Emerging Immune Context

Abstract

Historically, in the microbiological contamination control (MCC) of small molecule drugs (SMDs) and large volume parenterals (LVPs), the adaptive immune response was of little concern. Fever, as a systemic response, as measured first in a rabbit then as correlated in an in vitro horseshoe crab blood test, has been the gauge used to monitor endotoxin presence as a contaminant. The differences between biologics and small molecule drugs (SMDs) that came before are detailed in this chapter and the next chapter. Major differences to be borne in mind here include: (i) the size and complexity of biologic molecules that exert their therapeutic influence by interacting with receptors on the outside of cells, (ii) the propensity for immune related responses and (iii) the potential for PAMPs to act as co-stimulatory signals to adaptive immunity (i.e. the “second signal” as described in Janeway’s 1989 lecture and as, in some cases, synonymous with the adjuvant effect of vaccinology). The latter provides a way to understand the immune stimulating effects of endotoxin that is separate from its historical role as “only a pyrogen”.

Appendices

Milestone Publications in Bridging Innate and Adaptive Immunity

Listed here are some landmark papers of interest that revolve around the discovery of the connectedness of mammalian innate and adaptive immune responses.

The first presentation of the idea of non-clonal recognition in lymphocyte activation.

• Immune activation of B cells: evidence for ‘one nonspecific triggering signal’ not delivered by the Ig receptors, Coutinho, A. & Moller, G., Scand. J. Immunol. 3, 133–146 (1974)

Conceptualization of “pattern recognition receptors” in the detection of PAMPs

• Approaching the asymptote? Evolution and revolution in immunology, Janeway, C. A. Jr., Cold Spring Harb. Symp. Quant. Biol. 54, 1–13 (1989)

Discovery of first human TLR (TLR4) and activation of TLR4 to induce expression of cytokines and costimulatory molecules (B7.1) thus linking innate and adaptive immunity.

• A human homologue of the Drosophila Toll protein signals activation of adaptive immunity, Medzhitov, R., Preston-Hurlburt, P. & Janeway, C. A. Jr., Nature 388, 394–397 (1997)

Three papers contributing to the initial discovery of LPS as TLR4 activating ligand.

• Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene, Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B. & Beutler, B., Science 282, 2085–2088 (1998)

• Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4), Qureshi, S. T., Lariviere, L., Leveque, G., Clermont, S., Moore, K. J., Gros, P. & Malo, D., J. Exp. Med. 189, 615–625 (1999)

• Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product, Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K. & Akira, S., J. Immunol. 162, 3749–3752 (1999)

Demonstration of TLR gene polymorphism as influencer of human physiologic response.

• TLR4 mutations are associated with endotoxin hyporesponsiveness in humans, Arbour, N. C., Lorenz, E., Schutte, B. C., Zabner, J., Kline, J. N., Jones, M., Frees, K, Watt, J. L. & Schwartz, D. A., Nat. Genet. 25, 187–191 (2000)

Demonstration of critical function for TLRs in immune cell maturation and induction of adaptive immune responses.

• Toll-like receptors control activation of adaptive immune responses, Schnare, M., Barton, G. M., Holt, A. C., Takeda, K., Akira, S. & Medzhitov, R., Nat. Immunol. 2, 947 (2001).

Demonstration that immunoglobulin G2a-chromatin immune complexes synergistically engage antigen receptor and TLR9 to lead to antibody production.

• Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors, Leadbetter, E. A., Rifkin, I. R., Hohlbaum, A. M., Beaudette, B. C., Shlomchik, M. J. & Marshak-Rothstein, A., Nature 416, 603–607 (2002)

Determination of crystal structure of bound ligands (LPS PAMPs) in TLR4 complex activation

• Crystal Structure of the TLR4-MD-2 Complex with Bound Endotoxin Antagonist Eritoran, Ho Min Kim, Beom Seok Park, Jung-In Kim, Sung Eun Kim, Judong Lee, Se Cheol Oh, Purevjav Enkhbayar, Norio Matsushima, Hayyoung Lee, Ook Joon Yoo, and Jie-Oh Lee, October 2007, Cell 130(5):906–17

Appendix I

Foretelling of subsequent immune discoveries (Janeway’s 1989), paraphrased for brevity from: Approaching the asymptote: Evolution and Revolution in Immunology, Cold Spring Harb Symp Quant Biol. 1989. 54: 1–13, Janeway, Pillars of Immunology.

a	Infectious agents are highly variable in structure, and their short generation time allows them to alter their structure quickly. This is especially true of their protein structure, which can diversify remarkably even during an infection within an individual…
b	Burnet realized the necessity for the immune system to develop mechanisms to generate an essentially open and unlimited repertoire of receptors, since evolutionary selection could not provide the immune system with receptors that would recognize the hemagglutinin molecules of next year’s strain of virus.
c	…T cell receptors derive from a separate and unique set of rearranging gene segments… these two sets (TCR and BCR) of rearranging genes arose only once and by duplications they diverged…
d	…the ability to deliver the second signal is induced on host APCs by infectious agents as the result of a distinct type of immunological recognition specific for microorganisms, but not resulting from clonally distributed receptors.
e	I raise the possibility that the second signals arose prior to the development of specific antigen recognition. Under this construction, second signals may be viewed more as positive initiators of immunity than as late adaptations to avoid autoimmunity.
f	Why do we need to use adjuvants? …it seems likely that these substances are required to provide two attributes not found in soluble proteins. The first is effective antigen uptake into macrophages, thus increasing ligand density in the form of peptides derived from the foreign protein bound to class I MHC molecules. The second is the provision of costimulatory activity, induced in macrophages and/or B cells by the bacterial constituent of the adjuvant.
g	If effector mechanisms used by lymphocytes in contemporary vertebrate immune systems derived from primitive immune systems lacking the rearranging receptor gene families that allow for clonal selection, how was effector function regulated in primitive organisms? The most likely possibility is that primitive effector cells bear receptors that allow recognition of certain pathogen-associated molecular patterns (PAMPs) that are not found in the host. I term these receptors pattern recognition receptors.
h	I argue that PAMPs are still an important part of vertebrate immune systems… I propose that these pattern recognition systems activated effector functions of primitive immune systems prior to the development of rearranging gene families and continue to play a role in host defense today.
i	What kinds of ligands or patterns should such non-clonally distributed receptors recognize? I think it likely that such receptors will recognize general structural patterns in molecules found in many microorganisms, but not in the multicellular organisms… The pattern recognized should be the product of a complex and critical enzymology in the microorganism. Complex cell wall carbohydrates or LPS are likely ligands.
j	The well-known responses of B cells to polyclonal B-cell activators (also called mitogens), such as LPS, presumably represent the action of non-clonally distributed pattern recognition receptors on the B cell.
k	…lymphocyte-activating signals derived from other host cells, evolved prior to the development of rearranging receptor genes. The substances that trigger second-signal expression, such as LPS, do so very rapidly and do not require recognition by rearranging, clonally distributed receptors… Such receptors must be distributed non-clonally on all APCs.