Lack Of IL-7 And IL-15 Signaling Affects Interferon-gamma Production by, More than Survival of, Small Intestinal Intraepithelial Memory CD8⁺ T Cells

Abstract

Survival of antigen-specific CD8⁺ T cells in peripheral lymphoid organs during viral infection is known to be dependent predominantly on IL-7 and IL-15. However, little is known about a possible influence of tissue environmental factors on this process. To address this question we studied survival of memory antigen-specific CD8⁺ T cells in the small intestine. Here, we show that 2 months after vaccinia virus infection, B8R_20–27/H2-K^b tetramer⁺ CD8⁺ T cells in the small intestinal intraepithelial (SI-IEL) layer are found in mice deficient in IL-15 expression. Moreover, SI-IEL and lamina propria lymphocytes do not express the receptor for IL-7 (IL-7Rα/CD127). In addition, after in vitro stimulation with B8R_20–27 peptide, SI-IEL cells do not produce high amounts of IFN-γ either at 5 days (5d) or at 2 months post infection (p.i.). Importantly, the lack of IL-15 was found to shape the functional activity of antigen-specific CD8⁺ T cells, by narrowing the CTL avidity repertoire. Taken together, these results reveal that survival factors as well as functional activity of antigen-specific CD8⁺ T cells in the SI-IEL compartments may markedly differ from their counterparts in peripheral lymphoid tissues.

1. Introduction.

Gastrointestinal mucosa represents a major site of entry as well as initial replication for many viral and bacterial pathogens (including HIV). Massive depletion of CD4⁺CCR5⁺ memory T cells occurs in the mucosal tissues within the first two weeks of HIV-1 infection [1, 2]. In connection with this, vaccines providing protection against gastrointestinal infectious diseases must be able to induce long-term mucosal immune responses [3–9]. Previously, we demonstrated that long-lasting protection against mucosal viral transmission could be accomplished by CD8⁺ CTLs that must be present at the mucosal site of antigen exposure, although some mucosal memory CTLs maybe induced even after systemic vaccination [10–16]. This protective effect was ablated when CD8⁺ cells were depleted in vivo, and required that CTL were present in the gut mucosa, whereas splenic CTLs alone were unable to protect against mucosal viral challenge [10]. Thus, unless antibodies are able to completely prevent viral entry, induction of local long-lasting mucosal CD8⁺ CTLs in the gut should be considered essential to design a protective mucosal vaccine.

The maintenance of antigen-specific CD8⁺ T cells, their homeostatic proliferation and survival during the memory phase of immune responses are known to be predominantly dependent on IL-15 and IL-7, respectively [17–26]. Other cytokines elicited by infectious (or tumor) antigens may work during the primary response as endogenous adjuvants, and could contribute to the survival of long-lived memory CD8⁺ T cells [27–30]. Based on proliferative potential, cytokine production, and surface phenotype, memory CD8⁺ T cells can be divided into effector (EM) and central (CM) memory cells [31, 32]. One approach to discriminate between these two subsets is based on detection of surface IL-7Rα/CD127 and CD62L, where EM have CD127⁺CD62L^– phenotype whereas CM should be CD127⁺CD62L⁺. In peripheral lymphoid tissues both CD8⁺ T cell memory subsets may be found, but in the case of non-lymphoid tissues, EM CD8⁺ T cells are expected to be more prevalent due to the differences in migratory pathways used [33]. Mechanisms of CD8⁺ CTL survival in mucosal tissues, in particular, in the small intestinal IEL (SI-IEL) and lamina propria (SI-LP) are not well understood yet [33–35]. Here, we addressed a role that IL-15 and IL-7 play for survival of memory intraepithelial CD8⁺ T cells in the small intestine in mice that were mucosally vaccinated with Modified Vaccinia Ankara (MVA). We found that 2 months after MVA was intrarectally (IR) administered, virus-specific B8R_20–27/H2-K^b tetramer (B8R tetramer)⁺ CD8⁺ T cells were present in the SI-IEL. Surprisingly, these cells in WT mice were mainly CD127^–CD62L^–, in sharp contrast to the B8R tetramer⁺ CD8⁺ T cells found in the spleens of the same animals (EM and CM). Additionally, B8R_20–27-specific CTLs isolated from the SI-LP were CD127^–CD62L^– as well. Moreover, such long-living CD127^– CD8⁺ T cells were found in the SI-IEL isolated from IL-15 KO mice, indicating that some CD8⁺ T cell subpopulation is able to survive within the gut epithelial layer in the absence of both IL-7 and IL-15 signaling. To find out which surface markers could be associated with residual survival of memory CD8⁺ CTLs in the gut, cells from WT and IL-15 KO mice were analyzed by flow cytometry for surface expression of CD11a, CD11c, NKG2D, and CD8αα homodimer. We found that none of these markers was expressed by the majority of the gut memory CD8⁺ CTLs.

Thus, we may conclude that the antigen-specific memory CD8⁺ CTLs residing in the SI-IEL compartment may be maintained for a long-term independently of signaling via IL-7 and IL-15. Their survival may be dependent on soluble and/or cell contact signals which substitute for cytokines specific predominantly for lymphoid tissues. Identifying these tissue-specific pro-survival mechanisms may be crucial for development of new strategies for mucosal vaccination.

