Chapter 8: T-Cell mediated Immunity
Chapter 8: T-Cell mediated Immunity
Slide 1 – There are some errors on this, one really important error is it indicates that CTLs are only CD8, however TH1 cells are capable of lysing virally infected cells or tumors or allogenic cells expressing class II MHC antigens. CD4 TH2 cells are not cytolytic, TH17 are inflammatory but have not been shown to have killer activity, so the TH1 cells do have Fas – Fas ligand interactions and that is how they kill, they also secrete TNF α which is capable under the right circumstances, of killing cells. The classic CTLs that were described in the early 80’s were characterized as CD8 cells, and they chiseled it in stone that CD8 cells are cytolytic, but there are also regulatory CD8 cells which aren’t shown. CD8 will kill allogeneic cells, virus infected cells, and they are required for eliminating many tumors, viruses, as are CD4 TH1 cells.
It’s really important to have an effective way of dealing with invading pathogens. For viruses and intracellular parasites, T-cells are how you eliminate them. For other pathogens, generating antibody is important because antibody can neutralize toxins, opsonize bacteria, fix complement, and if it is a gram negative bacteria you can even punch holes. TH17 cells not only enhance the neutrophil response, but they are profoundly involved in inflammatory autoimmune diseases. In MS, it is the TH17 cell which is centrally involved in the sequential waves of inflammation which result in loss of neuron function and increased disability.
Now remember for every action, there is an equal and opposite reaction, one of the ways is through regulatory cells. These secrete, among other things, TGF β which is able to shut down immune responses. Now another way to shut down immune responses is to get rid of the antigen presenting cell, and the T regs are capable of eliminating APCs.
In your skin, if you have a break, bacteria can get in, the local dendritic cell, the langerhaans cell, picks up fragments of the bacteria or virus, brings it to the draining lymph node and in the draining lymph node T-cells are recruited. T-cells normally circulate and do immune surveillance but they are recruited to the lymph node when there are APCs. There are specific receptors on the high endothelial venules which are the capillaries of lymph nodes and these look different to lymphocytes than do the receptors which are expressed on the capillaries in tissues where inflammatory responses are occurring. There are different CAMs that are recognized. Naïve T-cells want to enter the lymph node because in the lymph node and the spleen they will see antigen, differentiate, and be released into circulation, or if there role is to assist B-cells, they will do so in the lymph node.
In any tissues you are going to have antigen specific T-cells released, they are going to walk along the walls of capillaries and when they find inflammation, the inflammatory signals including chemokines and cytokines activate the endothelial cells along the capillaries and they display different markers, so instead of simply walking along, these cells stop, are activated and enter the tissue. In order to move through the capillary wall it must be able to reorganize its cytoskeleton, it must be able to secrete matrix proteinases because there are many tight junctions to be opened and there is all this extracellular matrix that the lymphocyte must be able to get through. The cell does not simply swim, it must be able to destroy the extracellular matrix with specific proteases that are able to cleave the extracellular matrix. A naïve cell does not express the surface molecules which are capable of recognizing the activated tissues, it only expresses CAMs which recognize the HEV, but it’s a similar process because a naïve cell will recognize the HEV and it will be bound and go through to enter the lymph node just as a differentiated cell will go through activated endothelium. A naïve T-cell has L-selectin on its surface, and this can bind several different cell adhesion molecules. The L-selectin binds and recognizes terminal sugars on the CAMs, not by recognizing amino acid sequences, which is what happens with the other recognition molecules. So on the HEV there are these two cell adhesion molecules, GlyCAM and CD34 which are able to grab a naïve T cell, and those cells which are supposed to be living in the mucosa bind MadCAM on the mucosal endothelium.
If you don’t have either ICAM or LFA-1, which is the integrin which is αL β2, then T cells are incapable of recognizing antigen presenting cells, of entering the lymph nodes and the individual will have profound immunodeficiency. LFA-1 was defined many years ago, around 1980, as lymphocyte functional antigen. It was discovered using monoclonal antibodies in an attempt to determine which monoclonal antibody would prevent T-cell differentiation and APC recognition. Through this LFA-1 was discovered, and it was later found that the α and β chain was a common in the entire family of integrins, and there are many different alpha molecules and many different beta molecules, some of which are capable of interacting together, others of which have a very discrete interaction. But this one, VLA-4, very late antigen 4, is critical for T-cells to bind activated endothelium and then enter tissues. VLA-4 is so important, that when a monoclonal antibody is infused into an animal you can prevent the animal model of MS, and it was so effective that is was humanized and is now called tsaverin and in clinical therapy for MS and Crohns disease and possibly rheumatoid arthritis and other autoimmune diseases where T-cells are the critical mediators.
