus (LCMV) L_338–346:D^b-specific cells to 1500 murine cytomegalovirus M45:D^b-specific cells/mouse (Table I). These numbers translate to frequencies of 1–89 cells/million naive CD8⁺ T cells, assuming a total of 2 × 10⁷ CD8⁺ naive T cells/mouse (9). Again, a similar range of frequencies was reported for human CD8⁺ T cells (Table II).

An important conclusion from these studies is that naive T cell populations specific for different p:MHC vary in size in a predictable fashion in individuals that express the same MHC molecules. Our analysis of relatively small and large naive CD4⁺ T cell populations specific for different peptides bound to the same MHCII molecule revealed that the smaller population underwent more clonal deletion on cross-reactive self-p:MHCII ligands than did the larger population (24). Similarly, Day et al. (36) found that the reduction in the number of influenza acid polymerase 224–233:H-2D^b–specific CD8⁺ naive T cells in mice that expressed H-2D^b and H-2K^k compared with mice that expressed H-2D^b alone was explained, in part, by the deletion of influenza acid polymerase 224–233:H-2D^b–specific cells due to cross-reactivity with H-2K^k molecules. An abnormally low amount of deletion of T cells that cross-react on self-p:MHC may explain the extraordinary capacity of CD8⁺ T cells specific for HIV peptide:HLA-B57 complexes to control infection (37). HLA-B57 molecules bind a less diverse set of self-peptides than do other MHC class I (MHCI) molecules and, thus, may mediate less extensive negative selection of the CD8⁺ T cell repertoire. As a result, foreign peptide:HLA-B57–specific T cell populations may be larger and more promiscuous than average, thereby providing more effective immune responses to HIV.

In our studies, we found that a peptide recognized by a relatively large naive cell population contained tryptophan residues as TCR contacts, which, when changed to other amino acids, reduced the size of the population (23). Thus, foreign p:MHCII ligands that tend to be recognized by large naive populations may have chemical properties that allow favorable interactions with many different TCRs. The finding of Turner et al. (38) that CD8⁺ T cell populations with highly diverse TCRs recognize peptides containing TCR contact amino acids with large side chains is consistent with this possibility.

Variation in naive T cell population size is of more than academic interest, because it can determine the magnitude of the T cell response. Several studies demonstrated that naive T cells expand in proportion to their starting frequency following exposure to the relevant p:MHC ligand (7, 39). Work by Sette and colleagues (40) showed that variation in naive T cell population size is a partial explanation for the phenomenon of immunodominance. LCMV infection of B6 mice activates CD8⁺ T cells specific for one of ≥28 different p:MHCI combinations. However, three of these p:MHCI ligands account for about one third of the total response. This dominance was not completely explained by MHCI binding affinity, because several of the other peptides bound to MHCI in the same affinity range as did the dominant peptides. Rather, the best predictor of response magnitude was the size of the naive T cell population. The populations specific for the dominant peptides were ∼10 times larger than those specific for the less dominant peptides. This correlation between naive population size and immunodominance was also seen in studies of influenza-infected humanized HLA-transgenic mice (41) or hepatitis C virus-specific human peripheral T cells (42), as well as B. anthracis-vaccinated humans (32). Therefore, although Ag abundance, efficiency of peptide generation by Ag processing, MHC binding affinity, and stability of p:MHC complexes certainly influence immunodominance (5, 6), naive T cell population size is also an important factor. Large naive T cell populations are advantageous to the host because they can generate a fixed number of microbicidal effector cells more quickly than smaller populations. For example, it took 4 d to generate 10,000 effector cells from 200 naive phenotype 2W:I-A^b–specific CD4⁺ T cells but 8 d from 20 naive phenotype FliC:I-A^b–specific cells (7). Large naive T cell populations may be particularly important to aged individuals, because small populations are susceptible to extinction as the total preimmune repertoire contracts during aging (43).

Although the correlation between naive T cell population size and immune response magnitude has been substantiated by several studies (7, 32, 39–42, 44), this relationship was not observed in some recent comparisons of p:MHCI-specific CD8⁺ T cell populations during virus infection (45, 46). A disconnect between naive population size and response magnitude could occur if only a fraction of the population is recruited into the response. This situation could result if the number of APCs displaying the relevant p:MHC ligand is very low, such that some T cells by chance interact with a p:MHC⁺ APC, while other cells in the populations do not. Alternatively, low numbers of p:MHC ligands per APC could result in the response of only those T cells in the naive population with highest-affinity TCRs. In either case, a large naive population would produce a smaller-than-expected response.

Schumacher and colleagues (47) introduced unique molecular tags into each cell of a monoclonal naive CD8⁺ T cell population and then tracked the efficiency of naive T cell recruitment into the primary response. Following adoptive transfer, it was found that essentially all of the cells in the population were efficiently recruited into the primary response, regardless of the dose of Ag or type of infection. Therefore, naive CD8⁺ T cells with an identical affinity for a p:MHCI ligand were recruited into the response in an all-or-none fashion. This finding raises the possibility that, at a very low dose of Ag, no members of a large T cell population would respond, whereas at a slightly higher Ag dose, all of the members of the population would respond. To imagine how this situation could result in a disconnect between naive T cell population size and primary response magnitude, consider two p:MHC complexes, pA:MHC and pB:MHC, derived from the same virus and recognized by naive T cell populations containing 1000 or 100 members, respectively. If the amount of pA:MHC complexes produced during infection is very small, and the number of pB:MHC complexes is large, then it is possible that the 1,000 pA:MHC-specific cells will remain naive while the 100 pB:MHC-specific cells will proliferate to produce 100,000 progeny.

Differential T cell proliferation following initial recruitment could also produce a lack of correlation between naive T cell population size and primary response magnitude. This contention is supported by work from Zehn et al. (48), who used altered peptide ligands with variations in TCR binding affinity for a monoclonal TCR to model what would happen to clones within a polyclonal p:MHCI-specific CD8⁺ T cell population with varying affinities for the p:MHCI complex in question. The monoclonal T cells began to proliferate in response to each of the peptides. However, the high-affinity peptides induced larger expanded T cell populations by inducing longer periods of proliferation following initial activation. These findings are largely consistent with TCR repertoire analyses showing that TCR affinity-based immunodominance patterns are not apparent immediately after the first few rounds of cell division, but they emerge later on in the primary immune response (49). Taken together, these studies indicate that the initial recruitment of naive T cells from a p:MHC-specific population into a primary immune response is fairly uniform across all TCR affinities, but sustained proliferation and eventual effector cell population size depend on TCR signal strength. Again consider two p:MHC complexes, pA:MHC and pB:MHC, derived from the same virus and recognized by naive T cell populations containing 1000 or 100 members, respectively. If the pB:MHC-specific population contains a greater proportion of high-affinity clones than does the pA:MHC-specific population, then despite the initial response from all clones from both populations, the 100 naive cells from the pB:MHC-specific population will eventually undergo more extensive proliferation than will the 1,000 naive cells from the pA:MHC-specific population, resulting in 100,000 versus 10,000 effector progeny and creating a disconnect between naive population size and response magnitude. In contrast, if the two populations are represented by similar proportions of high-affinity clones and they see similar levels of p:MHC complexes, then they will proliferate largely in proportion to their starting numbers, thereby creating a situation in which the larger pA:MHC-specific naive population will produce more effector cells than will the smaller pB:MHC-specific population. Under these conditions, naive T cell population size will be the major determinant of primary immune response magnitude.

• Received December 22, 2011.
• Accepted February 27, 2012.

• Copyright © 2012 by The American Association of Immunologists, Inc.