Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity.

Cutaneous immune responses to contact sensitizers such as picryl chloride or oxazolone, are classical manifestations of T cell-mediated immunity in vivo. In fact, the first documentation of T cell-mediated immunity was the ability to adoptively transfer contact sensitivity (CS) responses. Although it is now clear that Ag/MHC-restricted alpha beta TCR positive effector T cells are responsible for 24 to 48 h CS responses, other subsets of Thy-1+ cells in mice also participate in the elicitation of CS. Thus, Thy-1+, CD5+, CD3-, B220+, hapten-specific, non-MHC-restricted early-acting cells are required to initiate CS responses by leading to local serotonin release, which allows for extravascular recruitment of the late-acting, alpha beta TCR+, CS effector T cells. This study describes another T cell population that is needed for the adoptive transfer of CS by alpha beta T cells. In vitro treatment of a mixture of CS effector cells with hamster mAb to gamma delta TCR, together with rabbit complement, or by panning on anti-hamster Ig-coated dishes, diminished substantially the subsequent transfer of CS reactivity without affecting either CS-initiating cells, or the later-acting, alpha beta TCR+ CS effector T cells. Immune cells treated with anti-alpha beta TCR mAb, or recovered as adherent cells from petri dishes after anti-gamma delta TCR panning (i.e., gamma delta TCR-enriched cells), reconstituted the ability of anti-gamma delta TCR-treated immune cells (i.e., alpha beta TCR-enriched cells) to transfer 24-h CS responsiveness. The phenotype of the gamma delta T cells that assisted CS effector alpha beta T cells was: CD3+, CD4-, and CD8+. The gamma delta T cells that assisted alpha beta T cells were not Ag-specific since anti-alpha beta-TCR-treated cells (gamma delta T-enriched) from picryl chloride immunized donors aided alpha beta T cells (anti-gamma delta TCR-treated) from oxazolone-immunized donors, and conversely gamma delta T cells from oxazolone-immunized donors aided alpha beta T cells from picryl chloride immunized donors. Furthermore, the CS-regulating gamma delta T cells were not MHC-restricted because gamma delta T cells from H2d or H2b donors could assist alpha beta T cells from H2k donors. It was concluded that a regulatory population of non-Ag specific, non-MHC-restricted gamma delta T cells was needed to assist immune effector, Ag/MHC-specific alpha beta T cells in the adoptive transfer of CS.     

A central role for alpha beta T cells in the pathogenesis of murine lupus.

We have previously shown that female transgenic mice expressing IFN-gamma in the epidermis, under the control of the involucrin promoter, develop inflammatory skin disease and a form of murine lupus. To investigate the pathogenesis of this syndrome, we generated female IFN-gamma transgenic mice congenitally deficient in either alpha beta or gamma delta T cells. TCR delta-/- transgenics continued to produce antinuclear autoantibodies and to develop severe kidney lesions. In contrast, TCR beta-/- IFN-gamma transgenic mice failed to produce antinucleosome, anti-dsDNA, or antihistone autoantibodies, and kidney disease was abolished. Both alpha beta- and gamma delta-deficient transgenics continued to develop IFN-gamma-associated skin disease, lymphadenopathy, and splenomegaly. The data show that the autoantibody-mediated pathology of murine lupus in IFN-gamma transgenic mice is completely alpha beta T cell dependent and that gamma delta T cells cannot drive autoantibody production. These results imply that production of antinuclear autoantibodies in IFN-gamma transgenic animals is Ag driven, and we identified clusters of apoptotic cells in the epidermis of the mice as a possible source of self Ags. Our findings emphasize the relevance of this murine lupus model to the human disease.     