VLA-4 recognizes V-CAM and you can see that V-CAM and I-CAM are similar in primary structure, but clearly the terminal domain is different because it is recognized by different integrins. I-CAM is also the receptor for the cold virus, rhinovirus, and another molecules derived from this family of proteins is the polio virus receptor, and only humans have the polio virus receptor.
This slide, pg. 4 slide 2, looks at the partners between the cell adhesion molecules and other molecules. ICAM is present on vessels and dendritic cells and its ligand is LFA-1 and also Mac-1 which is on macrophages. Mac-1, like LFA-1 is an integrin, but it has a different β chain. DC-SIGN is present on dendritic cells of the myeloid lineage and it’s highly unusual that ICAM3 can see DC-SIGN which has a very different structure from LFA-1 but LFA-3 sees CD2 and V-CAM and VLA-4 are a receptor ligand pair.
So you’ve seen this many times, pg. 5 slide 1, whether it’s a T-cell or a macrophage, the same basic thing is true. Normally, lymphocytes roll along the endothelial wall with these selectin molecules recognizing the cell adhesion molecules and this brings the cells close enough so that if there are activation molecules including chemoattractant molecules those lymphocytes are capable of recognizing them if they have a receptor for those chemokines. If they do, and they get activated this triggers a change in the integrins so that the integrins are then able to bind on ICAM and then the cell gets further activated and diapedesis takes place.
Now we are going to talk about the different antigen presenting cells; there are dendritic cells in tissues which have a resting phenotype until they get activated, so a resting dendritic cell is waiting for a pathogen to come and when those PAMPs interact with the toll like receptors or the damage receptors the dendritic cells are activated and leave the tissue and they enter the blood and in the blood they become more mature and activated and they reach the lymph node where they have a fully activated phenotype and start interacting with the t-lymphocytes and activating them and triggering an adaptive immune response. There are 3 different kinds of professional antigen presenting cells, the dendritic cell is the most effective APC, it stimulates immune responses by T-cells, there are different varities of dendritic cells (the myeloid and the plasmacytoid and several others which we won’t discuss) but when they leave the tissues, they enter the T-cell dependent regions of the lymph nodes. Macrophages are found in many different regions of tissues, they have different names, and like the dendritic cells they are in a resting state normally, they can also get activated by receptors and PAMPs and PRRs and activated toll like receptors activate the macrophages and they travel into the lymph nodes as well. B lymphocytes can also present antigen. They present antigen differently and very poorly because they are not phagocytic, they are not endocytitic but they constitutively express class II molecules, they constitutively express their unique antibody molecule, and if they encounter a pathogen that can bind that, it is taken in by receptor mediated endocytosis, it is broken up and it is presented in the lymph node to T-cells, so any other antigen that happens to be found on a complex with this specific antigen bound by the receptor, the antibody, can also be presented.
Pg. 6 slide 2: by conventional they mean myeloid dendritic cell and these express DC SIGN molecules, a number of different chemokines, they express B7.1 and B7.2 and I CAMs which are critical for the interaction between T cells and dendritic cells; they express MHC I and II molecules and they are very effective at presenting antigens. Plasmacytoid dendritic cells also express class I and II, they express a different variety of chemokine receptors and they express toll like receptors; these are the ones that are activated rapidly when a pathogen comes in, they secrete large quantities of interferon beta which is critical for the bridge between the innate and adaptive immune response. For a viral infection, within 24 hours there are high levels of interferon beta circulating in the host.
How do dendritic cells present antigen? There is receptor mediated endocytosis and phagocytosis, there is pinocytosis which is “drinking”, viruses can get in and infect and express their gene products in the cytoplasm of dendritic cells. Dendritic cells are also capable of taking up fragments of dying cells. So, for example, if in the region near a dendritic cell, another cell has been infected and its dying because its infected it will undergo apoptosis. During apoptosis, they release bubbles of their cytoplasm, and these get taken up by specific receptors on the dendritic cells and those dendritic cells present those antigens present in the cytoplasm. Of course, it is possible for one cell to transmit material to another. Both class I and class II molecules are critical for this antigen processing and presentation and if class II molecules are what is used to present the foreign peptide, then generally it will activate CD4 cells. If class I molecules are used then CD8 t cells are activated.
