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The Journal of Immunology, 2002, 169: 4998-5004.
Copyright © 2002 by The American Association of Immunologists

Murine Neonatal CD4+ Lymph Node Cells Are Highly Deficient in the Development of Antigen-Specific Th1 Function in Adoptive Adult Hosts1

Becky Adkins2, Yurong Bu and Patricia Guevara

Department of Microbiology and Immunology, University of Miami Medical School, Miami, FL 33136


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is well established that murine neonates are biased toward Th2 responses. Th2-dominant responses are observed following immunization with a variety of Ags, using different carrier/adjuvant systems, and are seen in both BALB/c and C57BL/6 mice. Therefore, Th2 skewing appears to be a universal phenomenon unique to the neonatal period. One important question about this phenomenon is whether these responses are due to T cell intrinsic properties or are regulated by the neonatal environment. Here we have addressed this issue by transferring neonatal or adult CD4+ lymph node cells to adoptive adult recombinase-activating gene 2-/- hosts and studied the development of Th responses. Neonatal CD4+ cells were highly deficient in the development of both primary and secondary Ag-specific Th1 responses. This did not appear to be due to anergy of a developed population, since exogenous IL-2 only marginally increased production of the Th1 cytokine IFN-{gamma}. This profound Th1 deficiency was observed despite similar proliferation by neonatal and adult cells within the recombinase-activating gene 2-/- hosts. Moreover, neonatal CD4+ cells up-regulated activation markers in a manner similar to adult CD4+ cells. Therefore, although their proliferation and phenotypic maturation proceeded normally, neonatal CD4+ cells appeared to be intrinsically deficient in the functional maturation of Th1 lineage cells. These results offer a candidate explanation for the reduced graft-vs-host responses observed following transplantation of cord blood cells or murine neonatal lymphoid cells to allogeneic adult hosts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Over the last decade it has become increasingly clear that murine neonates are immature in the development of Th function. Notably, murine neonates are prone to Th2 responses (1, 2, 3). For example, exposure to Ag during the neonatal period often leads to Th2-dominant memory responses much later in life, once the animals have grown to adulthood and are subsequently re-exposed to the same Ag. This Th2 skewing, as assessed by in vitro measures, is clearly physiologically relevant, since Ag-specific B cell responses that develop in vivo show skewing to the Th2-associated isotype IgG1 (4, 5). Moreover, there are important immunological ramifications to this Th2 skewing since 1) tolerance to alloantigens established during neonatal life is mediated by alloantigen-specific Th2 cells (6, 7, 8, 9, 10), 2) bystander effects can result in Th2 skewing to unrelated Ags (11), and 3) Th1-mediated protective immunity to viruses is severely compromised in neonates (12, 13).

While the observation that neonates are biased to Th2 function is well established, how this comes about is less clear. The particular question that arises is whether this process is regulated by the neonatal environment, T cell intrinsic properties, or both. Data concerning the ability of the neonatal environment to support Th1 function are mixed. Early studies (14, 15) indicated that neonatal APC may have limited capacity to promote T cell responses. More recently, reduced expression of class II MHC and costimulatory molecules has been reported for murine neonatal dendritic cells (DC),3 B cells, and monocytes (16, 17). Diminished production of the Th1-promoting cytokine IL-12 by neonatal monocytes has also been described (16). In contrast to those reports, however, phenotypically mature DC capable of promoting vigorous CTL responses (and presumably strong Th1 responses) have recently been isolated from the neonatal spleen, albeit in reduced numbers (18). Thus, at this point the contribution of APC function to neonatal Th2 skewing is unclear. Similarly, there are in vitro data that argue both for and against T cell intrinsic properties as a major contributor to the Th2 skewing seen in neonates in vivo. Over 10 yr ago we showed that freshly isolated murine neonatal lymph node T cells make high levels of IL-4, but reduced levels of IFN-{gamma}, in response to primary stimulation with anti-CD3 mAb (19, 20). This cytokine profile was obtained in the presence of adult APC, suggesting an intrinsic Th2 bias among the T cells themselves. However, it is clear that neonates are competent to mount mature Th1 responses because they can do so under the appropriate experimental conditions. For example, when IL-12 is added to in vitro cultures, both human (21) and murine (22) neonatal T cells dramatically increase IFN-{gamma} production to the levels made by adult T cells.

