James J. Kenny

TIM-4 is the ligand for TIM-1, and the TIM-1–TIM-4 interaction regulates T cell proliferation

The newly identified TIM family of proteins is associated with regulation of T helper type 1 (TH1) and TH2 immune responses. TIM-1 is genetically linked to asthma and is a receptor for hepatitis A virus, but the endogenous ligand of TIM-1 is not known. Here we show that TIM-4, which is expressed by antigen-presenting cells, is the ligand for TIM-1. In vivo administration of either soluble TIM-1–immunoglobulin (TIM-1–Ig) fusion protein or TIM-4–Ig fusion protein resulted in hyperproliferation of T cells, and TIM-4–Ig costimulated T cell proliferation mediated by CD3 and CD28 in vitro. These data suggest that the TIM-1–TIM-4 interaction is involved in regulating T cell proliferation.

Reciprocal generation of Th1/Th17 and Treg cells by B1 and B2 B cells

Regulatory T (Treg) cells are indispensable for maintaining peripheral tolerance, whereas T helper (Th)1 and Th17 cells induce inflammation and tissue destruction. Using Foxp3‐GFP knock‐in mice, we report a novel regulatory role for B cell subsets in influencing the differentiation of Treg versus Th1/Th17 cells. Peritoneal B1 cells strongly promoted T cell proliferation and cytokine secretion when presenting nominal or allogeneic antigens, as compared to conventional follicular B (B2) cells. However, peritoneal B1 cells largely failed to convert naive Foxp3–CD4+ T cells into Foxp3+ Treg cells in the presence of TGF‐β and IL‐2, in marked contrast to conventional B2 cells, which excelled in Treg conversion. Interestingly, under the same Treg conversion conditions, peritoneal B1 cells preferentially promoted Th1 and Th17 cell differentiation. Blockade of CD86 but not CD80 costimulation markedly enhanced Treg cell induction by B1 cells. Thus, B cell antigen presentation function is inversely correlated with de novo Treg cell induction for these B cell subsets. Our findings suggest that B1 and B2 cell subsets play distinct roles in immune regulation by promoting reciprocal differentiation of T cell lineages.

Specificity of CD4+CD25+ Regulatory T Cell Function in Alloimmunity

CD4+CD25+ regulatory T cells (TRegs) are critical for the acquisition of peripheral allograft tolerance. However, it is unclear whether TRegs are capable of mediating alloantigen-specific suppressive effects and, hence, contributing to the specificity of the tolerant state. In the current report we have used the ABM TCR transgenic (Tg) system, a C57BL/6-derived strain in which CD4+ T cells directly recognize the allogeneic MHC-II molecule I-Abm12, to assess the capacity of TRegs to mediate allospecific effects. In these mice, 5–6% of Tg CD4+ T cells exhibit conventional markers of the TReg phenotype. ABM TRegs are more effective than wild-type polyclonal TRegs at suppressing effector immune responses directed against I-Abm12 alloantigen both in vitro and in vivo. In contrast, they are incapable of suppressing responses directed against third-party alloantigens unless these are expressed in the same allograft as I-Abm12. Taken together, our results indicate that in transplantation, TReg function is dependent on TCR stimulation, providing definitive evidence for their specificity in the regulation of alloimmune responses.

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.

Regulation of T cell dependent immune responses by TIM family members.

The T cell immunoglobulin mucin (TIM) proteins are type I membrane glycoproteins expressed on T cells and containing common structural motifs. While our understanding on the distribution and functions of TIM family members is still incomplete, data from several recent reports indicate that these proteins, together with T cell receptor and costimulatory signals, regulate the expansion and effector functions of T helper cells. In the current review, we provide evidences indicating that TIM-3 is capable of modulating the function of CD4+CD25+ regulatory T cells and inhibiting aggressive Th1 mediated auto- and allo-immune responses. Similarly, additional data suggest that TIM-2 molecules function by negatively regulating Th2 immune responses. In contrast, TIM-1 appears to be an activation molecule for all T cells, although the mechanisms through which TIM-1 activates T cells remain to be elicited.

Tim-1 signaling substitutes for conventional signal 1 and requires costimulation to induce T cell proliferation.