Pg. 7 slide 2 – We’ve seen this before, the break in the skin, the dendritic cell takes up antigen and processes it, but what this is showing is that when the dendritic cell is activated by the pathogen it begins expressing B7, and B7 enables the CD28 expressing T cells to bind more avidly, provides the second signal and the third signal is cytokine.
Pg. 8 slide 1 – I don’t know that this tells us anything more than that the pathogen binds to the myeloid dendritic cell and this dendritic cell is activated by its PAMPs, starts to present antigen on its cell surface, the chemokine receptors change as the cell migrates into the lymph nodes, it follows a gradient of chemokines that the lymph nodes secrete to recruit those cells, it goes from not expressing b7 to expressing b7 and then being able to interact in a functional way with a t-cell.
As said before, B-cells are very good at presenting antigen that they are capable of binding with receptor mediated endocytosis. They have the same cathepsins in their endosome, they are able to cleave the peptides and the peptides are then able to bind onto class II with the assistance of H2D HLA DM, and then present those peptides on the surface.
Dendritic cells are the best, they constitutively express the co-stimulatory molecules, they are very good at presenting peptides of viral antigens, unfortunately they are good at presenting allergens as well and they are found through out the body in various tissues. Macrophages use phagocytosis to get things in, they do not generally express class II, but class II is inducible. Class I is constitutively expressed, they both make lots of IL-12 and IL-23 to stimulate t-lymphocytes. The co-receptors, the B7s are inducible but not constitutively expressed and they are found largely in the peritoneum but also in tissues, but in the peritoneum they are very important. B cells, the antigen receptor is critical, they constitutively express MHC, they constitutively express co-stimulatory molecules, but they are not nearly as efficient as dendritic cells. Macrophages also secrete so many other molecules including prostaglandins that can shut off immune responses and cause pathology with the release of reactive oxygen and nitrogen intermediates, as well as lots of the proteolytic enzymes that can be released into tissue, so you really don’t want lots of activated macrophages.
Pg. 10 slide 1 – this basically shows you what you had in that table a few slides ago. It’s the interaction between the T-cell and the APC and what’s on the APC and what’s on the T-cell.
Pg. 10 slide 2 – this talks about the necessity, because the MHC t-cell receptor interaction, even with the association of the co-receptors is so poor, that you really need to have the Velcro of the I CAM and LFA to enable those cells to get close enough to determine if there is going to be interaction, but you also need a second signal. You need the interaction between B7 and CD28, but then there is a cross-talk of cytokines, this third signal, is absolutely critical. If you don’t get the second and third signal the naïve t-cell will not divide and differentiate. If you have the interaction of B7 and CD28 without t-cell receptor interaction, there is no activation. If there is an absence of cytokines, there will be no differentiation.
We talked about some of the cytokine receptors and cytokine signaling before, but this (pg. 11 slide 1) talks about two very important things. One is that the alpha chain of the IL-2 receptor complex is an inducible gene product, and this interacting with the β and γ provides a high affinity receptor for IL-2, and so you only need a little IL-2 in the vicinity to turn that T-cell on. Since differentiated T-cells express this, they also secrete IL-2, they have an autocrine pathway promoting their division. IL-2 was originally described as T-cell growth factor and it is critical for mitosis of activated cells. Another important part of this slide is you see that the γ chain of the receptor is present in both naïve and activated t-cell and the γ chain is commonly used by several different cytokines. A number of cytokines have heterodimer or trimers as receptors and recycle one of the gene products for this. One of the forms of SCID lacks the gamma chain. You can have T-cell receptors and antibody expressed on B cells but there will be no response in the absence of an IL-2 signal and the cytokines that share this γ chain. A resting T lymphocyte expresses the low affinity receptor for IL-2, the T-cell gets activated and it induces the expression of the high affinity receptor, and this really drives the T-cell in cell division.