In addition to in vitro analyses, a number of investigators have shown that it is possible to redirect a neonatal Th2-biased response in vivo. Immunization of neonates with DNA vaccines (13, 23, 24, 25) or the use of strong Th1-promoting agents (26, 27, 28, 29) results in mature Th1 responses when analyzed in later life. These studies are important because they demonstrate that it is possible to elicit mature Th1 responses in intact neonates. However, these approaches do not directly test whether there are intrinsic differences between neonatal and adult T cells or APC because 1) the relative effects of these treatments on neonatal T cells and APC were not distinguishable in intact neonates; and 2) most responses were analyzed later in life, once the animals were past the actual neonatal period. Thus, what is critically needed is a separation of neonatal T cells from neonatal APC in vivo, such that the importance of each can be directly compared with their adult counterparts. The most direct test of this sort was recently reported by Siegrist and colleagues (18). They isolated neonatal and adult DC and compared their capacities to elicit T cell responses in young adult mice in vivo. On a per cell basis neonatal DC were as competent as adult DC in promoting specific CTL responses in vivo. This study indicates that neonatal DC may, in fact, be fully mature in their capacity to support naive Th1 responses. If so, the Th2 bias seen in intact neonates may be largely due to properties intrinsic to T cells. Here we have tested this idea by comparing the in vivo responses of neonatal and adult CD4+ cells adoptively transferred to mature adult hosts. Our findings show that neonatal CD4+ cells are highly deficient in the development of both primary and secondary Ag-specific Th1 responses. Since neonatal and adult CD4+ cells responded very differently within the context of the same environmental signals, these data argue that neonatal and adult CD4+ cells are intrinsically different.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c mice, originally obtained from Charles River Laboratories (Wilmington, MA), were bred and housed under barrier conditions at the Division of Veterinary Resources, University of Miami Medical School. Periodic screening showed the colony to be free of commonly occurring infectious agents. Females from timed matings were monitored closely from days 19–21 of gestation, and the date of delivery was recorded. The day of birth was considered day 0. Recombinase-activating gene 2-/- (RAG2-/-) mice were purchased from Taconic Farms (Germantown, NY) and similarly bred and housed under barrier conditions.

CFSE labeling

CFSE was purchased from Molecular Probes (Eugene, OR). Total lymph node cells were labeled with CFSE according to the manufacturer’s instructions, with the concentrations of cells and CFSE in the labeling reaction adjusted to 5.0 x 106/ml and 0.5 µM, respectively.

Donor cell preparations

Pools of tissues from >=10 neonatal (day 6) or >=2 adult (6- to 8-wk-old) BALB/c mice were used for the cell preparations. Mesenteric, inguinal, axillary, brachial, and cervical lymph nodes were collected to prepare total lymph node cell suspensions (5). Enriched (93–99%) CD4+ lymph node cells were positively selected (MS+ columns) using the Miltenyi Biotec (Auburn, CA) MACS system, precisely according to the manufacturer’s directions. Tests comparing CD4+ cells prepared by positive or negative selection showed similar patterns of cytokine secretion (data not shown).

For the experiment shown in Fig. 4Go, adult BALB/c CD4+ cell preparations were stained with anti-CD4 CyChrome (BD PharMingen, San Diego, CA) and anti-CD44 biotin (Caltag, Burlingame, CA), followed by fluorescein-coupled streptavidin (Jackson ImmunoResearch, West Grove, PA). CD4+CD44low cells were purified on a Vantage SE cell sorter (BD Biosciences, Mountain View, CA). Cells were defined as CD44low by comparison with 48 h anti-CD3-activated CD44high cells.



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  		FIGURE 4. Deficient Th1 function by donor neonatal CD4+ cells is not due to anergy. Chimeric animals were prepared and treated as described for Fig. 2Go. Donor CD4+ cells were cultured as described for Fig. 2Go, except that rIL-2 (R&D Systems, Minneapolis, MN) was added to some of the cultures, as indicated. One experiment representative of two independent experiments is shown.

