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Firefighter occupational exposures were categorized as a class 1 (known) carcinogen by the International Agency for Research on Cancer in 2022. As a result, firefighters have become heavily focused on identifying effective and easy to implement decontamination strategies to reduce their chemical exposures. Skin decontamination using wipes post-exposure is one decontamination strategy that every firefighter has available to them. However, firefighters have expressed concerns over the ingredients in the wipe solution increasing dermal absorption. The goal of this study was to determine if the ingredients in skin decontamination wipe solution had any enhancement effect on the dermal absorption of phenanthrene. To determine any enhancement effects, the additive solution of four skin decontamination wipe products was applied to porcine skin 15 min after chemical dosing. The absorption of phenanthrene was tested in vitro using a flow-through diffusion cell system over eight hours. The wipe solution effects on dermal absorption were determined by measuring multiple absorption characteristics including cumulative absorption (µg/cm2), absorption efficiency (% dose absorbed), lag time (minutes), flux (µg/cm2/h), diffusivity (cm2/h), and permeability (cm/h). No penetration enhancement effects were observed in any of the skin decontamination wipe solutions tested; rather, all wipe solutions decreased the absorption of phenanthrene. Slight differences in cumulative absorption among two pairings of skin decontamination wipe solutions, wipes 1 and 3 vs. wipes 2 and 4, were observed, indicating that some ingredients may impact dermal absorption. These findings show that firefighters should continue using skin decontamination wipes to reduce their dermal exposures to fireground contaminants with little concern of increasing the absorption of phenanthrene.

Firefighters face significant risks of exposure to toxic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), during fire suppression activities. PAHs have been found in the air, on the gear and equipment, and in biological samples such as the skin, breath, urine, and blood of firefighters after fire response. However, the extent to which exposure occurs via inhalation, dermal absorption, or ingestion is unclear. In this study, three PAHs, naphthalene, phenanthrene, and benzo[a]pyrene, were applied to porcine skin in vitro in an artificial sweat solution to better gauge firefighters’ dermal exposures while mimicking their sweaty skin conditions using an artificial sweat dosing vehicle. Multiple absorption characteristics were calculated, including cumulative absorption, percent dose absorbed, diffusivity, flux, lag time, and permeability. The absorption of the PAHs was greatly influenced by their molecular weight and solubility in the artificial sweat solution. Naphthalene had the greatest dose absorption efficiency (35.0 ± 4.6% dose), followed by phenanthrene (6.8 ± 3.2% dose), and lastly, benzo[a]pyrene, which had the lowest absorption (0.03 ± 0.04% dose). The lag times followed a similar trend. All chemicals had a lag time of approximately 60 min or longer, suggesting that chemical concentrations on the skin may be reduced by immediate skin cleansing practices after fire exposure.

Reagents that activate the signaling adaptor stimulator of interferon genes (STING) suppress experimentally induced autoimmunity in murine models of multiple sclerosis and arthritis. In this study, we evaluated STING agonists as potential reagents to inhibit spontaneous autoimmune type I diabetes (T1D) onset in non-obese diabetic (NOD) female mice. Treatments with DNA nanoparticles (DNPs), which activate STING when cargo DNA is sensed, delayed T1D onset and reduced T1D incidence when administered before T1D onset. DNP treatment elevated indoleamine 2,3 dioxygenase (IDO) activity, which regulates T-cell immunity, in spleen, pancreatic lymph nodes and pancreas of NOD mice. Therapeutic responses to DNPs were partially reversed by inhibiting IDO and DNP treatment synergized with insulin therapy to further delay T1D onset and reduce T1D incidence. Treating pre-diabetic NOD mice with cyclic guanyl-adenyl dinucleotide (cGAMP) to activate STING directly delayed T1D onset and stimulated interferon-αβ (IFN-αβ), while treatment with cyclic diguanyl nucleotide (cdiGMP) did not delay T1D onset or induce IFN-αβ in NOD mice. DNA sequence analyses revealed that NOD mice possess a STING polymorphism that may explain differential responses to cGAMP and cdiGMP. In summary, STING agonists attenuate T1D progression and DNPs enhance therapeutic responses to insulin therapy., (© 2019 John Wiley & Sons Ltd.)

