Cells In Peripheral Lymphoid Organs Research Articles

Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation

Rapamycin is an oral immunosuppressant drug previously reported to efficiently induce naturally occurring CD4 +CD25 +FoxP3 + regulatory T ( nT reg) cells re-establishing long-term immune self-tolerance in autoimmune diseases. We investigated the effect of rapamycin administration to SJL/j mice affected by PLP 139–151-induced relapsing–remitting experimental autoimmune encephalomyelitis (RR-EAE). We found that oral or intraperitoneal treatment at the peak of disease or at the end of the first clinical attack, dramatically ameliorated the clinical course of RR-EAE. Treatment suspension resulted in early reappearance of disease. Clinical response was associated with reduced central nervous system demyelination and axonal loss. Rapamycin induced suppression of IFN-γ, and IL-17 release from antigen-specific T cells in peripheral lymphoid organs. While CD4 +FoxP3 + cells were unaffected, we observed disappearance of CD4 +CD45RB high effector T (T eff) cells and selective expansion of T reg cells bearing the CD4 +CD45RB lowFoxP3 +CD25 +CD103 + extended phenotype. Finally, the dual action of rapamycin on both T eff and T reg cells resulted in modulation of their ratio that closely paralleled disease course. Our data show that rapamycin inhibits RR-EAE, provide evidence for the immunological mechanisms, and indicate this compound as a potential candidate for the treatment of multiple sclerosis.

Hyperactivable NFAT1 Ameliorates Autoimmune Encephalitis In Vivo.

Abstract 711Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells.NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases.We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls.To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells.To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease.In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures:No relevant conflicts of interest to declare.

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates

The thymus is the primary site of T-cell development and plays a key role in the induction of self-tolerance. We previously showed that the intrathymic (i.t.) injection of a transgene-expressing lentiviral vector (LV) in mice can result in the correction of a T cell-specific genetic defect. Nevertheless, the efficiency of thymocyte transduction did not exceed 0.1-0.3% and we were unable to detect any thymus transduction in macaques. As such, we initiated studies to assess the capacity of recombinant adeno-associated virus (rAAV) vectors to transduce murine and primate thymic cells. In vivo administration of AAV serotype 2-derived single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors pseudotyped with capsid proteins of serotypes 1, 2, 4, 5, and 8 demonstrated that murine thymus transduction was significantly enhanced by scAAV2/8. Transgene expression was detected in 5% of thymocytes and, notably, transduced cells represented 1% of peripheral T lymphocytes. Moreover, i.t. administration of scAAV2/8 particles in macaques, by endoscopic-mediated guidance, resulted in significant gene transfer. Thus, in healthy animals, where thymic gene transfer does not provide a selective advantage, scAAV2/8 is a unique tool promoting the in situ transduction of thymocytes with the subsequent export of gene-modified lymphocytes to the periphery.