 
Preparation and immunization of chimeric mice

Neonatal or adult CD4+ cells (1.5–2.0 x 106) were injected i.v. into 6- to 12-wk-old RAG2-/- mice. For all experiments unless indicated (see below), the mice were immunized on the same day with 100 µg keyhole limpet hemocyanin (KLH; Calbiochem, San Diego, CA) in PBS. Intact 6-day-old neonatal or adult BALB/c mice were immunized in parallel. Neonatal mice were weighed and immunized with 5 µg/g of KLH, and adult BALB/c mice (~20 g in weight) were immunized with 100 µg of KLH in PBS. To ensure Ag draining to the spleen, mesenteric lymph node, and the majority of peripheral lymph nodes, each mouse was injected at three sites, i.p., s.c. between the shoulder blades, and s.c. at the base of the tail. Adult BALB/c and RAG2-/- mice received 100 µl/site, and neonates received 10 µl/site. For those experiments analyzing secondary responses, mice were reimmunized 4 wk after the first immunization; all mice then received 100 µg of KLH in PBS. For the experiment shown in Fig. 3Go one group of chimeric mice was immunized with KLH in LPS (Sigma, St. Louis, MO); each mouse received 100 µg of KLH and 50 µg of LPS.



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  		FIGURE 3. Immunization with KLH in LPS does not substantially increase donor neonatal Th1 function. Chimeric mice were prepared as described for Fig. 1Go. One group of mice was immunized with KLH in PBS as described for Fig. 1Go, and a second group was immunized in parallel with KLH in LPS. Donor CD4+ cells were then restimulated with the indicated concentrations of KLH, and the levels of IFN-{gamma} secreted were analyzed as described for Fig. 2Go.

 
Flow cytometry for activation marker expression

Pools of lymph node and spleen cells were prepared from chimeric RAG2-/- mice or from BALB/c mice age-matched with the donor CD4+ cells. Cells were stained with anti-CD4 CyChrome and fluorescein-conjugated anti-TCR{alpha}{beta} and simultaneously with PE-coupled anti-CD44, anti-CD122, anti-CD69, or anti-Ly6C. All Abs were purchased from BD PharMingen. Gated CD4+TCR{alpha}{beta}+ cells were analyzed for expression of the activation markers.

Cell culture conditions for ELISA

CD4+ cells were prepared from pooled lymph node and spleen cells as described above. To prepare APC, total spleen cells from naive adult BALB/c were treated with anti-Thy-1 (mAb 42-21) plus complement, followed by treatment with 50 µg/ml mitomycin C as described previously (19).

CD4+ cells (2 x 105) were cultured with 4 x 105 APC in a 96-well culture dish in the presence or the absence of 50 µg/ml of KLH unless otherwise indicated. Culture medium consisted of RPMI 1640 (Life Technologies, Grand Island, NY) containing 1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine (Life Technologies), 5 x 10-2 mM 2-ME (Life Technologies), 1% penicillin-streptomycin (Life Technologies), and 10% heat-inactivated (56°C, 30 min) FCS (HyClone, Logan, UT). Culture supernatants were harvested at 72 h, and IFN-{gamma} and IL-4 contents were assessed using mouse-specific cytokine ELISA kits (Pierce Endogen, Rockford, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal CD4+ lymph node T cells are highly deficient in the development of Ag-specific Th1 memory responses in adoptive adult RAG2-/- hosts

To determine the relative contributions of the neonatal environment vs T cell intrinsic properties to Th2-biased neonatal responses, we isolated neonatal CD4+ cells to study their functional responses in mature adult hosts. CD4+ lymph node cells were prepared from day 6 neonatal and adult BALB/c mice, and 1.5–2.0 x 106 of these cells were injected i.v. into adult RAG2-/- hosts. The animals were then immunized twice with KLH in PBS, a regimen that results in clear Th2-skewing in intact neonates (5). Control animals of the same ages as the donor animals were immunized in parallel. Two weeks after the second immunization, spleen cell suspensions were made from individual chimeric animals. Because of the small sizes of the lymph nodes in RAG2-/- animals, pools of lymph nodes were made from all animals in each group. Aliquots of each tissue were taken for staining, and then all spleen and lymph node cells in each group were pooled for the functional analyses.

To test for chimerism, cells were stained with a combination of anti-CD4 and anti-TCR{alpha}{beta} mAb (Fig. 1Go and Table IGo). We found that the combination of mAb was critical for detecting donor cells, since either Ab alone gave relatively high background in uninjected RAG2-/- mice, while the two mAb together showed negligible background staining (Fig. 1Go, left panel). Although injected with equal numbers of similarly enriched CD4+ populations, RAG2-/- hosts injected with neonatal CD4+ cells contained 1.3- to 3-fold reduced percentages of donor cells in the spleens and lymph nodes compared with adult chimeras. These differences were highly significant within each experiment. However, differences in the absolute numbers of donor cells were either less significant or not significant at all (Table IGo). Taking these data together, there were very minor differences in the capacities of neonatal vs adult CD4+ lymph node cells to colonize RAG2-/- hosts.