Cytosolic DNA sensing activates the stimulator of IFN genes (STING) adaptor to induce IFN type I (IFN-αβ) production. Constitutive DNA sensing to induce sustained STING activation incites tolerance breakdown, leading to autoimmunity. In this study, we show that systemic treatments with DNA nanoparticles (DNPs) induced potent immune regulatory responses via STING signaling that suppressed experimental autoimmune encephalitis (EAE) when administered to mice after immunization with myelin oligodendrocyte glycoprotein (MOG), at EAE onset, or at peak disease severity. DNP treatments attenuated infiltration of effector T cells into the CNS and suppressed innate and adaptive immune responses to myelin oligodendrocyte glycoprotein immunization in spleen. Therapeutic responses were not observed in mice treated with cargo DNA or cationic polymers alone, indicating that DNP uptake and cargo DNA sensing by cells with regulatory functions was essential for therapeutic responses to manifest. Intact STING and IFN-αβ receptor genes, but not IFN-γ receptor genes, were essential for therapeutic responses to DNPs to manifest. Treatments with cyclic diguanylate monophosphate to activate STING also delayed EAE onset and reduced disease severity. Therapeutic responses to DNPs were critically dependent on IDO enzyme activity in hematopoietic cells. Thus, DNPs and cyclic diguanylate monophosphate attenuate EAE by inducing dominant T cell regulatory responses via the STING/IFN-αβ/IDO pathway that suppress CNS-specific autoimmunity. These findings reveal dichotomous roles for the STING/IFN-αβ pathway in either stimulating or suppressing autoimmunity and identify STING-activating reagents as a novel class of immune modulatory drugs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αβ). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b(+) DCs were the only cells to express IFN-β, whereas CD11b(+) non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-β expression by CD11b(+) DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αβ) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αβ release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αβ production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.

Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4→B7 and PD-1→PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.

Inflammation stimulates immunity but can create immune privilege in some settings. Here, we show that cutaneous Leishmania major infection stimulated expression of the immune regulatory enzyme indoleamine 2,3 dioxygenase (IDO) in local lymph nodes. Induced IDO attenuated the T cell stimulatory functions of dendritic cells and suppressed local T cell responses to exogenous and nominal parasite antigens. IDO ablation reduced local inflammation and parasite burdens, as did pharmacologic inhibition of IDO in mice with established infections. IDO ablation also enhanced local expression of proinflammatory cytokines and induced some CD4(+) T cells to express interleukin (IL) 17. These findings showed that IDO induced by L. major infection attenuated innate and adaptive immune responses. Thus, IDO acts as a molecular switch regulating host responses, and IDO inhibitor drugs are a potential new approach to enhance host immunity to established leishmania infections.

Foxp3(+) regulatory T (Treg) cells can undergo reprogramming into a phenotype expressing proinflammatory cytokines. However, the biologic significance of this conversion remains unclear. We show that large numbers of Treg cells undergo rapid reprogramming into activated T helper cells after vaccination with antigen plus Toll-like receptor 9 (TLR-9) ligand. Helper activity from converted Treg cells proved essential during initial priming of CD8(+) T cells to a new cross-presented antigen. Help from Treg cells was dependent on CD40L, and (unlike help from conventional non-Treg CD4(+) cells) did not require preactivation or prior exposure to antigen. In hosts with established tumors, Treg cell reprogramming was suppressed by tumor-induced indoleamine 2,3-dioxygenase (IDO) and vaccination failed because of lack of help. Treg cell reprogramming, vaccine efficacy, and antitumor CD8(+) T cell responses were restored by pharmacologic inhibition of IDO. Reprogrammed Treg cells can thus participate as previously unrecognized drivers of certain early CD8(+) T cell responses.

A discrete population of splenocytes with attributes of dendritic cells (DCs) and coexpressing the B-cell marker CD19 is uniquely competent to express the T-cell regulatory enzyme indoleamine 2,3-dioxygenase (IDO) in mice treated with TLR9 ligands (CpGs). Here we show that IDO-competent cells express the B-lineage commitment factor Pax5 and surface immunoglobulins. CD19 ablation abrogated IDO-dependent T-cell suppression by DCs, even though cells with phenotypic attributes matching IDO-competent cells developed normally and expressed IDO in response to interferon gamma. Consequently, DCs and regulatory T cells (Tregs) did not acquire T-cell regulatory functions after TLR9 ligation, providing an alternative perspective on the known T-cell regulatory defects of CD19-deficient mice. DCs from B-cell-deficient mice expressed IDO and mediated T-cell suppression after TLR9 ligation, indicating that B-cell attributes were not essential for B-lymphoid IDO-competent cells to regulate T cells. Thus, IDO-competent cells constitute a distinctive B-lymphoid cell type with quintessential T-cell regulatory attributes and phenotypic features of both B cells and DCs.