Differentiation and clonal expansion of Ag-activated naive T cells play a pivotal role in the adaptive immune response. T cell Ig mucin (Tim) proteins influence the activation and differentiation of T cells. Tim-3 and Tim-2 clearly regulate Th1 and Th2 responses, respectively, but the precise influence of Tim-1 on T cell activation remains to be determined. We now show that Tim-1 stimulation in vivo and in vitro induces polyclonal activation of T cells despite absence of a conventional TCR-dependent signal 1. In this model, Tim-1-induced proliferation is dependent on strong signal 2 costimulation provided by mature dendritic cells. Ligation of Tim-1 upon CD4+ T cells with an agonist anti-Tim-1 mAb elicits a rise in free cytosolic calcium, calcineurin-dependent nuclear translocation of NF-AT, and transcription of IL-2. Because Tim-4, the Tim-1 ligand, is expressed by mature dendritic cells, we propose that interaction between Tim-1+ T cells and Tim-4+ dendritic cells might ensure optimal stimulation of T cells, when TCR-derived signals originating within an inflamed environment are weak or waning.

Regulation of T-Cell Immunity by T-Cell Immunoglobulin and Mucin Domain Proteins

The ability of T helper (TH) precursor cells to differentiate into T effector populations confers the adaptive immune system with a means to protect the host from microbes and react to “foreign” antigenic tissues. T-cell immunoglobulin and mucin domain (TIM) proteins have recently been shown to be novel and critical regulators of T cell subset-driven dependent immune responsiveness. A dichotomy is emerging as to how Tim-3– and Tim-2– related signals respectively impact TH1 and TH2 responses. By comparison, the influence of the Tim-1 pathway seems to be broader and is probably not restricted to a specific type of T helper response. Beyond the mere control of the TH1/TH2 balance, Tim proteins are likely to target other regulatory components of the T cell response. Likewise, it is tempting to speculate that Tim proteins might also modulate the function of other T helper cell subsets such as TH3, TR1 and TH17 cells, among others.

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 JH region of chromosome 12. This T15H chain combines with a κ22–33 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, κ8–28 and κ19–15, were expressed in this population. The Vκ region of these L chains was recombined to Jκ5, 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:κ8–28L and T15H:κ19–15L Abs revealed L chain differences from the κ22–33 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:κ22–33L Ab.

Direct pathway T-cell alloactivation is more rapid than indirect pathway alloactivation.