Receptor revision in peripheral T cells creates a diverse V beta repertoire

In Vbeta5 transgenic mice, the age-dependent accumulation of Vbeta5(-)CD4(+) T cells expressing endogenous Vss elements represents an exception to the rule of strict allelic exclusion at the TCRbeta locus. The appearance of these cells is limited to the lymphoid periphery and is driven by a peripherally expressed tolerogen. Expression of the lymphoid-specific components of the recombinase machinery and the presence of recombination intermediates strongly suggest that TCR revision rescues tolerogen-reactive peripheral T cells from deletion. Here, we report that the appearance of Vbeta5(-)CD4(+) T cells is CD28-dependent. In addition, we find that the TCR repertoire of this unusual population of T cells in individual Vbeta5 transgenic mice is surprisingly diverse, both at the level of surface protein and at the nucleotide level within a given family of V(D)Jbeta rearrangements. This faithful recreation of the nontransgenic repertoire suggests that endogenous Vbeta-expressing populations do not arise from expansion of an initially rare subset. Furthermore, the undersized N regions in revised TCR genes distinguish these sequences from those generated in the adult thymus. The diversity of the revised TCRs, the minimal mouse-to-mouse variation in the expressed endogenous Vbeta repertoire, the atypical length of junctional sequences, and the CD28 dependence of the accumulation of Vbeta5(-)CD4(+) T cells all point to their extrathymic origin. Thus, tolerogen-driven receptor revision in peripheral T cells can expand the TCR repertoire extrathymically, thereby contributing to the flexibility of the immune repertoire.     

Expression of TCR alpha beta partly rescues developmental arrest and apoptosis of alpha beta T cells in Bcl11b-/- mice

Bcl11b(-/-) mice show developmental arrest at the CD44(-)CD25(+) double-negative 3 (DN3) or immature CD8(+)single-positive stage of alphabeta T cell. We have performed detailed analysis of sorted subsets of Bcl11b(-/-) thymocytes, DN3 and CD44(-)CD25(-) double-negative 4 (DN4) cells. Surface expression of TCRbeta proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRbeta proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRbeta or TCRalphabeta expression on the cell surface failed to promote transition from the DN3 to CD4(+)CD8(+) double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b(-/-) DN3 thymocytes showed normal DNA rearrangements between Dbeta and Jbeta segments but limited DNA rearrangements between Vbeta and DJbeta without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b(-/-) DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vbeta and Dbeta gene locus. Analysis of Bcl11b(-/-) DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-x(L) and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRalphabeta in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.     

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.     

Cutting edge: TCR alpha beta+ CD8 alpha alpha+ T cells are found in intestinal intraepithelial lymphocytes of mice that lack classical MHC class I molecules.

TCR alpha beta+ intestinal intraepithelial lymphocytes (IEL) can express either the typical CD8 alpha beta heterodimer or an unusual CD8 alpha alpha homodimer. Both types of CD8+ IEL require class I molecules for their differentiation, since they are absent in beta2m-/- mice. To gain insight into the role of class I molecules in forming TCR alpha beta+ CD8+ IEL populations, we have analyzed the IEL in mice deficient for either TAP, beta 2m, CD1, or K and D. We find that K-/-D-/- mice have TCR alpha beta+ CD8 alpha alpha+ IEL, although they are deficient for TCR alpha beta+ CD8 alpha beta+ cells. This indicates that at least some TCR alpha beta+ CD8 alpha alpha+ IEL require only nonclassical class I molecules for their development. Surprisingly, the TCR alpha beta+ CD8 alpha alpha+ IEL are significantly increased in K-/-D-/- mice, suggesting a complex interaction between CD8+ IEL and class I molecules that might include direct or indirect negative regulation by K and D, as well as positive effects mediated by nonclassical class I molecules.     

T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of T15i knockin mice.

T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the J(H) region of chromosome 12. This T15H chain combines with a kappa22-33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id-. Analysis of L chains expressed in these T15-Id-, PC-specific B cells revealed that two L chains, kappa8-28 and kappa19-15, were expressed in this population. The V(kappa) region of these L chains was recombined to J(kappa)5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id- B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id- B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:kappa8-28L and T15H:kappa19-15L Abs revealed L chain differences from the kappa22-33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:kappa22-33L Ab.     