Hematopoietic Stem Cells Are Primarily Involved in Pathogenesis of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by consistent expansion of B cells in peripheral lymphoid organs. CLL B cells frequently express CD5 antigen, and have clonal rearrangement of immunoglobulin heavy chain (IGH) gene with restricted usage of V1, V3 and V4 of the variant region. CLL has thus been believed to represent retention or proliferation of abnormal B cell clones presumably with anti-apoptotic potential, or with deregulated response to auto-antigens. In this study, we extensively search for CLL-initiating cells by utilizing the NOD/SCID/IL2rgnull (NOG) xenogeneic transplantation system, in which human hematopoietic stem cells (HSCs) can normally develop multi-lineage cells including polyclonal B and T cells. In the NOG xenotransplant system, neither CD34-CD19+ circulating B cells, nor CD34+CD38+ bone marrow (BM) progenitor populations from 12 CLL patients engrafted even after injection of >106 cells. We then transplanted CD34+CD38− BM HSC population from 7 CLL patients into 13 NOG mice. Injection of as few as 103 cells of the CD34+CD38− BM population resulted in multi-lineage reconstitution. Most of these mice, however, died within 12–24 weeks after xenotransplantation. In 9 mice analyzed by multi-color FACS, 7 mice possessed both CD5+ and CD5− B cell populations, and the remaining 2 mice had only CD5− B cells. These CD5+ or CD5− human B cell populations were purified separately by FACS, and tested for the IGH gene rearrangement. Strikingly, 16 out of 20 B cell populations were clonal with single IGH rearrangement irrespective of their CD5 expression, by multiplex PCR analysis. In contrast, CD34+CD38− HSC populations in CLL patients never had IGH rearrangement. We then directly sequenced PCR products of IGH gene in each B cell clones as well as those in the original CLL cells purified directly from patients' blood. Surprisingly, VDJ recombination in B cell clones developed in NOG mice were different from that of the original CLL clones in all 7 CLL cases. Interestingly, all of these clones used only V1, V3 and V4 regions for their VDJ recombination like primary CLL cells. Furthermore, when the CD34+CD38− BM HSC fraction from single CLL patients was transplanted into a set of 3 mice simultaneously, each mouse developed independent B cell clones with different VDJ recombination in all 3 experiments. The fact that CD34+CD38− HSCs from CLL patients but not those from normal individuals give rise to clonal B cell population in our xenograft model strongly suggests that some genetic abnormality for CLL progression is acquired already at the HSC level in CLL patients. HSCs in CLL patients are multipotent, but once they commit to the B cell lineage, they use preferentially the V1, V3 and V4 regions for IGH recombination. Our hypothesis is that such B cell clones may already be abnormal in that they clonally expand in response, for example, to auto-antigens (xeno-antigens in NOG mice), and they may possibly sequentially receive additional mutations to become clinical CLL. Although this xenograft model may not recapitulate full picture of CLL progression, our data clearly show that primary leukemogenic event occurs at the multipotent HSC stage in human CLL.

Instructive Role of the Transcription Factor E2A in Early B Lymphopoiesis and Germinal Center B Cell Development

The transcription factor E2A controls the initiation of B lymphopoiesis, which is arrested at the pre-pro-B cell stage in E2A-deficient mice. Here, we demonstrate by conditional mutagenesis that E2A is essential for the development of pro-B, pre-B, and immature B cells in the bone marrow. E2A is, however, dispensable for the generation of mature B cells and plasma cells in peripheral lymphoid organs. In contrast, germinal center B cell development is impaired in the absence of E2A despite normal AID expression and class-switch recombination. Molecular analysis revealed that E2A is required not only for initiating but also for maintaining the expression of Ebf1, Pax5, and the B cell gene program in pro-B cells. Notably, precocious Pax5 transcription from the Ikzf1 locus promotes pro-B cell development in E2A-deficient mice, demonstrating that ectopic Pax5 expression is sufficient to activate the B lymphoid transcription program in vivo in the absence of E2A.

Licensing of naïve T cells to produce TNF

TNF is a pro‐inflammatory cytokine that regulates numerous aspects of immune responses, including the generation of virus‐specific CD8 T cells during an infection. We have shown that naïve T cells in peripheral lymphoid organs produce TNF rapidly after TCR engagement (within 4 hrs) and prior to the acquisition of other effector functions. Given the immunoregulatory role of TNF and the ability of peripheral naïve T cells to produce this cytokine, we questioned at what stage of development do T cells become competent to produce TNF. Our findings indicate that, in contrast to peripheral naïve T cells, single positive thymocytes have a poor ability to produce TNF on ex‐vivo stimulation. Despite this inability to produce TNF, thymocytes express mature TNF message, confirming that TNF production can be post‐transcriptionally regulated. Thymocytes that are adoptively transferred into the periphery of congenic hosts become competent to produce TNF as early as 6 hrs after transfer and before undergoing division. Moreover, stimulation of thymocytes in the presence of splenocytes for 4 hours in vitro is sufficient to partially enable thymic lymphocytes to produce TNF. Together, these findings indicate that thymic lymphocytes require post‐thymic maturation in the periphery to become licensed to produce TNF.

Origin of dendritic cells in peripheral lymphoid organs of mice

Parabiosis experiments demonstrating that dendritic cells (DCs) do not equilibrate between mice even after prolonged joining by parabiosis have suggested that DCs are derived from self-renewing progenitors that divide in situ. However, here we found that unequal exchange of DCs between mice joined by parabiosis reflected uneven distribution of DC precursors in blood due to their short half-life in circulation. DCs underwent only a limited number of divisions in the spleen or lymph nodes over a 10- to 14-day period and were replenished from blood-borne precursors at a rate of nearly 4,300 cells per hour. Daughter DCs presented antigens captured by their progenitors, suggesting that DC division in peripheral lymphoid organs can prolong the duration of antigen presentation in vivo.