One of the ways of countering the activation by the B7 CD28 interaction is the alternative expression of T-cells of CTLA-4. CTLA-4 also binds B7 but it has an ITIM not an ITAM domain in the cytoplasm and depending on the expression level of CTLA-4 and the number of CD28 molecules on the surface of the cell, and whether or not the CTLA-4 gets engaged, although the T-cell receptor may be stimulated, that T-cell does not get activated because it will have too many negative signals. If there is no engagement of the CD28 and B7 then that T cell gets the signal that it should be anergic which means that it is incapable of response. So if you have a T-cell receptor interaction with the MHC, but you don’t have the second signal, you get anergy. If you don’t have the third signal you’ll still have a cell which can be activated, it won’t die or become anergic, but it will not divide and differentiate until it receives that signal.
What are the effector functions of T-cells? We know that lots of T-cells can secrete lots of cytokines and can have profound effects on the local environment of the surrounding cells, but they also have the differentiation of CD8 and CD4 TH1 cells that get activated and then express perforin and granzyme, and when this T cell is released from the lymph node or spleen, it is released to do immune surveillance, and it looks for a virally infected cell, and if it binds that virally infected cell and the t-cell receptor interacts with its antigen the T-cell forms a tight association with the infected cell and releases the contents of its vesicles at that tight junction. The perforin forms pores on the target cell which enables granzyme to get in, and granzyme activates the caspase cascade and that cell undergoes apoptosis. The T-cell can synthesize more granzyme and perforin, and go on to find another infected cell. The interaction that signals the T-cell to kill is highly specific, those CTLs do not kill uninfected neighboring cells when they recognize class I or II. There are, however, cells that can release TNF or lymphotoxin and these will bind to their respective receptors and that is capable of inducing the apoptotic cascade and killing neighboring cells.
If you want to go into a lymph node, if you are a resting cell, you must express the selectins that are bound by the cell adhesion molecules expressed on the high endothelial venule so L-selectin is found on resting cells but not activated cells, whereas VLA-4, which is going to be targeting differentiated cells to tissues, is expressed on activated and differentiated T-cells. Both express LFA-1 which is critical for interaction, but we know that when T-cells get activated and differentiate they express more LFA-1 and CD-2. The resting cell has the RA form of CD45, the RO form is expressed on the activated cell. There are different chemokine receptors that are expressed on either kind, and depending upon where the cells are going to be attracted, different chemokine receptors are important, but sphingomyelin 1 phosphate receptor (s1PR) is critical for resting cells and in fact there is a new series of therapies that are directed at the sphingomyelin 1 receptor, and it works to keep cells in lymph nodes and preventing them from egressing. Why would you want that? If you have a huge inflammatory response occurring and you want to shut it down, this may be beneficial transiently, but if this is done persistently there is no way to fight invading pathogens. Another therapy is to block VLA-4, but that also blocks any immune response against a pathogen. There are finer addressins that indicate tissue specificity, and those may be targeted in the future.
CTL’s recognize virally infected cells or tumors, and they kill it. They can be CD8 or CD4 TH1. Another very important thing that TH1 cells do is that they activate macrophages and macrophages can then destroy intracellular bacteria because they make nitric oxide, proteases and other inflammatory molecules and destroy the bacteria that are inside the vesicles, generally by combining with the lysosomes. TH1 cells also make a lot of cytokines, and they are responsible for delayed hypersensistivity. One example of delayed hypersensitivity is the reaction to poison ivy, oak or sumac. You only need 1 T-cell which is capable of recognizing the modified cell in the tissue where the person has been exposed to poison ivy, that one T-cell will bring the cascade of inflammation to the site, and you have over 48 hours the development of redness, itchiness, the vesicle formation and this is why antihistamines don’t work for poison ivy, because it is a T cell mediated hypersensitivity.
Both TH1 and TH2 cells secrete a variety of cytokines and when B cells are being activated by their specific antigen, and get those third signals from the Tcells then those B cells undergo class switch and affinity maturation and makes lots of specific antibody and some will be instructed to become memory cells and be their for the rest of your life.
When TH17 cells get activated they cause a huge inflammatory response. They make many cytokines, but IL17 is their characteristic cytokine and it is not made by any other cell type.
T regs secrete TGF β and this suppresses the differentiation of dendritic cells and because dendritic cells are unable to differentiate and present antigen, then there is no induction of new responses. T regs are generally highly specific during the initial interaction, but they make so much TGFβ that other cells get suppressed as well.