2. Results

2.1. Absence of IL-15 affects antigen-specific CD8⁺ T cell response in mucosally vaccinated mice

In order to estimate quantitative and qualitative effect of IL-15 of antigen-specific CD8⁺ T cell response WT and IL-15 KO mice were IR inoculated with MVA. As a readout we assessed MVA-specific CTL expansion and contraction as well as their functional activity during acute (d5) and memory (2 months) phases postinfection, respectively. To enhance sensitivity of our model, especially in IL-15 KO animals, we measured immune response against the immunodominant vaccinia virus B8R_20–27 epitope [36]. By flow cytometry, in WT mice during the acute phase (5d) of infection, a robust immune response was found, with high relative percentages of B8R tetramer⁺ CD8⁺ T cells in the spleen as well as in SI-IEL (Fig.1, Table 1). By d13 their frequencies both in SI-IEL and spleen decreased approximately two-fold compared with their level at d5, and contracted to as low as 1–2% by 2 months after immunization. In sharp contrast, in IL-15 KO mice, the presence of B8R tetramer⁺ CD8⁺ T cells was substantially lower compared with WT mice, throughout the experiment (compare WT vs. IL-15 KO: spleen, 5d – 20.62±1.12 vs. 9.97±2.03, p=0.0101; 13d – 10.70±0.78 vs. 1.47±0.15, p=0.0003; 2 months – 1.73±0.12 vs. 1.53±0.09, p>0.05; SI-IEL, 5d – 23.83±1.58 vs. 3.34±0.29, p=0.0002; 13d – 13.39±0.68 vs. 3.20±0.75, p=0.0006; 2 months – 1.59±0.17 vs. 1.19±0.36, p>0.05). Difference in kinetics between WT and IL-15 KO mice was more evident when we estimated absolute numbers of B8R tetramer⁺ CD8⁺ T cells in the spleen and the SI-IEL (Fig.1; compare WT vs. IL-15 KO: spleen, 5d – 1.49±0.09 (x10⁶) vs. 0.15±0.03 (x10⁶), p=0.0002; 13d – 0.64±0.03 (x10⁶) vs. 0.11±0.03 (x10⁶), p=0.0003; 2 months – 0.09±0.01(x10⁶) vs. 0.03±0.01(x10⁶), p=0.0017; SI-IEL, 5d – 1.76±0.07(x10⁶) vs. 0.08±0.02 (x10⁶), p<0.0001; 13d – 0.77±0.04 (x10⁶) vs. 0.15±0.01(x10⁶), p<0.0001; 2 months – 0.43±0.02 (x10⁶) vs. 0.05±0.01(x10⁶), p=0.0001).

Fig.1. Kinetics of B8R_20–27/H2-K^b tetramer⁺ CD8⁺ T cells in the spleen and SI-IEL of C57BL/6 and IL-15 KO mice.

Relative percentages (upper panel) and absolute numbers (lower panel) of B8R tetramer⁺ CD8⁺ T cells recovered from the spleen and SI-IEL of WT and IL-15 KO mice are presented. Lymphocytes were isolated 5d, 13d, and 2 months after IR immunization with MVA 10⁷ pfu/mouse. Cells were stained with PE-conjugated B8R_20–27/H2-K^b tetramer together with anti-CD8α (spleen) or anti-CD8β (SI-IEL) mAbs. Data shown are the mean+SD of three animals per group. Experiments were performed twice with comparable results. Data were analyzed by unpaired Student’s t-test. * p<0.05, **p<0.01, *** p<0.0001, ns: not significant.

Table 1.

Phenotype of memory B8R_20–27-specific CD8⁺ T cells in the SI-IEL and spleen from WT and IL-15 KO mice.

Tissue	Marker	Time after infection
		5 days		2 months
		WT	IL-15 KO	WT	IL-15 KO
SI-IEL	CD127+CD62L– CD127+CD62L+	1.62±0.16	2.23±0.38	ND	ND
		ND	1.17±0.46	ND	2.93±1.13
	CD11c+CD11a– CD11c+CD11a+ CD11c–CD11a+	1.11±0.48 7.90±0.49 71.00±3.51	11.27±0.71# 17.50±1.04## 37.63±1.45*	17.90±1.99 3.33±0.52 ND	12.70±1.46 3.12±0.19 ND
	NKG2D+	1.40±0.12	8.40±0.70**	11.37±0.45	10.40±2.45
SI-LP	CD127+CD62L–	0.07±0.07	0.07±0.03	ND	NEC
	CD127+CD62L+	ND	ND	ND	NEC
	CD11c+CD11a–	0.47±0.15	NEC	14.70±0.85	NEC
	CD11c+CD11a+	24.60±1.70	NEC	ND	NEC
	CD11c–CD11a+	61.00±2.08	NEC	ND	NEC
	NKG2D+	0.32±0.08	21.00±1.91	20.73±1.69	NEC
Spleen	CD127+CD62L–	1.73±0.07	1.53±0.05	73.33±3.28	66.90±3.77
	CD127+CD62L+	2.20±0.30	1.63±0.08	15.00±2.52	19.67±1.45
	CD11c+CD11a–	6.58±0.36	2.56±0.59	1.03±0.09	0.23±0.15
	CD11c+CD11a+	71.77±3.26	67.40±2.91	42.33±2.03	43.23±1.75
	CD11c–CD11a+	15.73±0.94	16.07±0.55	40.53±1.81	42.77±1.97
	NKG2D+	70.97±2.00	61.13±3.21	80.30±1.31	72.43±3.35

Comments: Lymphocytes from WT and IL-15 KO animals were isolated at 5d and 2 months after IR immunization with MVA 10⁷ pfu/mouse. Four-color flow cytometry was performed. Cells from tissues were stained with PE-conjugated B8R_20–27/H2-K^b tetramer together with CD8α mAbs. Then, cells were gated on tetramer⁺ CD8⁺ T cells, and further analyzed for expression of CD127, CD62L, CD11a, CD11c, and NKG2D markers expression. Data shown are the mean percent of gated cells and SEM of three animals per interval and are one representative experiment of two experiments performed with comparable results.

Abbreviations: ND – not detected; NEC – not enough cells: due to the paucity of the total isolated LP cells as well as recovered B8R_20–27/H2-K^b tetramer⁺ CTLs;

^#– p=0.00057;

^##– p=0.00434;

^*– p=0.00487;

^**– p=0.00623.