In the direct pathway of allore-cognition, recipient T cells recognize intact donor major histocompatibility complex (MHC) molecules on the surface of donor antigen-presenting cells (APCs), whereas the indirect pathway involves recognition of processed donor antigen presented in the context of self-MHC on the host’s own APCs (1, 2). Both pathways play important roles in the allograft response. Herein, we report on the kinetics of graft rejection when the alloantigen is presented by one of the pathways to B6.TEa.Rag-2−/− recipient mice whose T cells express a donor direct or indirect pathway responsive transgenic T-cell receptor (TCR) at a given time. This TEa-TCR recognizes the IE-alpha peptide 52–68 (ASFEAQGLANIAVDKA) presented in the context of IAb. C57BL/6.Rag-2−/− mice expressing the TEa transgenes were produced by crossing C57BL/6.Rag-2−/− and TEa transgenic mice (3) (a gift from Randy Noelle at Dartmouth University, Hanover, NH). Tail skin from BALB/c (IAd/IEd), CB6F1(IAbd/IEd), or C57BL/6(IAb/IE−) mice were grafted onto B6.TEa. Rag2−/− mice. BALB/c dendritic cells (DCs) produce IEd 52–68 peptide but cannot present it directly to B6.TEa. Rag2−/− mice T cells. The IEd 52–68 peptide must be processed by the B6.TEa.Rag2−/− IAb DCs before presentation to its T cells allowing for indirect pathway of alloantigen presentation. Conversely, CB6F1 skin graft alongside indirect antigen presentation allows for direct antigen presentation as CB6F1 DCs can both produce IEd 52–68 peptide and directly present it to TEa Rag2−/− T cells in conjunction with their own IAb molecules. Therefore, we studied the kinetics of rejection of BALB/c or CB6F1 donor strain skin transplants by B6. TEa.Rag2−/− mice to study direct and indirect alloantigen presentation. In comparison with CB6F1 donor graft, rejection of BALB/c grafts were delayed by 6 days (P=0.0067; Fig. 1A). Hence, these data indicate that indirect alloantigen presentation alone leads to graft rejection albeit graft rejection is significantly delayed. Natural killer (NK) cells kill allogeneic donor APCs (4), and anti-NK1.1 treatment strengthens rejection in some (4) but not all MHC incompatible skin allograft strain combinations (5). Treatment did not alter mean survival time in this Balb/C to Tea-TCR tg C57BL/6 model (unpublished observations). Indeed in accordance with our work, anti-NK1.1 treatment did not affect graft rejection by C57BL/6 recipients grafted with Balb/C skin (5). Moreover, the magnified frequency of donor-reactive T cells in the TEa-TCR-Tg host is almost certain to make the NK effect on donor APCs less crucial to graft acceptance. FIGURE 1 (A) In comparison with CB6F1 donor skin grafts, there is a delay in rejection of BALB/c donor skin grafts by B6.TEa.Rag2−/− recipients (n≥4). (B) Delayed proliferation of B6.TEa.Rag2−/− CD4+ T cells when the alloantigen ... We hypothesized that the delay in graft rejection is due to a delayed onset of immune response primarily due to delayed T-cell activation. To test this hypothesis, we studied in vitro B6.TEa.Rag2−/− T-cell proliferation in the mixed leukocyte reaction using CB6F1, BALB/c, or C57BL6 DCs. When activation of CFSE-labeled B6.TEa. Rag2−/− CD4 cells was triggered by CB6F1 DCs, the T cells start proliferating as early as day 2, and by day 4, approximately 75% of the cells were proliferating (Fig. 1B). In comparison, proliferation was not discerned using syngeneic C57BL/6 DCs or allogeneic BALB/c DCs as stimulator cells (Fig. 1B). However, when both BALB/c and C57BL/6 DCs are added to the same well to allow indirect antigen presentation, it leads to T-cell proliferation albeit with a delay as proliferation was noticed on day 3 with approximately 10% cells proliferating (Fig. 1B). Hence, in this particular experimental setting, these in vitro data are in line with our hypothesis, suggesting a delay in T-cell activation and proliferation as one of the many possible mechanisms leading to a delay in graft rejection. In a distinct adoptive transfer model study, where direct and indirect pathway T cells were injected into immune compromised mice, it has been shown that indirect pathway CD4 T cells proliferate more rapidly (6). The difference in observations can be attributed to the peculiarity of each model, and the observations cannot be generalized. We appreciate that the TEa-TCR transgenic model does not allow an analysis of the frequency of alloreactive T cells committed to the direct or indirect pathways. But the peculiarity of this model allows study of both direct and indirect pathway alloantigen recognition by a single clone of T cells. Thus, this TEa-TCR Tg model that is a reductionist model with a single clone of T cells able to mount antidonor responses allows understanding the mechanism by which delayed graft rejection occurs when antigen is presented only through the indirect pathway. It is well known that activation through the indirect pathway alone is sufficient for allograft rejection, but the rejection is significantly delayed. The classical work by Auchincloss et al. (7) using MHCII-deficient donor mice demonstrated the importance of indirect allorecognition alone in graft rejection. It is traditionally believed that direct alloreactivity is the driving mechanism behind early acute graft rejection. But the direct response subsides as the donor antigen disappears, meanwhile the indirect alloresponse appears and persists causing chronic rejection. Study in our specific TCRTg model is in line with this belief where indirect alloantigen alone can lead to delayed rejection. Our in vitro studies in this particular experimental setting further suggest that the time lag for foreign antigen processing and presentation by self-MHC on recipient APCs may delay the T-cell proliferation and hence indirect alloresponse-mediated rejection. Various studies have shown results contrary to our study, and we attribute the difference in these findings to the peculiarity of the different models studied and believe that no generalization can be drawn from any of these studies including ours.

The M603 idiotype is lost in the response to phosphocholine in terminal deoxynucleotidyl transferase-deficient mice.

The majority of anti‐phosphocholine (PC) antibodies induced by the PC epitope in Proteus morganii (PM) express the M603 idiotype (id), which is characterized by an invariant Asp to Asn substitution at the VH:DH junction. To elucidate the molecular basis by which M603‐like B cells acquire the mutations resulting in this invariant substitution, we analyzed the immune response to PC‐PM in terminal deoxynucleotidyl transferase (TdT) gene knockout (KO) mice. In the absence of TdT, T15‐id antibodies comprised 80–100% of the primary response to PC‐PM. Less than 10% of the response in wild‐type mice is T15‐id+. In TdT KO mice, the secondary response to PC‐KLH was higher than in wild‐type mice and was dominated by the germ‐line T15‐id. About 10% of this response, in both TdT KO and wild‐type mice, comprised M167‐id+ antibodies. Additionally, none of the functionally rearranged V1/DFL16.1/JH1 cDNA isolated from PC‐PM‐immunizedTdT KO mice showed the Asp/Asn substitution characteristic of PC‐binding, PC‐PM‐induced M603‐like antibodies. These data indicate that production of M603‐id antibody is TdT dependent, while generation of M167‐id antibody is TdT independent, and that in the absence of competition from M603‐like B cells, T15‐id B cells can respond to PC‐PM.