There are a whole variety of these ligands which can enhance immune responses. CD40 – CD40 ligand, 4-IBB – 4 IBB ligand is another pair and all of these are important for the stimulation of cells. So we talked about the third signal and how it can affect the differentiation of cells, so this (pg. 15 slide 2) some of those third signals and it lists the critical transcription factors that are characteristically in each of these cell types. So TH2 cell types all have GATA 3 as a transcription factor which is characteristic of TH2 cells. IL-4 is important for the production of GATA-3 and these cells make IL-4, IL-5, IL-10 and other cytokines as well. IL-12 is made by the dendritic cells and macrophages and this binds to the IL-12 receptor which causes a signal transduction cascade including STAT 4, STAT 4 is absolutely essential, and it induces the expression of T-bet, which enables that cell to make IFN-γ, and IFN-γ feeds back and makes more IL-12, so there is a positive feedback loop between IL-12 and IFN-γ and the dendritic cell or the macrophage and the TH1 cell. If the CD4 cell is differentiated in the presence of TGF β and IL-6 then ROR γT gets made and ROR γT is the transcription factor that is essential for the production of IL-17, these T cells also make IL-6 which is a differentiation factor for plasma cells. TGF β in the absence of IL-6 induces Fox P3, which induces more TGFβ, more IL-10, and this is T regulatory cell and this antagonizes both the IL-17 and IFNγ expressing cells. In the presence of IL-10 alone, which probably means that you had some TH2 cell and some Tregs in the vicinity, the IL-10 induces an unknown transcription factor which induces another kind of T-reg.
This (pg. 16 slide 2) has a number of different names, one of which is the supermolecular activation complex or the T-cell synapse, which was the original name. In the middle is the T-cell receptor, it is surrounded by a ring of ICAM interactions and then you have all the other CD40, CD40 ligand, CD28 B7 interactions, and this shows how much of the cell surface is involved in this transient tight junction, the “kissing” of those cells, if you don’t have the engagement of the T-cell receptor you will not trigger that cell, and if you don’t have the CD40 – CD40 ligand interactions or the CD28 – B7 interactions, because the cell being scanned by the T-cell does not express them, you get anergy. So a resting fibroblast, a resting endothelial cell, may have the viral antigen and certainly has ICAM but it doesn’t have the B7 or the CD40, the T-cell will be anergic, so only the professional APCs can stimulate a T cell.
The CTL, whether it’s a CD4 or a CD8 or an NK cell, the same basic thing happens. They bind a cell, the microtubular organizing center, the golgi, redistribute vesicles along the interface and then they get activated to release at that tight junction. The T-cell is immune from the perforin and the granzyme, but they are very effective at killing the target cells.
The table on pg. 18 slide 1 reiterates many of the important cytokines, not all of them, and I don’t expect you to memorize all aspects of this table, but clearly there are some things that they have highlighted here. We have said that IL-2 is T-cell growth factor and it is made by T-cells and it acts on T-cells. IFNγ can act on many cell types, it is made by TH1 cells and CD8 cells and its affect on B-cells (which depends on the B cell) can induce IgG2a synthesis in terms of class switching and on TH2 cells, the IFNγ receptor induces a negative signal and shuts them off. IFNγ activates TH1 cells, it activates CD8 cells, it activates macrophages to kill, it induces the expression of class II molecules on all of those cells capable of making class II MHC, it enhances the expression of class I on all those cells capable of making it (neurons, red blood cells, sperm are incapable) it activates NK cells. When you don’t have IFNγ individuals are very susceptible to infections. If you don’t have IL-2 then it is hard to differentiate and divide T-cells. If you don’t have IL-4 you don’t have TH2 cells and when you have IL-4 you get enhancement of mast cells, IgE antibodies and type I hypersensitivities, the standard allergies that people think about. IL-10 inhibits TH1 by inhibiting antigen processing and presentation, and in the absence of that inflammatory bowel disease occurs. IL-3 is one of those factors that goes to the bone marrow, so you have an immune response occurring, and the innate immune cells produce IL-3 which travels to the bone marrow and directs it to make more cells to help deal with the infection. TNF has many inflammatory consquences, and not shown in this table is its affect on appetite, it suppresses appetite and causes tumors to break down. A lot of TNF stimulates apoptosis, it activates endothelium, and in the absence of TNF you have poor differentiation of lymph nodes and high susceptibility to gram negative infections. TNF is a target of a number of monoclonal antibodies and soluble receptors, remicaid is one of those monoclonal antibodies, and they are used to modulate inflammatory responses, like in the case of rheumatoid arthritis and Crohn’s disease, it doesn’t work for MS. IL-17 stimulates fibroblast, endothelial cells, it is the inflammatory problem in a lot of autoimmune diseases.