It is well-known that IL-15 is a crucial cytokine necessary not only for survival of memory CD8⁺ T cells, but also for their functional activity. To ascertain functional avidity, we quantitated IFN-γ production by splenocytes and SI-IEL (by ELISpot assay). We found that splenocytes from WT animals (Fig. 2A, upper panel; Fig.2B and C show normalized and net IFN-γ response as percentaage of max. and total response, respectively) showed IFN-γ production in response to titrated amounts of B8R_20–27 peptide. This response was at peak during acute phase (5d) after immunization, and declined with time. In contrast, in IL-15 KO mice the magnitude of response to the highest dosage of B8R_20–27 peptide (1μM; characterizes total responders to peptide epitope), was significantly lower (compare WT vs. IL-15 KO: 5d. – 1358.67±311.10 vs. 430.00±164.62, p=0.0577). Furthermore, the immune response in IL-15 KO mice decreased with time more abruptly, as it was already markedly diminished at 13d after vaccination (WT vs. IL-15 KO: 506.67±116.09 vs. 35.33±5.24, p=0.0072). Importantly, in IL-15 KO mice we detected almost complete lack of CD8⁺ T cells having high functional avidity (recognizing 1pM of B8R_20–27 peptide), which was already evident during the acute phase (compare WT vs. IL-15 KO: 5d – 110.33±6.06 vs. 43.33±5.81, p=0.0624; 13d – 61.33±3.53 vs. 21.00±2.65, p=0.0059; 2 months – 41.00±2.31 vs. 10.00±0.58, p=0.0002). Thus, IL-15 was found to be important not only for maintenance of antigen-specific CD8⁺ T cells during the memory phase (2 months postinfection), but also for generation of cells with full functional capacity (high functional avidity).

Fig.2. Functional avidity of B8R-specific CD8⁺ T cells for IFN-γ production in the spleen and SI-IEL of C57BL/6 and IL-15 KO mice.

(A-C) Splenic (upper panels) and SI-IEL (lower panels) populations were isolated from WT and IL-15 KO animals 5d, 13d, and 2 months after infection, and treated with B8R_20–27 peptide to perform ELISpot for IFN-γ production. B8R_20–27 peptide (1μM, 100pM, 1pM) was added directly to the tissue leukocyte populations, which were placed in triplicates (see Materials and Methods). (A) Absolute numbers of responders/10⁶ total leucocytes to different peptide concentrations are shown. (B) Normalized data calculated against the response to 1μM of peptide are shown. Dotted line corresponds to 50%max.response (max.response=1μM peptide concentration). (C) Percentage of responders out of total response are shown [53]. Data are the mean+SD of three animals per group. Experiments were performed twice with comparable results.

In contrast to what was found in the spleen, IFN-γ production in the SI-IEL compartment from the WT animals was barely detectable 2 months after immunization (Fig.2, lower panel; compare 5d vs. 2 months 1μM: 666.67±370.33 vs. 18.33±3.18). Also, this response was characterized by skewing to the presence of cells recognizing high and middle concentrations of B8R_20–27 peptide (1μM and 100pM) that further distinguishes them from the splenic counterparts (compare spleen vs. SI-IEL, 5d: 1μM – 1358.67±311.10 vs. 666.67±370.33; 100pM – 506.67±116.09 vs. 421.33±239.88; 1pM – 41.00±4.00 vs. 0.90±0.17). Strikingly, although B8R tetramer⁺ CD8⁺ T cells can be detected in SI-IEL in IL-15 KO animals, these cells had almost completely lost capacity to produce IFN-γ even at the peak of infection (Fig.2; compare 5d vs. 13d: 1μM – 7.90±4.55 vs. 6.53±3.31, p>0.05).

2.2. High physical avidity and TCR expression level may not correspond to functional avidity of CTLs from non-lymphoid tissues

Based on the ELISpot data, we reasoned that physical avidity of TCR B8R tetramer⁺ CD8⁺ T cells might affect their functional capacity. To check this possibility we assessed by flow cytometry the level of TCR expression on B8R tetramer⁺ CD8⁺ T cells (Fig.3: panel A shows staining of naïve splenoctes and SI-IEL, panel B shows the strategy for gating cells as exemplified on WT cells 5d p.i.). We found that two uneven populations with high and low TCR expression were present both in the spleen and SI-IELs (Fig.3C). Interestingly, during the acute and memory phases, B8R tetramer⁺ CD8⁺ T cells in the spleen from both WT and IL-15 KO animals had comparable percentage of cells with high TCR expression (WT vs. IL-15 KO: 72 and 65 and 70 and 66%, respectively). In contrast, a different pattern was found in the SI-IEL. Whereas at the acute phase CTLs in both mouse strains highly expressed TCR at comparable, but still lower frequency compared to the spleen (WT vs. IL-15 KO: 55 and 50%, respectively), however, during the memory phase the cells from WT but not IL-15 KO expressed the TCR at the same or even higher level (WT vs. IL-15 KO: 72 vs. 32%, respectively). In fact, among memory CTL in the SI-IEL of the IL-15 KO animals, the majority of B8R tetramer⁺ CD8⁺ T cells expressed TCR at low levels (compare d5 vs. 2 months: 49 vs. 67%, respectively). Thus, MVA-specific SI-IEL from the IL-15 KO mice but not from WT animals had selective tissue-specific decline in frequency of B8R tetramer⁺ cells that expressed TCR at high level, that was progressing from the acute to the memory phase. The extensive and sustained TCR downregulation in the absence of IL-15 may be linked to a higher activation status of these cells and may be associated with a lower production of IFN-γ in IL-15KO animals. Additionally, by using B8R_20–27/H-2K^b tetramer, we measured mean fluorescence intensity of vaccinia virus-specific CTLs stained with different concentrations of tetramer, which reflects the relative physical TCR avidity [37, 38]. For this we isolated tissues from the WT animals 5 days after IR infection with MVA. Comparison of the curves for CD8⁺ T cells from the SI-IEL versus spleen (Fig.4, left panel) revealed that the MVA-specific CTLs expressed TCR with rather high physical avidity for B8R_20–27. Interestingly, the LP cells, which are anatomically adjacent to the IEL, had slightly lower TCR avidity, but still it was much higher compared with the spleen. Similar results were obtained when we depicted the same data as a % of max. MFI level, determining an average tetramer dilution factor giving 50% max.MFI (Fig.4, right panel): spleen – 1:60, SI-LP – 1:80, SI-IEL – 1:200.

Fig.3. TCR expression level on B8R-specific CTLs from SI-IEL and spleen isolated during acute and memory phase after IR MVA-immunization.