Acute systemic immune activation following vaginal exposure to staphylococcal enterotoxin B—Implications for menstrual shock

Menstrual toxic shock syndrome (mTSS) is an acute systemic inflammatory disease associated with the superantigenic exotoxin, toxic shock syndrome toxin (TSST)-1, produced by Staphylococcus aureus and the use of high absorbency tampons. Even though S. aureus is capable of elaborating several other superantigenic exotoxins, only TSST-1 has been implicated in the pathogenesis of mTSS possibly because most other superantigenic exotoxins are known enterotoxins. Nonetheless, we have shown recently that one of the enterotoxigenic staphylococcal superantigens, staphylococcal enterotoxin B (SEB), can cause robust systemic immune activation following exposure through non-enteric mucosa, including nasal or conjunctival routes. In a similar manner, we show here that vaginal administration of SEB in HLA class II transgenic mice can cause robust systemic immune activation characterized by profound elevation of proinflammatory cytokines in the serum, activation and expansion of SEB-reactive CD4 + and CD8 + T cells in peripheral lymphoid organs and SEB-induced deletion of immature thymocytes. Vaginal administration of SEB also caused leukocytic infiltration in major organs, such as liver and lung, reminiscent of human toxic shock syndrome. Systemic immune activation following vaginal superantigen delivery was independent of the stage of the estrus cycle in the mouse. Using HLA class II transgenic mice, we have shown that exposure to SEB through the vaginal canal can cause robust systemic immune activation. SEB could thus play a role in the pathogenesis of mTSS.

Increasing Flt3L availability alters composition of a novel bone marrow lymphoid progenitor compartment

AbstractWe have recently described a CD19– B220+CD117low bone marrow subpopulation with B, T, and myeloid developmental potential, which we have called “early progenitors with lymphoid and myeloid potential” or EPLM. These cells also expressed Fms-like tyrosine kinase 3, Flt3, or CD135. Treatment of mice with the corresponding ligand, Flt3L, showed a 50-fold increase in EPLM. In addition to the expected increase in dendritic cell numbers, Flt3L treatment had a reversible inhibitory effect on B lymphopoiesis. Limiting dilution analysis of sorted EPLM from Flt3L-treated mice showed that B-lymphocyte progenitor activity was reduced 20-fold, but that myeloid and T-cell progenitor activity was largely preserved. EPLM from treated mice transiently reconstituted the thymus and bone marrow of recipient mice, generating cohorts of functional T and B cells in peripheral lymphoid organs. Thus, Flt3L treatment results in a dramatic increase in a novel bone marrow cell with lymphoid and myeloid progenitor activity.

Golli Protein Negatively Regulates Store Depletion-Induced Calcium Influx in T Cells

Calcium influx is crucial for T cell activation and differentiation. The detailed regulation of this process remains unclear. We report here that golli protein, an alternatively spliced product of the myelin basic protein gene, plays a critical role in regulating calcium influx in T cells. Golli-deficient T cells were hyperproliferative and showed enhanced calcium entry upon T cell receptor stimulation. We further found that golli regulates calcium influx in T cells through the inhibition of the store depletion-induced calcium influx. Mutation of the myristoylation site on golli disrupted its association with the plasma membrane and reversed its inhibitory action on Ca2+ influx, indicating that membrane association of golli was essential for its inhibitory action. These results indicate that golli functions in a unique way to regulate T cell activation through a mechanism involving the modulation of the calcium homeostasis.

CD25− T Cells Generate CD25+Foxp3+ Regulatory T Cells by Peripheral Expansion

Naturally occurring CD4(+) regulatory T cells are generally identified through their expression of CD25. However, in several experimental systems considerable T(reg) activity has been observed in the CD4(+)CD25(-) fraction. Upon adoptive transfer, the expression of CD25 in donor-derived cells is not stable, with CD4(+)CD25(+) cells appearing in CD4(+)CD25(-) T cell-injected animals and vice versa. We show in this study that CD25(+) cells arising from donor CD25(-) cells upon homeostatic proliferation in recipient mice express markers of freshly isolated T(reg) cells, display an anergic state, and suppress the proliferation of other cells in vitro. The maintenance of CD25 expression by CD4(+)CD25(+) cells depends on IL-2 secreted by cotransferred CD4(+)CD25(-) or by Ag-stimulated T cells in peripheral lymphoid organs.