We talked a lot about receptors and receptor signaling before, and this (pg. 18 slide 2) emphasizes all of the different families of molecules and how they look. One of the points is that this γc chain, which we discussed for IL-2, is found in IL-4, IL-7, IL-9, IL-15, and without it you have SCID. There are a whole bunch of homodimeric receptors, there are a lot of heterodimeric receptors, including interferon receptors. There are others that have a common chain and a unique chain. The TNF family of receptors are always homotrimeric, but there are different ones, and there is whole family of these homotrimeric members. The seven transmembrane g-coupled protein receptors are found not only for all of the chemokines but many other molecules. The lipid molecules like leukotrienes and prostaglandins use this, cannabinoids signal through this, and also small fragments of complement, C3a, C4a and C5a, signal through similar families of molecules.
So a CTL kills by inducing apoptosis through granzyme. How does apoptosis work? There is a series of serine proteases which act on one another and on pro-enzymes to make active enzymes, the mitochondrion gets targeted and if there is an excess of the negative signals on the mitochondrion vs. BCL-2 family of molecules then the mitochondrion gets disrupted and cytochrome C gets released into the cytoplasm, cytochrome C then joins this, actisome and activates the destruction of DNA in between the nucleosomes, which results in fragmentation of DNA, what this doesn’t show you is the boiling of the cell membrane and the release, on the cell surface of the inner leaflet. There are many ways to look for the presence of apoptosis, one is to look for active proteases, and you can do that for looking for the fragments or by looking for their activity, you can look for the integrity of the mitochondrion, because if the mitochondria are alive they can metabolize protein and make a colorimetric protein where it had been uncolored before, you can look for the breakdown of DNA in a variety of ways, one is to look for more 3’ hydroxy ends using the tunnel assay, you can look for the turning of the inner leaflet onto the cell surface. When apoptosis occurs in the lung, there are stem cells below the epithelium that will be the source of brand new polarized epithelial cells which will replace the dead and dying cells, and after a few days or weeks the epithelium is brand new.
To get the activation of the infected macrophage, macrophages respond to infection, it presents some of the antigen, is probably making some IL-12, it has induced CD40 expression and when this antigen specific T cell binds this infected macrophage, the initial reaction is T-cell receptor, ICAM based and then CD40 and CD40 ligand get involved and the T cell secretes IFNγ which enables the macrophage to differentiate its lysosomes which fuse to the phagosomes and destroy the bacteria. So activated macrophages make lots of reactive oxygen and nitrogen intermediates, they induce the expression of CD40, they upregulate their Class I and class II molecules, they induce expression of B7 molecules, they make lots of TNF, IL-1 and IL-6. Some of the TNF will feed back on the macrophages TNF receptors, others interact with other cells. So activated macrophages can be quite harmful, they release many of the proteolytic enzymes into the vicinity and there is tissue destruction as a result.
Activated CD4 TH1 cells make many things, like IFN. They can kill cells, they make Fas ligand, they produce IL-2 which is not only an autocrine factor but can also affect CD8 cells, IL-3 and GMCSF go to the bone marrow and induces the production of more precursor cells for macrophages and neutrophils and even a few megakaryocytes for clotting ability. TNF and lymphotoxin in small amounts activate cells, in large amounts they kill cells. Chemokines of all sorts are important for chemoattraction. So a TH1 cell that sees poison ivy is going to activate the blood vessels, recruit cells, and cause inflammation.
Another thing that CD4 cells do is form granulomas, they make cysts. So if you have an infection, the body will want to wall that infection off so that the bacteria can’t enter other tissues and so this granuloma is one of the important functions of T-cells and if it is active for long enough time you will get fibrin deposition, it will get hard, there will be liquification in the middle as the bacteria kill the cells inside, but the bacteria will be stuck there. One problem with a longstanding granuloma is that the fibrin wall can prevent antibiotics from percolating through because the fibrin can be impermeable to some antibiotics.