Cells from WT and IL-15 KO mice were isolated during acute and memory phases as indicated in the Materials and Methods, and stained with PE-conjugated B8R_20–27/H2-K^b tetramer. Both splenic and SI-IEL cells were co-stained with anti-CD8α and CD8β (spleen) mAbs. Then, B8R tetramer⁺ CD8β⁺-gated CTLs were plotted against CD8α. The level of B8R tetramer staining is shown. (A) The control staining of lymphocytes from naïve animals was performed and a very low background was detected. (B) Originally, cells were gated on B8R tetramer⁺ CD8β⁺ T cells as exemplified on WT cells, day 5 post infection. (C) TCR level expression on B8R tetramer⁺ CTLs from spleen and SI-IEL is shown on density plots and presented as percentage out of total B8R tetramer⁺ CTLs. Data from a representative staining are shown. Comparable results were found in 3 mice studied.

Fig.4. Small intestinal B8R-specific IEL have high physical avidity for vaccinia virus B8R_20–27 epitope.

Cells from spleen (circles), SI-IEL (quadrants) and SI-LP (triangles) were isolated from WT mice 5 days after IR immunization with 10⁷ pfu MVA, and stained with anti-CD8β Abs and B8R tetramer at the indicated dilutions. Left, Mean fluorescence intensities (MFI) of CD8β⁺ T cells representative of 3 mice per tissue from two comparable experiments are shown. Right, Normalized MFI of CD8β⁺ T cells presented as a percentage out of max.MFI level.

Altogether, we may conclude that data on functional and physical avidity of MVA-specific CTLs isolated from spleen (lymphoid tissue) correspond to each other, whereas in the SI-IEL and SI-LP (non-lymphoid tissues) they do not. It may imply that not only cell-intrinsic features (physical avidity) may shape CTL functional activity, but cell-extrinsic parameters as well, especially in case of non-lymphoid tissues (cytokine milieu, co-stimulatory molecules). Our data on MFI of tetramer⁺ cells in IEL and LP compared to spleen in the memory phase cannot answer this question.

2.3. Memory Antigen-specific CD8⁺ T cells in SI-IEL and SI-LP from IL-15 KO mice lack IL-7Rα/CD127 expression

It is known that both IL-7 and IL-15 are of crucial importance for homeostatic proliferation of memory CD8⁺ T cells [17, 24, 39]. In particular, IL-15 signals are considered to be more important for homeostatic proliferation of memory CD8⁺ T cells, whereas IL-7 signals are responsible for their survival [31]. However, what defines pro-survival conditions for memory CD8⁺ T cells in different non-lymphoid tissues, including the gut, is not well understood. At least for the cells from the SI-LP it was shown that they may survive due to signals provided by non-hematopoietic cells via CD70 [40]. As was recently demonstrated by Jiang w. et al., [41] the residual c-Myc-deficient CD8 αα TCR αβ IEL display reduced proliferation and increased apoptosis, which correlate with significantly decreased expression of interleukin-15 receptor subunits and lower levels of the antiapoptotic protein Bcl-2 [41]. Thus, c-Myc controls the development of CD8αα TCR αβ IELs from thymic precursors apparently by regulating IL-15 receptor expression and consequently Bcl-2-dependent survival [41]. It is well accepted that IEL are slower proliferating cells compare to other lymphoid cells. In many experimental systems, IEL alone did not show activation-induced proliferation, but they significantly inhibited the proliferation of activated lymph node T cells in a cell number-dependent manner [42].

To determine whether the cells from the SI-IEL comply to the same pro-survival strategy, we first assessed by flow cytometry whether the B8R tetramer⁺ CD8⁺ T cells express CD127 (IL-7Rα). Memory CD8⁺ T cells in the spleens in both mouse strains developed towards EM (CD127⁺CD62L^–) and CM (CD127⁺CD62L⁺) subsets at comparable frequencies (Fig.5; Table 1). Surprisingly, however, very few memory B8R tetramer⁺ CD8⁺ SI-IEL both in WT and IL-15 KO mice (Fig.5; Table 1) expressed CD127. The same was true for the gut cells analyzed during the acute phase as well. In addition, SI-LPL from WT mice were also negative for CD127 expression during the entire experiment, and in IL-15 KO animals it was proved at least at the acute phase (Fig.5; Table 1). Thus, memory B8R-specific CD127^lo/–CD8⁺ T cells residing in the SI-IEL compartment can be found not only in WT mice but also in the IL-15 KO.To evaluate whether residual IL-7Rα/CD127⁺ lymphocytes in the SI-IEL were still able to transduce signals from IL-7, we isolated SI-IELs, and treated them in vitro with recombinant murine IL-7 cytokine (rmIL-7; 200 ng/mL, 20 min at 37°C), followed by subsequent staining for intracellular phosphorylated STAT5 protein. As CTLs from naïve and antigen-experienced mice contain comparable low frequency of CD127⁺ cells (our unpublished data) and due to the paucity of antigen-specific CD8⁺ CTLs recoverable from SI-IEL compartment especially at the memory stage, we used SI-IEL from naïve mice. As a control we used splenocytes. As shown in Fig.6A and B, a much smaller proportion of total SI-IELs was able to mediate IL-7-dependent STAT5 phosphorylation compared with total splenocytes (solid black and grey lines, respectively). The relative percentage of IL-7Rα/CD127⁺ cells almost completely corresponded to the frequency of IL-7-treated STAT5 (Y694)⁺ cells both in the SI-IEL (5±0.8 and 4.5±0.3, respectively) and the spleen (16.0±0.7 and 20.1±1.3, respectively). Thus, these results confirm that the cells from the spleen as well as SI-IEL express functional receptor for IL-7, but the proportion of TCRαβ⁺CD8αβ⁺ SI-IEL that express functional IL-7Rα/CD127 is miniscule and much smaller than in the spleen (Fig. 6B).

Fig.5. Memory B8R tetramer⁺ CD8⁺ T cells in SI-IEL from WT and IL-15 KO mice express CD127 at low level.