TLR ligands are effective vaccine adjuvants because they stimulate robust proinflammatory and immune effector responses and they abrogate suppression mediated by regulatory T cells (Tregs). Paradoxically, systemic administration of high doses of CpGs that bind to TLR9 ligands stimulated Tregs in mouse spleen to acquire potent suppressor activity dependent on interactions between programmed death-1 and its ligands. This response to CpG treatment manifested 8-12 h and was mediated by a rare population of plasmacytoid dendritic cells (CD19(+) pDC) induced to express the immunosuppressive enzyme IDO after TLR9 ligation. When IDO was blocked, CpG treatment did not activate Tregs, but instead stimulated pDCs to uniformly express the proinflammatory cytokine IL-6, which in turn reprogrammed Foxp3-lineage Tregs to express IL-17. Thus, CpG-induced IDO activity in pDCs acted as a pivotal molecular switch that induced Tregs to acquire a stable suppressor phenotype, while simultaneously blocking CpG-induced IL-6 expression required to reprogram Tregs to become Th17-like effector T cells. These findings support the hypothesis that IDO dominantly controls the functional status of Tregs in response to inflammatory stimuli in physiological settings.

Topical application of phorbol myristate acetate (PMA) elicits intense local inflammation that facilitates outgrowth of premalignant lesions in skin after carcinogen exposure. The inflammatory response to PMA treatment activates immune stimulatory mechanisms. However, we show here that PMA exposure also induces plasmacytoid dendritic cells (pDCs) in local draining lymph nodes (dLNs) to express indoleamine 2,3 dioxygenase (IDO), which confers T cell suppressor activity on pDCs. The induced IDO-mediated inhibitory activity in this subset of pDCs was potent, dominantly suppressing the T cell stimulatory activity of other DCs that comprise the major fraction of dLN DCs. IDO induction in pDCs depended on inflammatory signaling by means of IFN type I and II receptors, the TLR/IL-1 signaling adaptor MyD88, and on cellular stress responses to amino acid withdrawal by means of the integrated stress response kinase GCN2. Consistent with the hypothesis that T cell suppressive, IDO(+) pDCs elicited by PMA exposure create local immune privilege that favors tumor development, IDO-deficient mice exhibited a robust tumor-resistant phenotype in the standard DMBA/PMA 2-stage carcinogenesis model of skin papilloma formation. Thus, IDO is a key immunosuppressive factor that facilitates tumor progression in this setting of chronic inflammation driven by repeated topical PMA exposure.

Scurfy mice develop CD4 T-cell-mediated lymphoproliferative disease leading to death within 4 weeks of age. The scurfy mutation causes loss of function of the foxp3 gene (foxp3(sf)), which is essential for development and maintenance of naturally occurring regulatory CD4 T cells (nTregs). In humans, mutations of the foxp3 gene cause immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX). In most patients with IPEX and also in scurfy mice, T cells show hyperreactivity and levels of Th1- and Th2-associated cytokines are substantially elevated. We report that removal of CD28 expression rescued scurfy mice from early death. Longer-term surviving CD28-deficient scurfy mice still had lymphoproliferative disorder, but their CD4 T cells showed decreased interferon-gamma and no sign of interleukin-4 or interleukin-10 hyperproduction. Furthermore, injection of CTLA4-Ig to block CD28-B7 interactions substantially improved the survival of scurfy mice by blocking effector T-cell differentiation. These data support the hypothesis that CD28-B7 interactions play a critical role in the etiology of lethal autoimmune disease in scurfy mice by stimulating the differentiation of antigen-activated naive T cells into effector T cells.

Following CD80/86 (B7) and TLR9 ligation, small subsets of splenic dendritic cells expressing CD19 (CD19(+) DC) acquire potent T cell regulatory functions due to induced expression of the intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan. In CD19(+) DC, IFN type I (IFN-alpha) is the obligate inducer of IDO. We now report that IFN-alpha production needed to stimulate high-level expression of IDO following B7 ligation is itself dependent on basal levels of IDO activity. Genetic and pharmacologic ablation of IDO completely abrogated IFN-alpha production by CD19(+) DC after B7 ligation. In contrast, IDO ablation did not block IFN-alpha production by CD19(+) DC after TLR9 ligation. IDO-mediated control of IFN-alpha production depended on tryptophan depletion as adding excess tryptophan also blocked IFN-alpha expression after B7 ligation. Consistent with this, DC from mice deficient in general control of non-derepressible-2 (GCN2)-kinase, a component of the cellular stress response to amino acid withdrawal, did not produce IFN-alpha following B7 ligation, but produced IFN-alpha after TLR9 ligation. Thus, B7 and TLR9 ligands stimulate IFN-alpha expression in CD19(+) DC via distinct signaling pathways. In the case of B7 ligation, IDO activates cell-autonomous signals essential for IFN-alpha production, most likely by activating the GCN2-kinase-dependent stress response.