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  		FIGURE 1. Long term colonization of RAG2-/- adult hosts by neonatal or adult CD4+ lymph node cells. Day 6 or adult BALB/c CD4+ lymph node cells (2.0 x 106) were injected i.v. into adult RAG2-/- hosts. The animals were immunized on the same day with 100 µg of KLH in PBS. One month later the animals were reimmunized with 100 µg of KLH. Two weeks after the second immunization, spleen cells were prepared and stained with anti-CD4 and anti-TCR{alpha}{beta} mAb. The boxed area shows the cells staining positively for both Abs. One animal of each group, representative of nine individual animals from three independent experiments, is shown.

 

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  		Table I. Long term colonization of RAG2-/- hosts by CD4+ neonatal vs adult lymph node T cellsa

 
To analyze Th1/Th2 responses, CD4+ cells from chimeric and intact animals were cultured with splenic APC from adult BALB/c mice, in the presence of KLH (Fig. 2Go). The secondary responses of intact animals originally immunized as 6-day-old neonates (Fig. 2GoA) were very similar to those originally described by our laboratory for 1-day-old animals (5) and those reported by Siegrist and colleagues (4) for 7-day-old animals immunized with protein Ags such as measles hemagglutinin. In particular, cells from animals originally immunized as day 6 neonates made 3- to 4-fold less IFN-{gamma} and 3- to 4-fold more IL-4 than did cells from adult animals. IL-4 production by CD4+ cells prepared from the RAG2-/- chimeras was somewhat similar, with neonatal donor CD4+ cells producing ~2-fold more IL-4 than that produced by adult donor CD4+ cells (Fig. 2GoB). In contrast, adult donor CD4+ cells made >10-fold more IFN-{gamma} than did neonatal donor cells. This large discrepancy in IFN-{gamma} production was seen at all time points analyzed (48 and 72 h; data not shown) and over a wide range of KLH concentrations (Fig. 3Go, left panel).



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  		FIGURE 2. Neonatal CD4+ cells are highly deficient in the development of Th1 memory in adoptive adult hosts. Chimeric mice were prepared and immunized as described in Fig. 1Go. Intact day 6 neonatal or adult BALB/c mice were immunized in parallel. Two weeks after the second immunization, CD4+ cells were prepared from pools of lymph node and spleen tissues derived from three animals per group. The cells were cultured with adult BALB/c APC in the presence of 50 µg/ml of KLH. Seventy-two-hour supernatants were harvested, and IFN-{gamma} and IL-4 levels were measured by specific ELISA. In the RAG2-/- chimeras, background cytokine values in the presence of medium alone were as follows: IFN-{gamma}, undetectable for the neonatal chimeras and <=60 x 103 pg/ml for the adult chimera; IL-4, <=180 pg/ml for both the neonatal and adult chimeras. In the intact animals IFN-{gamma} levels were <=200 x 102 pg/ml in the absence of KLH, while spontaneous IL-4 levels were undetectable. One experiment typical of three independent set-ups is shown.

 
Robust responses to KLH immunization can generally be achieved using PBS as the carrier, since KLH itself appears to have adjuvant activity (30). Nonetheless, it was important to determine whether neonatal Th1 responses could be boosted to those of adults if an adjuvant known to have strong Th1-promoting activity was used. To test this, we immunized chimeric RAG2-/- mice with KLH in CFA. However, the mice did not tolerate this adjuvant well and developed severe anemia and diarrhea (data not shown). We next immunized chimeric RAG2-/- mice with KLH in either PBS, our standard carrier, or LPS. RAG2-/- mice were injected with day 6 neonatal or adult CD4+ cells and immunized twice, as described above, with KLH in either PBS or LPS. Donor CD4+ cells were then restimulated with different concentrations of KLH, and IFN-{gamma} production was measured (Fig. 3Go). Immunization with KLH in LPS resulted in increased IFN-{gamma} production by both neonatal and adult donor cells. However, the fold increase was similar (~2-fold), and neonatal donor cells remained highly deficient in IFN-{gamma} production.