Lymphocytes from WT and IL-15 KO animals were isolated at 5d and 2 months after IR immunization with MVA 10⁷ pfu/mouse. Four-color flow cytometry was performed. Cells from IEL, LP and spleen were stained with PE-conjugated B8R tetramer together with anti-CD8α mAbs. Then, cells were gated on tetramer⁺ CD8⁺ T cells, and further analyzed for CD127 and CD62L marker expression. Due to the paucity of cells isolated from the lamina propria of IL-15 KO mice at 2 months p.i., these data are not presented. One out of two representative experiments is shown.

Fig.6. Residual CD127/IL-7Rα expression on naïve CD8⁺ T cells from WT SI-IEL induces IL-7-specific STAT5 phosphorylation.

Lymphocytes from naïve WT animals were isolated as described in Materials and Methods. Then cells were either treated with recombinant murine IL-7 in DPBS/1%BSA (rmIL-7; 200 ng/mL, 20 min at 37°C) or kept untreated. (A) Histograms for intracellular phospho-STAT5 (Y694) expression on total splenoctes and SI-IEL before and after adding rmIL-7 representative of 2 mice per tissue from two comparable experiments are shown. (B) Relative percentages of IL-7-induced intracellular STAT5 (Y694)⁺ total splenic and IEL populations from naïve WT mice. Isolated cells were treated in vitro with IL-7 (see Materials and Methods). Data show representative example from one of two experiments. Error bars indicate mean+SD. Data were analyzed by Mann-Whitney test, **** p< 0,0001.

2.4. Memory Antigen-specific CD8⁺ T cells in the SI-IEL from IL-15 KO mice barely express CD11a, CD11c NKG2D, and CD8αα homodimer

The prevalence during both acute and memory phases after immunization of MVA-specific CTLs lacking IL-7 receptor in the SI-IEL from both WT and even IL-15 KO animals suggested that during the memory phase, SI-IELs might survive by using alternative pathways without IL-7 or IL-15 signaling. Until recently several attempts have been taken to phenotype memory CD8⁺ T cells in SI-IEL in WT as well as TCR transgenic mice [34, 43]. Based on these reports, we decided to study expression of plausible candidates (β2-integrins CD11a and CD11c, and NKG2D) that could be associated with the gut specific survival of memory CTLs.

At 5d after immunization, B8R tetramer⁺ CD8⁺ T cells in the SI-IEL from WT and, to a lesser extent, in IL-15 KO animals, abundantly expressed CD11a integrin (Fig.7, upper panel; Table 1), and a few of them were CD11a⁺CD11c⁺ (7.90±0.49 vs. 17.50±1.04 for WT vs. IL-15 KO, p=0.00434; whereas the majority were CD11a⁺CD11c^–: 71.00±3.51 vs. 37.63±1.45, p=0.00487). The same pattern was observed for the MVA-specific CTLs from the LP (Table 1 and data not shown). In sharp contrast, B8R tetramer⁺ CD8⁺ T cells from the spleen in both mouse strains revealed an inverse pattern of expression, where the CD11a⁺CD11c⁺ subset was dominant, and CD11a⁺ CD11c^– was subdominant. It is interesting that 2 months after immunization, the majority of B8R tetramer⁺ CD8⁺ T cells in SI-IEL lost surface CD11a expression, and few of them were positive for CD11c both in WT and IL-15 KO mice (Fig.7; Table 1). In contrast, in the spleen the vast majority of these cells was expressing CD11a⁺ (>80%) being either single positive (CD11a⁺) or double-positive (CD11a⁺,CD11c⁺) (Table 1). Thus, although at early stages both CD11a and CD11c may be induced on the SI-IEL population, during the memory phase they were downregulated irrespective of the presence or absence of IL-15 in vivo, and thus these markers could not be associated with their survival.

Fig.7. Memory B8R tetramer⁺ CD8⁺ T cells in SI-IEL from WT and IL-15 KO mice express CD11a and CD11c at low level.

Lymphocytes from WT and IL-15 KO animals were isolated at 5d and 2 months after IR immunization with MVA 10⁷ pfu/mouse. Four-color flow cytometry was performed. Cells from IEL, LP and spleen were stained with PE-conjugated B8R tetramer together with anti-CD8α mAbs. Then, cells were gated on tetramer⁺ CD8⁺ T cells, and further analyzed for CD11a and CD11c marker expression. Due to the paucity of isolated cells from the LPL of IL-15 KO mice at 5d and 2 months p.i. these data are not presented. One out of two representative experiments is shown.

Apart from the β2-integrins, the NKG2D molecule is also known to transduce co-stimulatory and/or pro-survival signals after binding to a number of its ligands expressed by the gut epithelial cells. At the acute stage few B8R tetramer⁺ CD8⁺ T cells in SI-IEL and SI-LP from the WT mice expressed NKG2D, whereas in IL-15 KO mice this frequency was modestly increased (Fig.8, upper panel; Table 1, compare WT vs. IL-15 KO: 1.40±0.21 vs. 8.40±0.70, p=0.00623). In contrast, in the spleen from both mouse strains, most antigen-specific CD8⁺ T cells were positive for NKG2D (Table 1), although in IL-15 KO mice their frequency was slightly lower. When the cells were checked during the memory phase, we saw that in all tissues (SI-IEL, spleen, and SI-LP,) from WT mice, especially in the gut, the relative percentage of NKG2D⁺ tetramer⁺ CD8⁺ T cells had increased compared to the acute phase (Fig.8, lower panel; Table 1) but was still much smaller than that found in the spleen. In contrast, in IL-15 KO animals, the percentage of NKG2D⁺ cells did not change with time.

Fig.8. Memory B8R tetramer⁺ CD8⁺ T cells in SI-IEL from WT and IL-15 KO mice express NKG2D at low level.

Lymphocytes from WT and IL-15 KO animals were isolated at 5d and 2 months after IR immunization with MVA 10⁷ pfu/mouse. Triple-color flow cytometry was performed. Cells from IEL, LP and spleen were stained with PE-conjugated B8R tetramer together with anti-CD8β mAbs. Then, cells were gated on tetramer⁺ CD8⁺ T cells, and further analyzed for NKG2D marker expression. Due to the paucity of isolated cells from the LP of IL-15 KO mice at 2 months p.i. these data are not presented. One out of two representative experiments is shown.