Absence of antitumor natural killer cell-mediated defense in the brain

To better understand the role of immunosurveillance in defense against brain tumors, the brain has been conceived as a “partially immunologically privileged” organ, meaning that antigens within the brain do not evoke an afferent response, probably because of effective maintenance of the blood-brain barrier (BBB) and subsequent influx of immune cells, lack of lymphatic drainage, and very low expression of major histocompatibility complex (MHC) molecules on most of the normal cells in the brain. During the earliest stage of oncogenesis, the BBB is intact and the immune system is normal. As the tumor grows, the accompanying histological or functional changes in vascular structures permit tumor antigens to leave the brain. As a result, the brain ceases to be an immunologically privileged organ. In response to the new antigenic stimulus, lymphocytes can proliferate and, most importantly, suppressor cells may be generated. The reason for the induction of suppressor activity remains unclear, but the result is a gradual loss of host immunocompetence. At this stage, the tumor becomes clinically evident, and the immune abnormalities are easily detected. Such suppressor cells may be generated by thymus-derived lymphocytes or macrophages. In addition, humoral suppression because of immune complexes may play a role in suppressor cell induction. It is postulated that in adult life the thymus maintains a regulatory population of T cells in peripheral lymphoid organs that suppress early T cell differentiation to cytotoxic effector cells and potentiate the development of immune memory. With regard to T cell-mediated immune responses, effector T cells of systemic origin have been shown to enter the brain in about 30% of glioma cases, for example, being manifested as perivascular accumulations of small lymphocytes. In contrast, natural killer (NK) cells are usually absent in brain tumors or present only in low amounts. However, the reason for the small number of NK cells in the brain is unclear, and there has been no definite evidence of NK cellmediated intracerebral immunosurveillance. Our recent experimental study (Yamasaki T et al. Experimental appraisal of the lack of antitumor natural killer cell-mediated immunosurveillance in response to lymphomas growing in the mouse brain. J Neurosurg 2003;98:599–606) based on a well-controlled, novel murine model provided the first data to suggest that the brain may lack an NK cell-mediated defense mechanism against tumors growing in the brain, representing another characteristic aspect of this immunologically privileged organ. It is accepted that NK cells are large, granular lymphocytes that mediate lysis of certain virally infected cells or tumor cells. The activity of NK cells is regulated by the expression of MHC Class I molecules on potential target cells. It has been proposed that a correlation exists between resistance to NK cell-mediated cytolysis and MHC Class I expression on tumor cells, although there are some exceptions. Thus, expression of MHC Class I molecules can protect target cells from NK cell cytotoxicity. This protection is mediated by inhibitory NK cell receptors that recognize MHC Class I molecules on target cells. Loss of MHC Class I expression can render cells sensitive to NK cell attack. Contact with sensitive target cells induces a series of signals in NK cells leading to target cell adhesion; polarization of surface receptors; and signaling molecules at the NK: target cell interface, followed by polarization and exocytosis of granules toward the target cell and the production of cytokines such as interferongamma. The interface that forms between NK and target cells is referred to as the “NK immunological synapse,” where adhesion molecules, other surface receptors, and cytoplasmic signaling molecules are recruited in an ordered manner.