Antigen stimulation of lymphocytes induces upregulation of phospholipase D (PLD) activity, but the biological significance of PLD-mediated signaling in T cells has not been well established. Here we demonstrate that PLD signaling is essential for proliferation of mouse CD8(+) T cells and CD4(+)CD25(-) T cells, but is not required for proliferation of CD4(+)CD25(+) regulatory T cells. We exploited this observation to develop an efficient method to enrich for regulatory T cells starting from preparations of total CD4(+) T lymphocytes. Inhibition of PLD signaling blocked effector T-cell proliferation after T cell-antigen receptor (TCR) engagement, but had no significant effect on the proliferation of CD4(+)CD25(+) T cells with regulatory functions. Consequently, cells expanded in vitro for one week by antigen receptor stimulation with PLD signal inhibition were markedly enriched for regulatory T cells.

CpG oligodeoxynucleotides (CpG-ODNs) stimulate innate and adaptive immunity by binding to TLR9 molecules. Paradoxically, expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is induced following i.v. CpG-ODN administration to mice. CpG-ODNs induced selective IDO expression by a minor population of splenic CD19+ dendritic cells (DCs) that did not express the plasmacytoid DC marker 120G8. Following CpG-ODN treatment, CD19+ DCs acquired potent IDO-dependent T cell suppressive functions. Signaling through IFN type I receptors was essential for IDO up-regulation, and CpG-ODNs induced selective activation of STAT-1 in CD19+ DCs. Thus, CpG-ODNs delivered systemically at relatively high doses elicited potent T cell regulatory responses by acting on a discrete, minor population of splenic DCs. The ability of CpG-ODNs to induce both stimulatory and regulatory responses offers novel opportunities for using them as immunomodulatory reagents but may complicate therapeutic use of CpG-ODNs to stimulate antitumor immunity in cancer patients.

By ligating CD80/CD86 (B7) molecules, the synthetic immunomodulatory reagent CTLA4-Ig (soluble synthetic CTLA4 fusion protein) induces expression of the enzyme indoleamine 2,3-dioxygenase (IDO) in some dendritic cells (DCs), which acquire potent T cell regulatory functions as a consequence. Here we show that this response occurred exclusively in a population of splenic DCs co-expressing the marker CD19. B7 ligation induced activation of the transcription factor signal transducer and activator of transcription (STAT1) in sorted CD19+, but not CD19(NEG), DCs. STAT1 activation occurred even when DCs lacked receptors for type II IFN (IFNgamma); however, STAT1 activation and IDO up-regulation were not observed when DCs lacked receptors for type I IFN (IFNalphabeta). Thus, IFNalpha, but not IFNgamma, signaling was essential for STAT1 activation and IDO up-regulation in CD19+ DCs following B7 ligation. Consistent with these findings, B7 ligation also induced sorted CD19+, but not CD19(NEG), DCs to express IFNalpha. Moreover, recombinant IFNalpha induced CD19+, but not CD19(NEG), DCs to mediate IDO-dependent T cell suppression, showing that IFNalpha signaling could substitute for upstream signals from B7. These data reveal that a minor population of splenic DCs expressing the CD19 marker is uniquely responsive to B7 ligation, and that IFNalpha-mediated STAT1 activation is an essential intermediary signaling pathway that promotes IDO induction in these DCs. Thus, CD19+ DCs may be a target for regulatory T cells expressing surface CTLA4, and may suppress T cell responses via induction of IDO.

The male-specific minor histocompatibility antigen H-Y plays an important role in both graft rejection and graft-versus-host disease following transplantation of male tissue into females that are completely matched at the major histocompatibility loci. The recent identification of two peptides that, in association with the mouse H-2Kk or human HLA B7 major histocompatibility class I molecules, are recognised by H-Y-specific T cells, has provided evidence for the molecular basis for such anti-H-Y responses. These peptides are encoded by the mouse and human homologues of a ubiquitously expressed Y chromosome gene, Smcy, whilst the equivalent peptides encoded by the X chromosome homologues of this gene fail to be recognised. Genetic studies have demonstrated that, as is the case for other minor histocompatibility antigens, peptide epitopes from several closely linked genes may be required to interact in order to elicit a response against H-Y. Definition of the peptides and the genes that encode these epitopes will allow the development of tolerogenic protocols that could specifically down-modulate the response to H-Y and perhaps even other minor histocompatibility antigens.