Along with that, previously it was shown that some of the memory CTLs may express CD8αα homodimer, which may ligate an MHC-class I-like molecule, thymus leukemia (TL) antigen, known to be abundantly expressed on the basolateral membrane of mouse intestinal epithelium [44, 45]. When we checked appearance of CD8αα homodimer within the B8R tetramer⁺ CD8⁺ T cells from the SI-IEL, we found that whereas 5d after infection SI-IEL expressed CD8αα homodimer at comparable frequency in both mouse strains (Fig.9, upper panel), spleen cells did not express it at all. Interestingly, the frequency of CD8αα homodimer-positive cells in IL-15 KO mice was approx. 3-fold higher compared with WT animals. During the memory phase no changes in relative percentage of CD8αα⁺ cells were seen in either mouse strains when compared with d5, nor were positive cells found in the spleens.

Fig.9. Memory B8R tetramer⁺ CD8⁺ T cells in SI-IEL from IL-15 KO mice contain a higher frequency of CD8αα-homodimer⁺ cells than WT mice.

Splenocytes and SI-IEL from WT and IL-15 KO animals were isolated at 5d and 2 months after IR immunization with MVA 10⁷ pfu/mouse. Triple-color flow cytometry was performed. Cells were stained with PE-conjugated B8R tetramer together with anti-CD8α and anti-CD8β mAbs. Then, cells were gated on tetramer⁺ CD8⁺ T cells, and further analyzed for expression of CD8αα-homodimer. One out of two representative experiments is shown.

Thus, memory B8R tetramer⁺ CD8⁺ T cells residing in the SI-IEL compartment in both WT and IL-15 KO animals modestly express CD127, CD11c and NKG2D, but not CD11a. The relative frequency of CD8αα homodimer-positive cells in the SI-IEL did not change with time, and has mouse-strain-specific features. In contrast, in both WT and IL-15 KO mice, the vast majority of memory B8R tetramer⁺ CD8⁺ T cells from the spleen expressed CD11a and NKG2D, and some of them were positive for CD11c.

CD103 may also be involved in the maintenance of memory cells at mucosal sites. However, a study by Masopust et al., concluded that memory CTL IEL are positive for CCR9, and less so for CD103 [46].

Altogether, the data obtained allow us to conclude that although memory CTLs can be found in the SI-IEL, which factors contribute to their survival in the absence of signaling via IL-7, IL-15, CD11a and/or CD11c integrins, NKG2D, and CD8αα homodimer remains unknown.

3. Discussion

Development of protective immunity is intrinsically connected to the route of infection [46–50]. However, generation and survival of antigen-specific CD8⁺ T cells after IR immunization is not well understood [10, 51, 52]. In this study we continued to characterize: 1) the kinetics of CD8⁺ T cell responses in the gut after IR immunization, 2) the dependency of functional activity and survival of antigen-specific CD8⁺ T cells from the SI-IEL compartment on IL-7 and IL-15 signals.

We found that the immune response in the spleen and SI-IEL compartment from WT mice was characterized by induction of B8R tetramer⁺ CD8⁺ T cells that paralleled each other during both expansion and contraction phases (Fig.1). Response against the immunodominant vaccinia virus B8R_20–27 epitope (also present in the MVA), may elicit as high as 12% total splenic CTLs or up to 10 × 10⁶ vaccinia-virus-specific CTLs [36]. These data perfectly fit to our results (Fig.1). However, we saw that the number of IFN-γ-producing cells (Fig.2) was much lower for the SI-IEL compartment despite the substantial presence of B8R tetramer⁺ CD8⁺ T cells. As was shown here and in our recent study [53] memory B8R_20–27-specific CD8⁺ T cells in the SI-IEL compartment modestly produced IFN-γ, but with low avidity, whereas in the spleen they were characterized by populations with low-, middle-, and high functional avidity. In addition, B8R_20–27-specific CD8⁺ T cells from the LP were previously shown to be uniquely enriched in the high avidity cells [53]. Thus, after mucosal immunization, CD8⁺ T cells with different functional avidity were distributed unequally in different mucosal and systemic sites. As shown in the current study even greater changes in functional activity were found in IL-15 KO mice, in which T cells tended to quickly loose their IFN-γ−producing capacity. Strikingly, in the SI-IEL compartment from IL-15 KO animals even at the peak of immune response a very modest response was detected (Fig.2, d5).

As functional avidity may be directly linked to the physical TCR avidity and high TCR expression level we compared them for B8R tetramer⁺ CTLs from SI-IEL vs. spleen. Interestingly, we found that the modest functional activity of B8R_20–27-specific CD8⁺ T cells from the WT SI-IELs did not correspond tightly to the TCR expression level by the B8R tetramer⁺ CD8⁺ T cells (Fig.3, lower panels labeled with dates). In particular, whereas at the acute phase (d5) the SI-IEL from the WT mice had a pronounced in vitro IFN-γ production, however, only half of them were characterized by high TCR level expression. In contrast, during the memory phase, although they had hardly detectable functional activity, still the vast majority of them expressed TCRs at high level that was even increased compared to the acute phase. In sharp contrast, whereas SI-IEL from the IL-15 KO animals had very low functional activity, the frequency of B8R tetramer⁺ CD8⁺ T cells with high TCR expression was comparable with that found in the WT mice. Strikingly, during the memory phase SI-IEL from IL-15 KO mice revealed an inverse proportion of high-to-low TCR expression subsets, where the latter comprised up to 67% compared to 27% for the WT mice. However, due to the fact that the physical avidity of B8R tetramer⁺ CTL in the SI-IEL was even higher compared with the spleen (Fig.4; WT mice), we may conclude that assessment of both functional and physical avidities maybe of special importance for antigen-specific CD8 T cells residing in non-lymphoid tissues. Altogether, IL-15 turned out to participate not only in expansion of memory CD8⁺ T cell precursors (see Fig. 1), but also in development of their full functional activity, which was evident during the acute phase of infection (decreased number of total and high avidity responders). In particular, the lack of IL-15 in vivo mostly affected functional activity/development of middle-to-high avidity CD8⁺ T cells and had less impact on low avidity T cells, consistent with our earlier finding that IL-15 promotes induction of high avidity CTLs [54].