The Absence of CD44 Ameliorates Faslpr/lpr Disease

The lpr mutation in the Fas gene leads to symptoms of autoimmune disease, including polyclonal B-lymphocyte activation and lymphoproliferation. The expanding T-lymphocyte populations in the disease are characterized by high expression levels of the memory marker CD44. It has not been known whether CD44 expression contributes to the pathophysiology of the condition or merely reflects a consequence of excessive lymphocyte activation. We therefore tested the role of CD44 gene products in Faslpr/lpr disease in gene targeted mice. We bred CD44 knockout mice and C57Bl/6-lpr mice to generate the Faslpr/lpr CD44+/+, and Faslpr/lpr CD44−/− genotypes and analyzed the disease manifestations around 215 days of age. The absence of CD44 substantially reduced the immunoglobulin secretion and lymphocyte expansion that are characteristic of the Faslpr/lpr syndrome. Surprisingly, the percentage of CD3+CD4−CD8− (double negative) cells in peripheral lymphoid organs increased in Faslpr/lpr CD44−/− mice almost to the same extent as in Faslpr/lpr CD44+/+ mice. These results indicate that the expansion of the fraction of double negative cells in spleens and lymph nodes, believed to be generated by down-regulation of CD8, does not depend on increased lymphocyte numbers. Furthermore, they corroborate an essential role for CD44 gene products in the T-cell expansion and polyclonal B-cell activation that constitute the Faslpr/lpr syndrome. The CD44 receptor may be a suitable therapeutic target for inhibition of lymphoproliferation in autoimmune diseases.

Identification of an HLA-A*0201-restricted epitopic peptide from human dystrophin: application in duchenne muscular dystrophy gene therapy

Dystrophin-based gene therapy treatments aimed at correcting the Duchenne muscular dystrophy phenotype require stable expression of normal dystrophin (DYST) protein in myocytes without immune responses, which would compromise long-term expression. To predict cytotoxic T-cell-mediated responses elicited by transgenes, we used here H-2-negative HLA-A*0201 transgenic mice and identified human DYST epitopes, which elicit HLA-A*0201-restricted cytotoxic T cell activities. Among a series of eight peptides predicted from the human DYST sequence, not shared with the endogenous mouse DYST sequence, four of them were able to bind to HLA-A*0201 molecules and to induce cytotoxic T lymphocyte (CTL) responses. After human DYST DNA transfer in muscle of HLA-A*0201 mice, only the human DYST1281 epitope, located in the spectrin-like repeat 9 domain, induced strong CD8+ CTL responses. Using the corresponding human DYST1281 peptide/HLA-A*0201 tetramer, we detected human DYST1281-specific CD8+ T cells in peripheral lymphoid organs and blood of HLA-A*0201 mice injected with human DYST DNA. Our results demonstrate that muscle injection with human DYST DNA systematically triggers CTL responses against this HLA-A*0201-restricted human DYST1281 peptide, which is present in long human DYST isoforms. Identification of such immunodominant human DYST epitopes and use of peptide/HLA tetramers will allow the immunomonitoring of CTL responses in HLA-phenotyped Duchenne muscular dystrophy patients undergoing gene therapy. Finally, the knowledge of HLA-A*0201-restricted human DYST peptides will be of importance to test, in mouse models, new immunomodulatory interventions allowing long-term engraftment of human dystrophin.

B Cell–specific Transgenic Expression of Bcl2 Rescues Early B Lymphopoiesis but Not B Cell Responses in BOB.1/OBF.1-deficient Mice

Mice deficient for the transcriptional coactivator BOB.1/OBF.1 show several defects in B cell differentiation. Numbers of immature transitional B cells in the bone marrow are reduced and fewer B cells reach the periphery. Furthermore, germinal center B cells are absent and marginal zone (MZ) B lymphocytes are markedly reduced. Increased levels of B cell apoptosis in these mice prompted us to analyze expression and function of antiapoptotic proteins. Bcl2 expression is strongly reduced in BOB.1/OBF.1-deficient pre–B cells. When BOB.1/OBF.1-deficient mice were crossed with Bcl2-transgenic mice, B cell development in the bone marrow and numbers of B cells in peripheral lymphoid organs were normalized. However, neither germinal center B cells nor MZ B cells were rescued. Additionally, Bcl2 did not rescue the defects in signaling and affinity maturation found in BOB.1/OBF.1-deficient mice. Interestingly, Bcl2-transgenic mice by themselves show an MZ B cell defect. Virtually no functional MZ B cells were detected in these mice. In contrast, mice deficient for Bcl2 show a relative increase in MZ B cell numbers, indicating a previously undetected function of Bcl2 for this B cell compartment.

New functions for the sialic acid-binding adhesion molecule CD22, a member of the growing family of Siglecs.

New functions for the sialic acid-binding adhesion molecule CD22, a member of the growing family of Siglecs.