Single amino acid disparities in MHC class I molecules can elicit transplantation responses. Since beta 2 microglobulin (beta 2m) is noncovalently associated with class I antigens on the cell membrane we investigated whether the single amino acid polymorphism at position 85 (Asp-->Ala) in the mouse beta 2m molecule can cause skin graft rejection. A B2mb transgene was introduced into CBA(B2ma) mice which subsequently expressed both forms of beta 2m. Skin from these CBA beta 2mb transgenic mice was not rejected by the parental CBA strain. Previous studies showed that cytotoxic T lymphocyte (CTL) responses directed against beta 2mb use H2Kb as a restriction element. We therefore produced mice expressing H2Kb and H2Ab as well as beta 2mb by crossing CBA.beta 2mb mice with either CBA.Kb (CBK) transgenic mice or C3H.SW mice and used these as skin graft donors for beta 2mb negative littermates. In both cases rejection of transgenic skin only occurred when mice had received both a beta 2mb graft and an H2-disparate allograft lying adjacent in the same site. Introduction of the male specific antigen, H-Y, as a helper determinant did not result in rejection of beta 2mb skin. Neither did two CTL determinants (P91A and beta 2mb) on the same graft complement one another to elicit a transplantation response. Prior immunisation with tissues expressing the beta 2m disparity alone did not generate in vivo or in vitro beta 2mb-specific CTL responses, suggesting that this single amino acid difference is not sufficient to elicit a CTL or helper T cell response.

Transgenic CBA (H-2k haplotype) mice expressing the H-2 Kb major histocompatibility complex (MHC) class I gene under control of transcriptional promoter elements from a milk protein gene display high-level H-2 Kb transcription in lactating mammary glands and low-level transcription in skin and thymus of male and virgin female transgenic mice. However, H-2 Kb antigen could be detected only in lactating mammary gland epithelial cells by immunohistological methods. All transgenic mice are tolerant of H-2 Kb since they fail to reject skin grafts from mice expressing H-2 Kb molecules. Furthermore, anti-H-2 Kb cytotoxic responses could not be generated using responder T cells from transgenic mice but T cells from the same mice proliferated, in the presence of interleukin-2, in response to stimulator cells expressing H-2 Kb. Tolerance to H-2 Kb is induced in the thymus since CBA mice grafted with thymus tissue from transgenic mice fail to reject H-2 Kb disparate skin grafts. However, experiments with double-transgenic mice also expressing a T cell receptor with anti-H-2 Kb specificity reveal that tolerance induction is not brought about by elimination of thymocytes bearing H-2 Kb-reactive receptors. Instead, a non-deletional mechanism which results in down-modulation of both CD8 and T cell receptor expression in peripheral T cells correlates with the induction of tolerance in these mice. These data reveal that extremely low levels of self-antigen expression in the thymus are sufficient to induce tolerance via non-deletional mechanisms.

Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

The secondary cytotoxic responses to the male specific antigen (H-Y) in in mice show H-2 restrictions so that cytotoxic female cells must share K and/or D end antigen with the male target cells. The production of cytotoxic cells is under the control of Ir genes, thus offering the possibility of studying the function of Ir genes in H-2-restricted cytotoxic responses. There are two kinds of Ir genes regulating this response; the dominant gene in the H-2b haplotype and complementary genes in other haplotypes. Now we have been able to map the dominant gene and some of the complementary genes: the dominant genes is in IAb, and in H-2k/H-2d complementation, the Ir genes are in ICk and ICd, and in H-2k/H2s and H-2k/H-2q complementations, at least the H-2k gene is in IC.

The ability of female mice to rapidly reject syngenic male skin grafts is largely determined by dominant genes in the IB region of the H-2b halotype, whereas the ability to produce anti-H-Y cytotoxic cells is determined by a dominant gene in the IA region the H-2b halotype, or by complementary genes in the IC region of some other haplotypes. Thus, it seems that H-2-retricted anti-H-Y cytotoxic T cells are not responsible for the rejection of syngeneic male skin grafts.