A special role for IL-7 and IL-15 for survival and homeostatic proliferation of memory CTLs has been documented in numerous studies [18–23, 25, 35, 55, 56]. As we were able to detect memory CTLs in the SI-IEL from the IL-15 KO animals we decided to check if their survival was linked to CD127 expression and IL-7 signaling. We showed by flow cytometry that during the acute phase the vast majority of B8R tetramer⁺ CD8⁺ T cells isolated from SI-IEL both in the IL-15 KO and WT mice were negative for CD127 expression (Fig.5), consistent with data published elsewhere [34]. Additionally, we found that the memory CTLs from LP were negative for CD127 as well (Fig.5), suggesting that some alternative pro-survival factor(s) specific to the epithelial layer of the small intestine might exist. Such assumption is substantiated by the finding that even in naïve C57BL/6 animals some non-specific “memory-like” TCRβ⁺CD8αβ⁺ T cells may be detected in the SI-IEL, which express CD127/IL-7Rα at very low levels [57]. Here, we proved that despite the low frequency of CD127⁺ SI-IEL, these few cells were capable of transducing signals from IL-7 in vitro, thus confirming the functionality of CD127/IL-7Rα on the gut CD8αβ⁺ T cell IEL (Fig.6). However, the vast majority of memory CD8⁺ T cells within intestinal epithelium was found to survive independently of CD127 expression, thus ruling out IL-7 as a necessary survival factor in this tissue. In line with this, previously it was shown that other cytokines, transducing signals via the common γ_c-chain/CD132, may influence SI-IEL development. In particular, the lack of signaling via IL-2, 4, 7, 9, 15 affected only TCRαβ⁺CD8αα⁺ and TCRγδ⁺CD8αα⁺ IEL subsets [58–62], seemingly having no major impact on TCRαβ⁺CD8αβ⁺ IELs. On the other hand, selective overexpression of either IL-7 [63] or IL-15 [64] by the gut epithelium did not result in increased frequency of CD8αβ⁺ T cells in SI-IEL, possibly because TCRαβ⁺ SI-IEL in naïve mice express few if any receptors for IL-2, 4, 7, 15 [65]. Similarly, splenic TCRαβ⁺CD8αβ⁺ T cells in naive mice do not entirely depend on the IL-15 signaling as they still contain CD44^loCD122^lo precursors of “conventional” TCRαβ⁺CD8αβ⁺ T cells that were described in IL-15 KO mice, thus, arguing for their IL-15–independent maintenance [66]. In fact, additional molecules might have had even bigger impact on their pro-survival program (e.g. itk and IRF-1 etc.; [67, 68]). Based on this, we reasoned that in contrast to the role for IL-7 and IL-15 documented in peripheral lymphoid tissues, some other cytokine/cell contact factors might be involved in long-term maintenance of memory CD8αβ⁺ T cells in non-lymphoid tissues (intestinal epithelium; [69]). Even though some memory intraepithelial CD8⁺ T cells can survive in the small intestine in IL-15 KO mice after mucosal vaccination with MVA, these mice will be significantly less protected against mucosal challenge with WR virulent vaccinia virus compared to WT mice. Some studies demonstrated already that IL-15 plays an important role in protection, especially in early activation of memory CD8⁺ CTL after reinfection [70].

As was shown above, not only survival but also functional activity of the gut memory CTLs was compromised in the IL-15 KO mice. It is known that the local tissue microenvironment (cytokine, chemokine, TLR-ligands, cell-cell contacts) can significantly influence the pathway of antigen presentation that elicits proliferation and differentiation of CD8 T cells, affecting cytokine profile and memory responses [71–76]. Inclusion of cytokines and other biological adjuvants into vaccine formulas can facilitate skewing immune responses both quantitatively and qualitatively in desirable directions [3, 76]. As a result of such strategy, previously we found a synergistic effect of cytokines and mucosal adjuvants for induction of mucosal and systemic CD8⁺ T cell responses together with protective immunity against mucosal viral challenge [12, 50]. Moreover, such protection was mediated by CD8⁺ T-cells and was associated with their presence at the mucosal site [77, 78]. Our studies provided a better understanding of the effects that local microenvironment (cytokines, cell contact signals) might have on generation of memory CTL, particularly at mucosal sites. An important factor for development of effective memory CD8⁺ T cells is the presence of tissue-specific pro-survival factors. Our current study, demonstrated that the mechanisms of CTL survival in mucosal tissues (small intestinal epithelium) may differ from those present in peripheral lymphoid tissues.

As memory CTL which reside in the gut SI-IEL are non-migrating cells [46] and can not recirculate with pool of systemic memory CTLs, they are unable to obtain pro-survival signals produced within lymphoid tissues, e.g. IL-7 and IL-15. Thus, it is important to pin-point what additional factors may contribute to their survival. Apart from different signaling molecules involved in this process, it is also plausible that the gut memory CTLs might merely depend on the abundancy of nutrient factors supplied from food, in particular, glucose. It is worth mentioning that in response to high glucose concentration some gut epithelial cell types are able to produce IL-10 [79], that may also contribute to IEL survival especially when they are deprived of growth factors [80].

Despite the fact that currently no cytokine/cell contact molecule(s) responsible for survival of TCRαβ⁺CD8αβ⁺ SI-IEL has been identified, still it is plausible to deduce that somehow they may integrate signals which elicit constitutive expression of anti-apoptotic factors including cIAP-1, XIAP, bcl-x_L, and Mcl-1 as it was found in naïve animals [81]. In fact, antigen-specific memory TCRαβ⁺CD8αβ⁺ from both spleen and SI-IEL do express bcl-2 at high levels [34].