Relatively poor Th1 responses are not due to anergy among neonatal CD4+ cells or to the presence of memory cells in the adult CD4+ population

The relatively poor Th1 responses of neonatal cells could arise in several possible ways. For example, the population as a whole may be deficient in generating Th1 memory. Alternatively, Th1 memory may develop, but the cells may be anergic. A hallmark of T cell clonal anergy is that it can be readily reversed by the addition of IL-2 (31, 32). To distinguish between these possibilities, cells from chimeric animals were stimulated with KLH in the presence of exogenous IL-2, using concentrations known to promote the proliferation and survival of anti-CD3-stimulated lymph node T cells (data not shown). As shown in Fig. 4Go, IFN-{gamma} levels secreted by neonatal CD4+ cells increased in the presence of IL-2 in a dose-dependent manner (343.9 ± 5.6 x 103 pg/ml with no rIL-2; 537.4 ± 3.0 x 103 pg/ml with 1 ng/ml rIL-2; 1598.5 ± 32.2 x 103 pg/ml with 10 ng/ml rIL-2). However, these levels remained >10-fold decreased relative to those produced by adult CD4+ cells. The amounts of IL-4 produced by donor neonatal as well as adult CD4+ cells also showed modest increases in the presence of IL-2, but the overall relative patterns of cytokine production remained the same (data not shown). Since the addition of IL-2 would be expected to "break" anergy (31, 32), these results indicate that the deficient Th1 function of neonatal CD4+ cells is unlikely to be due to anergy.

Another possibility is that differences in the composition of neonatal and adult CD4+ populations account for the functional differences observed. One of the major differences between neonates and adults is that adults may contain some memory T cells, whereas neonates do not. Our animals are bred under barrier conditions and are free of commonly occurring infectious agents. Therefore, it is unlikely that large numbers of memory cells are present in our adult animals. In fact, the expression of a number of activation/memory cell markers is no different on CD4+ cells from unprimed neonates and that on cells from adults in our colony (see below). Nonetheless, it was important to rule out the possibility that the large amount of IFN-{gamma} made by adult cells was being produced primarily by memory cells that cross-reacted with the immunizing Ag. To address this issue, naive phenotype CD44low CD4+ cells were purified by sorting of cells from adult donor animals. These cells were then similarly transferred to RAG2-/- hosts in parallel with unfractionated neonatal or adult CD4+ cells. As shown in Fig. 5Go, the Th1/Th2 responses of the adult CD44low CD4+ population were very similar to those of the total adult CD4+ cells. These experiments lead to the conclusion that compared with adult naive CD4+ cells, neonatal CD4+ cells are highly deficient in the development of Ag-specific Th1 memory.



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  		FIGURE 5. Neonatal CD4+ cells are also deficient in the development of Th1 function compared with naive adult CD4+ cells. Total CD4+ cells were prepared from day 6 neonatal or adult BALB/c mice. In addition, naive phenotype CD4+CD44low were sort-purified from the adult CD4+ population. Chimeric mice were prepared and treated as described for Fig. 1Go, and donor cells were cultured as described for Fig. 2Go. One experiment representative of two individual set-ups is presented.

 
Deficient primary Th1 function by neonatal CD4+ cells in adoptive adult hosts despite normal proliferation and up-regulation of activation markers

The responses we have analyzed in vivo to date are the cumulative result of many activities, including initial and subsequent activation, proliferation, development of primary function, conversion to memory, and cell death. To understand how these responses arise, we decided to eliminate the later events in memory development by focusing on the primary response phase only. Chimeric RAG2-/- animals were prepared and immunized as described above, except that the responses were analyzed 1 wk after cell transfer and a single immunization. As we had seen for the long term experiments, neonatal CD4+ cells showed modestly reduced (1.3- to 1.4-fold) chimerism compared with adult CD4+ cells (Fig. 6Go). However, this reduction was not statistically significant for either the percentage (p = 0.37) or number (p = 0.19) of donor neonatal compared with donor adult cells. In addition to similarities in chimerism, the primary cytokine profiles (Fig. 7Go) were remarkably similar to the memory responses generated after two immunizations and 6 additional wk of in vivo development (refer to Fig. 2Go). Thus, neonatal CD4+ cells are markedly deficient, compared with adult CD4+ cells, in the generation of both primary and memory Th1 function.



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  		FIGURE 6. Short term colonization of RAG2-/- adult hosts by neonatal or adult CD4+ lymph node cells. Chimeric RAG2-/- mice were set up as and treated as described for Fig. 1Go, except the animals were analyzed by flow cytometry 1 wk following cell injection and primary immunization. Each panel shows an individual experiment. Each symbol is the average of three animals per group.