MHC class II A alpha and E alpha molecules determine the clonal deletion of V beta 6+ T cells. Studies with recombinant and transgenic mice

Interactions between MHC class II genes and minor lymphocyte stimulating (Mls) associated products are responsible for clonally deleting self-reactive T cells in mice. Here we demonstrate the role of the intact I-A and I-E molecules as well as the individual A alpha and E alpha chains in the deletion of cells bearing the V beta 6 TCR. DBA/1 (H-2q, Mls-1a) mice were crossed with various inbred congenic, recombinant, and transgenic strains and the F1's were screened for V beta 6 expression. All I-E+ strains were fully permissive in deleting V beta 6+ T cells. I-E- strains expressing I-A b,f,s,k,p permitted only partial deletion, while I-Aq strains showed no deletion. Recombinant I-Aq and I-Af strains which expressed E kappa alpha chain in the absence of E beta chain showed a decrease in V beta 6+ T cells as compared to their H-2q and H-2f counterparts. Furthermore, transgenic mice expressing E kappa alpha Aq beta gene in an H-2q haplotype (E kappa alpha Aq beta?) gave similar results to that of the recombinants in deleting V beta 6 T-cells. The role of the 1-A molecule was also shown by the partial deletion of V beta 6+ T cells in H-2q mice expressing transgenic I-Ak molecules. These results demonstrate that the E alpha chain is important in the deletion of V beta 6 T-cells in Mls-1a mice. The role of A alpha chain is also implied by the permissiveness of E kappa alpha Aq beta but not Aq alpha Aq beta molecules in the deletion of V beta 6+ T cells.     

CD8+ T lymphocytes in double alpha beta TCR transgenic mice. II. Competitive fitness of dual alpha beta TCR CD8+ T lymphocytes in the peripheral pools.

We studied Rag2-deficient mice bearing two rearranged alphabeta TCR transgenes, both restricted to the MHC H-2D(b) class I molecule. We have previously shown that, in these DTg mice, most peripheral CD8 T cells express one TCRbeta chain associated with two TCRalpha chains, as in one-third of the mature T cells from normal mice. We examined the functional behavior of the dual-receptor CD8 T cells developing either in the absence or in the presence of self-Ag. The dual-receptor CD8 T cells, which develop in absence of self-Ag, show efficient responses to immunization and remain sensitive to induction of peripheral tolerance. In contrast to single TCR T cells, the dual-TCR cells, when tolerized upon exposure to high levels of self-Ag, are not deleted and therefore may exert important regulatory functions. When developing in the presence of self-Ag, the dual-receptor-expressing CD8 T cells escape central deletion, but are not fully competent to respond to cognate stimuli. Overall, we found that the dual-TCR CD8 T cells show a poor competitive value and can be out-competed by single-TCR cells, both in the course of immune responses and in reconstitution experiments. The decreased fitness of the dual-receptor cells may contribute to diminishing the autoimmune hazard that they could represent.     

Depletion of mouse alpha beta T cell antigen receptor bearing lymphocytes by neonatal monoclonal antibody treatment.

Neonatal treatment with a monoclonal antibody specific for the alpha beta TCR results in mice with a long term, severe depletion in the number of alpha beta T cells in the periphery. Significant numbers of T cells reappear in the periphery about age 65 days, but these cells tend to lack expression of CD4 or CD8. Splenocytes of antibody-treated mice are less sensitive to mitogen stimulation or stimulation with MHC allogeneic cells. The level of serum IgG but not IgM was decreased by the treatment. Anti-alpha beta TCR antibody treatment decreased single-positive T lymphocytes that express high levels of the CD3/alpha beta TCR complex from the thymus, suggesting that the treatment could act in part by affecting negative selection of alpha beta TCR+ thymocytes. This treatment does not, however, detectably affect either the homing or the numbers of gamma delta T cells which are abundant in the intestinal epithelium, but which remain a minor population in the spleen and lymph nodes. This supports the hypothesis that gamma delta T cells are developmentally autonomous from alpha beta T cells. These mice provide an excellent model system for assessing the developmental and functional role of gamma delta T lymphocytes in vivo.     