Besides classic surface markers that help to distinguish between naïve and memory CD8⁺ T cells [31], little is known about expression of the activating NKG2D molecule on the CD8⁺ T cells in the SI-IEL. As a number of its ligands are expressed by the gut epithelium, such ligation might be involved in providing pro-survival signals to the antigen-specific CTLs [82]. Previously, NKG2D expression was described only on a subset of splenic memory CD8⁺ T cells [83], which transduces activating signals by complexing with either DAP10, the only adaptor protein expressed on CD8⁺ T cells, and/or with DAP12 [83]. DAP10 under certain circumstances is considered to transduce co-stimulatory signals in CD8⁺ T cells, whereas DAP12 has direct stimulatory activity in NK cells. CD8⁺ T cells normally do not express DAP12; however, during in vitro stimulation with IL-2, at least CD8⁺CD44^lo T cells do express DAP12 [84]. Importantly, in naïve mice neither TCRαβ⁺ nor TCRγδ⁺ SI-IEL express NKG2D, i.e. that only cytokine-specific and/or antigen-specific activation may be responsible for NKG2D expression [65, 83]. Thus, we speculated that during antigen-specific stimulation, signaling via NKG2D in CD8⁺ SI-IEL might substitute for the lack of pro-survival IL-7 and IL-15. We found that the frequency of NKG2D⁺ cells was increased in IEL (and LP) from IL-15 KO mice at memory phase compared with acute phase (Fig.8). We may suppose that at least, in part, NKG2D expression may be associated with CTL survival both in the SI-IEL and SI-LP compartments.

Additionally, we investigated expression on B8R tetramer⁺ CD8 T cells of CD8αα homodimer (Fig.9), which binds to the thymus leukemia (TL) antigen, known to be abundantly expressed on the basolateral membrane of mouse intestinal epithelium [45]. Due to the fact that the CD8αα homodimer was shown to be transiently expressed on activated CTLs, we thought it might contribute to the survival and differentiation of CD8 memory T cell precursors. Although interaction of CD8αα homodimer with TL may be of some importance in antigen-experienced CTL, however, it was not involved in survival of non-specific CD8αα⁺ TCRαβ⁺ and CD8αα⁺ TCRγδ⁺ IEL [85]. In our study we saw that during both acute and memory phases the frequency of CD8αα⁺ B8R tetramer⁺ CTLs was increased only in the SI-IEL from IL-15 KO but not from WT mice (Fig.9). Collectively, around 16 and 21% of total memory CTL in the SI-IEL from WT and IL-15 KO mice were positive for either NKG2D or CD8αα, which could bind to the gut epithelium-specific ligands (Fig.8 and 9). These results raise the possibility that somehow memory CTL from SI-IEL compartment were shifting their programs towards higher frequencies of NKG2D⁺ and CD8αα⁺ that might be associated with their survival in the absence of IL-7 and IL-15 signaling.

Altogether, our findings emphasize that the local tissue environment in the intestinal mucosa is unique, and shapes and organizes very specific populations of different protective lymphocytes and allows CD8⁺ T cell survival without IL-7 or IL-15 signaling. Furthermore, an environmental condition in the intestinal epithelium does not promote IFN-γ production by SI-IEL long after vaccination, whereas in the adjacent lamina propria they are enriched with high avidity IFN-γ producing CD8⁺ CTLs [53]. What factors contribute to development of pro-survival conditions that facilitate maintenance of antigen-specific T cells in the gut mucosa and their unique pattern of functional activity remains to be investigated.

4. Materials and methods

4.1. Mice

Female C57BL/6 mice were purchased from the Frederick Cancer Research Center (Frederick, MD). IL-15 KO mice on H2-K^b genetic background were purchased from Jackson Laboratories.

4.2. Viruses and immunization protocol

MVA, developed by A. Mayr, University of Munich, Germany [86], was propagated and titrated in chicken embryo fibroblast cells. This virus was a gift of Drs. Bernard Moss, Patricia Earl and Linda Wyatt (NIAID). For immunization, mice were injected IR with 10⁷ pfu MVA as previously described [87].

4.3. Cell purification: Isolation of small intestinal intraepithelial and lamina propria lymphocytes, and lymphocytes from the spleen

Spleens were aseptically removed and single-cell suspensions were prepared by gentle passage of the tissue through sterile screens. SI-IEL and LPLs from mice were isolated as previously described with a minor modification [52].

4.4. Flow cytometry

Flow cytometry was performed by using a FACScalibur, and data were further analyzed with CellQuest software (BD Biosciences) [88]. The following mAbs were used: FITC-conjugated anti-CD62L (clone 53–5.8; obtained from BD Biosciences), APC-conjugated anti-CD127 (clone A7R34); APC-conjugated anti-NKG2D (clone CX5), APC-conjugated anti-CD8β (clone CT-CD8b; all obtained from eBiosciences). Soluble tetrameric B8R_20–27/H2-K^b complex was conjugated to PE-labeled streptavidin (made by the NIH Tetramer Core Facility).

4.5. IL-7 Bioassay

Lymphocytes from naïve WT animals were either treated with recombinant murine IL-7 (rmIL-7; PeproTech Inc.) in DPBS/1%BSA or kept untreated, and incubated for 20 min at 37°C. To verify rmIL-7-specific signaling, cells were stained for intracellular phosphorylated STAT5 protein with anti-phospho STAT5 (Y694) Alexa Fluor^®−488 mAbs according to the manufacturer (clone 47; BD™ PhosFlow) and studied by flow cytometry. Additionally cells were stained with mAbs against APC-conjugated anti-CD127 (clone A7R34; eBiosciences).

4.6. IFN-γ ELISpot

IFN-γ ELISpot were performed as previously described [89]. Cells were plated in triplicates in a volume of 200 μl, 0.2 × 10⁶ /well, to which we directly added titrated amounts of B8R_20–27 peptide (TSYKFESV), which is an immunodominant poxvirus CTL epitope restricted by H-2K^b [36]. Spots were counted in AID ELISpot Reader (Cell Technology, Inc.). Data are presented as the mean and SEM of three animals per interval. These experiments were performed twice with comparable results.

4.7. Statistical analysis

Statistical comparisons were assessed by unpaired Student’s t-test and Mann-Whitney test by using GraphPad Prism version 5.00, GraphPad Software (San Diego CA; www.graphpad.com).