Animal Models for Development of an AIDS Vaccine

We found that a homogenous CD4-/CD8- T cell bearing an invariant TCR encoded by Vα14Jα281 with a one-base N-region is highly dominant in the periphery (2-3% in spleen). Surprisingly, the high expression of this invariant Vα14 TCR is a general phenomenon in all laboratory strains irrespective of MHC haplotype and in some wild mouse subspecies. The majority of Vα14+ TCR is associated with Jα other than Jα281 during the neonatal stage, after which the frequency of invariant Vα14Jα281 TCR expression increases with time reaching a maximum at around 5-8 weeks after birth. The dominant expression of Vα14Jα281 TCR is found in both euthymic and athymic mice. These results indicate that homogenous Vα14Jα281 T cells are positively selected in the periphery without thymic influence and that their VJ junction is important for positive selection. We also demonstrate that Vα14⁺ TCR gene rearrangements take place at extrathymic sites, such as bone marrow, liver, and intestine, since frequent nonproductive Vα14 TCR products and Vα14-Jα281 gene mediated signal sequences in circular DNA are detected as a result of TCR rearrangements in extrathymic tissues rather than in the thymus. This indicates the extrathymic development of Vα14Jα281 T cells. Furthermore, the biological roles of homogenous T cells bearing Vα14Jα281 TCR and the human counterpart of this invariant TCR are also discussed.     

Expansion of CD56+ NK T and γδ T cells from cord blood of human neonates

A particular T cell population expressing NK cell markers, CD56 and CD57, exists in humans. Many CD56⁺ T and CD57⁺ T cells (i.e. NK T cells) exist in the liver and increase in number in the blood with ageing. They may be a human counterpart of extrathymic T cells, similar to NK1.1⁺ CD3^int cells seen in mice. We investigate here the existence of such NK T cells in human cord blood and the in vitro expansion of these cells by the stimulation of human recombinant IL-2 (rIL-2). There were very small populations (< 1.0%) of CD56⁺ T cells, CD57⁺ T cells, and γδ T cells in cord blood. However, all of these populations increased in number after birth and with ageing. When lymphocytes in cord blood were cultured with rIL-2 (100 U/ml) for 14 days, CD56⁺ T cells expanded up to 25% of T cells. CD57⁺ T cells were never expanded by these in vitro cultures. The expansion of γδ T cells (mainly Vγ9^? non-adult type) also occurred in the in vitro culture. A considerable proportion of CD56⁺ T cells was found to use Vα24 (i.e. equivalent to invariant Vα14 chain used by murine NK T cells) for TCR αβ. These results suggest that neonatal blood contains only a few NK T cells but CD56⁺ NK T cells and γδ T cells are able to expand in vitro.     

Regulation of immunological disorders by invariant Vα19-Jα33 TCR-bearing cells

We have previously shown that over-expression of the invariant Vα19-Jα33 TCR α transgene (Tg) using a natural TCR α promoter in mice induces the development of NK1.1⁺ T cells (Vα19 NKT cells) in lymphoid organs, including the liver and intestine. These cells produce different spectra of immunoregulatory cytokines such as IL-4, IL-10, IL-17, and IFN-γ depending on the duration and intensity of the invariant TCR stimulation. In this study, we examined the effects of over-expression of invariant Vα19-Jα33 TCR-bearing cells on disease progress in the models of immunological disorders. The introduction of invariant Vα19 TCR Tg into non-obese diabetic mice delayed the onset of the disease. In addition, delayed-type hypersensitivity (DTH) to sheep erythrocytes was suppressed in the Vα19 Tg mice. DTH was also suppressed in the wild type mice previously transferred with Vα19 Tg⁺ but not non-Tg cells. Thus, invariant Vα19 TCR-bearing cells are suggested to participate in the homeostasis of immunity to suppress disease progression resulting from Th1-immunity excess.     

