When first stimulated by antigen in tissue culture, naive T cells normally differentiate into effector phenotypes such as Th1 or Th2, particularly with the addition of IL-12 or IL-4. However, in the current studies we examined the phenotypes of T cells produced during an in vivo response and found that many antigen-primed T cells are uncommitted precursor cells that secreted IL-2 but not IL-4 or IFN-γ. This phenotype is similar to the proliferating precursor cells derived in vitro by stimulating naive mouse CD4 T cells with alloantigens in the presence of TGF-β and anti–IFN-γ 18
, although the exact equivalence of the in vitro and in vivo primed precursor cells has not yet been established.
Several lines of evidence suggest that the primed IL-2–secreting cells are not a transient intermediate stage during T cell differentiation. Previous studies have shown that memory T cells produce relatively high levels of IL-2 and lower levels of IL-4 or IFN-γ 232
. In our experiments, the frequency of cells secreting IL-2 remained at a relatively high level even 50 d after immunization with a low amount of soluble antigen. Although we expected that Ribi adjuvant would induce a stronger response and therefore cause more T cells to fully differentiate into effector cells (e.g., Th2), this adjuvant actually increased the proportion of cells secreting IL-2 but not IL-4 or IFN-γ. Immunization of mice with antigen plus CFA induced a Th1-biased response with both IL-2- and IFN-γ–producing cells in the spleen and draining LNs. Although cells producing only IL-2 could have been Th1 cells that failed to produce IFN-γ on that particular occasion 1235
, the IL-2–secreting cells were much more abundant than IFN-γ–secreting cells in the nondraining LNs, and the cloning experiments with TCR transgenic T cells () confirmed that uncommitted IL-2–secreting cells could be induced during Th1-biased responses. Taken together, these results suggest that the IL-2–producing cells are not a transient T cell population, but can survive for a long time and constitute an important part of the memory T cell reservoir.
Do the IL-2–producing cells secrete any other cytokines? The two-color Elispot results, and the Ribi adjuvant experiments, showed clearly that the majority of IL-2–secreting cells did not secrete either IL-4 or IFN-γ. Although some single-cell methods analyzing cytokine production at one time point (in situ hybridization, intracellular staining) have suggested that even T cell clones do not always express cytokines coordinately 835
, the Elispot method detects cytokine secretion over a longer period and so should be less subject to this reservation. Also, no IFN-γ–secreting cells were detected in most KLH-immunized populations, so the large excess of IL-2–secreting cells in all of our experiments strongly suggests that the IL-2–secreting cells are not just Th1 cells that did not secrete IFN-γ on that particular occasion. Even after immunization with partially purified derivative of Mycobacterium tuberculosis
or KLH plus Ribi adjuvant, which induced low numbers of IFN-γ–secreting cells, the great majority of the antigen-specific cytokine-secreting cells were IL-2+
. In preliminary experiments, we have also tested the antigen-specific secretion of IL-3, IL-5, and IL-6, and found that most of the IL-2–secreting cells did not secrete any of these cytokines (results not shown). Thus, the IL-2–secreting cells appear to secrete few if any of the characteristic Th1 or Th2 cytokines.
We have shown that IL-2–secreting cells primed in vitro 18
or in vivo (this study) are uncommitted, and can differentiate into either Th1 or Th2 cells. An analysis of human peripheral blood T cells revealed a subpopulation that may be the human equivalent 24
of the long-term primed IL-2–secreting mouse T cells that we have identified. These human T cells were CCR7high
and secreted high levels of IL-2 and low levels of IFN-γ when stimulated. After 10 d of stimulation in vitro, a substantial number of cells secreted IFN-γ and had lost expression of CCR7, suggesting that some of the CCR7+
cells could differentiate or selectively proliferate. The human CCR7+
IL-2–producing cells were found at long times after immunization, consistent with our data from mice immunized for 50 d.
Primed, IL-2–producing cells show different distribution patterns from effector cells. The expression of CCR7 and CD62L on the human IL-2–producing memory cells suggests that they home preferentially to LNs 24
. A LN population of mouse memory CD4 T cells generated in vivo by antigen plus LPS immunization secreted only IL-2, in contrast to the secretion of both IL-2 and IFN-γ by T cells from nonlymphoid tissue 23
. In addition, mouse IL-2–producing cells generated in vitro by a short antigenic stimulation 21
home mainly to LNs but not to spleen and other peripheral tissues, in contrast to polarized Th1 and Th2 cells. Together with our observation that the in vivo–primed IL-2–producing cells were enriched in nondraining LNs (), this suggests that the primed, uncommitted cells circulate preferentially through LNs, whereas effector T cells mainly enter the spleen and nonlymphoid tissues.
What conditions are required in vivo for the maintenance of these IL-2–producing cells? TGF-β (or anti–IL-4) and anti–IFN-γ are required for the maintenance of similar IL-2–producing cells in vitro, 1819
. TGF-β is produced by several cell types 36
and so may also influence T cell differentiation in vivo, and the cytokine antagonist functions may be supplied by soluble receptors instead of antibodies. Alternatively, IL-4, IL-12, and IFN-γ may exist at such low concentrations in some locations that T cells do not receive sufficient signals to induce differentiation and therefore remain as IL-2–secreting cells. Brief antigen stimulation also induced a similar population of primed, IL-2–producing cells 21
. Finally, it is possible that there are additional costimulatory or cytokine signals that maintain primed cells in an undifferentiated state.
The primed IL-2–producing CD4 T cells comprise the majority of some immune responses, even with strong adjuvants. The IL-2 may cause optimal proliferation of these cells, resulting in rapid expansion of the antigen-specific population. These primed, bipotential CD4 T cells might provide an expanded population of uncommitted antigen-specific precursor T cells that could include both recently activated and memory cells, as IL-2–producing T cells comprised the majority of KLH-specific cells for at least 50 d after immunization. Preferential migration of these cells into LNs during an immune response should provide an ideal environment for future reeducation and differentiation according to APC signals. Even if an appropriate immune response was mounted during the priming infection, the effector phenotype required during a secondary response might be different if the secondary infection involved a cross-reactive pathogen that was best attacked by different effector mechanisms. Thus, the induction of a substantial population of primed, uncommitted cells would give more flexibility for subsequent differentiation into Th1, Th2, or other phenotypes, allowing a more appropriate reaction to future infections.