Mechanisms involved in antithymocyte globulin immunosuppressive activity in a nonhuman primate model.

The mechanisms of action of polyclonal antithymocyte globulins (ATGs) are still poorly understood and the selection of doses used in different clinical applications (prevention or treatment of acute rejection in organ allografts, treatment of graft-versus-host disease, or conditioning for allogeneic stem cell transplantation) remains empirical. Low T-cell counts are usually achieved in peripheral blood during ATG treatment but the extent of T-cell depletion in lymphoid tissues is unknown. Experiments were conducted in cynomolgus monkeys using Thymoglobuline at low (1 mg/kg), high (5 mg/kg), and very high (20 mg/kg) doses. ATG treatment induced a dose-dependent lymphocytopenia in the blood and a dose-dependent T-cell depletion in spleen and lymph nodes but not in the thymus, indicating a limited access of ATG to this organ. T-cell apoptosis in peripheral lymphoid tissues was the main mechanism of depletion. Remaining T cells in peripheral lymphoid organs were coated by antibodies and had down-modulated surface expression of CD2, CD3, CD4, and CD8 molecules, whereas their responsiveness in mixed leukocyte reaction was impaired. The survival of MHC-mismatched skin and heart allografts was prolonged in a dose-dependent fashion, despite the occurrence of a strong anti-ATG antibody response resulting in the rapid clearance of circulating ATGs. The results indicate that T-cell depletion is achieved rapidly and primarily in peripheral lymphoid tissues at high ATG dosage. Short ATG treatments could therefore be clinically evaluated when major peripheral T-cell depletion is required.

Altered selection of CD4+ T cells by class II MHC bound with dominant and low abundance self-peptides.

We have investigated the development of CD4(+) T cells in mice expressing low levels of transgenic class II MHC molecules (A(b)) preoccupied with covalent peptide (Ep), which in the presence of invariant chain (Ii) is extensively cleaved and replaced with self-derived peptides. In these mice, the transgenic A(b) molecules, bound with predominant peptide (Ep) and with multiple self-peptides, selected more CD4(+) T cells than A(b)/self-peptide complexes expressed in wild-type mice. The enhanced outcome of thymic selection was a result of impaired negative selection, rather than more efficient positive selection by an overall lowered abundance of self-derived A(b)/peptide complexes. Peripheral CD4(+) T cells in the A(b)EpIi(+) mice had memory phenotype, often followed by polyclonal activation of B cells. The A(b)EpIi(+) mice preserved their good health and had a normal life span despite the profound number of activated CD4(+) T cells and B cells in peripheral lymphoid organs, moderate hypergammaglobulinemia, and deposited complexes in the kidneys. We propose that CD4(+) T cells positively selected due to low avidity for high abundant A(b)Ep complex avoid negative selection on A(b) molecules loaded with low abundant peptides and become self-reactive in the peripheral lymphoid organs.

Qa-2-dependent selection of CD8alpha/alpha T cell receptor alpha/beta(+) cells in murine intestinal intraepithelial lymphocytes.

Murine intestinal intraepithelial lymphocytes (iIELs) are made up of a heterogeneous mix of T cells with unique phenotypes. Whereas CD8(+) T cells in peripheral lymphoid organs use CD8alpha/beta and are selected on MHC class Ia molecules, a majority of iIELs use CD8alpha/alpha. Here, we report that the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is independent of classical MHC class I molecules K(b) and D(b), as illustrated by their presence in K(b)/D(b) double-knockout mice and in mice lacking a nonclassical MHC class I molecule, CD1d. Most strikingly, their presence is decreased by approximately 70% in mice lacking transporter associated with antigen processing (TAP). The TAP-dependent nonclassical MHC class I molecule Qa-2 is strongly implicated in the presence of these cells, as inferred from the low numbers of CD8alpha/alpha TCR-alpha/beta T cells in mice deficient in Qa-2 genes. Second, a Qa-2-transgenic mouse made in a Qa-2(-) strain showed an increase in the numbers of CD8alpha/alpha cells among its iIELs. Thus, the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is mainly dependent on the nonclassical MHC class I molecule Qa-2.

Autoreactive B cells escape clonal deletion by expressing multiple antigen receptors.

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2-/-) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2-/- knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.