Mouse γδ TCR+NK1.1+ thymocytes specifically produce interleukin-4, are major histocompatibility complex class I independent,and are developmentally related to αβ TCR+NK1.1+ thymocytes

Mouse T cells co-expressing an αβ T cell receptor (TCR) and the NK1.1 antigen have been shown to be major interleukin (IL)-4-producing cells and could therefore regulate cell-mediated immune responses. We have identified a related subset of thymocytes co-expressing a γδ TCR and NK1.1 which also produce IL-4. Unlike αβ⁺NK1.1⁺ thymocytes, the selection of γδ⁺NK1.1⁺ thymocytes is not dependent upon β2-microglobulin (β2m)-associated class I molecule expression because these cells are present in β2m-deficient mice. This suggests that γδ⁺NK1.1⁺ T cells may regulate immune responses to a different variety of antigens. However, the development of αβ⁺NK1.1⁺ and αβ⁺NK1.1⁺ thymocytes appears to be related. Analysis of different mutant mice lacking αβ⁺NK1.1⁺ thymocytes revealed a specific increase in γδ⁺NK1.1⁺ thymocyte production when the block in αβ⁺NK1.1⁺ thymocyte differentiation occurs after β TCR rearrangement.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

Shared amino acid sequences in the NDβN and Nα regions of the T cell receptors of tumor-infiltrating lymphocytes within malignant glioma

The purpose of this study was to assess the V-(D)-J junctional region of the T cell receptor (TCR), the CDR3 region, which is responsible for glioma-specific antigen contact in αβ TCR-mediated recognition. We sequenced the TCR α and β chians of Vα7, and Vβ13.1 cDNA derived from tumor-infiltrating lymphocytes (TIL) of 12 glioma patients and also the corresponding clones from the patients' peripheral blood lymphocytes (PBL). A shared Vβ13.1 DJ sequence of the CDR3 region, NDβN, was demonstrated in 49 of 66 Vβ13.1⁺ clones (74.2 %) from the glioma TIL, whereas only 4 of 33 clones (12.1 %) were observed in the Vβ13.1⁺ clones from the PBL (p < 0.001). A common VDJ sequence, FCASS (Vβ13.1)-YRLPWGTSDS (NDβN)-GELFF(Jβ2.2), was observed not only in the gliomas from each patient, but also among all the patients with a preference for Vβ13.1. In contrast, the amino acid sequences of the Vβ13.1⁺ PBL clones were diverse and random. Next, we sequenced subclones from other Vβ subfamilies randomly selected to compare their VDJ region rearrangements (Vβ3 and Vβ5.1). In contrast to Vβ13.1, the amino acid sequences of these junctional regions were completely different in these subclones. The V-J junctional region of the α chain is dominated by a few clones in some patients, and no shared amino acid sequences were detected in the TCR Vα junctional region. However, in the Nα region of the Vα7-bearing TIL clones, arginine was used in 27 of 44 clones (61.4%) compared to only 3 of 12 clones from the PBL (p < 0.05). These results are consistent with the hypothesis that a clonal expansion/accumulation of glioma lineage-specific T cells occurred in vivo at the tumor site and that these T cells may be recognizing glioma-specific antigens.     

Interleukin-4-producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down-regulated by Listeria monocytogenes

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)-12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL-4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4⁺NK1.1⁺ TCRα/β^intermediate (TCRα/β^int). Here we show that IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes are down-regulated early in listeriosis. We assume that curtailment of IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

A TCR α chain transgene induces maturation of CD4− CD8− α β+ T cells from γ δ T cell precursors

The proportion of CD4⁻ CD8⁻ double-negative (DN) α β T cells is increased both in the thymus and in peripheral lymphoid organs of TCR α chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to α β and γ δ T cells. We show that the transgenic DN cells are phenotypically similar to γ δ T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCRα genes nor been negatively selected by the Mls^a antigen, suggesting that they originate from a differentiation stage before the onset of TCR α chain rearrangements and CD4/CD8 gene expression. Neither in-frame VδDδJδ nor VγJγ rearrangements are over-represented in this population. However, since peripheral γ δ T cells with functional TCRβ gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to γ δ lineage-committed precursors can be delivered via TCR α β heterodimers.     