CD8+ alpha/beta or gamma/delta T cell receptor-bearing T cells from athymic nude mice are cytolytically active in vivo.

Phenotypic analysis of lymphocytes that mature extrathymically in congenitally athymic nude mice has revealed a large population of CD3+ CD8+ T cells that express gamma/delta-TCR. In euthymic mice, significant numbers of cells with this phenotype are found only in the intestinal epithelium. Intestinal intraepithelial lymphocytes have been shown to be cytolytically active in vivo, as measured by the redirected lysis assay. In this communication, freshly harvested T cell subsets obtained from pooled nude mouse spleen and lymph nodes and separated by flow cytometric cell sorting were assayed for their ability to lyse FcR+ P815 targets in the presence of mAb to the epsilon-chain of the CD3 complex. CD8+, but not CD4+ or CD4- CD8-, T cells in nude mice were cytolytically active. CD8+ alpha/beta- and gamma/delta-TCR-bearing T cells from the spleen and lymph nodes of nude mice demonstrated similar cytolytic activity. No cytolytic activity of purified cell subsets was apparent in the absence of anti-CD3 mAb, even when NK-susceptible target cells were used. These data indicate that, in contrast to euthymic mice, a large proportion of CD8+ cells from the spleen and lymph nodes of nude mice are cytolytically active in vivo. In addition, these results suggest that the intestinal epithelium is not the only anatomical location where constitutively cytolytic CD8+ alpha/beta- or gamma/delta TCR-bearing T cells may be found.     

Phenotype, ontogeny, and repertoire of CD4-CD8- T cell receptor alpha beta + thymocytes. Variable influence of self-antigens on T cell receptor V beta usage

We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.     

Size estimate of the alpha beta TCR repertoire of naive mouse splenocytes

The diversity of the T cell repertoire of mature T splenocytes is generated, in the thymus, by pairing of alpha and beta variable domains of the alpha beta TCR and by the rearrangements of various gene segments encoding these domains. In the periphery, it results from competition between various T cell subpopulations including recent thymic migrants and long-lived T cells. Quantitative data on the actual size of the T cell repertoire are lacking. Using PCR methods and extensive sequencing, we have measured for the first time the size of the TCR-alpha beta repertoire of naive mouse T splenocytes. There are 5-8 x 105 different nucleotide sequences of BV chains in the whole spleen of young adult mice. We have also determined the size of the BV repertoire in a subpopulation of AV2+ T splenocytes, which allows us to provide a minimum estimate of the alpha beta repertoire. We find that the mouse spleen harbors about 2 x 106 clones of about 10 cells each. This figure, although orders of magnitude smaller than the maximum theoretical diversity (estimated up to 1015), is still large enough to maintain a high functional diversity.     

Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "natural suppressor" cells

We developed a nonmyeloablative host conditioning regimen in a mouse model of MHC-mismatched bone marrow transplantation that not only reduces radiation toxicity, but also protects against graft-vs-host disease. The regimen of fractionated irradiation directed to the lymphoid tissues and depletive anti-T cell Abs results in a marked change in the residual host T cells, such that NK1.1+ or DX5+asialo-GM1+ T cells become the predominant T cell subset in the lymphoid tissues of C57BL/6 and BALB/c mice, respectively. The latter "natural suppressor" T cells protect hosts from graft-vs-host disease after the infusion of allogeneic bone marrow and peripheral blood cells that ordinarily kill hosts conditioned with sublethal or lethal total body irradiation. Protected hosts become stable mixed chimeras, but fail to show the early expansion and infiltration of donor T cells in the gut, liver, and blood associated with host tissue injury. Cytokine secretion and adoptive transfer studies using wild-type and IL-4(-/-) mice showed that protection afforded by NK1.1+ and DX5+asialo-GM1+ T cells derived from either donors or hosts conditioned with lymphoid irradiation is dependent on their secretion of high levels of IL-4.     