 


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  		FIGURE 7. Neonatal CD4+ cells are also highly deficient in the development of primary Th1 responses in adoptive adult hosts. Chimeric RAG2-/- mice were set up and treated as described for Fig. 2Go, except the animals were tested for cytokine production 1 wk following cell injection and immunization. Background cytokine levels in the absence of KLH were as follows: IFN-{gamma}, <10 x 103 pg/ml for the neonatal chimeras and <50 x 103 pg/ml for the adult chimeras; IL-4, undetectable for the neonatal chimeras and <100 pg/ml for the adult chimeras. One experiment typical of three independent experiments is presented.

 
When transferred to lymphopenic hosts, adult T cells proliferate in the absence of specific immunization (33, 34). This proliferation is apparently due to cross-reactivity with endogenous Ags and for both CD4+ and CD8+ cells appears to require signals through the IL-7R (35, 36). In our system the expectation is that there would be proliferation to endogenous Ags as well as proliferation to the specific immunizing Ag. Possibly, neonatal CD4+ cells may not proliferate as well as adult cells in this setting, resulting in greatly diminished Th1 function. To test this possibility, we first investigated the potential of these cells to proliferate by examining the expression of IL-7R on neonatal vs adult T cells (Fig. 8GoA). As assessed by flow cytometry, CD4+ cells from neonatal and adult lymph nodes expressed equivalent levels of IL-7R. We next directly compared the proliferation of neonatal and adult cells in vivo. Neonatal and adult lymph node cells were labeled with CFSE and injected i.v. into adoptive RAG2-/- hosts. As we had done in our functional experiments, the host animals were immunized with KLH. Five days later pools of lymph nodes and spleen were prepared and stained with anti-CD4 and anti-TCR{alpha}{beta} mAb. The CFSE profiles of the CD4+ TCR{alpha}{beta}+ cells are shown in Fig. 8GoB. By 5 days after injection, the majority of both neonatal and adult CD4+ cells had undergone extensive division. This result is consistent with many previous reports on adult cells from other laboratories (37). Moreover, the percentages of cells that had not divided or had divided only once were similar among the neonatal and adult donor populations.



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  		FIGURE 8. Neonatal CD4+ cells show normal expression of IL-7R and proliferate similarly to adult CD4+ cells in adoptive hosts. A, Day 6 or adult total lymph node cells from BALB/c mice were stained with anti-CD4 and anti-IL-7R or isotype control mAb. Shown is the staining of gated CD4+ cells with the isotype control (- - -) or anti-IL-7R (—-). The percentages of CD4+ cells that are IL-7R+ are indicated in the figure, with the mean fluorescence intensity of the IL-7R staining shown in parentheses. One experiment representative of two individual experiments is shown. B, Day 6 or adult total lymph node cells were labeled with CFSE and injected i.v. into RAG2-/- mice. On the same day the animals were immunized with 100 µg of KLH in PBS. Five days later, pools of spleen and lymph node tissues were prepared and stained with anti-CD4 and anti-TCR{alpha}{beta} mAb. The CFSE profiles of CD4+ TCR{alpha}{beta}+ cells are shown. One experiment typical of four independent experiments is shown.

 
Among adult cells, proliferation in lymphopenic hosts is accompanied by the up-regulation of activation/memory markers (33, 34). It seemed possible that although neonatal cells appeared to proliferate normally, they may not undergo phenotypic differentiation. To test this possibility, donor neonatal and adult CD4+ cells were examined for expression of the activation markers CD44, CD122, CD69, and Ly6C (Fig. 9Go). In parallel, naive animals that were age-matched to the donor cells were examined. As mentioned previously, the expression of all these activation markers was at low levels among cells from intact neonatal and adult animals. Among donor cells in the RAG2-/- chimeras, these markers were all up-regulated and to a similar extent by neonatal and adult CD4+ cells.



