Our findings support a model where IL-4 derived from PLZF+
cells regulates the pool of memory-like CD8+
T cells. Such cells are readily apparent in KLF2, ITK, and CBP gene deficiency models. They have also been observed in Id3-deficient mice, where they arise from a similar non-intrinsic mechanism (Barbara Kee, personal communication). The phenotype of ITK-deficient CD8+
T cells, in particular, has been studied extensively 21,31
. Because of ITK's involvement in TCR signaling, it was proposed that ITK is necessary for thymic selection of conventional CD8+
T cells. Thus in the absence of ITK, CD8+
T cells were thought to be diverted into an innate-immune lineage. We show here that this CD8+
phenotype is actually a cell extrinsic effect dependent on IL-4. Thus ITK, IL-4Rα and ITK, PLZF double-deficient mice will be a useful tool to study the direct effects of ITK deficiency on thymic selection.
We propose that the origin of the memory-like CD8+
T cell phenotype in these models is the expanded PLZF+
population. Mice lacking Id3 also have an expanded PLZF+
similar to mice lacking KLF2, ITK, and CBP. Are the mechanism(s) resulting in PLZF expansion shared or distinct in each model? In ITK- and Id3-deficient mice, the PLZF+
population is skewed toward increased γδ NKT cells19,24
. ITK's positive role in TCR signaling has led to the proposition that γδ progenitors that lack ITK avoid negative selection because of decreased signaling and instead become PLZF+
γδ NKTs. CBP deficiency might lead to the same process since CBP-deficient T cells have defects in ITK dependent genes following TCR stimulation27
. One way that ITK deficiency and KLF2 deficiency could be linked is if ITK deficiency led to decreased KLF2 expression in PLZF+
cells. However when Itk−/−
mice were crossed to KLF2-GFP reporter mice12
no difference in KLF2 expression was observed in any PLZF+
population (data not shown).
We believe the expansion of PLZF+
with KLF2 deficiency is unlikely to be due to altered selection. KLF2 does not drastically alter the repertoire of PLZF+
cells and KLF2 deficiency does not expand PLZF+
cells when the TCR is fixed. Also, the expansion of PLZF+
cells and subsequent bystander CD8+
effects with KLF2 deficiency does not seem to be caused solely by altered trafficking. S1P1
deficiency causes thymic retention of NKT cells, the major PLZF+
, yet we did not observe bystander effects with S1P1
deficiency. We favor a model where KLF2 deficiency causes increased expansion or survival of PLZF+
cells after selection.
PLZF deficiency leads to an overall reduction of NKT cells, yet it is interesting to consider that the CD1d tetramer-binding NKT that remain in PLZF-deficient mice express higher levels of CD62L and preferentially localize to lymph nodes instead of spleen and liver5,6
. This phenotype is reminiscent of KLF2-expressing naïve T cells, and might suggest a mutual antagonism between PLZF and KLF2 in an NKT precursor. When we bred PLZFLu/Lu
mice to the KLF2-GFP reporter mouse12
we found that PLZF-deficient NKT cells did indeed express higher levels of KLF2 (data not shown). In addition PLZF transgenic overexpression led to lower CD62L expression on T cells6,37
. Further studies into the interactions between KLF2 and PLZF should be helpful in understanding the dichotomy between conventional and NKT cells.
NKT cells have diverse functions from autoimmunity to responses to pathogens and tumors4
. The potential of NKT cells in therapeutics is also actively being investigated38
. We report a novel role for NKTs cells in the development of a memory-like CD8+
T cell population. We find evidence for this mechanism in both genetic deficiency models and “normal” inbred mouse strains. Variations in the number of CD1d-restricted NKTs and γδ NKT have been reported in inbred strains of mice28-30
. In the BALB/c compared to B6 mice, we found an increase in the proportion of all PLZF-expressing T cells. Importantly, we show that this correlated with an IL-4 dependent, memory-like CD8+
population in BALB/c mice. This effect is consistent with other studies showing that IL-439,40
, including IL-4 from activated NKT41
, can directly promote CD8+
T cell proliferation and differentiation. We were also able to show that CD1d deficient BALB/c mice had a decrease in memory-like CD8+
s. Thus, studying mice that are genetically deficient in NKTs might be complicated by the fact that those mice could also have an altered memory CD8+
T cell population.
Since differences exist between inbred mouse strains, it seems likely that there could be variations in the PLZF+
pool size within the genetically diverse human population. Indeed, humans might have more PLZF+
cells because of a species-specific difference in how CD4 T cells are selected in the thymus. MHC class II is expressed on human but not mouse thymocytes. Transgenic expression of MHC class II on mouse thymocytes allows selection of a unique T cell subset that shares many characteristics with NKT cells, including PLZF expression42
. Recent evidence suggests that MHC class II thymocyte-thymocyte selection does lead to the generation of PLZF+
cells with diverse TCR specificity in the human thymus43
, and humans have a higher proportion of PLZF+
peripheral T cells than mice (Derek Sant'Angelo, personal communication). Thus it will be interesting to determine to what extent PLZF-derived IL-4 shapes the human memory CD8+
T cell pool.
While we are not aware of any cases of human deficiency of IL-4 or PLZF, there has recently been a report of homozygous missense mutation in the ITK
gene in a pair of sisters44
. Both girls died from an Epstein-Barr Virus-associated lymphoproliferative disorder. Of interest to the current work, Eomes was upregulated in CD8+
T cells of both patients. While this finding could certainly be complicated by the pathological situation in the patients, it is consistent with memory-like CD8+
T cell formation in ITK-deficient mice.
The final consequence of the expanded PLZF+ population and increased IL-4 is more memory-like CD8+ T cells. Using bystander-effected OT-I transgenic T cells, we were able to demonstrate that such cells can subsequently promote both antigen-specific and non antigen-specific responses. Thus, the differentiation of memory-like CD8+ T cells by IL-4 might contribute to more potent innate and adaptive immune responses.
One question that arises from these findings is why would IL-4 production by PLZF+
T cells be coupled to memory-like CD8+
T cells capable of making IFN-γ? One possibility is the need for multiple cell types to participate in innate immune production of IFN-γ. Thus, by linking differentiation of memory-like T cells to the rapid cytokine-producing PLZF+
T cells ample IFN-γ-producing cells are generated. Innate IFN-γ production by memory CD8+
T cells has been demonstrated in multiple infection models and have been reported to have an increased innate protective ability compared to NK cells45-48
. PLZF-dependent, memory-like CD8+
T cells might be particularly important neonatally before the immune system matures and infections have generated pathogen-specific memory.