The role of alpha beta+ T cells and homeostatic T cell proliferation in Y-chromosome-associated murine lupus

Male BXSB mice develop an early life, severe lupus-like disease largely attributed to an undefined Y-chromosome-associated autoimmunity accelerator, termed YAA: Although the exact disease pathogenesis is uncertain, indirect evidence suggests that T cells play an important role in the male BXSB disease. We have developed TCR alpha-chain gene-deleted BXSB mice to directly examine the role of alphabeta+ T cells and the mode by which Yaa promotes disease in this strain. All disease parameters, including hypergammaglobulinemia, autoantibody production, glomerulonephritis, and the unique monocytosis of BXSB males, were severely reduced or absent in the alphabeta+ T cell-deficient mice. Adoptively transferred CD4+ T cells of either male or female BXSB origin showed equal homeostatic proliferation in alphabeta+ T cell-deficient male recipients. Moreover, deficient male mice eventually developed equally severe lupus-like disease after adoptive transfer and homeostatic expansion of T cells from wild-type BXSB males or females. The results directly demonstrate that the Yaa-mediated disease requires alphabeta+ T cells that are not, in themselves, abnormal in either composition or properties, but are engaged by a Yaa-encoded abnormality in a non-T cell component. In addition, homeostatic anti-self proliferation of mature T cells derived from a small number of precursors can induce systemic autoimmunity in an appropriate background.     

Suppressor T cells: presence in mice rendered tolerant by neonatal treatment with anti-receptor antibody or antigen.

Specific tolerance to phosphorylcholine (PC) was induced in BALB/c mice by two methods. Neonatal mice received a single injection of either: 1)PnC, the C-polysaccharide from S. pneumoniae, R36a vaccine which has PC as a major antigenic determinant or 2) ARA, an homologous antibody directed against the receptor for PC. Spleen cells from animals treated as neonates with either PnC or ARA were specifically suppressed for the response to PC antigens in vitro. In addition, cells from either group of unresponsive animals co-cultured with spleen cells of normal BALB/c mice markedly suppressed the response of the normal cells to PC. Greater than 90% inhibition of the plaque-forming cell response was obtained when unresponsive cells were mixed with normal cells in ratios of 1:1 or greater. Equal numbers of cells from animals made unresponsive by PnC or ARA produced an equivalent degree of suppression. Neither supernatants of cultures nor sera of animals of either unresponsive group suppressed the response of normal spleen cells to PC. Suppression by cells from both groups of tolerant mice was eliminated by treatment with anti-Thy 1.2 serum and C. Presumably, a common cell is responsible for suppression caused by cells from mice made unresponsive by either procedure.     

A comparison of random vs. chemotaxis-driven contacts of T cells with dendritic cells during repertoire scanning

Generating adaptive immunity after infection or immunization requires physical interactions within a lymph node (LN) T-zone between antigen-bearing dendritic cells (DCs) that arrive from peripheral tissues and rare cognate T cells entering via high endothelial venules (HEVs). This interaction results in activation of cognate T cells, expansion of that T cell lineage and their exit from the LN T-zone via efferent lymphatics (ELs). How antigen-specific T cells locate DCs within this complex environment is controversial, and both random T cell migration and chemotaxis have been proposed. We developed an agent-based computational model of a LN that captures many features of T cell and DC dynamics observed by two-photon microscopy. Our simulations matched in vivo two-photon microscopy data regarding T cell speed, short-term directional persistence of motion and cell motility. We also obtained in vivo data regarding density of T cells and DCs within a LN and matched our model environment to measurements of the distance from HEVs to ELs. We used our model to compare chemotaxis with random motion and showed that chemotaxis increased total number of T cell DC contacts, but decreased unique contacts, producing fewer activated T cells. Our results suggest that, within a LN T-zone, a random search strategy is optimal for a rare cognate T cell to find its DC match and maximize production of activated T cells.