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  		FIGURE 9. Neonatal and adult CD4+ cells show similar up-regulation of activation markers. Chimeric animals were prepared and treated as described for Fig. 5Go, except additional Abs were used to stain the cells. One week after cell injection and immunization, spleen and lymph node cells were stained with anti-CD4 and anti-TCR{alpha}{beta} mAb together with anti-CD44, anti-CD122, anti-CD69, or anti-Ly6C mAb. In parallel, spleen and lymph node cells from unmanipulated animals age-matched with the donor cells were stained. The staining profiles of each Ab on CD4+TCR{alpha}{beta}+ is shown. - - -, Intact neonates; —-, chimeric animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we have compared the capacities of neonatal and adult CD4+ cells to mount Th1/Th2 responses within the context of a mature adult environment in vivo. Our results show that neonatal CD4+ lymph node cells adoptively transferred to RAG2-/- mice are highly deficient in the capacity to mount both primary and memory Th1 responses compared with adult CD4+ cells. This deficiency does not appear to be due to the development of an anergic state, since exogenous IL-2 had little boosting effect on donor neonatal Th1 function. Moreover, the disparity between neonatal and adult Th1 responses did not arise from the presence of memory cells in the adult inoculum, since adult CD4+CD44low cells also produced up to 25 times more IFN-{gamma} than did neonatal CD4+ cells. Despite their poor functional responses, neonatal CD4+ cells proliferated as well as adult cells in the RAG2-/- hosts and showed equivalent up-regulation of memory/activation markers. Because neonatal and adult cells functioned differently within the same in vivo environment, these data argue that there are intrinsic differences between neonatal and adult CD4+ cells. In addition, these results indicate that neonatal CD4+ cells may have an uncoupling between proliferation and phenotypic differentiation and the acquisition of Th1 effector function.

In the mid-1990s, reports from the laboratories of Matzinger and colleagues (38), Sarzotti et al. (12), and Lehmann and colleagues (28) led to the idea that the differences between neonatal and adult T cell-mediated responses could be accounted for largely by differences in the absolute numbers of T cells, i.e., that the major differences in the neonatal and adult T cell compartments were quantitative. However, because those studies were all performed using intact animals, it was not possible to discriminate between the effects of the experimental treatments on T cells vs non-T cells. Here, we have separated neonatal and adult T cells from their intact environments and compared their relative capacities within the same context in vivo. These studies clearly indicate that in addition to quantitative differences, there are major qualitative differences in the capacities of neonatal and adult T cells to develop Th effector function. These in vivo results are consistent with several earlier studies from our laboratory showing that neonatal and adult T cells are inherently different in their cytokine-secreting potentials in vitro (19, 20). From these conglomerate studies we conclude that the Th1-deficient responses of intact neonates are at least partially due to T cell intrinsic qualitative properties.

In the RAG2-/- chimeric animals, both neonatal and adult CD4+ cells made more IFN-{gamma} than that made by CD4+ cells from the age-matched, intact animals. However, the fold increase was much greater for adult than for neonatal cells. For example, in the experiment shown in Fig. 2Go, neonatal cells from chimeric animals produced approximately twice as much IFN-{gamma} as did cells from intact neonates. In contrast, adult donor cells made >=20 times more IFN-{gamma} than did their counterparts from normal animals. How can we explain this phenomenon? As mentioned previously, we do not believe that this results from the selective outgrowth of cross-reacting adult memory cells, since naive (CD44low) adult CD4+ cells also developed very high levels of Th1 function in the chimeric mice (see Fig. 5Go). Another possibility is that non-CD4+ adult cells might negatively regulate the development of Th1 function; separating CD4+ cells from the rest of the population would then be expected to remove this negative influence. However, this possibility seems unlikely, since similar results were obtained when total neonatal or adult lymph node cells were transferred to RAG2-/- hosts (data not shown). Given these unlikely explanations, it is important to consider the acquisition of Ag-specific Th1 function within the specific RAG2-/- environment. In lymphopenic hosts, donor CD4+ cells not only proliferate, but also acquire Th1 effector function, even in the absence of specific immunization (39, 40). The IFN-{gamma} produced by Th1 cells developing in response to homeostatic signals may act to drive efficient Ag-specific Th1 development. In intact animals homeostatic development of Th1 function is almost certainly minimal. Thus, Ag-specific Th1 development would be expected to be more limited in intact animals than in lymphopenic hosts. This scenario may also provide an explanation for the relatively poor Th1 function acquired by neonatal CD4+ in the RAG2-/- hosts. If neonatal cells develop limited Th1 function in response to homeostatic signals, this may lead to their relatively poor development of Ag-specific Th1 function. We are currently investigating the acquisition of Th1/Th2 function by neonatal and adult CD4+ cells in RAG2-/- hosts in the absence of specific immunization.