α‐Galactosylceramide‐activated murine NK1.1+ invariant‐NKT cells in the myometrium induce miscarriages in mice

Innate immunity, which is unable to discriminate self from allo‐antigens, is thought to be important players in the induction of miscarriages. Here, we show that the administration of IL‐12 to syngeneic‐mated C57BL/6 mice on gestation day 7.5 (Gd 7.5), drives significant miscarriages in pregnant females. Furthermore, the administration on Gd 7.5 of α‐galactosylceramide (α‐GalCer), which is known to activate invariant natural killer T (iNKT) cells, induced miscarriages in both syngeneic‐mated C57BL/6 mice and allogeneic‐mated mice (C57BL/6 (♀) × BALB/c (♂)). Surprisingly, the percentages of both DEC‐205⁺ DCs and CD1d‐restricted NK1.1⁺ iNKT cells were higher in the myometrium of pregnant mice treated i.p. with α‐GalCer than in the decidua. IL‐12 secreted from α‐GalCer‐activated DEC‐205⁺ DCs stimulated the secretion of cytokines, including IL‐2, IL‐4, IFN‐γ, TNF‐α, perforin, and granzyme B, from the NK1.1⁺ iNKT cells in the myometrium, leading to fetal loss in pregnant mice. Finally, the i.p. administration of IL‐12 and/or α‐GalCer in iNKT‐deficient Jα18(‐/‐) (Jα18 KO) mice did not induce miscarriages. This study provides a new perspective on the importance of the myometrium, rather than the decidua, in regulating pregnancy and a mechanism of miscarriage mediated by activated DEC‐205⁺ DCs and NK1.1⁺ iNKT cells in the myometrium of pregnant mice.     

Characterization of Subpopulations of T-Cell Receptor Intermediate (TCRint) T Cells

CD1-autoreactive T cells of two types have been demonstrated among T cells expressing the T-cell receptor (TCR) alphabeta at intermediate levels (TCRint cells). One type constitutes a major fraction of the natural killer (NK)1.1+ TCRint population in C57BL/6 (B6) mice and carries a restricted TCR composed of an alpha-chain with an invariant Valpha14-J281 rearrangement, and a beta-chain using Vbeta8. 2, 7 or 2. The second type utilises a variety of TCR and was derived from CD4+ cells in mice lacking MHC class II. To increase our understanding of the two different CD1-reactive subsets, we have investigated and compared the populations of origin: NK1.1+ and NK1. 1- TCRint subsets from MHC class II-deficient mice and CD4+NK1.1+ T cells from B6 mice. The three TCRint populations shared a phenotype indicating previous activation, and contained low frequencies of cells expressing NK receptors of the Ly49 family. In contrast to control CD4+ cells, the three TCRint subsets produced high amounts of interleukin (IL)-4 and interferon (IFN)-gamma after activation. Importantly, no IL-10 could be detected in either TCRint population, implying a distinct function for these cells, different from those of conventional CD8+ and CD4+ cells, including the typical T-helper 2 (Th2) cell. Analysis of TCR expression indicated that the proportion of cells using the semi-invariant Valpha14/Vbeta8.2-type TCR was lower in NK1.1+ cells from MHC class II-negative mice than in CD4+NK1.1+ B6 cells. Further, usage of the Valpha14-J281 rearrangement was also demonstrated among NK1.1- TCRint cells.     

Fetal thymocyte potential for T cell receptor Vγ3-Jγ1 junctional modification

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3-Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3-Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3-Jγl join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3-Jγ1 joins.     

Peripheral Thy1+ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of αβ and γδ T lymphocytes. Surprisingly, using a new model of LAT‐deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat^Inv/Inv) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1⁺ cells. Thy1^dull cells expressed markers of NK lymphocytes and contained low frequency of TCR‐γ gene rearrangements without detectable TCR‐δ rearrangements. Thy1^high cells resembled immature CD44⁺CD25⁺ thymocytes and contained high frequency of non‐productive TCR‐γ and TCR‐δ rearrangements, indicating that cells displaying molecular signatures of commitment toward γδ T‐cell lineage can develop and populate lymphoid tissues of LAT‐deficient mice. Phenotypically similar Thy1^high cells were also found in lymph nodes of lymphocyte‐deficient (Rag2^?/?) mice but not in T lymphocyte proficient, heterozygous Lat^+/Inv mice suggesting that Thy1^high cells of LAT‐deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.