Why neonatal CD4+ cells are so deficient at acquiring Th1 function is not clear. One possibility to consider is that there may be extensive cell death within the neonatal Th1 population. This is difficult to measure directly within a normal, polyclonal population of cells. However, two pieces of indirect evidence address this possibility. First, neonatal and adult CFSE-labeled cells showed equivalent proliferation in the adoptive hosts. Second, most clearly for the short term experiments, there were no significant differences in the recoveries of absolute numbers of donor neonatal and adult cells. Together these observations suggest that apoptosis may be occurring similarly among the neonatal and adult populations. Another possibility is that there may be relatively poor interaction between neonatal T cells and the APC compartment, resulting in diminished Th1 development. In that regard it has been reported (41, 42) that DC are immature in RAG2-/- mice, but that these cells become mature in vivo following the adoptive transfer of T cells. It could be postulated that neonatal CD4+ cells are not able to efficiently drive the maturation of DC in the RAG2-/- hosts. Probably the single most important cell surface molecule for signaling by CD4+ cells to APC is CD40 ligand (CD40L) (43). Indeed, Goldman and colleagues (44) have shown that day 4 neonatal CD4+ cells are deficient, compared with adult cells, in the up-regulation of CD40L. In contrast, we have found normal up-regulation of CD40L by CD4+ cells from day 6 animals (data not shown), i.e., the age of the donor neonatal animals used in these experiments. Therefore, it seems unlikely that deficient up-regulation of CD40L by neonatal T cells leads to such poor relative Th1 function. Nonetheless, the neonatal T cells we have used are highly deficient in the production of IFN-{gamma} in adoptive hosts. IFN-{gamma} up-regulates MHC class II as well as costimulatory molecules (45) and thus has potent activating effects on host APC. Thus, the low levels of IFN-{gamma} made by neonatal CD4+ may result in limited maturation of DC and other myeloid lineage APC in the adoptive RAG2-/- hosts. We are planning experiments to examine the maturity of DC exposed in vivo to neonatal vs adult CD4+ cells.

What is clear is that neonatal CD4+ do not have a universal deficiency in the capacity to develop effector function, since they produced up to 4-fold more IL-4 than did donor adult CD4+ cells. This together with their relatively poor Th1 function produce Th2-skewed responses in adoptive adult hosts that are characteristic of the responses found in intact neonates. What could predispose neonatal T cells to underdevelop Th1 function while simultaneously mounting robust Th2 responses? One clear candidate set of molecules is the transcription factors that promote Th1 vs Th2 cytokine gene expression. For example, T-bet is a transcription factor that appears to be essential for Th1 lineage development (46). It is conceivable that neonatal T cells do not efficiently up-regulate T-bet, leading to poor Th1 function. Alternatively, the transcription factor GATA-3 is central to Th2 development (47). GATA-3 is expressed in resting T cells, but is down-regulated during Th1 development (48). One possible scenario is that neonatal T cells fail to down-regulate GATA-3, leading to predominant Th2 development. Our laboratory is currently conducting experiments to investigate these intriguing possibilities.

One of the hallmarks of cord blood transplantation is the limited graft-vs-host disease (GVHD), relative to that found following transplantation of adult bone marrow cells (49, 50). Similar observations have been made in a mouse model system in which neonatal blood cells are transferred to allogeneic hosts (51). The mechanisms underlying reduced GVHD in these systems are not known. The data we have presented here offer a potential explanation for this phenomenon. That is, neonatal CD4+ cells appear to be very limited in their capacity to develop Th1 responses in adoptive hosts. Since Th1 cell function promotes CTL generation, donor neonatal cells would also be expected to have limited CTL activity and, hence, reduced anti-host responses. If, as proposed above, limited IFN-{gamma} production by neonatal CD4+ cells results in poor APC maturation, a clear prediction is that treatment with exogenous IFN-{gamma} should restore GVHD levels to those seen upon transplantation with adult cells.


   Acknowledgments
 
We are grateful to Shawn Rose and Dr. Bonnie Blomberg for critical evaluation of the manuscript and stimulating scientific discussion.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1AI44923-02. Back

2 Address correspondence and reprint requests to Dr. Becky Adkins, Department of Microbiology and Immunology, R-138, University of Miami Medical School, P.O. Box 016960, Miami, FL 33136. E-mail address: radkins{at}med.miami.edu Back

3 Abbreviations used in this paper: DC, dendritic cells; CD40L, CD40 ligand; GVHD, graft-vs-host disease; KLH, keyhole limpet hemocyanin; RAG, recombinase-activating gene. Back

Received for publication May 30, 2002. Accepted for publication September 3, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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